yeast by thomas h. huxley i have selected to-night the particular subject of yeast for two reasons--or, rather, i should say for three. in the first place, because it is one of the simplest and the most familiar objects with which we are acquainted. in the second place, because the facts and phenomena which i have to describe are so simple that it is possible to put them before you without the help of any of those pictures or diagrams which are needed when matters are more complicated, and which, if i had to refer to them here, would involve the necessity of my turning away from you now and then, and thereby increasing very largely my difficulty (already sufficiently great) in making myself heard. and thirdly, i have chosen this subject because i know of no familiar substance forming part of our every-day knowledge and experience, the examination of which, with a little care, tends to open up such very considerable issues as does this substance--yeast. in the first place, i should like to call your attention to a fact with which the whole of you are, to begin with, perfectly acquainted, i mean the fact that any liquid containing sugar, any liquid which is formed by pressing out the succulent parts of the fruits of plants, or a mixture of honey and water, if left to itself for a short time, begins to undergo a peculiar change. no matter how clear it might be at starting, yet after a few hours, or at most a few days, if the temperature is high, this liquid begins to be turbid, and by-and-by bubbles make their appearance in it, and a sort of dirty-looking yellowish foam or scum collects at the surface; while at the same time, by degrees, a similar kind of matter, which we call the "lees," sinks to the bottom. the quantity of this dirty-looking stuff, that we call the scum and the lees, goes on increasing until it reaches a certain amount, and then it stops; and by the time it stops, you find the liquid in which this matter has been formed has become altered in its quality. to begin with it was a mere sweetish substance, having the flavour of whatever might be the plant from which it was expressed, or having merely the taste and the absence of smell of a solution of sugar; but by the time that this change that i have been briefly describing to you is accomplished the liquid has become completely altered, it has acquired a peculiar smell, and, what is still more remarkable, it has gained the property of intoxicating the person who drinks it. nothing can be more innocent than a solution of sugar; nothing can be less innocent, if taken in excess, as you all know, than those fermented matters which are produced from sugar. well, again, if you notice that bubbling, or, as it were, seething of the liquid, which has accompanied the whole of this process, you will find that it is produced by the evolution of little bubbles of air-like substance out of the liquid; and i dare say you all know this air-like substance is not like common air; it is not a substance which a man can breathe with impunity. you often hear of accidents which take place in brewers' vats when men go in carelessly, and get suffocated there without knowing that there was anything evil awaiting them. and if you tried the experiment with this liquid i am telling of while it was fermenting, you would find that any small animal let down into the vessel would be similarly stifled; and you would discover that a light lowered down into it would go out. well, then, lastly, if after this liquid has been thus altered you expose it to that process which is called distillation; that is to say, if you put it into a still, and collect the matters which are sent over, you obtain, when you first heat it, a clear transparent liquid, which, however, is something totally different from water; it is much lighter; it has a strong smell, and it has an acrid taste; and it possesses the same intoxicating power as the original liquid, but in a much more intense degree. if you put a light to it, it burns with a bright flame, and it is that substance which we know as spirits of wine. now these facts which i have just put before you--all but the last--have been known from extremely remote antiquity. it is, i hope one of the best evidences of the antiquity of the human race, that among the earliest records of all kinds of men, you find a time recorded when they got drunk. we may hope that that must have been a very late period in their history. not only have we the record of what happened to noah, but if we turn to the traditions of a different people, those forefathers of ours who lived in the high lands of northern india, we find that they were not less addicted to intoxicating liquids; and i have no doubt that the knowledge of this process extends far beyond the limits of historically recorded time. and it is a very curious thing to observe that all the names we have of this process, and all that belongs to it, are names that have their roots not in our present language, but in those older languages which go back to the times at which this country was peopled. that word "fermentation" for example, which is the title we apply to the whole process, is a latin term; and a term which is evidently based upon the fact of the effervescence of the liquid. then the french, who are very fond of calling themselves a latin race, have a particular word for ferment, which is 'levure'. and, in the same way, we have the word "leaven," those two words having reference to the heaving up, or to the raising of the substance which is fermented. now those are words which we get from what i may call the latin side of our parentage; but if we turn to the saxon side, there are a number of names connected with this process of fermentation. for example, the germans call fermentation--and the old germans did so--"gahren;" and they call anything which is used as a ferment by such names, such as "gheist" and "geest," and finally in low german, "yest"; and that word you know is the word our saxon forefathers used, and is almost the same as the word which is commonly employed in this country to denote the common ferment of which i have been speaking. so they have another name, the word "hefe," which is derived from their verb "heben," which signifies to raise up; and they have yet a third name, which is also one common in this country (i do not know whether it is common in lancashire, but it is certainly very common in the midland countries), the word "barm," which is derived from a root which signifies to raise or to bear up. barm is a something borne up; and thus there is much more real relation than is commonly supposed by those who make puns, between the beer which a man takes down his throat and the bier upon which that process, if carried to excess, generally lands him, for they are both derived from the root signifying bearing up; the one thing is borne upon men's shoulders, and the other is the fermented liquid which was borne up by the fermentation taking place in itself. again, i spoke of the produce of fermentation as "spirit of wine." now what a very curious phrase that is, if you come to think of it. the old alchemists talked of the finest essence of anything as if it had the same sort of relation to the thing itself as a man's spirit is supposed to have to his body; and so they spoke of this fine essence of the fermented liquid as being the spirit of the liquid. thus came about that extraordinary ambiguity of language, in virtue of which you apply precisely the same substantive name to the soul of man and to a glass of gin! and then there is still yet one other most curious piece of nomenclature connected with this matter, and that is the word "alcohol" itself, which is now so familiar to everybody. alcohol originally meant a very fine powder. the women of the arabs and other eastern people are in the habit of tinging their eyelashes with a very fine black powder which is made of antimony, and they call that "kohol;" and the "al" is simply the article put in front of it, so as to say "the kohol." and up to the 17th century in this country the word alcohol was employed to signify any very fine powder; you find it in robert boyle's works that he uses "alcohol" for a very fine subtle powder. but then this name of anything very fine and very subtle came to be specially connected with the fine and subtle spirit obtained from the fermentation of sugar; and i believe that the first person who fairly fixed it as the proper name of what we now commonly call spirits of wine, was the great french chemist lavoisier, so comparatively recent is the use of the word alcohol in this specialised sense. so much by way of general introduction to the subject on which i have to speak to-night. what i have hitherto stated is simply what we may call common knowledge, which everybody may acquaint himself with. and you know that what we call scientific knowledge is not any kind of conjuration, as people sometimes suppose, but it is simply the application of the same principles of common sense that we apply to common knowledge, carried out, if i may so speak, to knowledge which is uncommon. and all that we know now of this substance, yeast, and all the very strange issues to which that knowledge has led us, have simply come out of the inveterate habit, and a very fortunate habit for the human race it is, which scientific men have of not being content until they have routed out all the different chains and connections of apparently simple phenomena, until they have taken them to pieces and understood the conditions upon which they depend. i will try to point out to you now what has happened in consequence of endeavouring to apply this process of "analysis," as we call it, this teazing out of an apparently simple fact into all the little facts of which it is made up, to the ascertained facts relating to the barm or the yeast; secondly, what has come of the attempt to ascertain distinctly what is the nature of the products which are produced by fermentation; then what has come of the attempt to understand the relation between the yeast and the products; and lastly, what very curious side issues if i may so call them--have branched out in the course of this inquiry, which has now occupied somewhere about two centuries. the first thing was to make out precisely and clearly what was the nature of this substance, this apparently mere scum and mud that we call yeast. and that was first commenced seriously by a wonderful old dutchman of the name of leeuwenhoek, who lived some two hundred years ago, and who was the first person to invent thoroughly trustworthy microscopes of high powers. now, leeuwenhoek went to work upon this yeast mud, and by applying to it high powers of the microscope, he discovered that it was no mere mud such as you might at first suppose, but that it was a substance made up of an enormous multitude of minute grains, each of which had just as definite a form as if it were a grain of corn, although it was vastly smaller, the largest of these not being more than the two-thousandth of an inch in diameter; while, as you know, a grain of corn is a large thing, and the very smallest of these particles were not more than the seven-thousandth of an inch in diameter. leeuwenhoek saw that this muddy stuff was in reality a liquid, in which there were floating this immense number of definitely shaped particles, all aggregated in heaps and lumps and some of them separate. that discovery remained, so to speak, dormant for fully a century, and then the question was taken up by a french discoverer, who, paying great attention and having the advantage of better instruments than leeuwenhoek had, watched these things and made the astounding discovery that they were bodies which were constantly being reproduced and growing; than when one of these rounded bodies was once formed and had grown to its full size, it immediately began to give off a little bud from one side, and then that bud grew out until it had attained the full size of the first, and that, in this way, the yeast particle was undergoing a process of multiplication by budding, just as effectual and just as complete as the process of multiplication of a plant by budding; and thus this frenchman, cagniard de la tour, arrived at the conclusion--very creditable to his sagacity, and which has been confirmed by every observation and reasoning since--that this apparently muddy refuse was neither more nor less than a mass of plants, of minute living plants, growing and multiplying in the sugary fluid in which the yeast is formed. and from that time forth we have known this substance which forms the scum and the lees as the yeast plant; and it has received a scientific name--which i may use without thinking of it, and which i will therefore give you--namely, "torula." well, this was a capital discovery. the next thing to do was to make out how this torula was related to the other plants. i won't weary you with the whole course of investigation, but i may sum up its results, and they are these--that the torula is a particular kind of a fungus, a particular state rather, of a fungus or mould. there are many moulds which under certain conditions give rise to this torula condition, to a substance which is not distinguishable from yeast, and which has the same properties as yeast--that is to say, which is able to decompose sugar in the curious way that we shall consider by-and-by. so that the yeast plant is a plant belonging to a group of the fungi, multiplying and growing and living in this very remarkable manner in the sugary fluid which is, so to speak, the nidus or home of the yeast. that, in a few words, is, as far as investigation--by the help of one's eye and by the help of the microscope--has taken us. but now there is an observer whose methods of observation are more refined than those of men who use their eye, even though it be aided by the microscope; a man who sees indirectly further than we can see directly--that is, the chemist; and the chemist took up this question, and his discovery was not less remarkable than that of the microscopist. the chemist discovered that the yeast plant being composed of a sort of bag, like a bladder, inside which is a peculiar soft, semifluid material--the chemist found that this outer bladder has the same composition as the substance of wood, that material which is called "cellulose," and which consists of the elements carbon and hydrogen and oxygen, without any nitrogen. but then he also found (the first person to discover it was an italian chemist, named fabroni, in the end of the last century) that this inner matter which was contained in the bag, which constitutes the yeast plant, was a substance containing the elements carbon and hydrogen and oxygen and nitrogen; that it was what fabroni called a vegeto-animal substance, and that it had the peculiarities of what are commonly called "animal products." this again was an exceedingly remarkable discovery. it lay neglected for a time, until it was subsequently taken up by the great chemists of modern times, and they, with their delicate methods of analysis, have finally decided that, in all essential respects, the substance which forms the chief part of the contents of the yeast plant is identical with the material which forms the chief part of our own muscles, which forms the chief part of our own blood, which forms the chief part of the white of the egg; that, in fact, although this little organism is a plant, and nothing but a plant, yet that its active living contents contain a substance which is called "protein," which is of the same nature as the substance which forms the foundation of every animal organism whatever. now we come next to the question of the analysis of the products, of that which is produced during the process of fermentation. so far back as the beginning of the 16th century, in the times of transition between the old alchemy and the modern chemistry, there was a remarkable man, von helmont, a dutchman, who saw the difference between the air which comes out of a vat where something is fermenting and common air. he was the man who invented the term "gas," and he called this kind of gas "gas silvestre"--so to speak gas that is wild, and lives in out of the way places--having in his mind the identity of this particular kind of air with that which is found in some caves and cellars. then, the gradual process of investigation going on, it was discovered that this substance, then called "fixed air," was a poisonous gas, and it was finally identified with that kind of gas which is obtained by burning charcoal in the air, which is called "carbonic acid." then the substance alcohol was subjected to examination, and it was found to be a combination of carbon, and hydrogen, and oxygen. then the sugar which was contained in the fermenting liquid was examined and that was found to contain the three elements carbon, hydrogen, and oxygen. so that it was clear there were in sugar the fundamental elements which are contained in the carbonic acid, and in the alcohol. and then came that great chemist lavoisier, and he examined into the subject carefully, and possessed with that brilliant thought of his which happens to be propounded exactly apropos to this matter of fermentation--that no matter is ever lost, but that matter only changes its form and changes its combinations--he endeavoured to make out what became of the sugar which was subjected to fermentation. he thought he discovered that the whole weight of the sugar was represented by the carbonic acid produced; that in other words, supposing this tumbler to represent the sugar, that the action of fermentation was as it were the splitting of it, the one half going away in the shape of carbonic acid, and the other half going away in the shape of alcohol. subsequent inquiry, careful research with the refinements of modern chemistry, have been applied to this problem, and they have shown that lavoisier was not quite correct; that what he says is quite true for about 95 per cent. of the sugar, but that the other 5 per cent., or nearly so, is converted into two other things; one of them, matter which is called succinic acid, and the other matter which is called glycerine, which you all know now as one of the commonest of household matters. it may be that we have not got to the end of this refined analysis yet, but at any rate, i suppose i may say--and i speak with some little hesitation for fear my friend professor roscoe here may pick me up for trespassing upon his province--but i believe i may say that now we can account for 99 per cent. at least of the sugar, and that 99 per cent. is split up into these four things, carbonic acid, alcohol, succinic acid, and glycerine. so that it may be that none of the sugar whatever disappears, and that only its parts, so to speak, are re-arranged, and if any of it disappears, certainly it is a very small portion. now these are the facts of the case. there is the fact of the growth of the yeast plant; and there is the fact of the splitting up of the sugar. what relation have these two facts to one another? for a very long time that was a great matter of dispute. the early french observers, to do them justice, discerned the real state of the case, namely, that there was a very close connection between the actual life of the yeast plant and this operation of the splitting up of the sugar; and that one was in some way or other connected with the other. all investigation subsequently has confirmed this original idea. it has been shown that if you take any measures by which other plants of like kind to the torula would be killed, and by which the yeast plant is killed, then the yeast loses its efficiency. but a capital experiment upon this subject was made by a very distinguished man, helmholz, who performed an experiment of this kind. he had two vessels--one of them we will suppose full of yeast, but over the bottom of it, as this might be, was tied a thin film of bladder; consequently, through that thin film of bladder all the liquid parts of the yeast would go, but the solid parts would be stopped behind; the torula would be stopped, the liquid parts of the yeast would go. and then he took another vessel containing a fermentable solution of sugar, and he put one inside the other; and in this way you see the fluid parts of the yeast were able to pass through with the utmost ease into the sugar, but the solid parts could not get through at all. and he judged thus: if the fluid parts are those which excite fermentation, then, inasmuch as these are stopped, the sugar will not ferment; and the sugar did not ferment, showing quite clearly, that an immediate contact with the solid, living torula was absolutely necessary to excite this process of splitting up of the sugar. this experiment was quite conclusive as to this particular point, and has had very great fruits in other directions. well, then, the yeast plant being essential to the production of fermentation, where does the yeast plant come from? here, again, was another great problem opened up, for, as i said at starting, you have, under ordinary circumstances in warm weather, merely to expose some fluid containing a solution of sugar, or any form of syrup or vegetable juice to the air, in order, after a comparatively short time, to see all these phenomena of fermentation. of course the first obvious suggestion is, that the torula has been generated within the fluid. in fact, it seems at first quite absurd to entertain any other conviction; but that belief would most assuredly be an erroneous one. towards the beginning of this century, in the vigorous times of the old french wars, there was a monsieur appert, who had his attention directed to the preservation of things that ordinarily perish, such as meats and vegetables, and in fact he laid the foundation of our modern method of preserving meats; and he found that if he boiled any of these substances and then tied them so as to exclude the air, that they would be preserved for any time. he tried these experiments, particularly with the must of wine and with the wort of beer; and he found that if the wort of beer had been carefully boiled and was stopped in such a way that the air could not get at it, it would never ferment. what was the reason of this? that, again, became the subject of a long string of experiments, with this ultimate result, that if you take precautions to prevent any solid matters from getting into the must of wine or the wort of beer, under these circumstances--that is to say, if the fluid has been boiled and placed in a bottle, and if you stuff the neck of the bottle full of cotton wool, which allows the air to go through and stops anything of a solid character however fine, then you may let it be for ten years and it will not ferment. but if you take that plug out and give the air free access, then, sooner or later fermentation will set up. and there is no doubt whatever that fermentation is excited only by the presence of some torula or other, and that that torula proceeds in our present experience, from pre-existing torulae. these little bodies are excessively light. you can easily imagine what must be the weight of little particles, but slightly heavier than water, and not more than the two-thousandth or perhaps seven-thousandth of an inch in diameter. they are capable of floating about and dancing like motes in the sunbeam; they are carried about by all sorts of currents of air; the great majority of them perish; but one or two, which may chance to enter into a sugary solution, immediately enter into active life, find there the conditions of their nourishment, increase and multiply, and may give rise to any quantity whatever of this substance yeast. and, whatever may be true or not be true about this "spontaneous generation," as it is called in regard to all other kinds of living things, it is perfectly certain, as regards yeast, that it always owes its origin to this process of transportation or inoculation, if you like so to call it, from some other living yeast organism; and so far as yeast is concerned, the doctrine of spontaneous generation is absolutely out of court. and not only so, but the yeast must be alive in order to exert these peculiar properties. if it be crushed, if it be heated so far that its life is destroyed, that peculiar power of fermentation is not excited. thus we have come to this conclusion, as the result of our inquiry, that the fermentation of sugar, the splitting of the sugar into alcohol and carbonic acid, glycerine, and succinic acid, is the result of nothing but the vital activity of this little fungus, the torula. and now comes the further exceedingly difficult inquiry--how is it that this plant, the torula, produces this singular operation of the splitting up of the sugar? fabroni, to whom i referred some time ago, imagined that the effervescence of fermentation was produced in just the same way as the effervescence of a sedlitz powder, that the yeast was a kind of acid, and that the sugar was a combination of carbonic acid and some base to form the alcohol, and that the yeast combined with this substance, and set free the carbonic acid; just as when you add carbonate of soda to acid you turn out the carbonic acid. but of course the discovery of lavoisier that the carbonic acid and the alcohol taken together are very nearly equal in weight to the sugar, completely upset this hypothesis. another view was therefore taken by the french chemist, thenard, and it is still held by a very eminent chemist, m. pasteur, and their view is this, that the yeast, so to speak, eats a little of the sugar, turns a little of it to its own purposes, and by so doing gives such a shape to the sugar that the rest of it breaks up into carbonic acid and alcohol. well, then, there is a third hypothesis, which is maintained by another very distinguished chemist, liebig, which denies either of the other two, and which declares that the particles of the sugar are, as it were, shaken asunder by the forces at work in the yeast plant. now i am not going to take you into these refinements of chemical theory, i cannot for a moment pretend to do so, but i may put the case before you by an analogy. suppose you compare the sugar to a card house, and suppose you compare the yeast to a child coming near the card house, then fabroni's hypothesis was that the child took half the cards away; thenard's and pasteur's hypothesis is that the child pulls out the bottom card and thus makes it tumble to pieces; and liebig's hypothesis is that the child comes by and shakes the table and tumbles the house down. i appeal to my friend here (professor roscoe) whether that is not a fair statement of the case. having thus, as far as i can, discussed the general state of the question, it remains only that i should speak of some of those collateral results which have come in a very remarkable way out of the investigation of yeast. i told you that it was very early observed that the yeast plant consisted of a bag made up of the same material as that which composes wood, and of an interior semifluid mass which contains a substance, identical in its composition, in a broad sense, with that which constitutes the flesh of animals. subsequently, after the structure of the yeast plant had been carefully observed, it was discovered that all plants, high and low, are made up of separate bags or "cells," as they are called; these bags or cells having the composition of the pure matter of wood; having the same composition, broadly speaking, as the sac of the yeast plant, and having in their interior a more or less fluid substance containing a matter of the same nature as the protein substance of the yeast plant. and therefore this remarkable result came out--that however much a plant may differ from an animal, yet that the essential constituent of the contents of these various cells or sacs of which the plant is made up, the nitrogenous protein matter, is the same in the animal as in the plant. and not only was this gradually discovered, but it was found that these semifluid contents of the plant cell had, in many cases, a remarkable power of contractility quite like that of the substance of animals. and about 24 or 25 years ago, namely, about the year 1846, to the best of my recollection, a very eminent german botanist, hugo von mohl, conferred upon this substance which is found in the interior of the plant cell, and which is identical with the matter found in the inside of the yeast cell, and which again contains an animal substance similar to that of which we ourselves are made up--he conferred upon this that title of "protoplasm," which has brought other people a great deal of trouble since! i beg particularly to say that, because i find many people suppose that i was the inventor of that term, whereas it has been in existence for at least twenty-five years. and then other observers, taking the question up, came to this astonishing conclusion (working from this basis of the yeast), that the differences between animals and plants are not so much in the fundamental substances which compose them, not in the protoplasm, but in the manner in which the cells of which their bodies are built up have become modified. there is a sense in which it is true--and the analogy was pointed out very many years ago by some french botanists and chemists--there is a sense in which it is true that every plant is substantially an enormous aggregation of bodies similar to yeast cells, each having to a certain extent its own independent life. and there is a sense in which it is also perfectly true--although it would be impossible for me to give the statement to you with proper qualifications and limitations on an occasion like this--but there is also a sense in which it is true that every animal body is made up of an aggregation of minute particles of protoplasm, comparable each of them to the individual separate yeast plant. and those who are acquainted with the history of the wonderful revolution which has been worked in our whole conception of these matters in the last thirty years, will bear me out in saying that the first germ of them, to a very great extent, was made to grow and fructify by the study of the yeast plant, which presents us with living matter in almost its simplest condition. then there is yet one last and most important bearing of this yeast question. there is one direction probably in which the effects of the careful study of the nature of fermentation will yield results more practically valuable to mankind than any other. let me recall to your minds the fact which i stated at the beginning of this lecture. suppose that i had here a solution of pure sugar with a little mineral matter in it; and suppose it were possible for me to take upon the point of a needle one single, solitary yeast cell, measuring no more perhaps than the three-thousandth of an inch in diameter--not bigger than one of those little coloured specks of matter in my own blood at this moment, the weight of which it would be difficult to express in the fraction of a grain--and put it into this solution. from that single one, if the solution were kept at a fair temperature in a warm summer's day, there would be generated, in the course of a week, enough torulae to form a scum at the top and to form lees at the bottom, and to change the perfectly tasteless and entirely harmless fluid, syrup, into a solution impregnated with the poisonous gas carbonic acid, impregnated with the poisonous substance alcohol; and that, in virtue of the changes worked upon the sugar by the vital activity of these infinitesimally small plants. now you see that this is a case of infection. and from the time that the phenomenon of fermentation were first carefully studied, it has constantly been suggested to the minds of thoughtful physicians that there was a something astoundingly similar between this phenomena of the propagation of fermentation by infection and contagion, and the phenomena of the propagation of diseases by infection and contagion. out of this suggestion has grown that remarkable theory of many diseases which has been called the "germ theory of disease," the idea, in fact, that we owe a great many diseases to particles having a certain life of their own, and which are capable of being transmitted from one living being to another, exactly as the yeast plant is capable of being transmitted from one tumbler of saccharine substance to another. and that is a perfectly tenable hypothesis, one which in the present state of medicine ought to be absolutely exhausted and shown not to be true, until we take to others which have less analogy in their favour. and there are some diseases most assuredly in which it turns out to be perfectly correct. there are some forms of what are called malignant carbuncle which have been shown to be actually effected by a sort of fermentation, if i may use the phrase, by a sort of disturbance and destruction of the fluids of the animal body, set up by minute organisms which are the cause of this destruction and of this disturbance; and only recently the study of the phenomena which accompany vaccination has thrown an immense light in this direction, tending to show by experiments of the same general character as that to which i referred as performed by helmholz, that there is a most astonishing analogy between the contagion of that healing disease and the contagion of destructive diseases. for it has been made out quite clearly, by investigations carried on in france and in this country, that the only part of the vaccine matter which is contagious, which is capable of carrying on its influence in the organism of the child who is vaccinated, is the solid particles and not the fluid. by experiments of the most ingenious kind, the solid parts have been separated from the fluid parts, and it has then been discovered that you may vaccinate a child as much as you like with the fluid parts, but no effect takes place, though an excessively small portion of the solid particles, the most minute that can be separated, is amply sufficient to give rise to all the phenomena of the cow pock, by a process which we can compare to nothing but the transmission of fermentation from one vessel into another, by the transport to the one of the torula particles which exist in the other. and it has been shown to be true of some of the most destructive diseases which infect animals, such diseases as the sheep pox, such diseases as that most terrible and destructive disorder of horses, glanders, that in these, also, the active power is the living solid particle, and that the inert part is the fluid. however, do not suppose that i am pushing the analogy too far. i do not mean to say that the active, solid parts in these diseased matters are of the same nature as living yeast plants; but, so far as it goes, there is a most surprising analogy between the two; and the value of the analogy is this, that by following it out we may some time or other come to understand how these diseases are propagated, just as we understand, now, about fermentation; and that, in this way, some of the greatest scourges which afflict the human race may be, if not prevented, at least largely alleviated. this is the conclusion of the statements which i wished to put before you. you see we have not been able to have any accessories. if you will come in such numbers to hear a lecture of this kind, all i can say is, that diagrams cannot be made big enough for you, and that it is not possible to show any experiments illustrative of a lecture on such a subject as i have to deal with. of course my friends the chemists and physicists are very much better off, because they can not only show you experiments, but you can smell them and hear them! but in my case such aids are not attainable, and therefore i have taken a simple subject and have dealt with it in such a way that i hope you all understand it, at least so far as i have been able to put it before you in words; and having once apprehended such of the ideas and simple facts of the case as it was possible to put before you, you can see for yourselves the great and wonderful issues of such an apparently homely subject. bacteria in daily life by mrs. percy frankland fellow of the royal microscopical society; honorary member of bedford college, university of london; joint author of "micro-organisms in water," "the life of pasteur," etc. "spirits, when they please, can either sex assume, or both; so soft and uncompounded is their essence pure, not tied or manacled with joint or limb, nor founded on the brittle strength of bones, like cumbrous flesh; but, in what shape they choose, dilated or condensed, bright or obscure, can execute their aery purposes, and works of love or enmity fulfil." milton. longmans, green, and co. 39 paternoster row, london new york and bombay 1903 _all rights reserved_ transcriber's note: minor typographical errors have been corrected without note. irregularities and inconsistencies in the text have been retained as printed. words printed in italics are noted with underscores; _italics_. the cover of this ebook was created by the transcriber and is hereby placed in the public domain. preface the title of this little volume sufficiently explains its contents; it only remains to add that much of the text has already appeared from time to time in the form of popular articles in various magazines. it has, however, been carefully revised and considerably added to in parts where later researches have thrown further light upon the subjects dealt with. g. c. frankland northfield, worcestershire, _november, 1902_ contents page bacteriology in the victorian era 1 what we breathe 34 sunshine and life 65 bacteriology and water 93 milk dangers and remedies 118 bacteria and ice 149 some poisons and their prevention 168 bacteria in daily life bacteriology in the victorian era a little more than sixty years ago the scientific world received with almost incredulous astonishment the announcement that "beer yeast consists of small spherules which have the property of multiplying, and are therefore a living and not a dead chemical substance, that they further _appear_ to belong to the vegetable kingdom, and to be in some manner intimately connected with the process of fermentation." when cagniard latour communicated the above observations on yeast to the paris academy of sciences on june 12, 1837, the whole scientific world was taken by storm, so great was the novelty, boldness, and originality of the conception that these insignificant particles, hitherto reckoned as of little or no account, should be endowed with functions of such responsibility and importance as suggested by latour. at the time when latour sowed the first seeds of this great gospel of fermentation, started curiously almost simultaneously across the rhine by schwann and kützing, its greatest subsequent apostle and champion was but a schoolboy, exhibiting nothing more than a schoolboy's truant love of play and distaste for lessons. louis pasteur was only a lad of fifteen, buried in a little town in the provinces of france, whose peace of mind was certainly not disturbed, or likely to be, by rumours of any scientific discussion, however momentous, carried on in the great, far-distant metropolis. yet, some thirty and odd years later, there was not a country in the whole world where pasteur's name was not known and associated with those classical investigations on fermentation, in the pursuit of which he spent so many years of his life, and which have proved of such incalculable benefit to the world of commerce as well as science. thanks to pasteur, we are no longer in doubt as to the nature of yeast cells; so familiar, in fact, have we become with them, that at the dawn of the twentieth century we are able to select at will those particular varieties for which we have a predilection, and employ those which will produce for us the special flavour we desire in our wines or in our beers. large and splendidly-equipped laboratories exist for the express purpose of studying all kinds and descriptions of yeasts, for finding out their characteristic functions, and cultivating them with all the tenderness and care that a modern gardener bestows upon the rarest orchids. all this is now an old story, but some sixty years ago the great battle had yet to be fought which was to establish once and for all the dependence of fermentation upon life, and vanquish for ever those subtle arguments which so long refused to life any participation in the work of fermentation and other closely allied phenomena. when, however, pasteur finally cleared away the débris of misconception which had so long concealed from view the vital character of the changes associated with these processes, the bacterial ball, if we may so call it, was set rolling with a will, and information concerning these minute particles of living matter was rapidly gathered up from all directions. the recognition so long refused to bacteria was now ungrudgingly given, for it was realised at last that, in the words of m. duclaux, "whenever and wherever there is decomposition of organic matter, whether it be the case of a weed or an oak, of a worm or a whale, the work is exclusively performed by infinitely small organisms. they are the important, almost the only, agents of universal hygiene; they clear away more quickly than the dogs of constantinople or the wild beasts of the desert the remains of all that has had life; they protect the living against the dead. they do more; if there are still living beings, if, since the hundreds of centuries the world has been inhabited, life continues, it is to them we owe it." fortunately, the provisions made by nature for the preservation of the bacterial race are of so lavish a description that no fear need be entertained that this useful and indispensable world of life will be wiped out. the fabulous capacity for multiplication possessed by them (a new generation arising in considerably less than an hour), the powers of endurance which some of them exhibit in presence of the most trying vicissitudes of heat and cold (they have been known to survive exposure lasting for seven days to a temperature of about -200° c.), the inability of starvation or desiccation to undermine their constitution, combine to render the question of the extinction of bacteria as remote as it is undesirable. tempted by the prospects of exploring in this newly-revealed world of life, investigators rushed into the field, and the bacterial fever has been hardly less pronounced in these last years than that rush for a material golden harvest which has characterised so many enterprises in southern latitudes. the scientific results of this microbe fever have happily, however, been of a more solid and substantial character than can be said to have followed the more tangible but sordid ventures in south african mines. vague hypotheses have given place to facts, and bacteria have been brought more and more within the horizon of human knowledge, thanks to the genius and untiring zeal of investigators all over the world. by mechanical improvements in microscopes, and subtle methods for colouring bacteria, enabling us to study their form with precision, by ingenious devices for supplying them with suitable food materials, or, in other words, by the creation of bacterial nurseries, providing the means for watching their growth and observing their distinctive habits and character, this important branch of the vegetable kingdom has been raised from obscurity to one of the principal places in our catalogue of sciences, and bacteriology has won for itself an individual footing in the scientific curriculum of our great educational institutions, and is represented in literature by such famous serials devoted to the publication of bacterial and allied researches as the _annales de l'institut pasteur_, the _centralblatt für bakteriologie_, the _zeitschrift für hygiene_, the _annali d'igiene sperimentale_, and other well-known journals which constitute an essential but ever-increasing burden upon the library shelves as well as pocket of the investigator. museums of bacteria have been established where not only specimens of particular varieties of a permanent character for comparison and reference can be obtained, but living cultivations of hundreds of different micro-organisms are maintained; and only those who have had the charge of bacteria can realise the enormous amount of skilled labour involved in the catering for such a multitude, in which individual likes and dislikes in regard to diet and treatment must, if success is to be secured, be as carefully considered as is necessary in the case of the most delicate and highly pampered patient. bacteria, by means of these depôts, can, in fact, be bought or exchanged by collectors with as much facility as postage stamps, with the all-important difference that this collecting of bacteria is not a mere mania or speculation, but serves a most useful purpose. to the busy investigator who cannot afford either the time or space in which to maintain a large bacterial family, it is of immense convenience to be able to obtain at a moment's notice a trustworthy culture, say, of typhoid or tuberculosis, or specimens of obscurer origin from air or water for purposes of investigation. these bacterial cultures are all guaranteed pure, free from contamination or admixture with other and alien micro-organisms, and are strictly what they are represented to be. although such a declaration is attached to many commodities at the present day with ludicrous incongruity, in the case of micro-organisms such a breach of faith is unknown, and the antecedents of a microbe may be said to be regarded as of as much moment and to be as jealously preserved as is the pedigree of the most ambitious candidate for honours at a cattle or dog show! amongst some of the curiosities to be found on the shelves of microbe-museums may be mentioned bacteria which give out light, and thus, like glowworms, reveal themselves in the dark. these light-bacteria were originally discovered in sea-water and on the bodies of sea-fish, and cultures of them have been successfully photographed, the only source of light being that provided by the bacilli themselves. the amount of light emitted by a single bacillus might indeed defy detection by the most sensitive plate procurable, but when gathered together in multitudes, the magnitude of which even eight figures fail to express, these phosphorescent bacteria enable the dial of a watch to be easily read in the dark, whilst photographs of the face of a watch taken in such bacterial light have been so successful that the time at which the photograph was taken could be distinctly seen. of bacteria it may indeed truly be said, as has maeterlinck of the labours of bees--"though it be here the infinitely little that without apparent hope adds itself to the infinitely little, though our eye with its limited vision look and see nothing, their work, halting neither by day nor by night, will advance with incredible quickness!" mention may perhaps appropriately be made here of the highly interesting fact discovered by professor percy frankland, that ordinary bacteria which do not phosphoresce are capable of affecting a photographic film in absolute darkness, and can by this means produce a picture of themselves. if, however, a transparent piece of glass is placed between the bacteria and the film no photograph results, showing that glass interferes with their activity in this respect. the author points out that as this action upon the photographic film does not take place through glass, it is in all probability due to the evolution by the bacteria of certain volatile chemical substances which either directly or indirectly enter into reaction with the sensitive film. similar phenomena have been discovered in regard to many metals as well as organic substances, but this is the first observation which has been recorded of the action of living structures on sensitive films in the dark. we have already referred to the important services which pasteur has rendered by distinguishing between different varieties of yeast, and separating them out according to their functions and properties--pioneer work which has been followed up by and borne such splendid fruit in the hands of the renowned danish investigator, emil christian hansen of copenhagen. this work of isolating out individual varieties of micro-organisms has been not only pursued with the energy familiar to all in the case of bacteria associated with disease, but has been pursued in various other, though perhaps less well known, directions. a great deal of activity has lately been exhibited in so-called dairy bacteriology, and a long list has already been compiled of milk, cheese, and butter microbes; and agricultural authorities, even in this country, are slowly awakening to the fact that, in order to compete on modern lines with foreign dairy produce, dairy schools must be established, where bacteriology is taught, and where instruction is given in the principles of scientific butter and cheese making. but bacteria of the brewery and of the dairy are not the only useful germs which are to be found on the shelves of microbe museums. wine and tobacco manufacturers on application may respectively obtain the bacterial means of transforming the crudest must into the costliest claret, and the coarsest tobacco into the most fragrant havana. already considerable progress has been made in the isolation of particular varieties of wine-yeast, whilst highly encouraging results have been obtained by suchsland and others in the separation of various valuable tobacco-fermenting organisms. agricultural authorities, again, owe a debt of gratitude to those distinguished investigators whose labours have discovered the art of imprisoning the micro-organisms which play such an important part in the fertilisation of the soil. bacterial fertilisers are amongst the latest achievements which bacteriology has accomplished in this wonderful half-century, and the purchase of special varieties of bacteria to suit the requirements of particular kinds of leguminous plants is now fast becoming a mere everyday commercial transaction. but efforts for the amelioration of the conditions under which plant life is carried on have not been confined to providing plants with suitable bacterial friends; vigorous and successful efforts have been made to remove from their _entourage_ those bacterial enemies and undesirable parasites which have for so long played so important a part in the crop-returns of many an agriculturist. for the identification and separation of the plant-parasites of various kinds we have largely to acknowledge our indebtedness to american investigators, and the encouragement and support which dr. erwin smith, amongst others, has received from the government of the united states in the prosecution of these researches indicates how great is the public importance attached to them. there are in america alone fifty experiment stations where plant diseases are studied, whilst at a number of the colleges and universities more or less attention is given to the subject. some idea of the loss occasioned to agriculturists by these plant pests may be formed by a recent announcement that the department of agriculture in queensland was prepared to offer a reward of £5,000 for the discovery of a means to eradicate the prickly-pear disease. plant pathology has not yet had a distinct chair allotted to it in any of the great universities, but the subject is of such vast industrial importance, that doubtless before long some seat of learning will do itself the honour to establish one, and so set the example. a striking instance of the advantages of taking stock, so to speak, of the attributes of bacteria will occur to everyone in the revelation which has followed of their powers to solve one of the most knotty problems of the day--the efficient manipulation of those vast subterranean rivers of sewage which honeycomb every city of the world. the purification which sewage underwent by passing it through the pores of the soil, or, in other words, by filtration, was recognised about the year 1870, soon after the rivers pollution commissioners had begun to make their classical investigations on the land treatment of sewage; but although the rapid transformation of ammonia into nitrates which followed the passage of the sewage through a few feet of soil was noted, yet the mechanism of this nitrification process remained a mystery until 1877, when two french chemists--mm. schloesing and muentz--made the then astounding discovery that this change was dependent upon the vital energies of micro-organisms. the part played by bacteria in the purification of sewage thus became an established fact, and the later experiments have been devoted to studying the necessary conditions under which the maximum amount of work is obtainable from these novel bacterial labourers. two different classes of bacteria are required to carry on the purification of sewage: those which flourish in the _absence_ of air and are known as anaërobic bacteria, and those to which the _presence_ of air is essential for the exercise of their functions, the latter being therefore called aërobic bacteria. the work of the anaërobic labourers consists in breaking down the complex organic compounds present in sewage, whilst the completion of the process of purification is left to the aërobic varieties. in the ordinary course of nature both these processes are going on side by side, but it has been found advisable to separate these two different classes of bacteria as far as possible, and allot distinct premises to the anaërobic and aërobic varieties respectively engaged in the purification of sewage, for by so doing experience has shown that the work is not only more expeditiously, but also more efficiently, carried out. now the anaërobic bacteria are supplied along with the sewage, and the retention of their services offers practically no difficulty as long as an ample allowance of space and time is given them in which to carry on their labours. the aërobic bacteria, however, besides demanding space and time, insist upon their workshops being well ventilated, and if the supply of fresh air is in any way curtailed they stop work entirely. hence the ventilation of the aërobic workshops becomes a matter of primary importance if the valuable services of these labourers are to be retained. to ensure a sufficient supply of air being provided, it has been found advisable to have two or more aërobic workshops or bacteria contact beds, and the sewage is passed from one on to a second, and so on, until the purification is complete. under proper management the sewage should leave the works as an inodorous, almost pellucid liquid, incapable of putrefaction, which may be turned into rivers or other waterways without fear of rousing the wrath of local riparian authorities. but whilst the commercial side of bacteriology, so to speak, has made such great strides, the purely scientific applications which have been made of the facts it has furnished have by no means lagged behind. chemists, from pasteur downwards, have made use repeatedly of special bacteria to perform delicate operations in the laboratory which other methods have either failed to accomplish or have performed in a clumsy and less expeditious manner. there can be no doubt that, as our knowledge grows from day to day, we shall find more and more how much depends upon the work of individual bacteria, and how much importance attaches to the selection of just those varieties which are of value, and the banishment of those which are detrimental; and thus the many applications which bacteria already admit of render their easy access a matter of increasing consequence, enhancing the value of bacterial institutions such as already exist on the continent. but whilst the easy access of bacteria for experimental and scientific purposes is of great importance to the investigator, their indiscriminate distribution would equally be a source of uneasiness and danger to the community at large. already sensational fiction has made considerable capital out of the pathogenic microbe, and with the winged aid of penny publications it does not take long for suggestions of such kinds to spread in society and assume practical shape, and whilst the administration of bacterial poisons offers comparatively but little difficulty, their identification would be a far greater problem for experts than that presented by particular chemical poisons. to cope with this danger to the public, specimens of disease-germs from these bacterial depôts may not be supplied to applicants unless the latter can prove to the satisfaction of the director that they are connected with responsible public institutions. in recent times, indeed, one of the most remarkable practical uses to which bacteria have been put is that of poisoning-agents on a large scale, or in other words vermin exterminators; if this new rôle for bacteria becomes extended, as no doubt it will, the law for the sale of noxious drugs and preparations will also doubtless be amended to cover the distribution of bacterial-poisons. it was in the year 1889 that professor loeffler, while experimenting with mice in his laboratory at greifswald, discovered a micro-organism which was extremely fatal to all kinds of mice. the happy idea occurred to the professor that this lethal little microbe, which he christened _bacillus typhi murium_, might be turned to excellent account in combating plagues of field mice in grain-fields, where the devastation committed by these voracious rodents had become in parts of greece and russia a serious source of loss to agriculturists. experiments were accordingly made on a small scale to test the efficiency of this bacterial poisoner in destroying field mice, and so successful were the results that loeffler confidently announced the possibility of keeping down these pests by distributing food material infected with these bacteria over fields which were invaded by them. the greek government took up the question, and loeffler's method was applied with brilliant results; the disease was disseminated with extraordinary rapidity and severity, and the mice were readily destroyed. it is highly satisfactory to find that the character of this mouse-bacillus has stood the test of time, for after a period of more than ten years most encouraging reports concerning its efficiency still continue to be received. in one of the latest of these, drawn up by the director of the experimental agricultural institute in vienna, we read that in no less than seventy per cent. of the cases in which it was employed it was completely successful in its work of extermination, and it is interesting to note that in a considerable number of these instances it was the domestic mouse against which its energies were directed. the rat has, however, until recently escaped the hand of the bacterial executioner, but his knell has also now been sounded in the announcement that a rat-bacillus has been discovered. considering the undesirable notoriety which these rodents have of late obtained in connection with their undoubted culpability in the dissemination of plague, this discovery, if correct, should be warmly welcomed. that there is plenty of work awaiting such a micro-organism may be gathered from the fact that during the outbreak of plague in sydney the crusade against rats which followed led to the slaughter in one year of over 100,000. the discoverer of this useful member of the microbial community is tssatschenko, of the university of st. petersburg, and in his memoir he states that, whilst highly virulent as regards rats, it is quite harmless to domestic animals of various kinds. thus cats, dogs, fowls, and pigeons when fed with food infected with the bacillus suffered no ill effects whatever, whilst its administration in large quantities to farm stock, such as horses, oxen, pigs, sheep, geese, and ducks, was also without result; hence its distribution, according to its discoverer, offers no danger to other animals. this idea of employing bacteria as executioners was not original, for pasteur had already in 1888 suggested to the intercolonial rabbit commission in australia that chicken-cholera microbes should be employed for destroying the rabbits, which then, as now, are such a source of difficulty and pecuniary loss to the country. no active measures appear to have been taken, however, to carry out this suggestion, one of the principal objections raised being the undesirability of introducing a disease which was at that time believed to be a stranger to the colony. recently the idea has been revived by mr. pound, the government bacteriologist at brisbane, in consequence of his discovery that chicken-cholera, far from not existing in australia, has infested poultry yards more or less extensively for several years past, although it has only lately been accurately diagnosed as such. this chicken-cholera microbe is particularly well suited for the work in question, inasmuch as, whilst extremely fatal to rabbits, it produces, like loeffler's bacillus, no ill effect whatever on farm-stock of various kinds, and is perfectly harmless to man, so that its handling by the uninitiated is not attended with any personal danger. this brings us to what may be designated the human side of bacteriology, _i.e._ its relation to disease and its prevention. in these important departments of life the services already rendered by this infant prodigy of science can as yet be only approximately appreciated. anthrax, tuberculosis, cholera, typhoid, plague, influenza, tetanus, erysipelas, are only a few of the diseases the active agents of which bacteriology has revealed to us. bacteriology has, however, not been content to merely identify particular micro-organisms with particular diseases, it has striven to devise means by which such diseases may be mastered, and one of the most glorious achievements of the past sixty years is the progress which has been made in the domain of preventive medicine. the classical investigations of pasteur on the attenuation of bacterial viruses such as those of chicken-cholera and anthrax, and his elaboration of a method of vaccination with these weakened viruses whereby the power of the disease over its victim is removed or modified, are too well known to require repetition here. the success which followed pasteur's researches in this direction led him to undertake that great and difficult task, the prevention of rabies in the human subject--a task well-nigh superhuman in its demands, and one which only he could accomplish in whose life the pregnant words of a modern writer found expression--"il ne suffit pas de posséder une vérité, il faut que la vérité nous possède." the victory over this disease, which crowned a long life replete with brilliant achievements, has been universally recognised, and numerous institutes have arisen in all quarters of the globe for extending the benefits of this discovery for the relief of suffering humanity. these pasteur or bacteriological institutes also furnish highly important centres where original research work of various kinds is carried on, and the stimulus which has thus been given to experimental science in the remotest parts of the world cannot be overestimated. methods for the prevention of disease have, however, not been confined to the elaboration and employment of modified or weakened bacterial viruses; the subject has been still more recently approached from another and quite different side. this new departure we also originally owe to france, although its practical development has been worked out in germany. it was in 1888 that two frenchmen, richet and héricourt, communicated a memoir to the _comptes rendus_ of the academy of sciences, describing the curious results they had obtained with rabbits purposely infected with a disease microbe, the _staphylococcus pyosepticus_. some of the rabbits died after being inoculated with this micro-organism and some remained alive, and they proceed to point out how it was that such different results were obtained. before the inoculations were made some of the animals received injections of blood taken from a dog, which a few months previously had been infected with this same microbe, but had recovered. the rabbits which received the dog's blood all survived the inoculations, whilst those which did not, succumbed in every case to the action of the _staphylococcus pyosepticus_. so struck were the authors by these remarkable results that they repeated them, and their further investigations fully confirmed those originally obtained, proving that they were not "un fait exceptionnel." here we have the first steps in the direction of serum-therapy, that new treatment of disease which during the last few years has been so prominently before the public in the cure of diphtheria, tetanus, and other maladies, and for the development of which we owe so much to the labours of behring, roux, kitasato, and other investigators. the astounding fact that the blood of animals which have been trained to artificially withstand a particular disease becomes endowed with the power of protecting other animals from that disease is only in the earliest stages of its application. the results, however, which have already been accomplished are of so encouraging a character that the hope is justified that serum-therapy is destined to revolutionise the treatment of disease. one of the latest uses which has been made of this method of combating disease is the employment of serum for the cure of bubonic plague. during the recent outbreak of plague in india, yersin, formerly a student and assistant at the paris pasteur institute, was despatched to india to superintend the administration of this new remedy, and the serum he employed was that derived from horses which had been subjected to, and had recovered from, inoculations with the plague bacillus. the treatment of snake bites by means of curative serum will be dealt with in more detail later on; it only remains to cite it here as another instance of the success which is attending the new methods of protection against disease. another and highly ingenious application of serum has been brought forward by pfeiffer, gruber, widal, and others. this is the so-called sero-diagnosis of disease, and has been employed already with success in the identification of typhoid fever as such. the method sounds simple in the extreme, and consists in taking a few drops of blood from a patient supposed to be suffering from typhoid fever and mixing them with a recent cultivation in broth of genuine typhoid bacilli. if the blood is derived from a typhoid-infected person, then the bacilli should exhibit a curious and characteristic appearance when examined under the microscope. instead of moving about as individuals in various parts of the microscopic field, they should be seen gathering or clumping together in numerous small heaps, their movements the while becoming paralysed. the state board of health of massachusetts has recently taken up the official sero-diagnosis of typhoid fever, and issues in response to applications a simple outfit with instructions how to collect specimens of blood and a form which they request shall be returned filled in with all the details concerning the case under observation. only a few drops of blood are required for the examination, and these before being despatched to the state laboratory are collected on slips of paper and allowed to dry. if the addition of this suspected blood in the proportion of one to twenty to a young and vigorous culture of typhoid bacilli succeeds in paralysing their movements, producing the characteristic clumping together or agglutination of the bacilli, then the reaction is considered positive and the case one of typhoid fever. that, however, some risk attends the placing of too implicit a reliance on this method of diagnosis alone is evident from the fact that a _negative_ reaction, or in other words, absence of all agglutinising phenomena, is sometimes associated with blood throughout what is beyond all question a well-defined case of typhoid fever, whilst in the first week of this disease the test is frequently negative in character. rouget, who has made a very careful inquiry into the value to be attached to the sero-diagnosis of typhoid fever, states that he has found in a large number of examinations of blood derived from undoubted typhoid patients the agglutination phenomena fail altogether; it is, therefore, not surprising that the sero-diagnosis of this disease is still the subject of much discussion and investigation. an interesting example of how particular serums may be employed for the detection of particular poisons has been furnished by dr. calmette. in some districts of india the natives have an ugly custom of wreaking their vengeance on their enemies by poisoning their cattle, and to effect this both expeditiously and secretly they employ subtle poisons which they know can only be detected with great difficulty. serpent venom is a favourite substance, whilst abrine, a highly toxic vegetable poison, is another. the method adopted for the application of this abrine is highly original, and consists in taking small bits of wood shaped like miniature clubs, so diminutive in size that they can be concealed in the hand. in the head of the club small holes are bored, and tiny pointed rodlets of a hard greyish substance are fitted into them. armed with these crude instruments, the natives scratch the cattle in several places, and, although but little external sign of injury is to be seen, the rod-points penetrate the skin and are broken off, and the poison is left to work its lethal way through the animals' system. mr. hankin forwarded some of these broken-off rod-points to dr. calmette for the identification of their composition, and he diagnosed the material employed as abrine in the following original manner. he first introduced some of this rod material into animals, and found that their symptoms were suggestive of abrine poisoning. to confirm his suspicions, however, he took some more of this rod material, and, before inoculating it into animals, he mixed it with serum derived from animals which had been artificially rendered immune to abrine poison. instead of the animals into which this mixture of serum and "rod material" had been introduced dying like the previous ones, they remained alive. had the "rod material" consisted of some poison other than abrine, the abrine serum would not, according to dr. calmette, have negatived its action, and it has thus been indicated how protective serums may be successfully employed for the detection of poisons. foremost, however, among the beneficent reforms which have followed in the wake of bacteriology must be placed the antiseptic treatment of wounds, or listerism, as it is now universally designated in recognition of its renowned champion, the former president of the royal society. "lister comprend," in the words of dr. roux, "que les complications des plaies sont dues aux germes microbiens venus du dehors et il imagine les pansements antiseptiques. avec l'antiseptie commencent les temps nouveaux de la chirurgie." it only remains to add that, with the modesty characteristic of a great man, its brilliant author delights in repeating how any good which he may have been permitted to do he owes entirely to the inspiration which he received from the labours of louis pasteur. but if the victorian era has been productive of so many important applications of bacteriology to commerce and medicine, this period has been also fraught with results of the highest moment in the progress of hygiene. the terms of intimacy, so to speak, which we have been now able to establish with bacteria has enabled us to discover details of their life and habits which before were shrouded in mystery. their distribution in air has led to renewed endeavours on the part of sanitary authorities to procure efficient ventilation in our hospitals and public institutions; dust has acquired a fresh horror since it has been shown how disease germs may be disseminated by its means; whilst the important part which flies and lice may play in the spread of epidemics has opened up a new field for research, and made us conscious of a fresh source of danger in our daily life. the general public, however, is hardly yet fully alive to the capacity for mischief possessed and exercised by the common house-fly. true, it is universally execrated and regarded as a tiresome attendant upon the summer months, but it is not usually considered in any more serious light. that however, the comparative indulgence with which this homely insect pest has been treated is decidedly misplaced and fraught with danger to health, the researches of numerous scientists have now conclusively proved. as long ago as the year 1888 professor celli showed that the germs of consumption, anthrax, and typhoid fever could pass through the digestive organs of flies and reappear in the excreta of the latter not only alive but in full possession of their disease-producing powers. dr. sawtschenko made similar experiments with cholera germs. healthy flies were placed under glass shades and fed with broth in which these micro-organisms were growing, and the latter were not only subsequently recovered from the digestive organs of the flies but also from their excreta in a living and virulent condition. this is, however, not the only means whereby these insects can distribute deadly and other microbes, for it has been shown that in crawling over substances containing bacteria these may become attached to the feet of flies, and are in this manner transferred to other materials upon which they may alight, just as pasteur showed many years earlier silkworms can communicate the fatal plague of pébrine by crawling over each other's bodies, carrying in their disease-laden feet the infection from one worm to another. during the recent outbreak of bubonic plague in the east the part played by flies in disseminating the virus has been repeatedly emphasised. yersin was the first who called attention to the presence in large numbers of virulent plague bacilli within the bodies of flies which he collected in the vicinity of plague-stricken persons, and it was found that flies which had fed on plague-infected material and were then isolated lived for several days afterwards, during which time virulent plague bacilli were present in their bodies in immense numbers; thus were these insects converted into winged messengers of evil of the most repulsive type. i am not aware whether any experiments on the vitality and transmissibility of diphtheria and consumption germs by means of flies have been made; but in view of the overwhelming evidence of the culpability of these insects in spreading plague, it is not unreasonable to presume a responsibility on their behalf in regard to other diseases; indeed, in the report issued by the army medical commissioners of the spanish-american war, it is emphatically stated that flies played an important part in the dissemination of typhoid fever. there is no question as to the capability of certain micro-organisms to reside for considerable periods of time within the bodies of flies, and during this sojourn to abate no jot of their virulence. indeed, it has been shown that the bodies of these insects may constitute incubators of a most successful type, for some varieties of bacteria grow luxuriantly and multiply abundantly within them. in the hot days of summer, when flies abound, it would be well to banish these insects, as far as lies in our power, not only from our sick-rooms in particular, but from our general surroundings. the catholic taste of flies for garbage of all kinds is too well known to require entering into, but the consequences which may follow from their visits to dustbins and centres of disease, and then alighting upon our food or persons, has received too little attention in the past. in regard to the subject of insects as disease disseminators, it may be mentioned that mr. hankin, when studying plague conditions in india, expressed his belief that ants in bombay also assisted in spreading the scourge, for he found that when he inoculated mice with the excreta of ants, such insects having previously fed on plague-stricken rats, the mice succumbed to plague in a few hours. fleas have also been conclusively proved to be carriers of plague germs. there is no doubt that the revelations of hygienic science have aroused the vigilance and zeal of public authorities in various new directions to try and cope with the spread of zymotic disease. in no direction, perhaps, is the fruit of this energy so apparent as in the increasing supervision which it has incited over two of the greatest menaces to public health which hang over society--_i.e._ our water and dairy supplies. now that it has been proven beyond doubt that the germs of consumption, typhoid fever, and cholera can be and are distributed through the consumption of contaminated milk or water, not to mention other diseases such as diphtheria and scarlet fever, an ever-increasing demand is being made that these all-important articles of diet shall be protected from pollution, and that public authorities shall be made responsible for their distribution in a pure and wholesome condition. it is, however, undoubtedly in the matter of water that the greatest service has been rendered by bacteriology to sanitary science, and for the important advance in this department we are indebted to the beautifully simple and ingenious methods devised by robert koch. not yet twenty years have passed since the new bacterial examination of water was introduced and systematically employed, and the use which has been made of the opportunities thus opened up of investigating water problems on an entirely new basis is shown by the voluminous dimensions which the literature on this one branch of bacteriology alone has reached. considerably upwards of two hundred different water bacteria have been isolated, studied, and their distinctive characters chronicled. the behaviour of typhoid, cholera, and other disease-producing microbes in waters of various kinds has been made the subject of exhaustive experiments; the purification power of time-honoured processes in operation at waterworks and elsewhere has been for the first time accurately estimated. water engineers have through these bacteriological researches been provided with a code of conduct drawn up by the light of erudite scientific inquiries, which has now rendered possible the removal of the process of water purification from the rule of empiricism guided by tradition, and to raise it to the level of an intelligent and scientific undertaking. the above short sketch may serve to convey some idea of the rise and phenomenal development of bacteriology during the past sixty years. to record, even in outline, the individual triumphs of the various branches of this science would require volumes, whilst the astounding mass of work already accumulated by its devotees is but the earnest, the guarantee of yet greater achievements in the future. the progress which has been made in this brief period of time must not necessarily be expected to continue at this rapid rate; it may be that generations to come have yet the hardest and the longest tasks to accomplish; for in science, as in other walks of life, it is, as a rule, the easiest problems, which are first disposed of, and the farther we advance the more complicated, the more intricate become the questions to be attacked, the difficulties to be overcome. the late queen's reign has bestowed a splendid legacy of bacteriological discoveries upon those who, in the future as in the present, must inevitably follow in the footsteps of those great and brilliant leaders of bacteriological science belonging to this auspicious era, louis pasteur and robert koch. what we breathe few people realise that, with the advent of autumn, the great majority of the swarms of bacteria which have been circulating in the air during the hot summer months take their leave of us and disappear. practically, however, we are all conscious of this fact, for we know what greater difficulties attend the keeping of food sweet and wholesome in the summer than are met with in the winter; bacteria, not unlike some other armies of occupation, securing a footing rather by their numbers at this season of the year, than by virtue of the superior strategy or, in other words, special attributes of their units. bacterial operations are, however, distinctly favoured by the accident of temperature, the warmth of the summer encouraging their vitality and multiplication. when pasteur first announced his conviction that the familiar phenomena of putrefaction and decay were due to minute living particles present in our surroundings, his sceptical critics sought to ridicule his conclusions by declaring that, were this the case, the air must of necessity be so heavily laden with living forms that we should be surrounded by a thick fog--"dense comme du fer." we do not now, forty years later, require to recite the exquisitely simple experiments which, whilst sufficiently establishing his theories, served to effectually suppress those of his opponents. since pasteur's pioneering work was carried out, a vast number of investigations have been made in all parts of the world by scientists of almost every nationality on the subject of the distribution of bacteria in air, and not only on their distribution, but on their functions or the place they occupy in the economy of nature. with our increased knowledge concerning their distribution has come our ability to differentiate between individuals, and to adequately assess the value and importance of their work from various points of view. in the bacterial treatment of sewage we have not only one of the latest, but perhaps also one of the most successful examples of that system of division of labour, or specialisation of energy, which forms such a characteristic feature of work of all kinds at the present time. other familiar instances of the applications of individual and special bacterial labourers to the solution of industrial problems are to be found in the conduct of commercial undertakings of such national magnitude and importance as brewing and agriculture. but it is not with these beneficent or great industrial classes of bacteria that we are now more immediately concerned, but rather with the malevolent varieties, or the so-called "submerged tenth," for which no labour colony has at present been created to direct their energies into useful and profitable channels. we know that as regards mere numbers the bacteria in air may vary from 0 to millions in a couple of gallons, these extremes being dependent upon the surrounding conditions or relative purity of the atmosphere. out at sea, beyond the reach of land breezes, it is no uncommon thing to find none whatever; on mountains and even hills of humble elevation the paucity of bacteria is very marked if there are no abnormal or untoward circumstances contributing to their distribution. in illustration of this the recent investigations of the air on the summit of mont blanc by m. jean binot are of especial interest, inasmuch as the altitude at which they were carried out is the highest at which the search after bacteria has so far been pursued. this intrepid investigator spent no less than five days in the observatory, which is situated on the top of the mountain. as was to be anticipated, frequently no bacteria at all were found, and it was only when such comparatively large volumes of air as one thousand litres (about 200 gallons) were explored that microbes in numbers varying from four to eleven were discovered. the air of the country is far freer from microbial life than that of cities; whilst open spaces, such as those afforded by the london parks, are paradises of purity compared with the streets with their attendant bacterial slums. that it is no exaggeration to describe streets from the bacterial point of view as slums is to be gathered from the fact that much less than a thimbleful of that dust which is associated with the blustering days of march and the scorching pavements of summer may contain from nine hundred to one hundred and sixty millions of bacteria. but investigators have not been content to merely quantitatively examine street dust; in addition to estimating the numerical strength of these bacterial dust-battalions, the individual characteristics of their units have been exhaustively studied, and the capacity for work, beneficent or otherwise, possessed by them has been carefully recorded. the qualitative discrimination of the bacteria present in dust has resulted in the discovery of, amongst other disease germs, the consumption bacillus, the lock-jaw or tetanus bacillus, bacteria associated with diphtheria, typhoid fever, pulmonary affections, and various septic processes. such is the appetising menu which dust furnishes for our delectation. there can be no doubt, therefore, that dust forms a very important distributing agent for micro-organisms, dust particles, aided by the wind, being to bacteria what the modern motor-car, with its benzine or electric current, is to the ambitious itinerant of the present day. attached to dust, bacteria get transmitted with the greatest facility from place to place, and hence the significance of their presence in dust. mention has been made of the fact that the germs of typhoid fever have been discovered in dust, and the belief in the possibility of this disease being spread by dust is gaining ground. an interesting case in point is afforded by an outbreak of typhoid fever which occurred in athens a few years ago, and in which the starting-point or nucleus was discovered to be a group of labourers who were engaged upon excavating the soil in a street through which a sewer had once been taken. the epidemic subsequently spread to those districts of the city swept by the prevailing wind, which passed over the place where the soil had been turned up and exposed. m. bambas, who brought his observations before the international congress of hygiene at buda-pesth, was convinced from the inquiries he made that this outbreak of typhoid was due to the disturbance of the soil and the dissemination by means of the wind of typhoid-dust-particles to certain parts of the city. that this hypothesis is by no means without experimental justification is shown by the properties possessed by the typhoid bacillus in regard to its vitality in soil which have been discovered. thus numerous investigators have studied the important question of the behaviour of this micro-organism in soil, and have found that it can exist over periods extending from three to twelve or more months in the ground. this property of the typhoid bacillus may possibly explain the appearance over and over again of typhoid fever in particular localities, suggesting that the bacteria had become indigenous in the soil. dr. mewius, of heligoland, describes an epidemic of typhoid fever in the island, concerning which he made a most searching and elaborate inquiry. it appears that a case of typhoid occurred and was concealed from the medical authorities, so that no steps for disinfection could be taken in the first instance; and, following the primitive custom which prevails on the island, the dejecta was thrown over and upon the cliffs, this being the usual method of disposing of sewage. ample opportunity was thus given for its desiccation and subsequent distribution as dust. that this typhoidal matter did subsequently become pulverised and spread the infection dr. mewius has no doubt, the germs having been conveyed to the open rain-water cisterns which constitute the water-supply of the majority of the inhabitants. his theory is again supported by the coincidence between the prevailing direction of the wind and the quarter where the outbreak occurred. that diphtheria germs can remain for a long time in a living and, what is more, virulent condition in dust has been clearly demonstrated by germano, amongst other investigators, this organism being specially endowed with the capacity for resisting the, to other microbes, lethal effect of getting dried up. bacteria, however, survive this desiccation process much better when they are herded together in large numbers than when they have to face such untoward conditions as isolated individuals. this has been well illustrated in the case of diphtheria bacilli, and the difference in their powers of endurance under these respective conditions is very striking. thus when a few only were exposed to a very dry atmosphere on silken threads they disappeared after eight days; but when somewhat larger numbers were taken they contrived to exist for eighteen days, whilst when great multitudes of them were herded together even one hundred and forty days' starvation in these desert-like surroundings could not entirely stamp out their vitality. this dangerous property possessed by the germs of diphtheria should, if possible, increase the vigilance with which the outbreaks of this disease are watched and dealt with. abel cites an instance in which a wooden toy in the sickroom of a child suffering from diphtheria was found six months later to have _virulent_ diphtheria bacilli upon it. this reminds me of a case in which tetanus or lock-jaw ensued from the use of some old cobwebs in stopping the bleeding of a cut. the wound was a perfectly clean one, and nothing need have resulted from this obedience to a superstitious prejudice had not the cobwebs unfortunately arrested some tetanus germs, and these getting access to the wound set up the typical symptoms of lock-jaw. that this implication of the cobweb was no idle accusation was subsequently proved by portions of the same web, on being inoculated into animals, inducing in the latter well-defined symptoms of tetanus. that cobwebs readily catch dust is familiar to everyone who has the mortification of seeing them adorn ceilings and corners; that they also arrest bacteria follows as a natural consequence of the presence of dust, and hence these delicate filaments may become veritable bacterial storehouses, more especially as it is usually in the dark and remote corners that they best succeed in eluding the vigilance of the domestic eye, and are thus also out of reach of the lethal action of sunbeams; and hence their unwelcome lodgers may manage to maintain a very comfortable existence over long periods of time. that the bacillus of consumption should have been very frequently found in dust by different investigators is hardly surprising when it is realised that the sputum of phthisical persons may contain the tubercle germ in large numbers, and that until recently no efforts have been made in this country to suppress that highly objectionable and most reprehensible practice of indiscriminate expectoration. considering that the certified deaths from phthisis in 1901, in england and wales only, reached the enormous total of 42,408, and bearing in mind the hardy character of the _bacillus tuberculosis_ when present in sputum, it having been found alive in the latter even when kept in a dry condition after ten months, it is not too much to demand that vigorous measures should be taken by the legislature to cope with what is now regarded as one of the most fruitful means of spreading consumption. we know that in some of the states of america public opinion has permitted the enactment of laws penalising this practice. local rules to the same effect exist in our australian colonies. on the continent the trend of public opinion is evident by the prohibition found in the railway carriages and the notices to that effect conspicuously posted in public places. in this country public opinion moves so slowly that we are not yet ripe for any such strong step, and so far one of the few attempts at official activity in this respect is to be found in a circular issued by the local government board of ireland to the various local authorities stating that "tuberculous sputum is the main agent for the conveyance of the virus of tuberculosis from man to man, and that indiscriminate spitting should therefore be suppressed." the public exhibition of notices calling attention to the danger accruing from expectoration in public resorts is, as already pointed out, one means of educating the people, and it has been stated that such a notice is posted in every beerhouse in manchester. the question has also been raised of the inspection of beerhouses and the suggestion made that licences should be withdrawn in the case of those holders who did not wash the floors of their public rooms and keep them in a sanitary state. at the present time, in this country, it is perhaps more to the private conscience of the individual and the pressure of public opinion than to penal enactments that we must look for effective reform in this direction, for the objection of the english to official sanitary control is deeply rooted. it is to be hoped, however, that with the spread and popularisation of the knowledge acquired through the arduous labours of so many scientific authorities, it may come to be regarded as a matter for both public and private morality that every step should be taken which lies in the power of each member of society to minimise the opportunities for the spread of a disease which by its very familiarity we have until the last few years accepted as incurable and the ravages of which as inevitable.[1] [1] since the above was written, the first international conference of the central committee for the prevention of consumption has been held in berlin. the official report of the english national association for the prevention of tuberculosis was presented to the congress, and the encouraging announcement was made that the corporations of glasgow, manchester, and liverpool had made expectoration in tramcars a punishable offence; and that the glamorganshire county council had passed a bye-law providing as penalty for expectoration in public buildings a fine of £5, which enactment had been sanctioned by the secretary for the home department. now that we are considering the status of street dust in bacterial circles, it will not perhaps be out of place to inquire into the character of another waste product of streets, _i.e._ the discarded ends of cigars and cigarettes. that what is carelessly tossed away on the one hand may be as carefully collected on the other is well known, as is also the fact that such material may subsequently be raised once more to the dignity of a marketable commodity. under these circumstances, it is of hygienic interest and importance to ascertain whether disease germs, should they have obtained access to this tobacco refuse, are in a virulent or quiescent condition. some experiments to decide this question in connection with the tubercle bacillus have been recently carried out in padua by dr. peserico, who, whilst extending our knowledge on the subject of bacteria and tobacco, has also confirmed the earlier results obtained by kerez. portions of cigar-stumps smoked by phthisical persons in whose saliva the tubercle bacillus was known to be abundantly present were inoculated into guinea-pigs, with the result that fifty per cent. of the animals thus treated succumbed to tuberculosis. thus neither the fumes nor juice of the tobacco had destroyed the consumption bacillus. in these experiments the cigar ends were used directly they were discarded, in another series of investigations they were collected and kept in a dry place for from fifteen to twenty days before being tested; but even storage for this length of time did not prevent the animals inoculated with them from contracting tuberculosis. in another series of experiments dr. peserico kept the infected cigar-ends in damp surroundings, and it was satisfactory to find that under these conditions the tubercle bacillus at the end of ten days was entirely deprived of its virulence. encouraged by these results, inoculations were made with cigar-ends which had been left in the open and exposed to normal atmospheric conditions, which included falls of rain and snow, and in this case also no symptoms of tuberculosis followed their introduction into the guinea-pigs. these experiments show that the tubercle bacillus is prejudicially affected by contact with tobacco when the latter is kept in a moist condition, but that in a dry condition the properties in tobacco inimical to its vitality are not liberated and the bacillus can retain its virulent properties for a period of over twenty days. in view of the importance of this discovery on the destruction of the toxic character of the tubercle bacillus by contact with moist tobacco, further experiments were made in which emulsions of tobacco were infected with tuberculous sputum. it was found that the bacilli steadily declined in virulence as the length of time they were kept in the emulsion was prolonged. thus whereas after a few hours they were still armed with all their virulent properties, after three days, out of the four animals inoculated with the emulsion three succumbed to tuberculosis, after five days two out of four succumbed, whilst after eight days only one animal out of the four was infected, and after a period of ten days' immersion in the tobacco emulsion the tubercle bacillus failed to kill a single animal. cigarand cigarette-ends were collected from the streets and cafés of padua by peserico, but in spite of consumption being stated to be very prevalent in this city, in no single case could the presence of the tubercle bacillus be discovered, although, as in the other investigations, the surest method for its detection, _i.e._ animal inoculations, was employed. brief reference may be made also to the experiments conducted to ascertain if cigars and cigarettes, as sold, contain the tubercle bacillus. the more interest attaches to this investigation because it is well known that the operators employed in tobacco factories are, as a rule, an unhealthy class, diseases of the respiratory organs, and especially tuberculosis, being very prevalent amongst them. a german official report on this subject states that the average duration of life of such factory hands only reaches thirty-eight years. doubtless the lightness of the occupation encourages many to seek employment in these factories whose state of health would debar them from obtaining work under more trying circumstances. some of the conditions under which cigars and cigarettes are made, such as the workers using their saliva to facilitate the rolling of them and fixing of the leaves, and the testing of the "drawing" properties of a cigar by placing it in the mouth, with the facilities offered for the dissemination of dried tuberculous sputum as dust, contribute to make it highly probable that tobacco as it leaves the factory may contain the germs of consumption. before leaving the subject of tobacco and disease germs it may be of interest to inquire what justification in fact there is for the practice adopted by anxious mothers, when travelling in times of epidemics of zymotic disease, of thrusting themselves and their children into the sanctum of the other sex--the smoking compartment of a railway carriage. i have frequently seen this done, despite the voluble protests of its legitimate occupants. tassinari has made some very interesting experiments on the effect of tobacco smoke on the vitality of various descriptions of disease germs. he constructed an apparatus in which he suspended pieces of linen soaked in broth infected with the particular micro-organism to be tested. tobacco smoke was then admitted, and the microbes were retained in this stifling atmosphere for half an hour. in these surroundings cholera and typhoid germs were destroyed, and other bacteria, such as the anthrax bacillus and the pneumonia bacillus, were so prejudicially affected, that when subsequently transferred to their normal surroundings it was only with extreme difficulty that they could be revived. when, however, the tobacco smoke was made to pass through water before reaching the bacteria, its pernicious influence was entirely removed, and the latter suffered no detriment. hence the practice, so often seen in the east, of passing tobacco smoke through rose or other perfumed water before inhaling it, whilst doubtless rendering it less noxious to the smoker, deprives the exhaled tobacco fumes of all their bactericidal or disinfecting properties. to return, however, after this somewhat lengthy digression, to the question of dust and its bacterial properties, we have learnt enough to enable us to realise that the movement for the migration of the working-classes from crowded streets to rural districts, in which mr. george cadbury has played so practical and important a part in the creation of his model village, with its gardens and open spaces, some five miles from the city of birmingham, is, if only bacterially considered, a very real barrier against the dissemination of disease, for the denser the population, the greater will be the crowd of bacteria, and the greater the chance of pathogenic varieties being present amongst them. again, we know that sunshine is one of the most potent germicides with which nature has provided us;[2] and it requires no effort of the imagination to realise how, in the gloomy back courts and crowded tenements of our great smoke-laden cities, bacteria succeed in obtaining a firm hold on their surroundings, and, in the shape of spores, attaining an undesirable and hoary old age, in which they are in some cases almost indestructible. fräulein dr. e. concornotti has shown that this is no figment of fancy only, for she has recently made a special and very elaborate study of the distribution of pathogenic or disease bacteria in air, searching for them in the most varied surroundings, such as prisons, schools, casual wards, etc., with the result that, out of forty-six experiments in which the character of the bacteria found was tested by inoculation into animals, thirty-two yielded organisms which were pathogenic. dr. concornotti concludes her valuable memoir by stating that her investigations proved conclusively that the dirtier or more slumlike the surroundings, the greater was the frequency with which she found bacteria associated with disease in the air. [2] see "sunshine and life." messrs. valenti and terrari-lelli have quite recently been able fully to endorse these statements in the results they have obtained in their systematic study of the bacterial contents of the air in the city of modena. in their report they state that the narrower and more crowded the streets, the greater was the number of bacteria present in the air, and the more frequently did they meet with varieties associated with septic disease. numerous detailed investigations have also been made of the bacterial contents of the dust in hospitals. that cases of infection arising within hospital precincts are of no uncommon occurrence may be gathered from the observations made by lutand and hogg, who report no fewer than 2,294 such cases having arisen in the space of six years in certain paris hospitals, whilst solowjew records 1,880 cases as occurring in the space of four and a half months in the st. petersburg city hospital. solowjew made a special study of the bacterial contents of dust collected in hospitals, and states that 41·8 per cent. of the samples examined contained disease germs. the degree of infection possessed by dust in such surroundings must, of course, depend upon the degree of cleanliness which characterises the management of any particular institution; and such investigations as the above can only help to emphasise the immense importance of common cleanliness and the reasonableness of taking every precaution possible in the disinfection of utensils, etc. some years ago messrs. carnelley, haldane, and anderson carried out an elaborate series of investigations on the air of dwelling-houses in some of the poorest parts of dundee. the samples were taken during the night, between 12.30 a.m. and 4.30 a.m., and in their report the authors state that the one-roomed tenements were mostly those of the very poor; "sometimes as many as six or even eight persons occupied the one bed," whilst in other cases there was no bed at all. as regards the number of bacteria present in the air in these one-roomed houses, an average of several examinations amounted to sixty per quart; in two-roomed houses it was reduced to forty-six, and in houses of four rooms and upwards only nine micro-organisms in the same volume of air were discovered. on comparing the mortality statistics with the composition of the air of dwelling-houses of different dimensions, the authors arrive at the following conclusions: "that, as we pass from four-roomed to three-, two-, and one-roomed houses, not only does the air become more and more impure, as indicated by the increase in the carbonic acid and organic matter, and more especially of the micro-organisms, but there is a corresponding and similar increase in the death-rate, together with a marked lowering of the mean age at death."[3] [3] it is, of course, obvious that other circumstances besides overcrowding have to be reckoned with in considering these statistics. in the one-roomed houses the wages earned by the occupants must have been small, and the amount available for even the bare necessaries of life very limited, that, in fact, they were to be reckoned amongst the class defined by mr. rowntree as living in "primary poverty," whose earnings are insufficient to keep the body in a properly nourished condition. mr. rowntree has shown by statistics that the height, weight, and general condition of the poor are very much below those of the well-to-do labouring classes. mention may also here be made of the investigations made by these gentlemen on the air of board schools, which showed that in those buildings where mechanical ventilation was used the carbonic acid gas was three-fifths, the organic matter one-seventh, and the micro-organisms less than one-ninth of what was found in schools ventilated by the ordinary methods. in commenting upon this series of investigations, the authors write: "when we come to consider that the children who attend average board schools for six hours a day are during that time subjected to an atmosphere containing on an average nearly nineteen volumes of carbonic acid per 10,000, and a very large proportion of organic matter, and no less than 155 micro-organisms at least per quart, we need not be surprised at the unhealthy appearance of very many of the children. it must also be borne in mind that many of them are exposed for nine hours more to an atmosphere which is about five times as impure as that of an ordinary bedroom in a middle-class house. they are thus breathing for at least fifteen hours out of the twenty-four a highly impure atmosphere. the effects of this are often intensified, as is well known, by insufficient food and clothing, both of which must render them less capable of resisting the impure air. the fact that these schools become, after a time, habitually infected by bacteria renders it probable that they also become permanent foci of infection for various diseases, and particularly, perhaps, for tubercular disease in its various forms." further practical evidence of the manner in which the general death-rate for certain diseases is influenced by the conditions under which the poor are housed is afforded by statistics which have been collected at glasgow. in the case of zymotic diseases, whereas the death-rate in tenements consisting of one or two rooms was 4·78 per 1,000, it fell to 2·46 in those of three or four rooms, and to 1·14 per 1,000 in those of five rooms and upwards. again, in the case of acute diseases of the lungs, the death-rate was as high as 9·85 in the smallest tenements, and but 3·28 in the largest. of great interest are the certified mortality statistics of phthisis in the british army in the period 1830-46 and 1859-66 respectively; in the former it was 7·86 per 1,000, whilst in the latter period it had fallen to 3·1, this important difference being coincident with an increased cubic space per head in the barracks. such facts as these, if only fully realised, should surely serve to stimulate municipal and other local authorities to provide decent and wholesome accommodation for the poor. it has been recently estimated that in london the total number of persons living in tenements of one to four rooms is 2,333,152, and of these nearly half a million live the life of the one-room tenement of three to a room and upwards. in the stirring words of mr. john burns, m.p.: "at least a million of people who live thus on wages that barely sustain decent life, are but prisoners of poverty, whose lot in life is but a funeral procession from the cradle to the grave ... for these, as soon as practicable, better homes should be provided at once in the interest of physique, of morals, of industrial efficiency, and municipal health." yet, despite all these facts and the overwhelming evidence which has been collected on the dire results which follow in the wake of overcrowding and insanitary dwellings, we find a prominent magistrate in one of our great industrial cities publicly expressing himself as follows at a municipal banquet: "the town council sometimes attempted too much. for instance, they had been far too anxious to get quit of the slums. now slums, in his opinion, were one of the necessities of all large towns, and it was impossible in the present state of civilisation to dispense with slums unless they could take the people living in them, who were not fit to live anywhere else, and drown them wholesale, as would have been done in the time of the french revolution." we have seen how bacteria may be distributed by dust, how they may linger in crowded tenements and badly ventilated buildings, that insanitary surroundings provide, in fact, for the scientist a well-stocked bacterial covert, where he may with ease bag his thousands of germs of various descriptions. the fact already referred to, that the bacteria of consumption may be released in the sputum of phthisical persons, has perhaps already suggested the possibility of other bacteria being likewise discharged into the surrounding air, but it is no doubt difficult to realise that the utterance of even a few words may liberate a variety of bacteria, the mischievous or harmless character of which depends upon the condition of the speaker's health. but even the health of a speaker if satisfactory is not necessarily a safeguard against his dissemination of disease germs, for it is well known that the mouth secretions of healthy people may frequently contain the _staphylococcus pyogenes aureus_, and also, though less frequently, the _diplococcus lanceolatus_, both virulent microbes; whilst that diphtheria bacilli may be present in the mouths of people who are not suffering from the disease has been demonstrated repeatedly. what a capacity, then, for spreading evil does the public orator possess! it makes one tremble to think of the aërial condition of the house of commons when a big debate is on, for it has been found that the sharper the enunciation of the consonants, and the louder the voice, the larger is the number of organisms discharged and the farther they reach! if this danger attends the speaking of healthy people, what must be the risk accompanying the listening to speeches from persons suffering from consumption, influenza, or any other disease which specially affects the air passages! what applies to speaking applies to a still greater degree to the act of coughing or sneezing. to schäffer we owe the discovery that leprosy bacilli may be disseminated in immense numbers by the coughing of leprosy patients, whilst it has been estimated that a tuberculous invalid may discharge a billion tubercle bacilli in the space of twenty-four hours, whilst the dried sputum of consumptive persons has actually engendered tuberculous symptoms in the lungs of animals which were made to inhale it. plague bacilli have been found in masses in the mouths of plague patients, and were found, moreover, before any symptoms of the disease had declared themselves; and the sputum of infected persons is regarded by some authorities as one of the most important vehicles by which plague is spread. the culpability of air in the dissemination of tuberculosis amongst animals has been made the subject of some very exhaustive and valuable investigations by kasselmann. in as many as 71 per cent of bovine tuberculosis cases the respiratory organs, kasselmann found, were the seat of the disease. the undoubted contamination of the air which takes place in the surroundings of tuberculous animals is not, however, due to the bacilli being exhaled by such cattle in the mere process of respiration, for it has been repeatedly found by various investigators that the air expired by infected animals is free from the dreaded tubercle bacteria. as in man, so in animals--it is by the act of coughing that tuberculous secretions are discharged through the mouth and nasal passages, some of which in the form of spray may enable the bacilli to remain suspended in the air for periods of five hours or more, whilst other portions of such secretions fall on the ground or in the feeding troughs, and later on, as dust, may again relentlessly claim their toll of victims. in other cases of tuberculosis the excrementitious matter becomes, of course, a fertile source of infection to the surroundings. the dire results which may follow the introduction of a single tuberculous animal into a healthy stall of cows may be realised from the fact that in one instance a whole herd of twenty-eight animals became in the course of one year infected in consequence of the admission of one diseased cow, the cow-house having previously had a perfectly clean bill of health in this respect. on the continent the risk of wholesale infection by such means is greater than in this country; for abroad the animals are to a much greater extent stall-fed, and kept shut up both winter and summer. a case is mentioned by the well-known veterinary authority, m. nocard, of a whole stall of animals becoming infected through the cow-man who tended them being consumptive. he slept in a loft over the cows, and his tuberculous sputum in the form of dust was conveyed to the stalls beneath and so spread the infection. it has been stated on high authority that domestic pets such as parrots may contract consumption from their masters, and that no less than thirty-six per cent. of these birds brought to the veterinary college in berlin are found to be suffering from tuberculosis. in that much-dreaded south african cattle disease, rinderpest, the infection, contrary to what is found in the case of tuberculous animals, is principally spread by the _materies morbi_ being liberated in the air expired by afflicted cattle, the contagious area surrounding an infected animal extending to as much as a hundred yards and more. again, as regards pleuro-pneumonia in cattle, the contagion is given off in the air expired, and owing to the length of time which elapses before the lung becomes completely healed and healthy, even after a period of from six to nine months, the expired air may still prove a source of infection. in an official report on the open-air treatment of consumption in germany a case is mentioned in which the patient, a farmer by occupation, had contracted the disease from some tuberculous cattle which he had on his farm. the writer goes on to say, "this case is worthy of special attention, inasmuch as it indicates that in addition to the danger of contracting the disease from the use of milk or meat derived from tuberculous animals, the tending of such animals may serve to convey the infection to man possibly much more frequently than has hitherto been supposed." in addition to the above instances of the responsible part played by air in the dissemination of consumption many others might be cited, but perhaps the most striking is that in which a scientific assistant of tappeiner contracted the disease, and succumbed to it, in the course of some experiments which were being made to ascertain whether consumption could be communicated to animals by spraying them with an emulsion of the sputum of consumptive patients. it is of historical interest to note that these experiments were being conducted by tappeiner three years before robert koch made the now classical announcement to the scientific world that he had succeeded in identifying, isolating, and in cultivating outside the human body the specific cause of consumption in the shape of the now familiar _bacillus tuberculosis_. the opinion expressed by koch at the congress on tuberculosis recently held in london, that human and bovine tuberculosis are distinct diseases, is still the subject of contention and experimental investigation. even if the opinion of this great authority is correct, and in this connection it is interesting to note that already in 1896 this opinion was brought forward by smith in the _medical record_ at a time when koch was maintaining the _identity_ of human and bovine tuberculosis--granted that koch is correct, it should not, as so many fear, cause any relaxation in the efforts which have been at last made to safeguard our dairy produce by reasonable hygienic precautions; for even if tuberculosis is not transmissible from the cow to man, we know that in the hygienic supervision of our dairy industry we place a great barrier between us and the _bacillus tuberculosis_ and those numerous other disease germs which can and do gain access to milk from the _personnel_ of a dairy and so spread infection. with the alarming prevalence of consumption is it not justifiable to regard as certain that a definite proportion of the people engaged in milking, for example, are consumptive? and knowing, as we now do, how such persons can give off the germs of the disease in the simple act of speaking, the contamination of our milk with human tubercle bacilli must be regarded almost as a certainty. would it not be reasonable that a code of simple precautions to be taken, coupled with a few of the more cogent facts concerning consumption and its distribution, should be drawn up and circulated amongst all engaged in the dairy industry? the national health society has done much for the prevention of disease by disseminating, through leaflets and lectures, simple facts concerning health and its preservation; might it not make itself the vehicle for the transmission of some such code which, whilst instructing, should impress upon its readers the responsibility which rests upon each and every individual member of society, by his or her own personal efforts, to assist in the great task of combating disease? a fact which urgently needs the widest recognition is the possible dissemination of disease germs by individuals not themselves suffering from the disease in question, but who have resided in the immediate surroundings of infected persons. dr. koch was the first to call attention to this danger when he discovered, during the hamburg cholera epidemic, that _perfectly healthy_ persons were infected with cholera vibrios, and were the unconscious means of spreading the disease. still more recently it has been found that true typhoid germs may similarly be present in persons not suffering from typhoid fever but sharing the same living-rooms. huxley has said "science is nothing but trained and organised common sense," and it is in this spirit that we must endeavour to make use of the discoveries which have been made in the prevention of disease, in which the science of bacteriology has played so great and important a part. sunshine and life it was nearly a century ago that a german physician incidentally wrote, "our houses, hospitals, and infirmaries will, without doubt, some day be like hot-houses, so arranged that the light, even that of the moon and stars, is permitted to penetrate without let or hindrance." this was spoken long before the world of micro-organisms had been discovered, but curiously has found an echo in the writings of a distinguished bacteriological chemist in recent years. "laissons donc entrer largement partout l'air et le soleil," writes m. duclaux; "c'est là une maxime bien ancienne, mais si les mots sont vieux l'idée qu'ils revêtent est nouvelle." the interpretation of this ancient maxim is indeed very modern, and we must turn to the investigations made within the past few years to learn with what justification m. duclaux thus expresses himself, for it is only comparatively recently that we have learnt the novel fact that sunshine, whilst essential to green plant life, is by no means indispensable to the most primitive forms of vegetable existence with which we are acquainted, _i.e._ bacteria. in fact, we have found out that if we wish to keep our microbial nursery in a healthy, flourishing condition, we must carefully banish all sources of light from our cultivations, and that a dark cupboard is one of the essential requisites of a bacteriological laboratory. that light had a deleterious effect upon micro-organisms was first discovered in this country by messrs. downes and blunt, and their investigations led professor tyndall to carry out some experiments on the alps, in which he showed that flasks containing nutritive solutions and infected with bacteria when exposed in the sunshine for twenty-four hours remained unaltered, whilst similar vessels kept in the shade became turbid, showing that in these the growth of bacteria had not been arrested. in these experiments mixtures of micro-organisms were employed, and the interest of the french investigations which followed lies in the use of particular microbes--notably the anthrax bacillus and its spores,[4] roux demonstrating very conclusively that the bacillar form was far more sensitive to light than the spore form, while momont, in a classical series of experiments, not only fully confirmed these observations, but showed also that the intensity of the action of light depends to a very large extent on the environment of the organism. thus, if broth containing anthrax bacilli is placed in the sunshine, the latter are destroyed in from two to two and a half hours, whilst if blood containing these organisms is similarly exposed, their destruction is only effected after from twelve to fourteen hours of sunshine. this difference in resistance to insolation was also observed in the case of _dried_ blood and broth respectively--eight hours' exposure killing the bacilli in the former, whilst five hours sufficed in the latter. [4] in the interior of some bacilli there appears a round or oval body, having a very bright and shining lustre, which is known as a _spore_, and plays a most important part in the propagation of many kinds of bacilli. these spores are capable of resisting many hardships, which would be immediately fatal to the parent bacilli from which they have sprung. this is an instance of the apparent idiosyncrasies possessed by micro-organisms, which render their study at once so fascinating and so difficult, and it is through being thus constantly confronted with what, in our ignorance, we mentally designate as "whims," that we can hardly resist the impression of these tiny forms of life being endowed with individual powers of discernment and discrimination. indeed, these powers of selection and judgment are in certain cases so delicately adjusted that in some of the modern chemical laboratories micro-organisms have become indispensable adjuncts, and by their means new substances have been prepared and fresh contributions made to the science of chemistry. momont is not able to give any satisfactory explanation of this different behaviour of the anthrax bacilli in these two media, but goes on to show that yet another factor plays an important part during insolation. in the above experiments air was allowed to gain access to the vessels containing the broth, but if the precaution be taken of first removing the air and then exposing them to the sunshine, a very different result was obtained, for instead of the anthrax bacilli dying in from two to two and a half hours, they were found to be still alive after fifty hours' insolation. there appears, therefore, to be no doubt that sunshine in some way or other endows atmospheric oxygen with destructive power over the living protoplasm of the bacterial cells; indeed, there is considerable reason to believe that the bactericidal effect is due to the generation of peroxide of hydrogen, which is well known to possess powerfully antiseptic properties. numerous investigations have been also made to determine whether all the rays of the spectrum are equally responsible for the bactericidal action of light. geisler's work in st. petersburg is especially instructive in this respect, for by decomposing with a prism the sun's light, as well as that emitted by a 1,000-candle-power electric lamp into their constituent rays, he was able to compare the different effects produced by the separate individual rays of both these sources of light. the organism selected was the typhoid bacillus, and it was found that its growth was retarded in all parts of the two spectra excepting in the red, and that the intensity of the retardation was increased in passing from the red towards the ultraviolet end of the spectrum, where it was most pronounced of all. but whereas from two to three hours of sunshine were sufficient to produce a most markedly deleterious effect upon the typhoid bacillus, a similar result was only obtained by six hours' exposure to the electric light. dr. kirstein, of the university of giessen, in the course of some experiments he made to ascertain how long different varieties of bacteria can exist when they obtain access to the air in the form of fine spray, and subsequently, as happens under ordinary circumstances, get dried up, noted also the effect upon their vitality of exposure in daylight and darkness respectively. for this purpose the apparatus in which the experiments were carried out was in some cases kept in a dark cellar, whilst in others it was left standing in the laboratory in ordinary daylight. delicate bacteria, such as the fowl-cholera bacillus, it was found, could not survive exposure to daylight in this dried-up condition for more than ten hours, but when they were put in the dark their lease of life was prolonged for more than twice that length of time; whilst as regards varieties of tougher constitution, such as diphtheria and tubercle bacilli, whose initial vitality was very considerably greater under these adverse circumstances, confinement in the cellar enabled them to exist more than four times as long as they were able to in the healthy atmosphere of the well-lighted laboratory. dr. onorato, of the university of genoa, has recently shown, also, that influenza bacilli are entirely destroyed after the sun has been shining on them continuously for three and a half hours. such facts indicate how essential to health is plenty of light in our dwelling-rooms, and how important it is that in the designing of houses the trapping of the maximum amount of sunshine should be very carefully considered. architects might indeed with advantage be compelled to include in their qualifications a knowledge of the fundamental facts of sanitary science. the fashion of shutting the sunshine out by barriers of blinds and curtains drawn across the windows, a practice which seems to be almost entirely independent of the habitual gloom of the surroundings or general scarcity of sunshine, might possibly be modified were it but known that by thus excluding light we are conferring an inestimable benefit upon the members of the microbial community, which may at any moment comprise some of the subtlest and most dangerous antagonists with which we have to reckon in the struggle for existence. from a hygienic point of view, also, the question of the potency of sunshine in regard to the bacteria present in water is both important and interesting, for it is to water at the present time that we look for the dissemination of some of the most dreaded zymotic diseases. comparatively little has been done in this direction, but those results which have been obtained are exceedingly suggestive. professor buchner has published some preliminary experiments which he made with particular micro-organisms. in these investigations boiled tap-water was used to ensure the absence of all bacteria except those which were subsequently introduced, and, whilst some of the vessels were exposed to the sunshine, others were simultaneously preserved in the dark. it was found that typhoid, cholera, and various other bacilli were most deleteriously affected by insolation. perhaps an example will best serve to illustrate the nature of the results obtained. some boiled water contained in a flask was inoculated with an immense number of a bacillus, closely resembling the typhoid organism, normally present in the body and frequently found in water, the _bacillus coli communis_. so many were introduced that nearly one hundred thousand individuals were present in every twenty drops of the water. this flask then, containing water so densely sown with microbes, was placed in the sunshine for one hour, whilst another and similar flask was kept during the same time in the dark. on being subsequently examined it was ascertained that whereas a slight increase in the number of bacilli had taken place in the "dark" flask, in the insolated flask _absolutely no living organisms whatever_ were present. professor percy frankland has also investigated the action of sunshine on micro-organisms in water, and in one of his reports to the water research committee of the royal society an account is given of the effect of insolation on the vitality of the spores of anthrax in thames water. these experiments show again what an important influence the surroundings of the organism have on the bactericidal potency of the sun's rays, for the remarkable fact was established that when immersed in water anthrax spores are far less prejudicially affected by sunlight than when exposed in ordinary culture materials such as broth or gelatine. thus it was only after one hundred and fifty-one hours' insolation in thames water that these spores were entirely destroyed, whilst a few hours' exposure in the usual culture media is generally sufficient for their annihilation. in water not subjected to insolation anthrax spores were found to retain their vitality for several months. in case the reader should be tempted to compare these results with those obtained by buchner, it must be borne in mind that whereas those experiments were made with _bacilli_, these were directed to determine the behaviour of _spores_ in water, which are some of the hardiest forms of living matter with which we are acquainted. this alone would sufficiently explain the results obtained, whilst each variety of microbe may be, and doubtless is, differently affected during insolation. we know now that a remarkable improvement takes place in the bacterial condition of water during its prolonged storage in reservoirs, and although, no doubt, the processes of sedimentation which have been shown to take place during this period of repose are to a large extent responsible for the diminution in the number of bacteria present, yet it is also highly probable that insolation assists considerably in this improvement, at any rate, in the upper layers of the water. as the depth of the water increases the action of light is necessarily diminished. indeed, exact experiments conducted in the lake of geneva to ascertain by means of photographic plates the depth to which the sun's rays penetrate showed that they did not reach beyond five hundred and fifty-three feet, at which depth the intensity of the light is equal to that which is ordinarily observed on a clear but moonless night, so that long before that their bactericidal potency would cease. it is the more important that this limit to the powers of sunshine in water should be duly recognised, inasmuch as solar enthusiasts, when first the fact became known, rashly jumped at the convenient hypothesis, based on very slender experimental evidence, that the sun's rays were possessed of such omniscient power to slay microbes, that they might safely be relied upon to banish all noxious organisms from our streams, and that local authorities might therefore comfortably and without any qualms of conscience turn sewage into our rivers and so dispense with the cost and labour of its treatment and purification. this was actually suggested in a proposal made for dealing with the sewage of the city of cologne. fortunately further investigations have removed these most erroneous and dangerous ideas; and whilst all due credit may be given to sunshine for what it really does accomplish in the destruction of bacteria in water, there is now no doubt as to its potency being confined to the superficial layers of water. perhaps dr. procacci's experiments will most clearly convey some idea of this limitation, for he made a special study of this particular phenomenon. some drain water, containing, of course, an abundance of microbial life, was placed in cylindrical glass vessels, and only the perpendicular rays of the sun were allowed to play upon it. the column of water was about two feet high, and whilst a bacteriological examination at the commencement of the research showed that about two thousand microbes were present in every twenty drops of water taken from the surface, centre, and bottom of the vessel respectively, after three hours' sunshine only nine and ten were found in the surface and centre portions of the water, whilst at the bottom the numbers remained practically unchanged. professor buchner, of munich, demonstrated the same impotence of the sun's rays to destroy bacteria much beneath the surface of water, in some ingenious experiments he made in the starnberger see, near munich. he lowered glass dishes containing jelly thickly sown with typhoid bacilli to different depths in the water during bright sunshine; those kept at a depth of about five feet subsequently showed no sign of life, whilst those immersed about ten feet developed abundant growths; in both cases the exposure was prolonged over four and a half hours. in our own rivers thames and lea frequently about twenty times more microbes have been found in the winter than in the summer months, but it would be extremely rash to therefore infer that the comparative poverty of bacterial life was due to the greater potency of the sun's rays in the summer than in the winter. doubtless it may contribute to this beneficial result; but we know as a matter of fact that, in the summer, these rivers receive a large proportion of spring water, which is comparatively poor in microbes, and that this factor also must not be ignored in discussing the improved bacterial quality of these waters at this season of the year. another point which must be taken into consideration in regard to the effective insolation of water is its chemical composition, for it has been shown[5] that the action of sunshine in destroying germs in water is very considerably increased when common salt is added to the water, and this opens up a wide field for experimental inquiry before we can accept sunshine as a reliable agent in the purification of water. [5] percy frankland, _our secret friends and foes_, 4th edition, p. 188. again, we must remember that a great deal depends upon the condition of the microbe itself. if it is present in the spore or hardy form, then considerably longer will be required for its annihilation. this fact has been abundantly shown in the case of anthrax, which in the condition of spores will retain its vitality in water flooded with sunshine for considerably upwards of a hundred hours, the bacilli being far more easily destroyed. we must also bear in mind that the individual vitality of the microbe is an important factor in determining its chance of survival; if it is in a healthy, vigorous condition, it will resist the lethal action of sunshine for considerably longer than when its vitality has been already reduced by adverse surroundings. it is, therefore, sufficiently obvious that the power of insolation to bacterially purify water is by no means easy of estimation, and that numerous and very varied factors have to be taken into account when we attempt to endow it with any measure of practical hygienic importance. in connection with the vitality of anthrax germs in water, which has afforded material for so many laboratory investigations, it is of interest to consider what chance exists of anthrax being communicated by water. until a few years ago, as far as i am aware, no instance had been recorded of anthrax having been actually communicated by water, until an outbreak of anthrax on a farm in the south of russia was distinctly traced by a skilled bacteriologist to the use of water from a particular well, in the sediment of which the bacillus of anthrax was discovered. anthrax bacilli have also been detected in the water of the river illinois in the vicinity of chicago, one of the chief sources of pollution of which is the slaughtering of cattle and the discharge of their offal into the river. the likelihood of such contamination taking place through the drainage of soil makes it of importance to ascertain what may become of the bacilli of anthrax derived from the bodies of animals which have died of this disease, and whose carcasses have been buried and not burnt. the anthrax bacillus cannot produce the hardy spore form within the bodies of animals, but it does outside. now it has been shown that the bacilli of anthrax taken from the blood of an animal dead of anthrax are destroyed rapidly in ordinary river thames water, for example, but that if the temperature of the water to which they gain access is somewhat higher than usual, such bacilli are able to sporulate or produce spores in the water, and in that hardy form can retain their vitality and virulence for several months. that anthrax bacilli can produce spores in water under certain conditions has not hitherto been dwelt upon in discussing the question of their vitality in water, and it is of obvious importance in connection with the action of sunshine on anthrax germs in water, knowing as we now do the very different manner in which the spores and bacilli respectively behave when under the influence of the sun's rays. it was not, perhaps, unnatural that rash assumptions as to the efficacy of sunshine should have been readily accepted when such remarkable feats performed on microbes by sunshine were being continually put forward. thus it has been found that insolation, even when it does not destroy, may effect profound changes in the physiological character of certain micro-organisms. dr. lohmann, of rostock, discovered that some hours' exposure to bright sunshine entirely destroys yeast cells, whilst even feeble and intermittent sunshine is capable of paralysing them, and that they only recover their vitality when removed from this obnoxious influence. this recuperative power is not, however, shared equally by all varieties of yeast, some possessing it in a far greater degree than others. dr. lohmann also found that yeast cells, after being exposed to sunshine, assumed a shrunken and distorted appearance, showing that insolation had produced a striking physiological effect upon the structure of these cells. professor hansen published some years ago a most interesting memoir on some of the characteristic features of the moulds which are to be found on manure heaps, in which he records how light exerts a very important influence on the manner in which the spore or fruit of these lowly vegetables is set free or distributed. all the various phases in the fructification process of some of these moulds were carefully watched by dr. hansen. he kept his caged specimens near a window with an eastern aspect, and he states that in the first instance the stalks inclined towards the light, but that afterwards they assumed an upright position. darkness was nearly always chosen for the liberation of the spores, but in a few instances a small number were released during the daytime, and it was noticed that when this did occur they were invariably discharged on the side away from the source of light. in various other ways he confirmed this interesting observation, and found that the fruit of the mould was invariably discharged in the opposite direction to that in which the stalk had previously inclined under the influence of light. the force with which the spores were discharged varied very considerably, sometimes being cast to a distance of four inches or more from the stalk, and sometimes being found close to and even on the stalk. the manner in which sunshine may also modify the pigment-producing powers of micro-organisms is remarkable. many microbes are able to elaborate when grown on various culture media, such as gelatine or slices of potato, most brilliant and beautiful pigments ranging from intense blood-red to the most delicate shades of pink, and embracing every gradation of yellow, as well as browns, greens, and violets. now it has been found that some of these pigment-producing bacteria, when exposed to sunshine on these nutritive materials, fail to exhibit their characteristic colour, although the duration of insolation may not have sufficed to destroy their actual vitality. one of these organisms originally obtained from water has been specially studied in this respect by m. laurent. if slices of potato are streaked with a small number of this particular bacillus (_bacille rouge de kiel_) a magnificent patch of blood-red colour makes its appearance in the course of a day or two, but if, on the other hand, similar slices of potato are exposed to three hours' sunshine, a colourless growth subsequently develops, except where here and there a few isolated spots of pale pink are visible. when the insolation is prolonged for five hours nothing whatever appears on the potato, the bacilli having been entirely destroyed. but this is not all. m. laurent found that if he took some of the colourless growth and inoculated it on to potatoes he obtained again, but without insolation, a colourless vegetation--in fact, three hours' insolation had so modified the physiological character of the bacillus that _a new race had been generated, a race deprived of its power of producing this red pigment_. in what numerous directions the character of microbes may be and are being modified, even by simple exposure to sunshine, opens up a wide field for speculation and research, whilst the tractability of these minute and most primitive forms of life, if we only approach their education with sufficient insight and patience, may enable us to make them serve where they now are masters. the remarkable discoveries on the modification of the disease-producing properties of certain bacteria by sunshine may perhaps encourage the idea that we are making some progress towards the attainment of this desirable millennium. that diminution of the virulence or disease-producing power of such deadly microbes as those of cholera, anthrax, and tuberculosis can be brought about through simple exposure to the sun's rays seems almost inconceivable, yet it has been discovered that by placing the cholera bacillus, for example, in the sunshine its virulent character undergoes such a profound modification that it is actually reduced to the condition of a vaccine, and may be employed to protect animals from infection with its still virulent brethren. yet this is what has been undoubtedly shown by dr. palermo in very carefully conducted investigations. he was, moreover, able to indicate, within a very narrow margin, the precise amount of insolation necessary to bring about this result: for if the cholera cultures were only exposed for three hours, their toxic properties were not reduced to the condition of vaccine; but if the insolation was continued for three and a half hours up to four and a half hours, they became endowed with the requisite immunising properties, and animals treated first with the so-called sunshine-cholera-vaccine were able subsequently to withstand otherwise fatal doses of virulent cholera cultures. dr. palermo also found that, besides producing this subtle modification in the character of cholera bacilli, sunshine exerted a remarkable physiological change in these organisms, for when examined under the microscope they no longer exhibited their typical activity, having been deprived of all powers of movement, whilst those kept during the same length of time in the dark had not abated one jot of their customary mobility. but sunshine not only controls in this wonderful manner the action of the living bacillus, but it also operates upon the products elaborated by disease organisms. thus the microbe producing lock-jaw or tetanus may be grown in broth, and the latter may be subsequently passed through a porcelain or a berkefeld filter, so that the resulting liquid is entirely deprived of all germ life. this tetanus-filtrate, as it is called, is endowed with very powerful toxic properties, and it will retain its lethal action even when kept for upwards of three hundred days, providing it is screened from all light; but place such filtrates in diffused light, and they lose their poisonous properties, requiring, however, upwards of ten weeks to become entirely harmless; if, on the other hand, they be exposed to sunshine, they are completely deprived of their toxic character in from fifteen to eighteen hours. again, as little as five hours' sunshine is sufficient to greatly modify the toxic action of diphtheria cultures. it is of interest also to note that even the venom of the rattlesnake, that most potent of all poisons, cannot emerge unscathed from an exposure to sunshine maintained during a fortnight. interesting as all these isolated observations are, they indicate what an immense amount yet remains to be done before we can hope to have any connected conception of the mechanism, so to speak, of insolation. at present there is too large an allowance, which we are compelled to make, for the unknown to permit of our adequately manipulating this marvellous agency in relation to bacteriological problems. but who shall say what part has been, and is being still, played by sunshine in determining the individual character of microbes, operating as it has done from time immemorial upon countless generations of these minute germs of life? the problem of insolation has been attacked from an entirely novel point of view by dr. masella, who has endeavoured to find out whether sunshine plays any part in the predisposition of animal life to infection. now sunshine has long been credited with possessing therapeutic powers, and, indeed, traditions of cures effected by the ancients by means of insolation have been treasured up and handed down to the present day. even as late as the beginning of the present century we may read of a french physician seriously recording his claim to have cured a dropsical patient within two weeks by placing him daily for several hours in the sunshine, and many medical journals of recent years contain communications on the beneficial results derived from the use of sunshine in the treatment of various diseases. it seems curious, therefore, that whilst so much has been done to test the action of light on disease microbes in _artificial_ surroundings, such as are to be found in laboratory experiments, hardly any investigations have been made to try and define more precisely how sunshine may affect their pathogenic action within the animal system. dr. masella's researches, undertaken with the express object of, if possible, elucidating this question, are therefore of special interest and importance. the first series of experiments was carried out to ascertain whether exposure to sunshine increases or reduces an animal's susceptibility to particular diseases, those selected for investigation being typhoid fever and cholera. for this purpose guinea-pigs were exposed to the full rays of the sun during a period of from nine to fifteen hours for two days, whilst other guinea-pigs, for the sake of comparison, were not permitted to have more light than that obtainable in a stable where only diffused light was admitted. both these sets of animals were subsequently infected with virulent cultures of cholera and typhoid germs respectively, and were in neither case exposed to sunshine. the results which dr. masella obtained were remarkable, for he found that those animals which previous to infection had been placed in the sunshine died more rapidly than those which had been kept in the stable, and that the exposure to the sun's rays had so increased their susceptibility to these diseases that they succumbed to smaller doses, and doses, moreover, which did not prove fatal to the other guinea-pigs. still more striking was the part played by insolation in the course of these diseases in animals exposed to sunshine _after_ inoculation, for instead of dying in from fifteen to twenty-four hours, they succumbed in from three to five hours. here, then, we find sunshine, in some mysterious manner not yet understood, far from benefiting the animal and assisting it in combating these diseases, actually contributing to the lethal action of these bacteria. it has been asserted on the authority of some medical men that in cases of small-pox recovery is rendered more easy and rapid when light is excluded from the patient's room; whether dr. masella's experiments will permit of any such interpretation being placed upon them remains to be seen; they are, at any rate, extremely suggestive. that it is possible for temperature to have some determining influence upon the course of certain diseases has been shown by o. voges, who, experimenting with a minute bacillus which he isolated from tumours characteristic of a cattle disease very prevalent in south america, found that although this bacillus was the undoubted _fons et origo_ of the disease, he could not produce fatal results in animals if he kept them in cold surroundings; only when the temperature was raised to from 35-45 degrees centigrade did the infected animals succumb. the dependence of the activity and virulence of this micro-organism upon temperature is also borne out in actual experience, the disease being the more prevalent and the more fatal the hotter the climate of the country. it may be mentioned in passing that this bacillus has the distinction of being the smallest yet discovered; the influenza bacillus hitherto held the palm in this respect, but it must yield its position to its more successful rival, for voges states that when magnified about fifteen hundred times it is only just discernible in the microscopic field. even the smoke-laden atmosphere of our great cities, our leaden skies and dreary fogs and mists, may after all, then, if we can only learn to look at them from dr. masella's point of view, become a source of benefit and a subject for congratulation; yet our inherent love of light and sunshine would cause us willingly to hand over our murky climate had we but the chance of obtaining in exchange that of any of the sunny cities of the south. moreover, in the case of tubercular disease experience is daily impressing upon us the wisdom, and indeed necessity, of absorbing as much sunshine as possible, and hence the pilgrimage which is now recommended to davos and other resorts where invalids can get the maximum amount of bright sunshine. and not only is this the outcome of practical experience, but de renzi has shown by actual experiment that sunshine acts beneficially in cases of tuberculosis in animals. thus, guinea-pigs were infected with tuberculous material and exposed in glass boxes to the sun for five or six hours daily, others being similarly infected but protected from sunshine. the animals which had received the sunshine died in 24, 39, 52, and 89 days respectively, whilst those which had not been sunned succumbed in from 29, 25, 26, and 41 days; or, in other words, de renzi found that insolation had very materially increased the infected animals' power of coping with tuberculosis. the part which sunshine plays, or may be made to play, in disease is very obscure, but it would appear at least justifiable to assume that it is an agent which further investigation may show we cannot afford to disregard, contributing as it may to the production of a healthy tone in the system, and thereby materially assisting the body to defy the insidious attacks made upon it from without. the so-called open-air treatment of consumption which has made such giant strides in the last few years is an example of how, by contributing to the general health of an individual, the powers for resisting a localised disease may be so increased that the latter can, in many cases, be thrown off altogether. in no country has more progress been made in the establishment of institutions for the cure of consumption on these lines than in germany. at the end of the year 1899 there were forty-nine such institutions in germany, with four thousand beds; in a little more than twelve months later there were no less than sixty such, with accommodation for altogether five thousand patients. it is of interest to note that amongst the earliest of these institutions to be founded was that erected and endowed by the famous badischen anilin and soda fabrik company, for the exclusive benefit of those of their workpeople who were suffering from tuberculous disease. we have learnt that sunshine is endowed with distinctly lethal action as regards particular bacteria, that it can modify the subtle properties of toxic solutions, and we are asked to believe that it may exercise an important influence on the animal system in determining the power of the latter to deal with the agents of disease; but, as we have seen, the mechanism of it all is shrouded in mystery, and we are at a loss to divine how it works. might not some fresh light be thrown upon this problem if we could ascertain the effect of sunshine on some of these natural fluids of the body, which recent brilliant research has shown to be endowed with such wonderful protective or immunising properties? so far as i am aware, the action of sunshine on these anti-toxins or protective fluids has not yet been investigated. can sunshine interfere with the therapeutic effect of diphtheria-serum, for example? if simple insolation can so profoundly modify the character of toxic fluids, it is not unreasonable to anticipate some action on these anti-toxins, and their study in this connection would appear to offer an important step in the direction of unravelling the mystery attending the action of light on life. bacteriology and water whilst the hamburg cholera disaster of 1892 will certainly rank in the annals of epidemiology as one of the great catastrophes of recent times, it will also be memorable as one of the most instructive which has ever taken place. it is perhaps not unnatural that this should be the case, for since the last european epidemic of importance our study of the principles of sanitation has received a new impetus, and this impetus must be in great part ascribed to the science of bacteriology, which has sprung into existence within the past two decades. we have now no longer to confront mysterious and unknown morbific material, but have been brought face to face with some of the most dreaded foes of the human race. we are no longer groping, as it were, in the dark, but have a definite object, in the shape of well-recognised micro-organisms associated with specific zymotic diseases, for our common crusade. but it is the light which has been thrown for the first time upon numerous intricate problems connected with the sanitary aspects of public water-supplies which constitutes not the least important of the many services rendered by bacteriology to the public. perhaps one of the most striking of these may be considered the insight which it has afforded into the value of various processes of water-purification, furnishing us with the most subtle and searching tests, surpassing in delicacy those of the most refined chemical methods. thus for years the processes of sand-filtration, as practised at waterworks in dealing with river and other surface waters, were regarded by chemical experts as of but little or no value, because, on chemical analysis, but little or no difference was found to exist between the filtered and unfiltered samples respectively. water engineers started this method of water treatment in london as far back as the year 1839, with no other object than the distribution of a water bright and clear on delivery, but, unknown to themselves, they were carrying out a system of water-purification the nature and extent of which has been left to the infant science of bacteriology to unravel and reveal. it was in the year 1885 that dr. koch's new bacteriological water-tests were introduced, and systematically applied for the first time to the london water-supply by professor percy frankland, and the entirely unexpected result obtained, that whereas the river thames water at hampton contained as many as 1,644 micro-organisms in about twenty drops, this water, after passing through the sand-filters, possessed as few as thirteen in the same number of drops. the remarkable purification effected in the treatment of the water was thus very clearly shown, and an entirely new aspect was given to the processes of sand-filtration. the importance of these results was quickly appreciated by the official water-examiner, the late sir francis bolton, and at the request of the local government board regular monthly bacteriological examinations of the london water-supply were conducted. it is amusing to recall that, at the time when these results were first published, the public, instead of being reassured by these facts, were greatly alarmed, and it is a matter of history that the mere demonstration of the presence of micro-organisms in drinking-water caused a fall in the price of several of the water companies' stocks! these investigations, which have since been confirmed by others both in this country and on the continent, have clearly shown, then, that sand-filtration, when carefully carried out, offers a most remarkable and obstinate barrier to the passage of microbes, and there was every justification in presuming that if disease organisms should at any time be present in the raw untreated water, they would also undergo a similar fate, as there was no reasonable ground for supposing that they would behave any differently from the ordinary harmless water bacteria. but this was a hypothesis only, and, however satisfactory experiments in this direction made in the laboratory might prove, there was always the uncertainty attaching to a fact which had not passed through the ordeal of practical experience. the answer to this searching and all-important question has been furnished in the most conclusive manner by the history of the cholera epidemic in hamburg and altona respectively in the year 1892. these two cities are both dependent upon the river elbe for their water-supply, but whereas in the case of hamburg the intake is situated _above_ the city, the supply for altona is abstracted below hamburg _after it has received the sewage of a population of close upon 800,000 persons_. the hamburg water was, therefore, to start with, relatively pure when compared with that destined for the use of altona. but what was the fate of these two towns as regards cholera? situated side by side, absolutely contiguous, in fact, with nothing in their surroundings or in the nature of their population to especially distinguish them, in the one cholera swept away thousands, whilst in the other the scourge was scarcely felt; in hamburg the deaths from cholera amounted to 1,250 per 100,000, and in altona to but 221 per 100,000 of the population. so clearly defined, moreover, was the path pursued by the cholera, that although it pushed from the hamburg side right up to the boundary line between the two cities, it there stopped, this being so striking that in one street, which for some distance marks the division between these cities, _the hamburg side was stricken down with cholera, whilst that belonging to altona remained free_. the remarkable fact was brought to light that in those houses supplied with the hamburg water cholera was rampant, whilst in those on the altona side and furnished with the altona water not one case occurred. we have seen that the hamburg water, to start with, was comparatively pure when compared with the foul liquid abstracted from the elbe by altona, but whereas in the one case the water was submitted to exhaustive and careful filtration through sand before delivery, in hamburg the elbe water was distributed in its raw condition as drawn from the river. but further testimony was afforded later to the truth of these results, for during the winter, whilst the cases of cholera had almost completely died out in hamburg, suddenly a most unexpected and unaccountable recrudescence of the epidemic occurred, and this time in altona. this outbreak could not be traced to any direct infection from hamburg, but must have arisen in altona itself. in all about forty-seven cases were recorded between december 23rd, 1892, and february 12th, 1893. a searching inquiry was instituted, and it was ascertained that the number of bacteria found in the filtered water, usually about fifty, had during these months risen to as many as 1,000 and more in about twenty drops of water, clearly indicating that the filtration of the water was not being efficiently carried out. that this was actually the case was proved by the fact that one of the sand-filters which had been cleaned during the frost had become frozen over, and was not able to retain the bacteria. that the outbreak did not become more serious koch ascribes to the fact that this, to all intents and purposes raw untreated water, was largely diluted with efficiently filtered water before delivery. dr. koch, who personally superintended this inquiry in altona, traced another local outbreak of cholera in the city to the use of a well-water obviously open to pollution, which was used by about 270 persons. in one of the houses employing this water, and in the immediate vicinity of the well, a boy died of cholera on january 23rd, and during the week following a number of cases occurred amongst persons using this source. on discovering the cholera bacilli in this polluted water, its contamination was placed beyond doubt, and five days after the well was closed all cases ceased in the locality. there cannot be any longer a doubt as to the value of sand-filtration as a means of water-purification, but the responsibility which we have seen attaches to this treatment of water cannot be exaggerated, for whilst when efficiently pursued it forms a most important barrier to the dissemination of disease germs, the slightest imperfection in its manipulation is a constant menace during any epidemic. it is, as a rule, during the winter months that the largest number of bacteria are present in the filtered water, and it is therefore of especial importance that during this season, when the raw river water is generally richest in bacterial life, and when, therefore, the filters are most taxed and the consequences of frost are most to be apprehended, that those entrusted with this responsible task should be unremitting in their endeavours to obtain a good filtrate. that waters submitted to exhaustive natural filtration, such as those derived from deep wells sunk into the chalk, and usually almost entirely devoid of bacterial life, may at times become the carriers of disease has been proved by the disastrous outbreak of typhoid fever which occurred some years ago at worthing. this town has long been supplied with water of the very finest quality for dietetic purposes, and nothing could have been more unexpected than this most fatal epidemic. it must, however, be borne in mind that such deep-well waters are not necessarily immaculate, for in the event of any fault in the water-bearing strata occurring, the filtration becomes inefficient, and the water may then, as in the case of worthing, be the bearer and disseminator of zymotic disease. the bacteriological methods for the examination of water, although when first introduced but a few years ago were lightly looked upon, and by many opposed, have now become of paramount importance in all questions of water-purification. the immense mass of evidence of a purely bacteriological character which was taken, and indeed required by the royal commissioners of 1893 on the london water-supply, indicates clearly enough the change which has taken place in the public estimation of the value of these methods; and it is highly significant that in their report the commissioners lay stress upon the importance of extensive storage and efficient filtration, two factors the meaning and worth of which rest almost entirely on the results of bacteriological research. cholera is not, however, the only water-carried disease which has borne eloquent testimony to the services rendered by the efficient purification of public water-supplies. the experience of the state of massachusetts in america, in regard to typhoid fever and drinking-water, is also exceedingly instructive. massachusetts has, by creating a board of health, and affording the same every facility for the prosecution of hygienic investigations of the greatest importance, laid the whole scientific world under a deep obligation. the reports issued have a very wide circulation, and embrace a variety of subjects, but second to none in interest and importance is the account of the experimental work carried out by the officials of the board on the purification of water and sewage. these experiments have become classical, and have been conducted with a zeal and thoroughness which deserve the highest praise. it is in looking at the results achieved by the city of lawrence in regard to its water-supply that some conception can be obtained of the immense importance to the community of the scientific experiments conducted in the state laboratory. no expense has been spared, and for years past elaborate and costly experiments on a large scale have been carried out to determine the most efficient manner in which water may be rendered fit and safe for drinking. now the death-rate in a community from typhoid fever may be taken as an index of the general sanitary conditions prevailing in such a community, the character of the public water-supply, not without justification, being regarded as a prime factor in its determination. one of the most significant points in the sanitary history of the state of massachusetts is the almost uniform decline in the mortality from typhoid fever in proportion as measures have been taken to introduce better water-supplies and to improve those which already exist. thus in the twenty years, from 1856 to 1876, the death-rate from this disease was 8·6 per 10,000 of population, whilst in the period from 1876 to 1895 it had fallen to 4·1 per 10,000, the improvement in respect of typhoid-mortality being coincident with the improvement made during the last twenty years in providing public water-supplies. in the words of one of the state reports, "the death-rate from typhoid fever has generally fallen as the per cent. of the population supplied with public water has risen, for the reason that the majority of the deaths from this disease have occurred among communities and portions of communities _not supplied with public water_." that this improvement is being maintained is seen from the fact that in the four-year period 1896-99 the deaths from typhoid fever in massachusetts were further reduced to 2·6 per 10,000. it is, however, in the city of lawrence that the most striking insight is obtained as to the manner in which typhoid fever may be controlled by conditions surrounding the water-supply to the community. thus, whereas the death-rate from typhoid fever reached a mean of 11·2 per 10,000 in 1886-90, it fell to 7·7 in 1891-95 and to 2·5 in 1896-99. it was in the autumn of the year 1893 that the raw river-water supplied to the city from the merrimac river was first begun to be filtered, and since that time the sand-filters have been subjected to systematic and most elaborate bacterial supervision, and improvements have been constantly introduced so as to secure the most efficient purification possible of the water before distribution, and the results are reflected in the marked diminution in typhoid fever which has followed these strenuous efforts to obtain the best water-supply available. the splendid example set by the state of massachusetts, in promoting the welfare of the people by the encouragement of original researches in practical hygiene, has stimulated other american states to create boards of health and enact laws for the protection of their rivers and streams. in view of all that has been done to promote sanitary science in the united states, it is surprising to learn that lake michigan, which receives the untreated sewage of municipalities and small towns aggregating over two million people, still furnishes chicago with its drinking water, and undergoes no preliminary purification before distribution. the city of chicago, by constructing the chicago drainage and ship canal, opened in january, 1900, has diverted its own sewage from lake michigan, but this great sewer has only been made possible because of its advantages as a commercial waterway; and it has been stated, on high authority, that every project for the drainage of chicago into the illinois which has not recognised the waterway features has been predestined to failure. dr. egan, of the illinois state board of health, however, points out that "with the present increase in population the great lakes, if they continue to be used as common sewers, will soon become totally unfit for use as drinking water, ... and one of two alternatives must be followed--either every source of water-supply must be filtered, or the sewage of the towns must be efficiently purified before it is allowed to flow into the lakes." doubtless this seeming inertia of the citizens of chicago in the matter of filtering their water is attributable to the fact that already the authorities have expended eighty-five million dollars in their waterworks and sewerage systems, which represents an investment of something over fifty dollars per head of population, and that plans in connection with the great canal which has been described as "the greatest feat of sanitary engineering in the world," and to which reference has already been made, will, when carried out, involve an expenditure of thirty or forty million dollars more. in the face of such burdens even so prosperous a community as chicago does not care to contemplate further capital charges, at any rate until the unsatisfactory conditions shadowed by dr. egan become more pressing in regard to the source of their water-supply. the systematic investigations carried out in the great institutes of health on the continent and elsewhere should surely make the sporadic work, as by comparison it must be designated, produced in this country an eloquent argument for the creation of a british imperial board of health adequately endowed by the state, manned by the ablest investigators, and forming a centre for the prosecution of researches which in other countries, as in our own, have contributed so greatly to the health and welfare of mankind. why should england for ever have to knock at the door of foreign institutes for information and guidance in matters in which once she was the leader and enlightened example to every civilised country? the question of how far polluted water-supplies, besides possessing the potentiality for spreading cholera and typhoid, may disseminate consumption, has been approached in a very instructive manner by dr. musehold, of the german imperial board of health. some ten years ago the discovery of the tubercle bacillus in water for the first time was announced by a spanish investigator, fernandez. the water containing the bacillus tuberculosis was derived from an open ditch, and hence had been doubtless exposed to contamination of divers kinds. in the course of the elaborate experiments on london sewage and its treatment, carried out by professor frank clowes, chief chemist to the london county council, an instance was recently met with in which a guinea-pig, inoculated with a portion of coke-deposit derived from a bacterial sewage bed, died from typical tuberculosis, and sections of its organs showed the presence of numerous tubercle bacilli. dr. musehold has now submitted the whole question of the vitality of virulent tubercle bacilli present in the expectorations of consumptive persons in sewage, in river water, and on cultivated land respectively, to an exhaustive examination, and the novelty as well as importance of these researches merit their being carefully considered. in the first instance tuberculous sputum was introduced into river water in its natural condition, and as this water was abstracted from the river spree, in berlin, it was exposed at any rate to a certain degree of surface contamination. in this water, kept in ordinary daylight, the tubercle bacilli remained alive and in a virulent condition for over five months; in ordinary sewage for six and a half months. some of the sewage-infected samples were left in the open air and exposed to ordinary meteorological conditions, but even the ordeal of getting frozen up in their surroundings did not in the slightest shorten the lease of virulence possessed by the tubercle bacilli. some of this tubercle-infected sewage was poured over garden soil in which radishes were growing, and after the bacilli had spent eighty-eight days in these surroundings, during which time they had experienced frost, snow, rain, and sunshine, they still retained their virulence. of special interest are the investigations dr. musehold made to ascertain if tubercle bacilli could be detected on the fields attached to a hospital for consumption and irrigated with the sewage from the same. not only were tubercle bacilli found, but they were also, as was to be expected from the laboratory experiments cited above, discovered in a highly virulent condition. that disease germs may be distributed with the vegetables grown on municipal sewage farms is not a mere whim or fancy of the faddist, but is a very real danger, and must be regarded as a menace to the health of all who consume such articles as lettuces, radishes, celery, and other vegetables which are not first cooked before being placed on the table. this forcibly suggests the desirability of all expectorations from consumptive patients being thoroughly disinfected, or, in other words, deprived of their virulence before being admitted to sewage. the importance of such precautions being taken is borne out by the examinations of the clear effluent derived from the treatment of the sewage of a consumptive hospital which revealed the presence of virulent tubercle bacilli, whilst they were also discovered in the bottom of a ditch conducting the effluent away. such facts as these deserve the earnest attention of all public authorities, and it is to be hoped that the overwhelming evidence which is now available regarding the distribution and spartan character of the tubercle bacillus will lead to serious efforts being made to bestow upon it that measure of consideration which in the case of recognised zymotic diseases leads to the enactment of rules and regulations for the restriction at least of the fateful activities of these malignant foes of mankind. before leaving the subject of bacteria in relation to water, it will be interesting to glance at what is known regarding the attitude taken up by these minute forms of life towards that large and ever-increasing class of waters vaguely grouped together under the synonym of mineral waters. the fortunes made in manufacturing artificially aërated waters and the mine of wealth contained in a new mineral spring are sufficient evidence of the popularity enjoyed by this description of beverage. the beer and spirit statistics of the country and their contributions to the national revenue do not, however, permit us to indulge in the belief that this large consumption of harmless drinks is due to their displacing the use of intoxicants--the increasing sale of non-alcoholic beverages cannot in fact be taken as an index of the growing sobriety of a nation; far more must the greater demand be attributed to the improvements in manufacture which have cheapened production and placed what was formerly an article of luxury almost prohibitive in price, and hence reserved for the few, within comparatively easy reach of the many. perhaps also an increased sale may be assisted by a prevailing impression that by substituting carbonated for ordinary potable water, the risk of contracting zymotic disease is, if not altogether removed, at any rate very materially diminished. it will be therefore instructive to see how far this assumption is justified by actual facts. the first fact to be recognised is that the number of bacteria present may and does fluctuate between such wide limits as is represented by as few as three, and as many as 100,000 being found in about twenty drops of artificially aërated waters. seltzer water, manufactured from well water, was found by sohnke to contain numbers varying from 200 to 6,000, whilst when only distilled water was used, _i.e._ water previously deprived of all bacterial life, only from ten to thirty microbes were present. but an important and far too little recognised factor in the manufacture of aërated waters is the contamination which so frequently takes place subsequent to the initial purification of the water by sterilisation. in some instances this contamination is due to the storing of water before use in reservoirs, where an excellent opportunity is offered for microbial multiplication. merkel found water which originally only boasted of from four to five bacteria per cubic centimetre, subsequently, when ready for distribution as seltzer water, contained considerably over 3,000. in this case storage had been resorted to. again, insufficient importance is attached to the efficient cleansing of the syphons on their return to the factory. the experiments made by slater in this country and abba in italy have conclusively shown that the gaseous aëration of water exerts an inhibitory action on the growth of at least some varieties of water bacteria, for both these investigators found that in proportion as the amount of gas present was diminished by being allowed to escape, so was the multiplication of the bacteria present promoted and their numbers increased. unsavoury as may be the idea of swallowing down myriads of even harmless microbes, yet the real significance of the whole question from a hygienic point of view lies in the evidence as to the fate of disease germs in aërated waters. on this important matter there fortunately exists some precise and conclusive information in regard to the bacteria associated with two essentially water-borne diseases, _i.e._ typhoid fever and cholera. the investigations made to test the vitality of the anthrax bacillus are of significance as again emphasising the superior degree of vitality possessed by the spore over the bacillar form of this micro-organism, but the chances of this disease being disseminated by water are usually regarded as too remote to excite much interest in the fate of the _b. anthracis_ in seltzer water. it may, however, be mentioned that whereas the bacilli succumbed after being in the seltzer water from fifteen minutes to an hour, the spores were still living after one hundred and fifty-four days. investigations on the vitality of cholera bacilli in aërated waters have been made by hochstetter in germany, by slater in england, and by abba in italy, and these various authorities all agree that the lease of life of these micro-organisms is a very short one in ordinary unsterilised carbonated waters, and that they are in fact destroyed in from half an hour to three hours. as regards typhoid bacilli the case is different, for the same investigators found that in ordinary unsterilised aërated water these bacteria can live as long as eleven days. in seltzer water their vitality is not so marked, but even then it greatly exceeds that of the accommodating cholera microbes, extending to five days. thus supposing typhoid bacilli to be present in the water employed for the manufacture of aërated waters--and we cannot afford to disregard such a possibility--we have no guarantee that such waters will be safe for drinking purposes unless a considerable period has been allowed to elapse between their production and consumption. it was considerations of this kind which led m. duclaux, the accomplished director of the paris pasteur institute, to write now some years ago: "contentons-nous de conclure que l'usage de l'eau de seltz, recommandé en temps d'épidémie peut en effet être recommandable, surtout si on laisse vieillir l'eau quelques jours. on a chance d'y voir diminuer ou même périr les germes nuisibles." on the whole, therefore, the scientific report on bacteria and artificially aërated waters may be regarded as a reassuring one. it is to be regretted, however, that in england we do not follow the example set by italy, where the aërated water manufacturers are closely looked after by the state, and no factory may be opened unless a satisfactory guarantee can be given of the chemical and bacteriological purity of the water which is intended to be used, whilst the authorities must also be assured that the methods employed are satisfactory from a hygienic point of view. the sale of all aërated waters prepared from insanitary water-supplies is strictly prohibited by the state. it will now be of interest to ascertain what is the result of the endeavours which have been made to explore the bacterial flora of those highly prized and largely circulated natural mineral waters, which abound in so many parts of the world and are practically the making of so many health resorts. perhaps the most exhaustive examinations of mineral water which have been so far made are those published by dr. eugenio fazio, who studied the bacterial condition of some of the celebrated springs situated near naples at castellamare, telese, acetosella, and muraglione, care being taken to select examples of different types of water, samples being collected from chalybeate, carbonated sulphur, and alkaline springs respectively. all these various mineral waters were characterised by a remarkable paucity of bacteria; in the chalybeate and alkaline springs sometimes as few as two microbes only in a cubic centimetre were found, and the largest number recorded only amounted to forty-five. the satisfactory significance of such figures will be appreciated when we realise that they rival very closely the numbers which characterise the purest spring and the deepest well water, and which are usually regarded as the aristocracy among drinking-waters. of special interest is dr. fazio's discovery that the variety of bacteria present in these waters is extremely restricted, as a rule only three, or at most four, different kinds of bacteria being detected. this is also characteristic of the pure water derived from deep wells sunk into the chalk, usually but very few different kinds of bacteria being found amongst the limited number of their lilliputian inhabitants, whilst in samples collected from rivers or other surface sources, especially those which have been polluted with sewage or similar refuse matters, the bacterial population is frequently as diverse as it is unwieldy. from the exacting point of view of the uncompromising bacteriologist the most satisfactory waters in existence for drinking purposes should be those derived from sulphur springs. dr. fazio and other investigators have frequently found absolutely no bacteria whatever in these waters, and often only four in a cubic centimetre. when we remember the high temperature of so-called thermal sulphur waters, which in many cases reaches more than fifty degrees centigrade, it is perhaps surprising that even four individuals can be found in a cubic centimetre capable of withstanding the nauseous atmosphere of sulphuretted hydrogen in addition to such hot environment. perhaps in the bacterial community these hot sulphur springs provide that place of punishment which figured so largely in the imagination of the early christian fathers; certain it is that in this bacterial hell, in the picturing of which so many of the old masters seem to have revelled, but very few individuals are to be found, and those which are there are almost entirely derived from one family. in giving weight to the highly satisfactory results of these bacterial examinations in forming an estimate of the microbial quality of natural mineral waters, it must be borne in mind that these investigations were all made of the said waters in a state of nature straight from the source, and before they had undergone the barbarous ordeal of commercial manipulation such as the process of bottling. we are all of us sufficiently acquainted with the first principles of germ life to realise how deftly and how directly any inattention to hygienic details is reflected in the larder or the store-room; and it requires but little stretch of the imagination to picture the bacterial armaments which would at once invade these peaceful waters on the first suggestion of relaxed vigilance, or removal of that rigid surveillance so essential for their protection and preservation. milk dangers and remedies it may with justice be said that in no department of applied bacteriology is more activity apparent than in that which has for its object the building up of a scientific basis for dairy practice. although this is undoubtedly true, yet, unfortunately, unlike its continental neighbours, the british public, with whom practically rests the control of our dairy industries, has hitherto held itself strangely aloof, evincing little or no sympathy in researches which, even if they fail to interest, should surely impress with a sense of the great hygienic importance attaching to them. but this apathy is not only to be deprecated in the interests of health, but also on economic grounds. we have only to turn to the reports issued by the board of agriculture to realise what this characteristic british apathy has brought about in the dairy industry of this country. thus in the year 1898 we are officially informed that we imported 359,425,136 pounds of butter, the little country of denmark alone sending over to us 163,883,360 pounds! our cheese imports reached the enormous total of 262,018,624 pounds, whilst 817,274 cwts. of condensed milk and 10,691 of milk and cream were supplied to us from without. if we glance at the energy and enthusiasm displayed by other countries, and notably denmark, in the prosecution and scientific development of the dairy industry, we shall not wonder at the high standard of excellence achieved, or at the readiness displayed by great britain to absorb their produce. thus, whilst in england it may be questioned whether in a single dairy the artificial souring of cream by pure cultures of bacteria is carried out, in denmark the use of so-called special bacterial butter-starters is rapidly gaining ground. thus, whereas in 1888 at the odense exhibition not a single sample of butter was exhibited in which pure bacterial cultures had been employed, in 1894, 46·7 per cent. of the samples shown were thus produced, in 1896, 89·2 per cent., in 1897, 94·4 per cent., 1898, 95·9 per cent, and in 1899, _every sample_, and since this year nearly every dairy of importance in the country employs special bacterial butter-starters. the danes are enlightened and shrewd enough to realise that in order to retain their existing markets and acquire fresh ones, it is necessary to take advantage of every improvement in methods of manufacture which scientific research has placed at their disposal, and their reward is justly reaped in the prosperity of their dairy industry and the high reputation enjoyed by their produce. if we contrast the adaptability and elasticity of the continental mind in regard to new discoveries with the crude conservatism of the british manufacturer, then, indeed, is the success of our rivals and corresponding decline of our own prosperity most perfectly intelligible. again, we are informed that the recent visit to london of a deputation representing russian agricultural interests is already bearing fruit, and contracts have been signed for the regular importation of large quantities of russian dairy produce. the english market is already well supplied with russian eggs, but an opening has now been found here for the disposal of russian butter and cheese. finland, again, the total population of which is less than half that of london, exports to this country no less than 12 million marks' worth of butter annually. as a writer recently put it: "foreigners and colonists have captured our butter markets; if the consumption of milk sterilised in bottles becomes the fashion, they will likewise capture our milk markets." and this is no fanciful suggestion, for whilst the production of pasteurised milk does not involve any considerable outlay in apparatus, its transport may be effected with the greatest ease. indeed, frozen milk has been introduced into england from norway and sweden. it is first pasteurised, then frozen in large wooden boxes, and shipped in the congealed condition, in which state it remains unchanged for a long period of time. but it is undoubtedly with the public that the responsibility really rests, for as long as it does not care to create the demand for pasteurised dairy products all the efforts of enlightened agricultural authorities in this country must inevitably end in failure. on the continent and in america dairy-bacteriology, as already pointed out, has made enormous strides, and has practically revolutionised the conduct of dairy work; and if we could but rouse ourselves from our lethargy we likewise should be able not only to boast of progress, but also to better hold our own ground in this important branch of agriculture; and one result would be that dairy troubles, which for so long have been accepted as more or less necessary evils, would yield here, as they have done elsewhere, to a more rigid attention to details, the significance of which scientific research has so successfully shown. some of the most easily preventable, but at the same time most aggressively assertive, dairy troubles are undoubtedly directly dependent upon the conduct of milking operations. in the first place, the cow itself is only too frequently in an uncleanly condition, and as its coat offers exceptional facilities for the harbouring of dust and dirt, the danger of foreign particles falling into the milk is always present unless precautions are taken to negative, or at least minimise, all such chances of contamination. professor h. l. russell, of the wisconsin agricultural experiment station, cites in his little volume on _dairy bacteriology_ an instructive experiment which brings home very forcibly the importance of such precautions. a cow pastured in a meadow was selected for the experiment, and the milking was done out of doors, so as to eliminate as far as possible any intrusion of disturbing foreign factors into the experiment, such as the access of microbes from the air in the milking-shed. the cow was first partially milked without any precautions whatever being taken, and during the process a small glass dish containing a layer of sterile nutrient gelatine was exposed for one minute beneath the animal's body, in close proximity to the milk-pail. the milking was then interrupted, and before being resumed the udder, flank, and legs of the animal were thoroughly cleansed with water; a second gelatine surface was then exposed in the same place and for the same length of time. the results of these two experiments are very instructive. when the cow was milked without any special precautions being taken, 3,250 bacteria were deposited per minute on an area equal to the surface of a ten-inch milk-pail; after, however, the animal had been cleansed, only 115 bacteria were deposited per minute on the same area. thus a large number of organisms can, by very simple precautions and very little extra trouble, be effectually prevented from obtaining access to milk. even in the event of the milk being subsequently pasteurised, clean milking is of very great importance; but still more imperative is it when it is destined for consumption in its raw, uncooked condition. if we consider how cows become covered with dirt and slime, that obstinately adhere to them when they wade through stagnant ponds and mud, and realise the chance thus afforded for malevolent microbes to exchange their unsavoury surroundings for so satisfactory and nourishing a material as milk, then indeed precautions of cleanliness, however troublesome, will not appear superfluous. that a very real relationship does exist between the bacterial and dirt contents of milk has been clearly shown by actual investigation. a german scientist has made a special study of the subject, and has determined in a large number of milk samples the amount of foreign impurities present per litre, and the accompanying bacterial population per cubic centimetre. the following results may be taken as typical of those obtained: in milk containing 36·8 milligrammes of dirt per quart as many as 12,897,600 bacteria were present per cubic centimetre; in cleaner samples, with 20·7 milligrammes of dirt per quart, the number of bacteria fell to 7,079,820; whilst in a still more satisfactory sample, containing 5·2 milligrammes of dirt per quart, there were 3,338,775 bacteria per cubic centimetre. such results indicate how important a factor is scrupulous cleanliness in milking operations in determining the initial purity of milk, for there is no doubt that bacterial impurities in milk are in the first instance, to a very great extent, controlled by the solid impurities present. i do not know of any determinations which have been made of the actual amount of such solid impurities present in our public milk-supplies, but such estimations have been made in many of those belonging to large cities in germany. thus, professor renk found in a litre of milk supplied to halle about 75 milligrammes, whilst in another sample as much as 0·362 grammes per litre were detected. in berlin 10 milligrammes, and in munich 9 milligrammes per litre, were found. dr. backhaus has estimated that the city of berlin alone consumes daily with its milk no less than 300 cwt. of cow-dung. if we associate these amounts of solid impurities with their consequent bacterial impurities, then we shall obtain some idea of what the microbial population of these milk-supplies may amount to. these impurities are almost wholly preventable, but, unfortunately, but little importance is apparently attached to their presence in milk as a rule by dairymen. in a letter published in the _sussex daily news_ a correspondent and well-known authority on dairy matters sounds a timely note of warning to our dairy managers:- "i happen to know," he writes, "for a fact that americans who visited one of our dairy shows at islington were so disgusted at the method--or rather lack of cleanly method--exhibited there as our ordinary way of milking cows, that these visitors stated that nothing would induce them to drink milk while in england. i mention this circumstance so as to bring home to the minds of english dairy-farmers who may read this letter how very backward we are in this country as compared with more studious and careful foreign competitors. it is insisted upon by our foreign teachers that our cow-stalls are too short and not roomy enough, and our cow-houses badly constructed; that we do not (1) groom our cows or (2) clean the teats, nor (3) sponge their udders, bellies, and sides before milking with clean, tepid water; (4) that the milkers do not tie up the cow's tail nor clean their own hands and persons, nor (5) cover their clothes with a clean, well-aired blouse during milking; that (6) they generally milk in a foul atmosphere (bacterially), tainted with the odour of dung, brewer's grains, or farmyard refuse. i am sorry to state that there is too much solid fact about the contentions which, based upon bacteriology, are given as causes of injury to quality.... cleanliness is now a matter requiring the primary attention of english dairy-farmers. the study of bacteria proves that such inattention is greatly the cause of foreign butters beating ours." it follows as a natural sequence that all the cans and vessels used for dairy purposes should be absolutely beyond suspicion of contamination. professor russell has shown by actual experiment that, even where the vessels are in good condition and fairly well cleaned, the milk has a very different bacterial population when collected in them and in vessels _sterilised by steam_. two covered cans were taken, one of which had been cleaned in the ordinary way, and the other sterilised by steam for half an hour. previous to milking the animal was carefully cleaned, and special precautions were taken to avoid raising dust, whilst the first milk, always rife with bacteria, was rejected. directly after milking bacterial gelatine-plates were respectively prepared from the milk in these two pails, with the following results: in one cubic centimetre of milk taken from the sterilised pail there were 165 bacteria; in that taken from the ordinary pail as many as 4,265 were found. another experiment illustrates perhaps even more strikingly the effect of cleanly operations in milking upon the initial bacterial contents of milk. the preliminary precautionary measures were carried out by an ordinary workman, and are in no sense so refined as to be beyond the reach of ordinary daily practice. "the milk was received in steamed pails, the udder of the animal, before milking, was thoroughly carded, and then moistened with water, so as to prevent dislodgment of dirt. care was taken that the barn air was free from dust, and in milking the first few streams of milk were rejected. the milk from a cow treated in this way contained 330 bacteria per cubic centimetre, while that of the mixed herd, taken under the usual conditions, contained 15,500 in the same volume. the experiment was repeated under winter conditions, at which time the mixed milk showed 7,600 bacteria per cubic centimetre, while the carefully secured milk only had 210 in the same volume. in each of these instances the milk secured with greater care remained sweet over twenty-four hours longer than the ordinary milk." an organism which has exceptional opportunities for finding its way into cows' milk is the _bacillus coli communis_, normally present in the fæces of all animals. this microbe is a very undesirable adjunct to milk, and may greatly interfere with the souring process, by multiplying extensively, and so producing a change in the milk which renders it impossible for the particular souring bacteria to carry on their work, resulting in their collapse and ultimate extinction. but this is not the only injurious effect which these coli bacilli can produce in milk, for there is a growing conviction that their presence is responsible for many intestinal disturbances with which young children are specially troubled. quite recently determinations of the bacterial contents of cow-dung have been made, and it has been ascertained that _a single gramme_,[6] freshly collected, of this material may contain as many as 375,000,000 bacteria, of which the majority were found to be the above undesirable organism, the _b. coli communis_. [6] one gramme = 15 grains. milk may also contain bacteria characterised by their remarkable resistance to heat, which is due to their possessing what is known as the hardy spore in addition to the ordinary rod form. the numbers in which they are present in milk varies with different samples; but they may be taken as a sort of index as to the care observed in milking, for they are always present in great quantity in uncleanly-collected milk. careful studies have been made of this class of milk bacteria by professor flügge and others, and it has been found that when added to milk upon which puppies were subsequently fed the latter succumbed under symptoms of violent diarrhoea. the danger of even a few bacteria gaining access to milk is serious, on account of the fabulous rapidity with which they multiply when they find themselves in such congenial surroundings. professor freudenreich has made very exhaustive investigations to show how milk microbes may multiply in the time which elapses between milking and the receipt of the milk by the consumer. the following example will convey some notion of what bacterial propagation under these circumstances is capable of. the sample of milk in question was found to possess on reaching the laboratory, two and a half hours after milking, a little over 9,000 bacteria in a cubic centimetre. the sample was divided into three portions, which were kept at different temperatures, and after definite intervals of time they were examined. the following table shows at a glance the results obtained:- number of bacteria in about twenty drops of milk. +-----------------+---------------------------------------+ | | temperature. | | when examined. +------------+-------------+------------+ | | 15° c. | 25° c. | 35° c. | +-----------------+------------+-------------+------------+ | after 3 hours | 10,000 | 18,000 | 30,000 | | after 6 hours | 25,000 | 172,000 | 12,000,000 | | after 9 hours | 46,000 | 1,000,000 | 35,280,000 | | after 24 hours | 5,700,000 | 577,500,000 | 50,000,000 | +-----------------+------------+-------------+------------+ thus, after being kept in the laboratory for three hours the original 9,000 bacteria had in one case doubled, and in another more than trebled themselves. it will be seen that the temperature most favourable to the multiplication of these bacteria was 25 degrees centigrade. if a sample of milk containing originally such a comparatively small number of bacteria--for a figure under 10,000 per cubic centimetre sinks into utter insignificance when we read of samples containing 2,500,000--if such relatively bacterially pure samples may support such prodigious numbers of these lilliputians, what the microbial population of less satisfactory samples may amount to well-nigh baffles our powers of calculation. professor russell writes: "if we compare the bacterial flora of milk with that of sewage, a fluid that is popularly, and rightly, supposed to be teeming with germ life, it will almost always be observed that milk when it is consumed is richer in bacteria by far than the sewage of our large cities. sedgwick, in his report to the massachusetts board of health for 1890, found that the sewage of the city of lawrence contained at the lowest 100,000 germs, whilst the maximum number was less than 4,000,000 per cubic centimetre.[7] this range in numbers is much less than is usually found in the milk-supply of our large cities." [7] american sewage, it must be noted, is usually weaker and poorer in bacterial life than that of our country, by reason of the greater amount of water with which it is diluted. numerous researches have been carried out during the last half-dozen years to try and localise the origin of some of the principal dairy troubles, with a view to their possible extinction, or at least control. in the course of these investigations quite a number of the bacteria found in milk have been successfully hunted down, and their offences brought home to them. thus, from so-called "bitter" milk a bacillus has been isolated by professor weigmann, and found responsible for this particular change. another microbe was discovered in bitter cream whose office apparently consisted in rendering milk strongly acid and extremely bitter. again, that objectionable condition of milk known as slimy, ropy, or stringy, is brought about by certain bacteria which render it viscous; whilst another crop of microbes are occupied in conferring upon it the power of sticking to everything that touches it, making it capable of being drawn out into threads from several inches to several feet in length. although we object in this country to slimy milk, in holland it is in special request for the production of a certain cheese known under the name of edam. in norway this kind of milk forms a popular drink called taettemjolk, and to produce it artificially they put the leaves of the common butter-wort (_pinguicula vulgaris_) into milk. professor weigmann has discovered a micro-organism which frequents the leaves of this plant endowed with particular powers of producing slimy milk, and doubtless the credit of furnishing taettemjolk is really due to this microbe, and not to the innocent butter-wort. "soapy" milk, again, has been traced to a specific germ discovered in large numbers in straw used for bedding, whilst it was also detected in the hay that served for fodder. during milking these sources had supplied the infection, and the peculiar fermentation was distinctly shown to be microbial in origin. so-called red and blue milk, and those various hues ranging from bright lemon to orange and amber, are also now known to be directly attributable to bacterial activity. but of even greater significance than all these bacterial dairy troubles is the risk of spreading disease which is furnished by milk contaminated with pathogenic micro-organisms. "there can be no shadow of doubt," said the _lancet_ now many years ago, "that the contagia of typhoid and scarlet fever are disseminated by milk, and that boiled milk enjoys a much greater immunity from the chance of conveying disease." this was written at a time when the study of bacteria was yet in its infancy, and before any direct experimental evidence had been obtained on the behaviour of microbes in milk or concerning the part played by them in the dissemination of disease. the writer evidently did not venture to cast further aspersions on the character of milk, or he might have included diphtheria amongst the diseases which can be spread by its means; but there is another omission which still more conclusively indicates the remote age in the history of bacterial science at which this correspondent to the _lancet_ wrote, and that is the absence of all reference to the tubercle bacillus in relation to milk. at the present day hardly a bacteriological journal is published which does not contain some reference to the question of tuberculosis and milk, and the transmissibility of this disease when present in cattle to man. as regards the dissemination of various zymotic diseases by milk, the evidence which has been collected points very conclusively to the responsible part which may be played by milk in this connection. many instances have been cited, also, of the culpability of milk in distributing typhoid germs. a striking case which occurs to me, and which may be mentioned in passing, is one which occurred in a city in america a few years ago, in which an outbreak of this disease was traced to a dairy in which the vessels had been washed out with typhoidal-polluted water. no less than 386 cases of typhoid declared themselves in six weeks, and of this number over 97 per cent. occurred amongst families obtaining their milk from the same dairy. a careful inspection revealed the fact that the milk-cans had been rinsed out with water from a shallow well contaminated with typhoid dejecta. diphtheria is also justly associated with infected milk, and if we take into consideration the now established fact that diphtheria bacilli thrive and multiply with particular facility in milk, even more so than in ordinary broth cultures; that they have been found in air in a vital and virulent condition, and may be scattered far and wide attached to dust particles; and if we remember the numerous opportunities offered for the infection of milk by persons handling it, who either themselves are suffering from this disease or are in diphtheria surroundings--then indeed we can readily understand how milk becomes a diphtheria-carrier of the first order. tuberculosis in cattle, and how this disease may affect the character of dairy produce, is, as already pointed out, a subject which is attracting the attention of a large number of investigators. the general public is perhaps hardly aware of how widespread this disease is amongst cattle, and it is only of late years that very careful inquiries have elicited the fact that it is not only very extensively distributed, but may be present in animals to all outward appearance in perfect health. in germany it was asserted a few years ago that every fifth cow was tuberculous, and even this was regarded as a moderate estimate. the distinguished danish pathologist, professor bang, is responsible for the announcement that during the years 1891-3 17·7 per cent. of the animals slaughtered in copenhagen were infected with tuberculosis. in paris we have been told that, of every thirteen samples of milk sold, one was infected with tubercle bacilli, whilst in washington one in every nineteen samples of milk was stated to be similarly tainted. the existence of tubercular disease in cows, and its transmission to other animals fed with their milk, has been brought out in a striking manner in investigations published by the massachusetts society for the promotion of agriculture. in one case as many as over 33 per cent. of the calves fed with milk from tuberculous cows succumbed to the same disease. according to hirschberger, 10 per cent. of the cows living in the neighbourhood of towns where the conditions of their environment are not generally the most satisfactory or conducive to health suffer from tuberculosis, and 50 per cent. of these animals yield milk containing tubercle bacilli. the demand which is being made by municipal authorities to be invested with the power of inspecting the country farms from whence their cities are supplied with milk and other agricultural produce could not have received stronger support than was recently supplied by a case tried in edinburgh, and as this is only a sample of what is doubtless a daily, although undetected occurrence in many municipalities, it will not be out of place to quote the following from the published report of the proceedings:- "a cow was brought into the city for sale as food, and the evidence showed it to be in the last stages of tubercular disease. 'its head was hanging down; it breathed with difficulty, and it had frequent fits of coughing; while its udder was swollen with the disease.' all the organs were diseased, and the milk teemed with bacilli. yet, it seemed, the milk from this animal had been regularly sent into edinburgh for sale. in face of facts like these, it is difficult to see on what grounds the claim of towns to inspect country dairies doing a town business can be resisted. at least the towns should have the power to refuse admission to milk from sources not open to inspection. it is not enough for the county authorities to say that they inspect the dairies in their own areas. in this case the condition of the animal was only found out when it was brought into the town to be sold for food." further comment is unnecessary! some german investigators have discovered the interesting fact that the centrifugal method of separating milk not only has a remarkable effect upon its bacterial contents, but also upon tubercle bacilli when present. on examining the so-called "separator slime," it is found to contain not only large quantities of solid matters, but also masses of bacteria which have been thrown out during the operation. this method of treating milk has, curiously, a particular effect upon tubercle bacilli present, for professor scheurlen has found that they are nearly all left in the slime. naturally his observation was not slow in being tested by other investigators; but professor bang has quite independently confirmed scheurlen's discovery, and, still more recently, moore purposely infected milk with these bacilli, and found that they were deposited in the slime to a most remarkable extent. coupled, however, with this peculiar behaviour of tubercle bacilli in separated milk is the fact called attention to by ostertag, that tuberculosis is much more prevalent among swine in denmark and north germany, where the centrifugal process in creaming is extensively used, and where, until recently, this slime was given to the animals in its raw, uncooked condition. before leaving this subject of separated milk, reference may be made to a danger, which has recently been publicly called attention to, surrounding the use which is made of skim milk. by an arrangement with the farmers who supply the milk, those clients who principally use it for producing butter return the skim milk to them after it has been through the separator, when it is employed for stock-feeding purposes. the milk in large dairies derived from different farmers is mixed, and hence the skim milk which is returned is also mixed. thus, in the event of the milk from one farm being infected, not only is the whole milk-supply of a particular dairy infected, but, in returning the mixed skim milk likewise infected in its proper proportion to the different farmers, the virus is distributed over several farms. so real is this danger, and such unfortunate results have followed this practice of returning mixed infected skim milk, that since 1894 the prussian government has issued special orders for its disinfection by means of heat, in the hope of coping with this difficulty. the longevity of the tubercle bacillus and its remarkable vitality under all kinds of untoward circumstances have not unnaturally added fresh significance to this frequent discovery of its presence in milk; moreover, laboratory experiments have shown that these germs can live for upwards of one hundred and twenty days in butter, and from sixty to seventy days in cheese. it is not surprising, therefore, to find a royal commission appointed in 1890 with the express object of inquiring and reporting upon "what is the effect, if any, of food derived from tuberculous animals on human health?" in the summary appended to the report we read: "tuberculous matter in milk is exceptionally active in its operation upon animals fed either with milk or with dairy produce derived from it. no doubt the largest part of the tuberculosis which man obtains through his food is by means of milk containing tuberculous matter." that the commissioners were alive to the great importance of this means of spreading disease is further shown by the following significant paragraph: "in regard to milk, we are aware of the preference by english people for drinking cow's milk raw, a practice attended by danger on account of possible contamination by pathogenic organisms." the commissioners spared no pains in endeavouring to throw light upon the important question they were appointed to report upon, and five years elapsed before they published the results of their inquiries. a decade ago the opinions expressed by them represented the current opinions of the leading bacteriological authorities in scientific circles at home and abroad, and these opinions were gradually filtering down to the general public, which is so conservative in clinging to traditions and popular delusions, when, like a flash out of the blue, the bacteriological jove, professor robert koch, hurled his thunderbolt into the arena, and at the british congress on consumption, held in london in the summer of 1901, declared his belief that bovine and human tuberculosis were distinct diseases. the significance of such a challenge to current scientific opinion, and its far-reaching influence if proved to be correct, was quickly appreciated by the distinguished audience who had gathered to hear what so great an authority as dr. robert koch had to say on consumption and its distribution. the vital question raised by the original discoverer of the tubercle bacillus is still the subject of discussion, experimental inquiry, and much controversy, and we cannot here attempt to discuss the _pros_ and _cons_ for the acceptance or rejection of this new theory concerning the character of tuberculosis. it would, however, be regrettable in the extreme if the publication of this opinion were to encourage dairy authorities to relax in the slightest the efforts now so tardily being made by them to protect their dairy produce and ensure its safety for food-supply. before leaving this branch of the subject reference must be made to some very important researches recently published by professor ostertag, of berlin, on the presence of tubercle bacilli in the milk derived from cows which, whilst reacting to the tuberculin test, exhibit no _clinical_ symptoms of tuberculosis. the importance of this investigation to farmers and all breeders of stock is evident, for it has not infrequently been urged that all the milk from such tuberculin-reacting cows should be discarded for dietetic purposes. professor ostertag, at the request of the german government, has carried out a most elaborate and very extensive series of investigations to determine the question as to whether such milk is dangerous to health. i cannot do better than quote the conclusions appended to the original memoir, in which professor ostertag expresses himself as follows: "the milk of cows which only react to tuberculin does not contain tubercle bacilli; calves and pigs can be fed during weeks and months with milk derived from such cows without contracting tuberculosis." a very important rider, however, is added, in which it is pointed out that inasmuch as no doubt exists as to the highly infectious character of the milk derived from cows the _udders of which are tuberculous, and from animals in which the disease is clinically recognisable_, the weeding out of all such animals must be regarded as the most important measure for the prevention of the dissemination of tuberculosis through milk. we must now pass on to a consideration of some of the methods which are available for obtaining germ-free milk, some of which are, however, attended with too great labour and inconvenience to admit of practical application. thus, wishing to prepare some sterile milk without altering its chemical composition to feed certain microbes with, i had to patiently heat it for from one to two hours on five successive days, watching the while that the temperature remained between 58° and 65° centigrade. the milk was sterile, and i kept it for months, but such a process, of course, is impossible for domestic purposes. the addition of chemicals to milk is both undesirable and ineffectual; amongst such substances boracic acid, borax, and salicylic acid are employed, but whilst the two former have been found to produce but little effect upon disease germs present in milk, salicylic acid hinders curdling more than other substances, and even if added in the small proportion of twelve grains per quart is said to impart a taste to the milk, and is, moreover, incapable of destroying typhoid bacilli if present. authorities are, moreover, not agreed as to the harmlessness of this ingredient, and in france the employment of salicylic acid in the preservation of food is strenuously opposed by doctors, who consider its habitual use injurious to health. a departmental committee of the local government board was appointed in this country to inquire into the use of preservatives in foods. in their report they state that from 42 up to 126 grains of boracic acid were detected in milk offered for sale, and that on one occasion no less than 80 grains of this material were present in a pint of milk sold to their inspector. it is pointed out that as long as preservatives are permitted there is no guarantee against the addition of excessive amounts to milk, and that evidence has been obtained pointing to an injurious effect of boracised milk upon the health of young children. the committee report that in denmark the use of preservatives is strictly prohibited, and the prohibition is strongly enforced; neither are preservatives permitted in belgium. the application of heat to milk is, in fact, the only advisable and reliable method for rendering it free from germs, but a great deal depends upon the manner in which the heat is applied and the cleanly condition or otherwise of the milk employed. the difficulties which have to be overcome in producing efficiently sterilised milk are due, in the first place, to the remarkable power of resisting heat which characterises not only some disease germs, but also some of the microbes which are particularly partial to milk; secondly, to the sensitiveness of milk to heat, as exhibited by its alteration in taste and other respects through exposure to high temperatures. to overcome these difficulties many ingenious pieces of apparatus have been devised, based upon a process originally introduced by pasteur for preventing certain defects in wine and beer, and which consists in the application of a temperature of about 60° centigrade. this process is known as pasteurisation, after its renowned initiator. so-called "pasteurised" milk has become during the last year or so increasingly popular in this country, whilst on the continent it has been largely dealt in for several years past, and has commercially proved a great success. indeed, so strong is the prejudice amongst our neighbours across the channel against using unboiled milk that in leipzig and other cities in germany endeavours have been made by charitable and other societies to encourage the use of sterile milk amongst the poorer classes, whilst it has been stated that the introduction of pasteurised milk among the poor of new york city, through the philanthropic efforts of mr. nathan straus, has done much to reduce the high rate of mortality amongst infants during the hot summer months. in france, _i.e._ in paris and grenoble, in order to reduce if possible the lamentable mortality amongst infants from diarrhoea in the summer months, which was largely attributed to the use of unboiled milk, sterilised milk was distributed to the poor at the cost of the community in general. in grenoble, according to statistics collected by berlioz during the years 1894-6, the death-rate of infants under a year old in the months of july, august, and september fed on raw milk reached 69·3 per 1,000, whilst amongst those supplied with sterilised milk it was reduced to 27·9 per 1,000. just, however, as all is not gold that glitters, so all sterilised milk so-called is not necessarily free from bacteria. indeed, according to a recent german authority, "the complete and certain sterilisation of milk is not yet to hand." dr. weber examined the sterilised milk as supplied by various companies in the city of berlin. as many as 150 bottles were tested from eight different sources, with the result that not one of these eight companies was found to be supplying milk free from bacteria, or, in other words, what it professed to be--sterile. true, the percentage of sterile bottles varied from 5 per cent. in some of the supplies to 86 per cent. in others. thus it may be realised how, as has been already pointed out, difficult a matter it is to devise an efficient apparatus for the reliable sterilisation of milk. so far it appears that the best results have been obtained with an apparatus devised by flaack, a director of the brunswick sterilising milk company, and known as the flaack apparatus. exhaustive examinations made during the course of a whole year in the hygienic institute at würzburg never once showed a failure, all the samples tested being germ-free. some supervision is, therefore, necessary in the case of these milk-sterilising companies to ensure that the public is obtaining what it is paying for, as it has been shown by professor flügge, a world-renowned authority on the subject of milk and its sterilisation, that the bacteria left over in these so-called sterilised milk samples are by no means invariably a harmless residue, but, on the contrary, may consist of individuals which he has gathered together in a class under the heading of poisonous peptonising bacteria, and which owe this unfortunate designation to the rapidity and energy with which they can engender the putrefaction of albumen. as indicating how essential it is that every detail in the sterilisation of milk should be adequately assessed, i may mention a paper recently published by h. l. russell and e. g. hastings, of the wisconsin agricultural experiment station in the united states, on the importance of pasteurising milk in closed rather than in open vessels, bacteria having been found more resistant in milk when heated in contact with the air than in closed vessels, this variation being attributed to the formation of a surface pellicle, which readily forms on milk when heated in open vessels to a temperature of about 60° centigrade or above. experiments showed that organisms present in this pellicle or skin were capable of retaining their vitality when exposed to a temperature _six degrees higher_ than that of the milk beneath the membrane in which they were destroyed. objections to the use of boiled milk have been frequently made on the grounds of its being more difficult of digestion, and hence less wholesome than the raw article. i may only point out that in this, as in most other matters where opinions may be made or unmade, and in consequence of the facts available being scanty must be more or less arbitrary in character, dr. duclaux, the successor to pasteur as director of the pasteur institute in paris, has expressed himself as follows in an article on "la digestibilité du lait stérilisé." after reviewing the various special researches which have been made on the subject, he says:- "ceci nous amène à une conclusion qu'il faut bien avoir le courage de tirer, c'est que ces études chimiques sur la digestibilité du lait ne sont pas adéquates à la question à résoudre.... en attendant, tenons-nous-en à cette conclusion générale, que le lait pasteurisé, chauffé ou stérilisé, est encore du lait, devant la science comme devant la pratique, et que si son emploi présente parfois des inconvénients, ceux-ci sont légers et amplement compensés par les avantages." bacteria and ice the fate of bacteria when frozen excited the curiosity of investigators already in the early years of bacteriology, for in 1871 we find burdon sanderson recording the fact that water which he had obtained from the purest ice contained microzymes, or, as we now prefer to call them, micro-organisms. it is quite possible that at the time this announcement was made it may have been received with some scepticism, for it was undoubtedly difficult to believe that such minute and primitive forms of vegetable life, seemingly so scantily equipped for the struggle for existence, should be able to withstand conditions to which vegetable life in more exalted circles so frequently and lamentably succumbs. the tormented agriculturist realises only too well what havoc is followed by a return in may to that season "when icicles hang by the wall, and dick the shepherd blows his nail and tom bears logs into the hall, and milk comes frozen home in pail." again, with what solicitude those of us who have gardens wait to see what will have survived the iron grip of winter in our favourite flower borders, and how frequently we have to face blanks in the ranks of some of its most cherished occupants! numerous bacteriologists, however, have now confirmed this fact, the fields of ice and snow have been repeatedly explored for micro-organisms, and it has been shown how even the ice on the summit of mont blanc has its complement of bacterial flora, that hailstones as they descend upon the earth contain bacteria, that snow, the emblem of purity, is but a whited sepulchre, and will on demand deliver up its bacterial hosts. quite apart from its general scientific interest, the bacterial occupation of ice is of importance from a hygienic point of view, and a large number of examinations of ice as supplied for consumption have been made. thus, professor fraenkl and also dr. heyroth have submitted the ice-supply of the city of berlin to an exhaustive bacteriological examination. these investigations showed that the bacterial population of ice as supplied to berlin is a very variable one, and fluctuates between great extremes, rising to as many as 25,000 bacteria in a cubic centimetre (about twenty drops) of ice-water, and falling to as few as two in the same measure. there are numerous circumstances which come into play in determining the density of the bacterial population in ice. first, of course, the initial quality of the water from which the ice is derived is a factor of great importance, for the purer the water the fewer will be the bacteria found in the resulting ice. again, if the ice field is wind-swept by air bearing an unduly rich complement of bacteria, as may be expected in the vicinity of populous cities, for example, then the ice will reflect in its bacterial contents the undesirable neighbourhood in which it was produced. water in repose, again, yields purer ice than water in movement during freezing, for during rest opportunity is given for the bacteria present in suspension to subside, the process of sedimentation or deposition of bacteria which takes place under these conditions playing an important part in water-purification; when, however, the water is disturbed by swift currents, or agitated by storms, this process is interrupted, and the bacteria become entangled in the ice and frozen _in situ_. the importance attaching to the physical conditions under which ice is produced in enabling an estimate to be formed of the safety or otherwise of the same for consumption may be gathered from the following extract from an american report on the subject:- "on the whole it is evident that the conditions surrounding water when it freezes are very important factors in determining the purity of the ice formed. if there is a considerable depth of water in portions of a somewhat polluted pond or river, and the ice is formed in these portions in comparatively quiet water with but little matter in suspension, this ice will probably be entirely satisfactory for domestic use. on the other hand, ice formed in shallow portions of such ponds or rivers, even during still weather, or in any portion if there is a considerable movement of the water by currents or wind while it is forming, may be rendered by these conditions entirely unfit for domestic use." we have learnt that ice contains bacteria, that its bacterial contents are to a certain extent dependent upon the bacterial quality of the water before crystallisation, and that an important factor in determining its purity is afforded by the physical conditions prevailing at the time of freezing. it will be of interest to ascertain in more detail what effect the process of freezing has upon the number of bacteria present in the water--what is the degree of bacterial purification effected during the conversion of water into ice. now professor uffreduzzi, in his investigations on the ice-supply of turin, part of which is derived from a much-polluted portion of the river dora, found that about 90 per cent. less bacteria were present in the ice than were present in the water from which it was produced. in the making of ice, therefore, a remarkable removal of bacteria may be effected which approaches very nearly the degree of bacterial purification which is achieved during the best-conducted sand-filtration of water. uffreduzzi's results have been repeatedly confirmed by other researches. thus, in regard to ice obtained from the river merrimac, water which contained originally about 38,600 bacteria per cubic centimetre, on its conversion into ice had only from three to six. sewage, again, containing about a million and a half bacteria per cubic centimetre after being frozen only contained under 74,000. it should be mentioned that this last figure represented the number of bacteria obtained by thawing the _outside_ of the sewage ice-cake; _inside_ the cake there were more found--about 121,000. the difference in these figures is due to the fact that, whereas the outer layers of ice looked quite clear, towards the centre the ice contained sewage sludge and hence more bacteria had become arrested; but in spite of this the bacterial purification effected is very striking, although not sufficient to render the use of ice from such a polluted source either palatable or desirable. it is, of course, a well-known fact that water possesses the power of purifying itself during its transformation into ice, and that the process of crystallisation not only prevents a considerable proportion of the matters in suspension from becoming embodied in the ice, but also eliminates a large percentage of the matters in solution, the latter being driven from the water which is being frozen into the water beneath. if, therefore, ice in the act of forming can get rid of matters in solution, it is not difficult to understand how it can eject bacteria, which though so minute are yet bodies of appreciable dimension and in suspension. but that there are limits to this power of excluding bacteria, and that, as in the case of other mechanical processes, an overtaxing of the available resources is at once reflected in the inferiority of the product, is shown by the frozen sewage experiment, in which the ice, having had too large a supply of bacteria in the first instance to deal with, was unable to get rid of more than a certain proportion, and was obliged to retain a very considerable number. hence great as is the degree of purification achieved by ice in forming, yet it must be recognised that its powers in this direction are limited, and that the fact of water being frozen does not necessarily convert a bad water into immaculate ice. it is worthy of note that the city of lawrence, in massachusetts, obtains the greater portion of its ice from a river which in its raw, unpurified condition was rejected for purposes of water-supply in consequence of the numerous and severe epidemics of typhoid fever which accompanied its use. since the application of sand-filtration to this water, however, the death-rate from typhoid in this city, instead of being abnormally high, has fallen abnormally low, and this improvement is attributed to the excellent quality of the water supplied to the city, and has taken place despite the use which still continues of ice from the polluted river. the authorities consider the city's immunity from typhoid amply justifies their sanctioning the distribution of this river-ice, the freezing of the water having rendered it sufficiently pure to remove all danger to health from its consumption. so far we have been considering the effect on bacteria of freezing carried on under more or less natural conditions; but much interesting work of a more detailed character has been carried out with reference to the behaviour of particular varieties of micro-organisms when frozen under more or less artificial conditions. thus dr. prudden froze various bacteria in water at temperatures ranging from -1° c. to -10° c., and he found that different varieties were very differently affected by this treatment; that, for example, a bacillus originally obtained from water, and introduced in such numbers as represented by 800,000 individuals being present in every twenty drops, after four days' freezing had entirely disappeared, not one having survived. on the other hand, similar experiments in which the typhoid bacillus was used resulted in the latter not only enduring a freezing of four days' duration, but emerging triumphant after it had been carried on for more than 103 days! in these experiments it should be borne in mind that, as the ice was frozen to a solid block or lump, there was no opportunity for the mechanical committal of the bacteria during freezing to the water beneath; all the bacteria present were imprisoned in the ice, and the fact that the typhoid bacteria were not destroyed by being frozen shows that they can withstand exposure to such low temperatures, although, as we have seen, the other variety of bacillus employed was destroyed. dr. prudden, however, discovered an ingenious method by which even typhoid bacilli were compelled to succumb when frozen. in the course of his investigations he found that bacteria which had offered the stoutest resistance under freezing were extremely sensitive to this treatment if the process was carried on intermittently, or, in order words, if the temperature surrounding them was alternately lowered and raised. in this manner the bacteria may be said to be subjected to a succession of cold shocks, instead of being permitted to remain in a continuously benumbed condition. the vitality of typhoid bacilli was put to the test under these circumstances, the freezing process being carried on over twenty-four hours, during which time, however, it was three times interrupted by the ice being thawed. the effect on the typhoid bacteria was striking in the extreme; from there being about 40,000 present in every twenty drops, representing the number originally put into the water, there were only ninety at the end of the twenty-four hours; and after a further period of three days, during which this treatment was repeated, not a single bacillus could be found. this signal surrender to scientific tactics forms a marked contrast to the stout resistance maintained for over 103 days under the ordinary methods of attack. but, although the typhoid bacillus appears to submit and meekly succumb to this plan of campaign, yet the conclusion must not be rashly drawn that all descriptions of bacteria will be equally feeble and helpless in these circumstances. doctors percy frankland and templeman have shown that the spore form of the anthrax bacillus is able to successfully challenge all such attempts upon its vitality. thus when put into water and frozen at a temperature of -20° c., the process being extended over a period of three months and interrupted no fewer than twenty-nine times by thawings, when examined even after this severe series of shocks, it showed no signs of submission and clung to life as tenaciously as ever. the more sensitive form of anthrax, however, the bacillus, was readily destroyed; for after one freezing its numbers were already so much reduced that it was only with difficulty that even one or two could be found, and after the second freezing every one out of the large number originally present had died. renewed interest has been of late revived in the question of the behaviour of bacteria at low temperatures, in consequence of the possibility of obtaining, by means of liquid air and liquid hydrogen, degrees of cold which were undreamt of by the scientific philosophers of fifty years ago. public interest has also been quickened in such inquiries on account of the important part which low temperatures play in many great commercial developments, their application rendering possible the transport from and to all parts of the world of valuable but perishable foodstuffs, thus encouraging local industries by opening up markets, and bringing prosperity to countries and communities which before were seeking in vain an outlet for their surplus produce. the application of cold storage for preservation purposes is, however, no novelty; for nature ages ago set us the example, and of this we have been lately reminded afresh by the discovery announced by dr. herz of a mammoth in siberia, which, despite the thousands of years which have elapsed since it was originally overwhelmed and frozen, is described as being in a marvellous state of preservation. thus we are told that "most of the hair on the body had been scraped away by ice, but its mane and near foreleg were in perfect preservation and covered with long hair. the hair of the mane was from four to five inches long, and of a yellowish brown colour, while its left leg was covered with black hair. in its stomach was found a quantity of undigested food, and on its tongue was the herbage which it had been eating when it died. this was quite green." considering that certainly more than eight thousand years had elapsed since this creature was peacefully consuming what proved to be its last meal, nature's method of cold storage must indeed be regarded as unsurpassable in the excellence of its results. i believe it was in the year 1884 that the first attempts were made to follow more closely and in greater detail the precise effect upon different bacteria of submitting them to temperatures of such a low degree as -130° c., obtained by means of solid carbonic acid. these experiments were carried out by pictet and young, and are recorded in the _comptes rendus_ of the paris academy of sciences. they differ from those which we have so far been considering, inasmuch as the bacteria were not frozen in water, but in culture-material, or, in other words, like the mammoth, whilst enjoying a midday meal! one of the micro-organisms experimented with was a bacillus known at that time as the rinderpest bacillus, capable of producing disease in animals when inoculated into them and existing both in the spore and bacillar form. pictet and young specially state that the spore form was present in the specimens employed by them, and hence the fact that this micro-organism was alive after being frozen and exposed to this low temperature of -130° c. for the space of twenty hours is not, perhaps, so surprising when we bear in mind the remarkable feats of endurance exhibited by spores which have with justification obtained for them a prominent place amongst the so-called curiosities of bacteriology. but of more interest than their mere survival in these circumstances is the fact that, on being restored to animation--or, in other words, released from their ice-prison--these bacteria were discovered to have retained all their pathogenic properties, this period of enforced rigidity having in no way affected their disease-producing powers. such results naturally only served to whet the scientific appetite for more, and the liquefaction of air and of hydrogen placing much lower temperatures at the disposal of investigators, those bacteriologists who were fortunate enough to command a supply were not long in availing themselves of the opportunity thus given them of further testing the vitality of micro-organisms. botanists had already shown that exposure to liquid air, which means a temperature of about -190° c., and to liquid hydrogen, which means a temperature of about -250° c., did not impair the germination powers of various descriptions of seeds, such as those of musk, wheat, barley, peas, vegetable marrow, and mustard, and that their actual immersion in liquid hydrogen for the space of six hours did not prevent them coming up when sown just as well as ordinary seeds which had not undergone this unique experience; hence the opportunity of submitting other members of the vegetable kingdom to these low temperatures was eagerly sought for by bacteriologists. dr. macfadyen found this opportunity in the laboratories of the royal institution, and, professor dewar having placed a generous supply of liquid air and liquid hydrogen at his disposal, he submitted specimens growing in various culture-materials, such as gelatin, broth, potatoes, etc., of typhoid, diphtheria, cholera, anthrax with spores, and other bacteria, for twenty hours and seven days respectively, to a temperature of about -190° c. in no instance, however, whether exposed when growing in fluid or solid media, could any impairment of their vitality or the slightest alteration in their structure be observed. similar results were obtained when liquid hydrogen, or a temperature of about -250° c., was applied. the question of the retention or otherwise of the disease-producing powers of these bacteria was not investigated, and in this connection much interest attaches to mr. swithinbank's investigations on the vitality and virulent properties of that notorious malefactor amongst micro-organisms, the _bacillus tuberculosis_, when exposed to the temperature of liquid air. the specimens of the consumption bacillus employed were originally obtained from the human subject, and they were exposed for periods varying from six hours to six weeks to -190° c. in each case the malignant properties of the tubercle bacillus after exposure were tested by their direct inoculation into animals, and the results compared with those which followed similar inoculations made with bacilli which had not been frozen in this manner, but had been grown in ordinary circumstances. in no single case, mr. swithinbank tells us, were these frozen tubercle bacilli deprived of their virulence, and the length of exposure, at any rate as far as could be judged after six weeks, appeared to make no difference in this respect. it is true that the pathogenic action of the frozen bacilli appeared to be somewhat retarded--that is, they took rather longer to kill animals than the ordinary unfrozen bacilli--but in every case their inoculation produced the typical tuberculous lesions associated with them. of particular interest, however, in view of what has been already discovered about the lethal effect upon bacteria of violent alternations of temperature, are mr. swithinbank's observations on the vitality of the tubercle bacillus when exposed to such extreme variations of temperature as are represented by a passage from -190° c. to that of 15° c. the _bacillus tuberculosis_ is admittedly a tough antagonist to deal with, and enjoys an unenviable notoriety for its robust constitution amongst the pathogenic members of the microbial world; hence a knowledge of its behaviour in these trying circumstances, as we now know them to be to bacterial life, becomes of special interest. great must have been the investigator's satisfaction, then, when he discovered that the vitality of the consumption bacillus had been so seriously impaired by this treatment that its pathogenic properties collapsed, and the animals which were inoculated with these specimens, instead of with the continuously frozen bacilli, suffered no inconvenience, and remained in good health. but although no appreciable change either in the structure, vitality, or malignant properties of the particular bacteria investigated have been noted as resulting from their exposure to extremely low temperatures, yet there is no doubt that a certain proportion of the individual micro-organisms present--those probably whose constitution is less robust than their more fortunate associates--do succumb under these trying conditions. this fact has been well brought out by dr. belli, of the university of padua, in the experiments which he made with the fowl-cholera bacillus and the anthrax bacillus in the presence of very low temperatures. thus he exposed a large number of fowl-cholera bacilli in broth to the temperature of liquid air, as many as 396,000 bacilli being present in every twenty drops of the liquid. after exposing them continuously for nine hours to -190° c., he had the curiosity, after thawing them, to count how many were left alive, and he found that an enormous mortality had taken place amongst them; for, instead of nearly 400,000 bacilli being present in one cubic centimetre, there were only about 9,000. on the other hand, in the broth tubes kept during that time in ordinary surroundings, the bacilli had flourished remarkably, and had greatly increased in numbers. thus not only had no multiplication amongst these bacilli taken place, which circumstance is always regarded as indicative of their vital condition--not only, then, had their vitality been arrested--but a very large number of them had been actually destroyed in consequence of this severe treatment; but that the residue were not only alive, but unimpaired in their energies on being restored to animation, was proved by their being able to destroy animals, not having parted with any of their malicious propensities. dr. belli carried out similar experiments with the bacilli of anthrax and obtained very similar results. with regard to both these varieties of pathogenic bacteria, he mentions that their action upon animals was not quite so rapid as is characteristic of normal specimens of these micro-organisms, thus confirming the experiments in this direction made with frozen tubercle bacilli. not content with the exhibition of their powers of endurance, dr. belli determined to make yet another demand upon the vitality of these bacilli. for this purpose he immersed them in the liquid air itself, thus bringing them into direct contact with it, effecting this by lowering into the liquid strips of filter-paper soaked in broth containing these bacilli. but, in spite of remaining for the space of eight hours in these surroundings, they emerged triumphant, exhibiting no modification whatever either in their structure or pathogenic properties. there are doubtless many other trials yet awaiting bacteria, to which they will most certainly be submitted before the limits of their powers of endurance have been adequately tested, but it is difficult to conceive of a severer strain upon their vital resources than the imposition of the conditions of which the above is but a brief sketch. the triumphs achieved in this direction by micro-organisms are, however, closely approximated by the remarkable record established, according to the recent researches of dr. krause, by typhoid, anthrax, tubercle, and some other bacteria of preserving unimpaired not only their vitality but their virulence after having undergone for a period of twenty hours a pressure of no less than that of 500 atmospheres. when we reflect that a pressure of 500 atmospheres is equal to a pressure of about 7,500 pounds to the square inch, and that the normal pressure under which life is maintained upon this planet is approximately that of fifteen pounds to the square inch, this bacterial victory over physical conditions will be more readily appreciated. the more intimate becomes our knowledge of bacteria, the more must we marvel at the equipment with which they have been provided for enabling them to maintain themselves in the struggle for existence--a struggle which is as severe and as remorseless in this lowly region as it is in those domains the inhabitants of which have risen to far loftier heights on the great ladder of life. some poisons and their prevention little did the learned dutchman leeuwenhoek dream when, more than two hundred years ago, he recorded, in his _arcana naturæ_, that he had found "viva animalcula" in his saliva, that this, the first beginning of bacteriology, would lead, a couple of centuries later, to the inauguration of a new era in the treatment of disease, in which these so-called animalcula, from being considered as curiosities, would come to be regarded as powers for good and evil of the first importance. protective inoculation or serum therapy, of which the public have lately heard so much in connection with diphtheria, is the direct outcome of bacterial investigations which during the last two decades have been pursued with such zeal in every part of the globe. the vast domain of immunity, which until recently was an undiscovered country, is now being bit by bit annexed, and in all directions workers are engaged upon opening up new tracts, in overcoming difficulties, in changing chaos into order. the problems which surround immunity are of so complex and subtle a character that their mastery is by no means either easy or rapid, and many recondite researches appear at frequent intervals on this subject in foreign and other scientific journals, rendering it a difficult matter to keep pace with the new discoveries and the latest theories. the interest in this country in toxins and anti-toxins not unnaturally centres round that branch of the subject which deals with diphtheria, this disease having of late years figured so prominently in our mortality tables, whilst the production of diphtheria and other anti-toxic serums has been so finely elaborated abroad that it already constitutes an article of commerce, and doubtless helps to swell the exports of our great continental commercial rival. in this connection the following statistics, published by dr. jalzer, of the mülhaus hospital, are of interest regarding the mortality from diphtheria before and after the introduction and application of diphtheria anti-toxin. the death-rate from this disease, writes dr. jalzer, which in 1892 and 1893 was fully 50 per cent., fell in 1895 to 38·5 per cent., in 1896 to 28·8 per cent., in 1897 to 16 per cent., to 20 per cent. in 1898, 15·15 per cent. in 1899, and 18·75 per cent. in 1900. so far the efforts which have been made to mitigate _human_ suffering have attracted most attention; but it will be remembered that pasteur, before he commenced the study of hydrophobia, had already won his laurels in combating disease in the victory he gained over anthrax, the ravages of which so frequently decimated the herds of the french farmer and robbed him of his well-earned return on his capital and labour. in summoning the brilliant director of the german imperial board of health to south africa to investigate the nature of rinderpest, and, if possible, discover a means of protecting cattle from its onslaught, the cape government afforded another opportunity for the scientific study of a disease associated with animals, upon the successful mastery and limitation of which the agricultural prosperity of south africa is so largely dependent, being as it is one of the most fatal and contagious maladies to which cattle are subject. apart from the great commercial importance attending dr. koch's discovery of a device whereby cattle can be immunised or protected from contracting rinderpest when exposed to its contagion, this discovery is of great scientific interest, inasmuch as it has inaugurated a new departure in methods of immunisation. the previous methods in vogue for inducing immunity in animals from a particular disease consisted in converting the virus itself into a vaccine, as was done by pasteur in his classical investigations on anthrax and its prevention; and secondly, the employment of anti-toxic serums, in which the virus is not directly inoculated into the animal to be protected, but in which an intermediary is employed between the virus and its victim. this intermediary, or living machine for the generation of the anti-toxin, is usually a horse, which is artificially trained by being given gradually increasing doses of the virus or toxin, until it ultimately withstands doses which in the first instance would infallibly have killed it. when the animal has arrived at this satisfactory stage or condition of complete immunity, some of its blood is from time to time drawn off, and the serum thus obtained constitutes the anti-toxin which now figures so prominently in modern therapeutics. besides diphtheria-anti-toxic serum there are also those of tetanus, or lock-jaw, plague, the famous anti-venene serum, about the discovery and preparation of which greater detail is given later on, and many others which are still the subject of experimental inquiry. now koch's method for the compassing of rinderpest differed from both the systems above mentioned, inasmuch as he neither employed artificially weakened cultures of the virus, or an anti-toxic rinderpest-serum; instead he took one of the natural secretions of an animal infected with rinderpest, and by injecting this into a healthy animal it was discovered that the latter, as is the case with a vaccine, suffered only local and temporary discomfort, and acquired pronounced immunity from the active virus. the secretion selected by dr. koch and his assistant, dr. kolle, for this purpose was the gall, and it might be supposed, from the fact that its inoculation into healthy animals did not communicate the disease, that the rinderpest bacteria were absent from the gall. but this is not so, for dr. kolle has succeeded in isolating the latter from the gall of infected animals, and, moreover, has proved them on isolation to possess their full complement of virulence. further investigations made by koch and kolle have shown that the explanation of this seeming anomaly is to be found in the fact that the gall of an animal suffering from rinderpest contains a substance which prevents the migration of the rinderpest bacteria, with which it is associated, from the point of inoculation. hampered in their movements by the controlling influence of this special substance which has been generated in the gall, the bacteria remain rooted to the spot where they are first situate, and only a passing and exceedingly slight local affection results, which on its departure leaves the animal with an immunity from rinderpest lasting some four months. a number of interesting investigations have not unnaturally been stimulated by this remarkable discovery, and researches on the properties inherent in the gall of healthy animals of various kinds have been recently carried out by dr. neufeld, of the institute for infectious diseases in berlin, which are, however, of a too technical nature to deal with here. as an illustration of the practical use to which koch's gall immunisation method may be put in dealing with outbreaks of rinderpest, reference to a recent report furnished by the health officer of shanghai may be of interest. dr. arthur stanley describes the outbreak as follows:- "a large herd of cattle infected with cattle-plague was brought to shanghai from the tanyang district, around the grand canal, for export to the allied troops in the north of china. the disease spread to an adjacent dairy, most of the cattle dying. on this dairy becoming infected a police cordon was established round it to prevent ingress and egress of cattle and ingress of persons unconnected with the dairy, while the outside infected herd was removed to an isolated part of the settlement. having been previously convinced of the futility of police cordons in the prevention of cattle-plague, i was not surprised to find, within a short time, that the disease had spread, by the meeting together of cattle-coolies at a common tea-house, to three other dairies at a distance of a quarter, a half, and two miles from the original source of infection. "as the animals are not, as a rule, taken away from the immediate vicinity of the dairy, there being no grazing fields, and as neither fodder nor dung is taken from one dairy to another, it is practically certain the infection was carried by the dairy-coolies. "immediately on this second series of dairies becoming infected it was resolved to apply the gall immunisation method of koch as being the means at hand. about 1,500 cubic centimetres were collected from the gall-bladder of a rinderpest animal, and 10 cubic centimetres were injected into the dewlap of each of the twenty remaining cattle in the dairy. "the injection caused slight local swelling and tenderness, but no constitutional symptoms and no alteration in the milk-supply, an important matter in a dairy. in all sixty-eight cattle were injected with cattle-plague gall. of these, seventeen were among isolated uninfected herds; the remaining fifty-one belonged to infected herds, and among the latter eleven died of cattle-plague subsequent to the injection." dr. stanley points out that ten of these animals, judging by the time which elapsed _after_ the injection, when they showed the first symptoms of the disease, _must have been already infected when the injections were made_; the eleventh animal, however, undoubtedly contracted the disease after and in spite of the injection. "considering," continues dr. stanley, "the usual excessive mortality during an outbreak of this disease, the result may almost be compared to the success of vaccination against small-pox. three young bullocks, each having received 20 cubic centimetres of cattle-plague gall, were purposely exposed to severe infection. they remained well, while unprotected animals around them died of the disease." in the domain of immunity there is, however, no more fascinating or interesting story than that which deals with the discovery and elaboration of a cure for snake-bites, a discovery which, while attracting but comparatively little attention in this country, should prove of paramount importance to our fellow-subjects in the great indian empire. the significance to india of professor calmette's discovery of a specific cure for snake-poison may be gathered, indeed, from the statistics which have been compiled of the number of deaths attributed by indian officials to this cause alone, amounting, it is said, to some 22,000 annually. the pasteur institute in paris has despatched many pioneers of science to various quarters of the globe, but perhaps no scientific missionary has produced more fruitful results than has dr. calmette. it was while acting in the double official capacity of médecin de 1st classe du corps de santé des colonies and director of the bacteriological institute of saïgon, in cochin china, in the autumn of 1891, that calmette first commenced his experiments on the neutralisation of serpent venom in the animal system. he had, indeed, exceptional opportunities in the matter of serpent venom wherewith to carry out his investigations, for during the rainy season a village in the neighbourhood of bac-lieu (cochin china) had been attacked by a band of most venomous serpents. these creatures, driven by the floods into the very huts of the natives for shelter, created a terrible panic, and no fewer than forty individuals were bitten by them. the panic was certainly not without justification, for these serpents belonged to the species known as _naja tripudians_, or _cobra de capello_, renowned for the deadly nature of their venom, and widely distributed over india, burmah, sumatra, java, malacca, and cochin china; but until calmette set to work to systematically study the nature of this reptile's venom but little precise or reliable information had been obtained as to its character. the governor of the district gave orders that as many as possible of the reptiles were to be captured alive and forwarded to the director of the bacteriological institute, and a plucky annanite actually succeeded in securing ninety specimens, which were forwarded in a barrel to dr. calmette. this formidable gift was received with enthusiasm by the director, who realised the importance and scope of the inquiry, which he at once set himself to systematically work out. forty of these reptiles arrived alive, and several were at once sacrificed to secure their venom glands. each gland, resembling both in size and shape a shelled almond, contains about thirty drops of venom, and in this transparent limpid liquid is embodied a toxin of extraordinary strength. it was, of course, necessary in the first instance to ascertain, within as narrow a limit as possible, the exact degree of toxic power inherent in the venom, and to determine, if possible, the precise lethal dose in respect of each variety of animal experimented upon. a correct calculation of the quantity of venom required in every case was, however, found to be quite impossible, for so virulent is the poison that a single drop of an emulsion produced by pounding up eight glands in 300 grammes of distilled water is sufficient, when introduced into the vein of a rabbit's ear, to kill it in five minutes. all the mammals to which calmette administered this cobra venom, such as monkeys, dogs, rabbits, guinea-pigs, rats, succumbed more or less quickly, according to the size of the dose. small birds and pigeons die very rapidly, but the domestic fowl is more fortunate, being somewhat less susceptible. frogs also fall a prey to the venom, but they are far more refractory than rabbits, for it takes thirty hours to kill a frog with a dose of venom which would infallibly destroy a rabbit in ten minutes. toads, curiously, do not enjoy to the same extent this power of resisting its toxic action, for they die more quickly than frogs, whilst it makes short work of lizards and chameleons. fish form no exception to the rule, and even invertebrates, such as leeches, are killed by minute traces of venom. whilst calmette has found that the venom of different kinds of reptiles exhibits marked differences in its toxic character, he has also discovered that the venom secreted by one and the same serpent varies considerably, according to the length of time the animal has fasted. he describes how he kept a _naja haje_ (cleopatra's asp) in his laboratory, which during the whole eight months that it lived never took any food whatever, although it was offered the most diverse dainties. on its arrival it was made to bite on a watch-glass, this being one method adopted for collecting the venom; the liquid was at once dried, and 0·7 milligramme was found to kill a rabbit weighing nearly four pounds in four hours. two months later on, when the venom was again collected, 0·25 milligramme proved a fatal dose. on the death of the animal, at the end of eight months, the venom extracted from the glands was so toxic that it only required 0·1 milligramme to kill a rabbit of about the same weight as the previous one. the same curious fact was noted in the case of a cobra's venom. another circumstance which appears to control the degree of toxicity inherent in serpent venom is the interval of time which elapses between two successive bites. the longer the interval the more virulent is the venom; and calmette points out that these observations are in accordance with what has for a long time been known in france with respect to indigenous vipers--that their bites are far more dangerous and far more fatal in the spring, after the winter period of torpor is over, than in the autumn. until quite recently it was thought that the only creatures which could resist the fatal action of this poison were serpents, both poisonous and non-poisonous. calmette was led to this conclusion because, although he inoculated large doses, as much as ten drops, into cobras, they suffered absolutely no inconvenience, and the same results were obtained with harmless snakes. on repeating these experiments, however, and using much larger quantities of venom, calmette has found that they do ultimately succumb. that their susceptibility in comparison with other animals is very slight, may be gathered from the fact that a lethal dose of venom for reptiles is roughly estimated to amount to as much as three times the quantity of venom normally present in their respective poison glands. these animals, therefore, although very refractory, are not absolutely immune from the action of venom-toxin. there are, however, other animals which enjoy a relative although not absolute immunity to snake poison, and amongst these may be mentioned swine, hedgehogs, and the mongoose. swine, it is well known, will greedily devour reptiles, and in some countries they are specially trained up and employed for this purpose. of particular interest, however, are some experiments which were carried out to test the traditional immunity towards this toxin ascribed to the mongoose. these animals are very useful in sugar plantations, and are largely employed to keep down the serpents and rats with which they abound, for the carnivorous little mongoose is extremely partial to such prey. attempts have been made by sugar planters to introduce them into martinique, where they are not found in the wild state, as in the island of guadeloupe. six specimens of the mongoose were forwarded to calmette from martinique, and these particular animals, it was stated, had never been set at liberty since they were imported, so that they had had no previous experience of snakes or venom. on arriving at the laboratory, one of these little creatures was placed in a glass cage along with a large cobra. the cobra, at once rising up and dilating its neck, darted with fury upon the mongoose; but the latter, thanks to its extraordinary agility, escaped being caught, and took refuge, stupefied and terrified for the moment, in a corner of the cage. this stunned condition, however, did not last long, for just as the incensed cobra was preparing to make a fresh attack upon its insignificant little victim, the latter, with wide-open mouth, rushed and jumped upon the head of its enemy, viciously bit through its upper jaw, and broke its skull in a few seconds. thus, although in size but a little larger than a squirrel, this tiny creature was more than a match for a cobra two yards long. artificial inoculations of cobra venom into the mongoose fully substantiated all the observed facts as to its remarkable immunity from this poison. a dose sufficient to kill a large rabbit in three hours was absolutely without effect; only when the venom was introduced in quantities amounting to as much as eight milligrammes was it followed by fatal results. thanks, therefore, to their extraordinary agility and remarkable power of resisting the effects of this lethal toxin, these little animals are able to battle successfully with the most dangerous reptiles. the rapidity with which serpent venom becomes absorbed by the system is almost incredible, and is well illustrated by the following experiment. a rat was inoculated with venom near the tip of its tail. _one minute later_ the latter was cut off a short distance above the point of inoculation; but this operation was quite unable to save the animal's life, for even in that brief interval the poison had accomplished its fatal work, and a few hours later claimed its victim. this rapid diffusion of the venom helps to explain the difficulty which is experienced in arresting the course of the poison by local treatment, for its passage is too rapid to permit of its being overtaken by superficial measures of even the most stringent character. but calmette points out that local precautions are not to be neglected, for although they cannot nullify the action of the venom, they undoubtedly do delay its progress, and thus create a longer interval or respite, during which an opportunity is afforded for administering the anti-toxin. before, however, passing on to the investigations which have culminated in the production of a specific antidote for this terrible toxin, there are a few more details which calmette has furnished as to its character which are of interest. serpent venom is characterised not only by its intensely virulent properties, but also by the tenacity with which it retains them under diverse circumstances. thus it may be stored up for a whole year, and yet at the end of that time be as active as ever; and even after several years, although its toxic powers are somewhat reduced, it still retains them to a very appreciable extent. unlike the bacterial toxins, this venom toxin can stand exposure to considerable temperatures without injury to its activity, and that of the cobra only suffers after it has been submitted to 98° centigrade for twenty minutes. sensitiveness to temperature varies, however, with the snake from which the venom is derived. thus the venom of the so-called "tiger-snake" of australia will stand being exposed for ten minutes to from 100° to 102° degrees centigrade, and its virulence only disappears when this temperature has been applied for twenty minutes. the venom of the "black snake," another australian variety, loses its toxicity at a temperature of between 99° and 100° centigrade; whilst an exposure to only 80° centigrade for ten minutes is sufficient in the case of viper venom, according to messrs. phisalix and bertrand, to profoundly modify its lethal action. a continuous exposure for a fortnight to a temperature of 38° centigrade does not affect cobra venom in the least; but if during that same time it has been placed in the sunshine, it entirely loses all its lethal properties. thus, a pigeon was inoculated with about thirty drops of venom which had been exposed to the sun's rays for fourteen days, and it survived; whilst another pigeon was inoculated with a little over six drops of similar venom which had been kept during this time in the dark, and it died in a quarter of an hour. all these elaborate researches as to the character of serpent venom were essential to enable the next step to be taken in the elaboration of the antidote. before this great achievement could be accomplished it was necessary to first succeed in artificially immunising animals against the effects of this powerful toxin, so that the serum of such animals could be applied for the protection and cure of other animals from the effects of snakebites. it may be readily conceived that the task of artificially rendering animals immune from snake poison was not an easy one, for the process depends upon training the animal to gradually withstand larger and larger doses of the venom; and considering the intensely toxic character of the substance which had to be handled, the danger was ever present of the animal succumbing to venom poison before its serum had acquired the requisite pitch of protective power to render it of service as an anti-toxin. dr. calmette tells us that he carried out a very large number of experiments before he met with success. but it is not necessary here to discuss his various efforts; suffice it to say that at length his labours were rewarded, and the following extract from one of his memoirs describes the methods which he adopted for this purpose:- "the best method of procedure for the purpose of vaccinating large animals destined to produce anti-venomous serum consists in injecting them from the outset with gradually increasing quantities of the venom of the cobra mixed with diminishing quantities of a one-in-sixty solution of hypochlorite of lime.[8] the condition and the variations in the weights of the animals are carefully followed, in order that the injections may be made less frequently if the animals do not thrive well. quantities of stronger and stronger venom are in turn injected, first considerably diluted, and then more concentrated; and when the animals have already acquired a sufficiently perfect immunity, the venoms derived from as large a number of different species of snakes as possible are injected. the duration of the treatment is of considerable length--at least fifteen months--before the serum is sufficiently active to be used for the purposes of treatment." [8] more recently the snake venom employed by dr. calmette for the immunisation of his horses consists of a mixture of colubrine and viperine poisons, the former making up about 80 per cent. of the mixture. a solution of this mixture is heated at about 73° c. for half an hour and then filtered, and injected into horses. an immense number of animals have been vaccinated by this method at the pasteur institute at lille, where dr. calmette is now director; and in one of his memoirs we are told that they have horses there which have yielded during a period of eighteen months serum extremely active against venom. these horses receive in a single inoculation, without suffering the least inconvenience, doses of venom sufficient to kill fifty horses fresh to the treatment. large quantities of this serum have been forwarded from the lille institute to various parts of the world where venomous serpents are most frequently met with, and already important evidence has been collected as to its efficacy in cases of human beings bitten by dangerous reptiles. so impressed with its importance are indian medical authorities, that its preparation has been included in the work which the new great bacteriological institute at agra is carrying on. the importance of the production _in situ_ of this anti-venomous serum has been recently demonstrated by the experiments which have been conducted in the plague research laboratory, bombay, by mr. lamb and his colleagues, on the keeping properties of such serums in india. from the careful investigations which have been made on this subject, these gentlemen state that anti-venomous serum undergoes a progressive and fairly rapid deterioration when stored in hot climates, and that this deterioration is greater and more rapid the higher the mean temperature to which it is subjected. the protective potency of this horse-serum may be gathered from the fact that it suffices to inject a rabbit, for example, with a quantity amounting to about one two-hundred-thousandth of its weight to ensure the latter acquiring complete immunity from a dose of venom capable of otherwise killing it in twelve hours. the rapidity with which it acts is also extremely remarkable. thus, if a rabbit receive two cubic centimetres (about fifty drops) of anti-venomous serum in the marginal vein of one of its ears, it will suffer with absolute impunity an injection of venom into the marginal vein of the other ear capable of killing it under ordinary circumstances in a quarter of an hour. its curative powers are not less remarkable, for it is possible to inject venom sufficient to kill an animal in two hours, and to let one hour and three-quarters elapse before administering the antidote, and yet at this late stage to save the victim's life, although it is necessary where such a long interval has occurred between the respective venom and serum injections to employ the latter in larger quantities than is usually required. dr. calmette believes that the anti-toxin may be applied at an even more advanced stage of the disease if it is employed in yet larger doses. another novel and important feature about this anti-venomous serum is the fact that it not only protects animals from one species of very active venom, such as that of the cobra and other poisonous snakes, but it also affords protection from the dreaded venom of scorpions. this is a very remarkable and significant discovery, for hitherto the opinion has been stubbornly held that each toxin requires its specific anti-toxin for its correction. dr. calmette has, however, frequently indicated by his researches that this view cannot be considered so completely proven as is claimed by its supporters, and his latest investigations support the theory that particular toxins may be counteracted by several anti-toxins of different origin. thus it has been shown by calmette and roux that rabbits hyper-vaccinated against rabies acquire the power of resisting venom-poison, and that the serum of horses vaccinated against tetanus or lock-jaw also nullifies the action of serpent venom. the practical bearing of this discovery is obvious, and the hope is justified that the at present cumbrous appliances required for the elaboration of anti-toxins of such varied origin will ultimately give way to simpler and less costly methods, which will admit of these new antidotes being more widely circulated and applied. we have seen that although most animals fall an easy prey to serpent venom, yet there are a few notable exceptions, amongst which may be mentioned hedgehogs, swine, and the mongoose. now the very natural question arises why, if these animals are already in such a high degree immune from this poison, should not they be employed to furnish forth protective serum, instead of laboriously training up susceptible animals to become artificially immune and supply this venom anti-toxin? this brings us face to face with one of the many problems connected with the subject of immunity which so far have successfully eluded all attempts made to solve them. experience has shown repeatedly that although _artificially_ acquired immunity from a particular poison can be handed on by means of an animal's serum, yet the _natural_ immunity from a given poison enjoyed by one species of animal cannot be similarly transferred to less-favoured varieties. this fact has long been recognised in the case of poisons of bacterial origin. thus, white rats are absolutely immune from diphtheria, but wassermann showed some years ago that the serum of these animals has no power whatever to counteract the action of diphtheria-toxin in other animals. guinea-pigs were inoculated with fatal doses of diphtheria toxin along with white-rat serum; but although other guinea-pigs treated with the same toxin mixed with the ordinary artificially elaborated anti-diphtheritic serum survived, those which received the rat serum died in every case. now very similar results have been obtained by calmette in respect to the serum of animals naturally immune from serpent venom. the serum of the refractory little mongoose, as well as that of the hedgehog, is wholly unable to save other animals from the lethal effect of venom poison, and similar results have been noted in respect to swine serum. but a very curious fact has also been discovered by calmette--_i.e._ that these so-called naturally immune animals very frequently are quite incapable of being artificially trained to elaborate a serum possessing protective powers which can be transferred to another animal. how splendid a domain for beneficent research lies before the scientific investigator is apparent to all, and the important work already accomplished is but an augury of yet greater discoveries awaiting the labours of such leaders as calmette. it is not surprising, therefore, that the scientific interest in toxins and anti-toxins shows no signs of abatement. on the contrary, the competition for obtaining and working the new "claims" which pioneer research enthusiasts are constantly engaged in "pegging out" remains as keen as ever. despite, however, the extraordinary interest which this subject has aroused in scientific circles all over the world, nearly ten years elapsed before any notice was taken of the curious discovery made by two brothers that the blood of eels contained a highly poisonous principle, and the memoir containing this remarkable announcement remained until comparatively recently buried in the italian journals where it was first published. calmette was, we believe, the first to call attention to this discovery of the brothers mosso and give it the prominence it deserves, and both he and other investigators have not only fully confirmed it, but have greatly added to our knowledge concerning the character of the poison contained in eel serum. now the venerable izaak walton, in one of his quaint and most fascinating discourses, which although written more than two centuries ago have a freshness as if penned but yesterday, waxes enthusiastic over the eel, and supplies an elaborate recipe for its preparation for the table, telling us "it is agreed by most men that the eel is a most dainty fish; the romans have esteemed her the helena of their feasts, and some the queen of palate-pleasure." the announcement that the blood of eels is poisonous will hardly, despite its scientific interest, form a comfortable subject for reflection to the modern votaries of this novel helena. indeed, in the present timid temper of the public, this article of diet would not improbably share the ill-odour which befell the unfortunate oyster and be practically banished from our tables; but although the oyster is perhaps justifiably at present ostracised from our _menus_, taking the majority of its breeding-grounds into consideration, it would be the height of injustice to measure out similar drastic treatment to the eel. that the oyster bred in sewage-contaminated beds may revenge itself upon its consumer by infecting him with the germs of typhoid has been repeatedly contended, but that the eel, although its unsavoury surroundings are proverbial, can be held responsible for poisoning those who eat it has never, we believe, been seriously maintained, although there is an old italian saying which bids us "give eels and no wine to our enemies." public confidence, however, in the eel as an article of food need not be shaken, for it is satisfactory to learn that researches which, on the one hand, condemn eels as living generators of a highly poisonous substance, on the other hand allay any alarm which they may have reasonably raised by showing that this toxic principle is entirely destroyed in the processes of digestion, and that, therefore, taken through the mouth it is rendered harmless, and only when introduced into the system by inoculation beneath the skin or injected into the peritoneum can it assert its dangerous properties. that the blood of eels is, however, justifiably to be in future classed amongst the toxins, the number of which has of late been so increased, is at once apparent when we learn that about a dozen drops inoculated into a dog weighing about fourteen pounds will destroy the latter in less than ten minutes, whilst pigeons, rabbits, and guinea-pigs similarly treated, only with smaller quantities, also invariably succumb to its lethal action. quite recently an endeavour has been made to determine precisely the degree of toxicity possessed by eel's blood, or, in other words, to standardise the poisonous principle contained in it, so as to afford a guide to those experimenting on the subject; and it has been asserted that one cubic centimetre, or about twenty drops, injected into the veins of a rabbit weighing four pounds, may be regarded as a fatal dose for such an animal. but many difficulties surround such an attempt to exactly define the degree of toxic action possessed by such a substance, for, in the first place, the blood varies in respect to this property in different eels, whilst it also differs widely in character at different stages of the life of the fish. this seasonable variation in toxic character has been noticed in the case of viper venom, which it will be remembered was shown to be far more lethal in action when collected from snakes in the spring of the year than in the winter months. the toxic substance contained in eel serum was originally called by its discoverers, the mosso brothers, _ittio-tossina_; and they record the fact that the blood of rabbits and frogs, which animals had succumbed to its action, did not coagulate after death, whilst, curiously, in the case of dogs this abnormal phenomenon was not observed. there are various means which may be resorted to for destroying the poisonous principle contained in eel blood, and from a dietetic point of view it is satisfactory to know that heat-exposure for a quarter of an hour to a temperature of from 57·7° to 77·7° cent. entirely removes it, whilst its virulence is greatly modified by submitting it for a longer period, twenty-four hours, to a much lower temperature, _i.e._ 37° cent. it also gradually loses its toxic properties eight days after it has been collected, even when carefully shielded from light, a feature which contrasts favourably with viper venom, which can be kept for more than a year and remains as active as when first derived from the snake. we have seen also that its toxic properties invariably succumb to the processes of digestion, so that even if fashion or fad or advertising speculators, backed by scientific names, were to decree that a wealth of nourishment and support was contained in raw eel "juice," and the edict went out that it was a desirable and highly important article of invalid diet, the general public may, according to its wont, innocently accept the edict and in this case suffer no evil consequences. but another and very remarkable method of mitigating the virulence of eel blood, and one which so far has received no explanation, is mentioned by dr. wehrmann, of moscow, who has been lately studying the character of this fish's blood in dr. calmette's laboratory at the pasteur institute at lille. dr. wehrmann found that if blood serum be taken from animals previously rendered artificially immune to the action of serpent venom, and if some of this so-called anti-venomous serum be injected under the skin of eels some hours before they are killed, the lethal properties of their blood after death are considerably reduced. thus, an eel weighing about six ounces received subcutaneous injections of five cubic centimetres of anti-venomous serum; after the lapse of four-and-twenty hours it was killed and bled, and its serum inoculated into animals in the usual way. but whereas two cubic centimetres of normal eel blood sufficed to kill a guinea-pig, this eel's blood had to be administered in _twice_ that quantity to produce a fatal result, so that its toxic character had been reduced to a very appreciable extent. the readiness with which eel serum parts with its lethal properties, and the restricted conditions under which they can operate, sufficiently assure us that in the present state of our knowledge there is no danger to be apprehended from this fish, and in the absence of any experiments to show what is the effect on human beings of subcutaneous inoculations of such blood, there is no call for this substance to be scheduled under the poisons act. we have, however, by no means exhausted the extremely curious properties which characterise this material, and these properties are brought to light in a remarkable manner in connection with the investigations which have been carried out to artificially protect animals from its lethal influence, and also in some interesting experiments which have been made to compare the toxicity of eel blood with that of vipers. it is far from an easy matter to secure for experimental purposes an adequate supply of eel serum, for even a big fish weighing nearly five pounds is not capable of yielding more than about twenty-five cubic centimetres of blood, and from this only from ten to twelve cubic centimetres of serum are obtainable. calmette has shown that not only the venom glands of reptiles contain toxic substances, but that the blood of such snakes also possesses lethal properties, only in a far less degree. curiously, the serum of eels is no less than three times as toxic as the serum of the most vicious viper, and, moreover, produces far more discomfort and pain to the animals into which it is introduced than accompanies the injection of viper blood. in the case of viper blood its introduction is followed by no symptoms of discomfort, the animal remains quite quiet, growing more and more somnolent, a condition which is followed by an abnormal fall of temperature, ultimately ending in complete collapse, symptoms which in a much more modified degree characterise the injection of _heated_ eel serum into animals. this heated eel serum, which we have seen is deprived of the objectionable characteristics of ordinary eel serum, produces but very transitory symptoms in animals, occasioning some degree of somnolence, and now and again a reduction in temperature, a condition from which, however, the animals rapidly recover in from two to three hours. animals, however, treated with this heated eel serum acquire a power of resisting the lethal action of unheated or ordinary eel serum, and this artificially induced condition of immunity continues for about three days after the completion of the treatment. the protective properties of this heated serum are not restricted to animals subsequently inoculated with eel serum, but are extended also to animals which afterwards receive injections of viper serum; but of much greater interest and importance is the remarkable fact that heated eel serum, as well as weak doses of the latter not heated but diluted with water, are capable of protecting animals from the fatal consequences of the far more potent viper venom. it is interesting to note that, although diluted eel serum can protect an animal from so deadly a poison as viper venom, the serum of vipers is quite unable to afford any such service in the case of animals inoculated with ordinary eel serum. the full complement of protective power obtainable from this treated eel serum is only able to slowly assert itself, for it is necessary for a period of as long as twenty-four hours to elapse after its introduction to ensure the animal's system being thoroughly impregnated with it and enable it to withstand a lethal dose of viper venom. in this respect, what may be designated treated or protective eel serum differs very markedly from anti-venomous serum, which we have seen is serum derived from animals trained up to withstand fatal does of serpent venom, for anti-venomous serum acts immediately, and at once confers immunity on an animal from the lethal effects of such venom. the rapidity with which it acts is indeed one of the most astonishing properties of this particular anti-toxin. thus if two cubic centimetres of anti-venomous serum be inoculated into the marginal vein of a rabbit's ear, it at once confers upon the latter complete immunity from snake poison. immediately after the injection of the serum, venom sufficient to destroy an ordinary rabbit in a quarter of an hour may be injected with impunity into the vein of the other ear. but not only are the _protective_ powers of this serum so remarkable in their degree, but its _curative_ powers, a much more difficult property to establish in a substance, are extraordinarily intense, as may be gathered from the following example. four rabbits were inoculated with a quantity of venom calculated to destroy them in the space of two hours; one of these four animals was abandoned to its fate, but the other three received, practically at the eleventh hour, viz. just fifteen minutes before the expiration of the calculated two hours' respite, an intravenous injection of a small quantity of anti-venomous serum, only amounting to one four-hundredth part of the weight of each animal respectively. the rabbit which received only the venom died at the end of two hours, whilst the other three remained in perfect health. but although eel serum can be persuaded to part with its poisonous character and even exercise protective powers over otherwise doomed victims, it is not able to stretch forth a healing hand to the afflicted, for, when once the poison has been introduced, whether it be eel or viper blood, or the venom of snakes, it is absolutely powerless to mitigate or stop in any way the deadly progress of the toxin. thus whilst eel blood may acquire _protective_ properties it cannot acquire _curative_ properties, and, therefore, treated eel serum cannot be legitimately enrolled with the anti-toxins which have been elaborated, as, for example, anti-venomous serum, for, to be worthy of such rank, a substance must be capable of wielding both protective and curative powers. but, although eel serum may under certain conditions protect from the lethal action of serpent venom, eels are not themselves under ordinary circumstances endowed with any power to withstand the influence of this poison, for a good-sized eel will succumb to a dose of venom which is sufficient to kill a guinea-pig. considerable interest is attached to the fact that anti-venomous serum not only acts as an anti-toxin towards serpent venom, but also towards a poison of quite a different character, such as that present in the normal blood of eels, for this fact tends to confirm the view upheld by some authorities, that specific toxins do not necessarily only yield to specific anti-toxins, and that a particular anti-toxin may act as such towards divers toxins of varied origin and character. calmette has brought this point out very clearly in his later investigations on the vegetable poison abrine, a very powerful toxin, furnished by the active principle of the seeds or beans of a leguminous plant common in india and south america, and frequently used, as already mentioned, by the natives in india to revenge themselves on their enemies in poisoning their cattle. immunising serums of various kinds were selected for testing their protective action on animals poisoned with abrine, and it was found that anti-tetanic, anti-diphtheritic, anti-anthrax, and anti-cholera serums all individually exerted a decided immunising action with regard to this powerful vegetable poison. the hope is, therefore, perhaps not beyond the realm of possibility, that at some future time the complexity of drugs which now figure in the chemists' pharmacopoeia may be replaced by a few substances the application of which will come within the means and understanding of all. so far we have not dealt with the artificial immunisation of an animal from the action of eel poison, but this apparently offers very little difficulty, and is accomplished by introducing very small and gradually increasing doses of eel serum into the system, care being taken to proportion the quantity given according to the weight and general condition of the animal to be immunised. a rabbit, for example, treated in the above manner, subsequently yielded a serum which was proved to possess both preventive and curative powers in respect to both eel poison, and viper venom and blood, entitling this so called anti-eel serum to take its place amongst the anti-toxins, and furnishing yet another instance of a substance exercising its immunising influence over various toxins. this process of gradually acclimatising, as it were, animals to a particular poison by repeated doses of the same poison, recalls the old proverb, "seek your salve where you got your sore," and brings us to a consideration of some of the primitive antecedents of a practice which, at the present time, promises to bring about so profound a revolution in the art of medicine. the modern system of inoculation has, however, arisen quite without reference to such antecedents, which latter were not based upon any scientific laws or considerations, but owed their evolution to local customs and experience handed down from age to age by tradition, and in many cases preserved through a simple faith in the superstitions which surrounded them. to such a category must be added the curious superstitions indulged in by the native population of tunis regarding methods of preventing hydrophobia in persons bitten by rabid animals. dr. loir refers to these primitive ideas on the art of healing in a report of the work carried out at the anti-rabic institute at tunis, one of the many centres for the prevention of rabies by pasteur's method which have been established in every quarter of the globe except great britain, the inhabitants of this "great conservative island-empire," as a renowned foreign scientist describes it, still preferring a trip to paris to countenancing the establishment of an anti-rabic institute in their own country. the arab physicians in tunis have from time immemorial sought to specially identify themselves with cures for this disease, which is so prevalent as to be a veritable scourge to the country. a much-vaunted remedy advocated by the profession consists in pounding up the charred head of a rabid dog with vinegar, and administering an emulsion of the same to the patient. the dung of camels is also highly prized as a remedy, as also the water of certain wells which the simple faith of the natives has endowed with supernatural curative properties. but the strangest prescription of all consists in broth made from lambs a year old, to which is added a peculiar kind of beetle, but in such a small quantity that the latter ingredient only equals the weight of a grain of corn. this concoction is given to the unfortunate patient twenty-three days after he has been bitten. in the urine, according to the arabian doctors, seven small worms should be found which represent the embryos of dogs engendered by the virus in the human body, and which when once got rid of the patient recovers! in the face of such crude traditions upheld with so much tenacity by the native population, it is surprising that the tunisian anti-rabic institute has met with such a large measure of support in the shape of applicants for admission, which, on an average, number over one hundred annually. the mortality amongst those treated closely approaches the satisfactory results obtained at the paris institute, where the death-rate amounts to about 0·38 per cent. of the persons treated. there is perhaps no more interesting chapter in the history and literature of medicine than might be compiled by searching out the early uses of drugs and the primitive application of methods in the art of healing, and tracing their connection, if possible, with the practices which are in vogue at the present day. in the matter of toxins and anti-toxins, or in respect to the modern theories of preventive medicine, there would appear to be a curious link between the methods based upon elaborate scientific inquiries and those which arose through simple experience and expediency. the idea of a poison, as the old proverb above tells us, being a corrective for itself is no new idea, for we read how in ancient times, for example, the ophiogenes of the hellespont were renowned for their immunity to snake poison, and one account of them states particularly that they fed upon serpents, and that to this diet they probably owed their reputed magical art in withstanding the action of serpent venom. again, a traveller in egypt, hasselquist, tells us how the serpent-charmers there eat serpents, making them into a kind of broth, and that invariably before starting off to catch these reptiles they partake of some of it. in a paper by mr. t. r. rao on the yánádés tribe of the nellore district, madras presidency, the author mentions that these strange people have, amongst other characteristics, absolutely no fear in catching cobras, which they draw out of their holes without any alarm as to their fangs, and that they appear to protect themselves against the effects of snake-bites by swallowing the poison-sacs of snakes. bruce describes how he saw a serpent-charmer in cairo who allowed himself to be bitten by a viper between the forefinger and the thumb, and made no endeavour whatever to apply remedies, neither did he exhibit the slightest anxiety as to the consequences. that this was no trick, and that the viper was really possessed of all its deadly faculties at the time it bit the man, was proved by the fact that a pelican subsequently bitten by the same animal died in thirteen minutes. bruce also tells of a man who "with his naked hand took a viper from a number of others lying at the bottom of a tub. he put it on his head, then in his breast, and tied it about his neck like a necklace. next it was made to bite a hen, which died in a few minutes; and, to complete the experiment, the man took it by the neck, and, beginning at the tail, ate it as one does a carrot or a stick of celery, without any seeming repugnance." a most interesting account of snake-charmers is given by drummond hay, in his book on _western barbary_, in which he relates his experiences with some of these wonderful individuals belonging to the sect called eisowy. members of this sect, he mentions, frequently handled scorpions and poisonous reptiles without fear or hesitation, and they were never attacked by them. he was present at one of their exhibitions of feats with snakes in which they both allowed themselves to be bitten and provoked the snake to bite them. the charmer thus bitten then in his turn ate or chewed the reptile, which, he remarks, writhing with pain, bit him in the neck and hands till it was actually destroyed by the eisowy's teeth. in south africa snake poison is actually taken as a protection against snake-bites, and if we turn to the _lancet_ of the year 1886, we shall find a letter from mr. alfred bolton stating that his curiosity had been aroused by the fact that while in south africa cattle and horses frequently died from the effect of snake-bites, the natives themselves seldom or never appeared to suffer any inconvenience from such injuries other than would follow any accident which would set up local inflammation. on inquiry he found that they were in the habit of extracting the poison gland from the snake immediately it is killed, squeezing it into their mouths and drinking the secretion, thereby apparently acquiring absolute immunity from snake-bites. so impressed was mr. bolton by what he observed that he adds: "i can no longer refuse to believe in the efficacy of the snake virus itself as a remedy against snake poison." savage tribes have learnt from bitter experience how to protect themselves from snake-bites, and it is well known that they have a method of inoculation which they employ with success. the creoles of surinam use an ointment as a protection against snake-bites, which is regarded as highly efficacious. it is reputed to consist principally of the pounded head of a rattlesnake, which concoction would therefore include the contents of the venom glands. this is then mixed with the juices of a certain plant, which addition probably mitigates the intensity of the venom by acting as a diluent. this substance is generally applied by making an incision in the wrist or forearm and rubbing it in, after which individuals thus treated appear to enjoy security from the venom of snake-bites. what applies to serpent venom would also appear to hold good in regard to other poisons, such as that contained in the sting of a bee. this poison is extraordinarily tenacious of its irritant properties, and, unlike eel poison, retains its virulence even when exposed to high temperatures. an interesting memoir on the immunity of the bee-keeper from the effects of bee poison was published a short time ago by dr. langer in a german scientific journal. he issued a number of circulars with questions to be answered, and sent these to more than a hundred bee-keepers in different parts of the country, with the result that a hundred and forty-four stated that they were now immune to bee poison, nine having been fortunately endowed with a natural immunity to this irritant, whilst only twenty-six out of the whole number applied to stated that they were still susceptible. this condition of immunity to bee poison is obtained after a varying number of stings have been inflicted; in some cases thirty, at the rate of from three to four a day, are sufficient to ensure freedom from further discomfort, but the inoculations may have to be prolonged up to one hundred stings to secure complete immunity. in experiments carried out on animals this immunity to bee poison has been also induced by repeated application of the irritant. it was formerly generally supposed that the irritant nature of a bee's sting was due to the presence of formic acid; but inasmuch as bee poison can retain its poisonous character in spite of being submitted to heat, which would effectually volatilise the formic acid present, this assumption must be abandoned, and opinion is more inclined now to regard this irritant substance as partaking of the nature of an alkaloid. before closing this brief review of some of the most recent discoveries which have been made in the domain of immunity, we must mention some extremely suggestive and important researches on the poison of tetanus, or lock-jaw, which have emanated from dr. roux's laboratory at the institut pasteur in paris. it will perhaps be remembered that pasteur, when working at hydrophobia, experienced the greatest difficulty in exciting rabies in animals with certainty, and that it was only when the fact of its being a disease which essentially affects the nervous system of the animal was taken into account that it occurred to him to cultivate the virus in the medium for which it had seemingly the greatest affinity, viz. the nervous tissue of an animal; it was only on taking this step that he succeeded in invariably provoking rabies in the animals under experiment. in the case of tetanus we have another disease affecting the nerve-centres of the body, and although many authentic cases have been cited in which the treatment with anti-tetanic serum has been entirely successful, a great many instances have occurred in which it has been of no avail at all, more especially when the disease has obtained a firm hold on its victim. now dr. roux has not only been carrying out experiments to ascertain what is the result of directly attacking, as pasteur did in the case of rabies, the nerve-centres of an animal with the tetanus toxin, but he has also taken another and very important step further, and has investigated, not only the action of the toxin, but also that of the anti-toxin on the nerve-centres of an animal suffering from tetanus. in describing the cerebral inoculations which he has conducted on animals, dr. roux points out that the operation, in itself, is attended with no pain or even inconvenience to the animal in question, that subsequently it eats with its usual appetite, and shows no signs of discomfort. first, as regards the infection of an animal with the tetanus virus introduced directly into the brain, it has been found that very much smaller quantities produce a fatal result than when subcutaneously inoculated. thus, a rabbit which received two cubic centimetres of the poison under the skin took four days to succumb to tetanus, whilst one-twentieth of the quantity inoculated into the brain sufficed to kill another rabbit of the same size in less than twenty hours. another very instructive example of this susceptibility of the nerve-centres for certain poisons is afforded in the case of rats and the toxin of diphtheria. rats possess a natural immunity from this substance, and can successfully withstand a dose of diphtheria poison introduced under the skin which would infallibly kill several rabbits. this state of immunity, however, entirely disappears when the toxin is brought directly in contact with nervous tissue, for a very small quantity of diphtheria poison--insufficient to cause under ordinary circumstances even a passing swelling at the seat of inoculation--will, when introduced into the brain of a rat, kill the animal. again, rabbits are generally credited with possessing high powers of resisting the action of morphia, a large dose of this substance introduced subcutaneously producing no result whatever. a cerebral inoculation, however, of a minute quantity of morphia causes an immediate reaction, and the animal, after remaining in a more or less dazed condition for several hours, finally succumbs to this drug. dr. roux is inclined to regard this difference in the susceptibility exhibited by animals to one and the same poison as being due to a good deal of the toxin, when subcutaneously introduced, failing to reach the nerve-centres, it having been destroyed or arrested in the system before it could attack them. what is the nature of the subtle forces which may so beneficially intervene between the toxin and its victim has long been a problem which has excited the interest and ingenuity of some of the most brilliant scientific authorities of the day, and it is one which, even in the hands of men like metchnikoff, is still awaiting a satisfactory solution! the important point was next approached by dr. roux as to whether an animal, successfully trained to withstand large doses of the poison, as ordinarily introduced, could also resist it when directly inoculated into the brain. is, in fact, the undoubted immunity to tetanus poison which may be possessed by an animal due to the nerve-centres having become insensible to this substance? the answer to this question would appear to be in the negative, for animals artificially protected from tetanus poison introduced under the skin succumbed to a small dose inoculated direct into the brain, which would otherwise have not produced even a slight passing tetanic affection of the limb where the inoculation was made. immense numbers of experiments were made under varying conditions, but the result was fully confirmed, showing that the nerve-centres had not acquired any immunity to the poison, although the blood serum of the victims to such cerebral inoculations was proven over and over again to be endowed with strong protective properties against tetanus poison. the endeavour was then made to, in dr. roux's words, "place the anti-toxin where the toxin is working," and preserve the vital force of the nervous tissue. to arrest tetanus by substituting cerebral for subcutaneous inoculations of the anti-tetanic serum was the next feat attempted. several guinea-pigs and rabbits were inoculated subcutaneously with virulent doses of tetanus poison sufficient to kill them in about seventy hours; some were subsequently treated with anti-toxic serum introduced in the ordinary way under the skin, whilst others were inoculated with from six to seven drops of this protective serum direct into the brain. the results were extraordinarily successful. although but a few drops of the anti-toxin were used for the _cerebral_ inoculations, the animals survived the otherwise fatal doses they had received of the toxin; whilst out of seventeen guinea-pigs which received _subcutaneous_ inoculations of the anti-toxin only two recovered, and the quantity of the anti-toxin employed reached as much as from ten to twenty cubic centimetres in some of the experiments, contrasting in a remarkable manner with the few drops which sufficed in the case of the cerebral inoculations. dr. roux sums up this splendid result in the following modest words: "il ne suffit pas de donner de l'anti-toxine, il faut la mettre au bon endroit." the significance and far-reaching application of this most important discovery cannot easily be overestimated. hitherto the preparation of an anti-toxin has been the chief point considered, but dr. roux and his able coadjutor, m. a. borrel, have shown how great may be the results which attend its method of administration, and have opened up an entirely new direction for investigation. although the subject of immunity is not, as we have seen, by any means wholly a latter-day creation, yet its approach and consideration from a modern point of view, assisted by the resources and equipment provided by modern scientific methods, justifiably entitles the nineteenth century to claim it as its own discovery. however brilliant and successful the labours may be of those who will follow in the future, subsequent generations will know how to venerate those great leaders of scientific thought, amongst whom we must rank pasteur, to whose genius the world owes so great a debt of gratitude, and the vast extent of whose labours cannot be adequately measured at the present day by reason of the restricted scientific horizon which encircles public opinion in this country. the end plymouth william brendon and son printers the story of germ life by h. w. conn professor of biology at wesleyan university, author of evolution of to-day, the living world, etc. preface. since the first edition of this book was published the popular idea of bacteria to which attention was drawn in the original preface has undergone considerable modification. experimental medicine has added constantly to the list of diseases caused by bacterial organisms, and the general public has been educated to an adequate conception of the importance of the germ as the chief agency in the transmission of disease, with corresponding advantage to the efficiency of personal and public hygiene. at the same time knowledge of the benign bacteria and the enormous role they play in the industries and the arts has become much more widely diffused. bacteriology is being studied in colleges as one of the cultural sciences; it is being widely adopted as a subject of instruction in high schools; and schools of agriculture and household science turn out each year thousands of graduates familiar with the functions of bacteria in daily life. through these agencies the popular misconception of the nature of microorganisms and their relations to man is being gradually displaced by a general appreciation of their manifold services. it is not unreasonable to hope that the many thousands of copies of this little manual which have been circulated and read have contributed materially to that end. if its popularity is a safe criterion, the book has amply fulfilled its purpose of placing before the general reader in a simple and direct style the main facts of bacteriology. beginning with a discussion of the nature of bacteria, it shows their position in the scale of plant and animal life. the middle chapters describe the functions of bacteria in the arts, in the dairy, and in agriculture. the final chapters discuss the relation of bacteria to disease and the methods by which the new and growing science of preventive medicine combats and counteracts their dangerous powers. july, 1915. contents. i.--bacteria as plants historical.--form of bacteria.--multiplication of bacteria.--spore formation.--motion.--internal structure.--animals or plants?-classification.--variation.--where bacteria are found. ii.--miscellaneous uses of bacteria in the arts. maceration industries.--linen.--jute.--hemp.--sponges.--leather. --fermentative industries.--vinegar--lactic acid.--butyric acid.-bacteria in tobacco curing.--troublesome fermentations. iii.--bacteria in the dairy. sources of bacteria in milk.--effect of bacteria on milk.-bacteria used in butter making.--bacteria in cheese making. iv.--bacteria in natural processes. bacteria as scavengers.--bacteria as agents in nature's food cycle.--relation of bacteria to agriculture.--sprouting of seeds. --the silo.--the fertility of the soil.--bacteria as sources of trouble to the farmer.--coal formation. v.--parasitic bacteria and their relation to disease method of producing disease.--pathogenic germs not strictly parasitic.--pathogenic germs that are true parasites.--what diseases are due to bacteria.--variability of pathogenic powers.-susceptibility of the individual.--recovery from bacteriological diseases.--diseases caused by organisms other than bacteria. vi.--methods of combating parasitic bacteria preventive medicine.--bacteria in surgery.--prevention by inoculation.--limits of preventive medicine.--curative medicine. --drugs--vis medicatrix naturae.--antitoxines and their use.-conclusion. the story of germ life. chapter i. bacteria as plants. during the last fifteen years the subject of bacteriology [footnote: the term microbe is simply a word which has been coined to include all of the microscopic plants commonly included under the terms bacteria and yeasts.] has developed with a marvellous rapidity. at the beginning of the ninth decade of the century bacteria were scarcely heard of outside of scientific circles, and very little was known about them even among scientists. today they are almost household words, and everyone who reads is beginning to recognise that they have important relations to his everyday life. the organisms called bacteria comprise simply a small class of low plants, but this small group has proved to be of such vast importance in its relation to the world in general that its study has little by little crystallized into a science by itself. it is a somewhat anomalous fact that a special branch of science, interesting such a large number of people, should be developed around a small group of low plants. the importance of bacteriology is not due to any importance bacteria have as plants or as members of the vegetable kingdom, but solely to their powers of producing profound changes in nature. there is no one family of plants that begins to compare with them in importance. it is the object of this work to point out briefly how much both of good and ill we owe to the life and growth of these microscopic organisms. as we have learned more and more of them during the last fifty years, it has become more and more evident that this one little class of microscopic plants fills a place in nature's processes which in some respects balances that filled by the whole of the green plants. minute as they are, their importance can hardly be overrated, for upon their activities is founded the continued life of the animal and vegetable kingdom. for good and for ill they are agents of neverceasing and almost unlimited powers. historical. the study of bacteria practically began with the use of the microscope. it was toward the close of the seventeenth century that the dutch microscopist, leeuwenhoek, working with his simple lenses, first saw the organisms which we now know under this name, with sufficient clearness to describe them. beyond mentioning their existence, however, his observations told little or nothing. nor can much more be said of the studies which followed during the next one hundred and fifty years. during this long period many a microscope was turned to the observation of these minute organisms, but the majority of observers were contented with simply seeing them, marvelling at their minuteness, and uttering many exclamations of astonishment at the wonders of nature. a few men of more strictly scientific natures paid some attention to these little organisms. among them we should perhaps mention von gleichen, muller, spallanzani, and needham. each of these, as well as others, made some contributions to our knowledge of microscopical life, and among other organisms studied those which we now call bacteria. speculations were even made at these early dates of the possible causal connection of these organisms with diseases, and for a little the medical profession was interested in the suggestion. it was impossible then, however, to obtain any evidence for the truth of this speculation, and it was abandoned as unfounded, and even forgotten completely, until revived again about the middle of the 19th century. during this century of wonder a sufficiency of exactness was, however, introduced into the study of microscopic organisms to call for the use of names, and we find muller using the names of monas, proteus, vibrio, bacillus, and spirillum, names which still continue in use, although commonly with a different significance from that given them by muller. muller did indeed make a study sufficient to recognise the several distinct types, and attempted to classsify these bodies. they were not regarded as of much importance, but simply as the most minute organisms known. nothing of importance came from this work, however, partly because of the inadequacy of the microscopes of the day, and partly because of a failure to understand the real problems at issue. when we remember the minuteness of the bacteria, the impossibility of studying any one of them for more than a few moments at a time --only so long, in fact, as it can be followed under a microscope; when we remember, too, the imperfection of the compound microscopes which made high powers practical impossibilities; and, above all, when we appreciate the looseness of the ideas which pervaded all scientists as to the necessity of accurate observation in distinction from inference, it is not strange that the last century gave us no knowledge of bacteria beyond the mere fact of the existence of some extremely minute organisms in different decaying materials. nor did the 19th century add much to this until toward its middle. it is true that the microscope was vastly improved early in the century, and since this improvement served as a decided stimulus to the study of microscopic life, among other organisms studied, bacteria received some attention. ehrenberg, dujardin, fuchs, perty, and others left the impress of their work upon bacteriology even before the middle of the century. it is true that schwann shrewdly drew conclusions as to the relation of microscopic organisms to various processes of fermentation and decay--conclusions which, although not accepted at the time, have subsequently proved to be correct. it is true that fuchs made a careful study of the infection of "blue milk," reaching the correct conclusion that the infection was caused by a microscopic organism which he discovered and carefully studied. it is true that henle made a general theory as to the relation of such organisms to diseases, and pointed out the logically necessary steps in a demonstration of the causal connection between any organism and a disease. it is true also that a general theory of the production of ail kinds of fermentation by living organisms had been advanced. but all these suggestions made little impression. on the one hand, bacteria were not recognised as a class of organisms by themselves--were not, indeed, distinguished from yeasts or other minute animalcuise. their variety was not mistrusted and their significance not conceived. as microscopic organisms, there were no reasons for considering them of any more importance than any other small animals or plants, and their extreme minuteness and simplicity made them of little interest to the microscopist. on the other hand, their causal connection with fermentative and putrefactive processes was entirely obscured by the overshadowing weight of the chemist liebig, who believed that fermentations and putrefactions were simply chemical processes. liebig insisted that all albuminoid bodies were in a state of chemically unstable equilibrium, and if left to themselves would fall to pieces without any need of the action of microscopic organisms. the force of liebig's authority and the brilliancy of his expositions led to the wide acceptance of his views and the temporary obscurity of the relation of microscopic organisms to fermentative and putrefactive processes. the objections to liebig's views were hardly noticed, and the force of the experiments of schwann was silently ignored. until the sixth decade of the century, therefore, these organisms, which have since become the basis of a new branch of science, had hardly emerged from obscurity. a few microscopists recognised their existence, just as they did any other group of small animals or plants, but even yet they failed to look upon them as forming a distinct group. a growing number of observations was accumulating, pointing toward a probable causal connection between fermentative and putrefactive processes and the growth of microscopic organisms; but these observations were known only to a few, and were ignored by the majority of scientists. it was louis pasteur who brought bacteria to the front, and it was by his labours that these organisms were rescued from the obscurity of scientific publications and made objects of general and crowning interest. it was pasteur who first successfully combated the chemical theory of fermentation by showing that albuminous matter had no inherent tendency to decomposition. it was pasteur who first clearly demonstrated that these little bodies, like all larger animals and plants, come into existence only by ordinary methods of reproduction, and not by any spontaneous generation, as had been earlier claimed. it was pasteur who first proved that such a common phenomenon as. the souring of milk was produced by microscopic organisms growing in the milk. it was pasteur who first succeeded in demonstrating that certain species of microscopic organisms are the cause of certain diseases, and in suggesting successful methods of avoiding them. all these discoveries were made in rapid succession. within ten years of the time that his name began to be heard in this connection by scientists, the subject had advanced so rapidly that it had become evident that here was a new subject of importance to the scientific world, if not to the public at large. the other important discoveries which pasteur made it is not our purpose to mention here. his claim to be considered the founder of bacteriology will be recognised from what has already been mentioned. it was not that he first discovered the organisms, or first studied them; it was not that he first suggested their causal connection with fermentation and disease, but it was because he for the first time placed the subject upon a firm foundation by proving with rigid experiment some of the suggestions made by others, and in this way turned the attention of science to the study of micro-organisms. after the importance of the subject had been demonstrated by pasteur, others turned their attention in the same direction, either for the purpose of verification or refutation of pasteur's views. the advance was not very rapid, however, since bacteriological experimentation proved to be a subject of extraordinary difficulty. bacteria were not even yet recognised as a group of organisms distinct enough to be grouped by themselves, but were even by pasteur at first confounded with yeasts. as a distinct group of organisms they were first distinguished by hoffman in 1869, since which date the term bacteria, as applying to this special group of organisms, has been coming more and more into use. so difficult were the investigations, that for years there were hardly any investigators besides pasteur who could successfully handle the subject and reach conclusions which could stand the test of time. for the next thirty years, although investigators and investigations continued to increase, we can find little besides dispute and confusion along this line. the difficulty of obtaining for experiment any one kind of bacteria by itself, unmixed with others (pure cultures), rendered advance almost impossible. so conflicting were the results that the whole subject soon came into almost hopeless confusion, and very few steps were taken upon any sure basis. so difficult were the methods, so contradictory and confusing the results, because of impure cultures, that a student of to-day who wishes to look up the previous discoveries in almost any line of bacteriology need hardly go back of 1880, since he can almost rest assured that anything done earlier than that was more likely to be erroneous than correct. the last fifteen years have, however, seen a wonderful change. the difficulties had been mostly those of methods of work, and with the ninth decade of the century these methods were simplified by robert koch. this simplification of method for the first time placed this line of investigation within the reach of scientists who did not have the genius of pasteur. it was now possible to get pure cultures easily, and to obtain with such pure cultures results which were uniform and simple. it was now possible to take steps which had the stamp of accuracy upon them, and which further experiment did not disprove. from the time when these methods were thus made manageable the study of bacteria increased with a rapidity which has been fairly startling, and the information which has accumulated is almost formidable. the very rapidity with which the investigations have progressed has brought considerable confusion, from the fact that the new discoveries have not had time to be properly assimilated into knowledge. today many facts are known whose significance is still uncertain, and a clear logical discussion of the facts of modern bacteriology is not possible. but sufficient knowledge has been accumulated and digested to show us at least the direction along which bacteriological advance is tending, and it is to the pointing out of these directions that the following pages will be devoted. what are bacteria? the most interesting facts connected with the subject of bacteriology concern the powers and influence in nature possessed by the bacteria. the morphological side of the subject is interesting enough to the scientist, but to him alone. still, it is impossible to attempt to study the powers of bacteria without knowing something of the organisms themselves. to understand how they come to play an important part in nature's processes, we must know first how they look and where they are found. a short consideration of certain morphological facts will therefore be necessary at the start. form of bacteria. in shape bacteria are the simplest conceivable structures. although there are hundreds of different species, they have only three general forms, which have been aptly compared to billiard balls, lead pencils, and corkscrews. spheres, rods, and spirals represent all shapes. the spheres may be large or small, and may group themselves in various ways; the rods may be long or short, thick or slender; the spirals may be loosely or tightly coiled, and may have only one or two or may have many coils, and they may be flexible or stiff; but still rods, spheres, and spirals comprise all types. in size there is some variation, though not very great. all are extremely minute, and never visible to the naked eye. the spheres vary from 0.25 u to 1.5 u (0.000012 to 0.00006 inches). the rods may be no more than 0.3 u in diameter, or may be as wide as 1.5 u to 2.5 u, and in length vary all the way from a length scarcely longer than their diameter to long threads. about the same may be said of the spiral forms. they are decidedly the smallest living organisms which our microscopes have revealed. in their method of growth we find one of the most characteristic features. they universally have the power of multiplication by simple division or fission. each individual elongates and then divides in the middle into two similar halves, each of which then repeats the process. this method of multiplication by simple division is the distinguishing mark which separates the bacteria from the yeasts, the latter plants multiplying by a process known as budding. fig. 2 shows these two methods of multiplication. while all bacteria thus multiply by division, certain differences in the details produce rather striking differences in the results. considering first the spherical forms, we find that some species divide, as described, into two, which separate at once, and each of which in turn divides in the opposite direction, called micrococcus, (fig. 3). other species divide only in one direction. frequently they do not separate after dividing, but remain attached. each, however, again elongates and divides again, but all still remain attached. there are thus formed long chains of spheres like strings of beads, called streptococci (fig. 4). other species divide first in one direction, then at right angles to the first division, and a third division follows at right angles to the plane of the first two, thus producing solid groups of fours, eights, or sixteens (fig 5), called sarcina. each different species of bacteria is uniform in its method of division, and these differences are therefore indications of differences in species, or, according to our present method of classification, the different methods of division represent different genera. all bacteria producing streptococcus chains form a single genus streptococcus, and all which divide in three division planes form another genus, sarcina, etc. the rod-shaped bacteria also differ somewhat, but to a less extent. they almost always divide in a plane at right angles to their longest dimension. but here again we find some species separating immediately after division, and thus always appearing as short rods (fig. 6), while others remain attached after division and form long chains. sometimes they appear to continue to increase in length without showing any signs of division, and in this way long threads are formed (fig. 7). these threads are, however, potentially at least, long chains of short rods, and under proper conditions they will break up into such short rods, as shown in fig. 7a. occasionally a rod species may divide lengthwise, but this is rare. exactly the same may be said of the spiral forms. here, too, we find short rods and long chains, or long spiral filaments in which can be seen no division into shorter elements, but which, under certain conditions, break up into short sections. rapidity of multiplication. it is this power of multiplication by division that makes bacteria agents of such significance. their minute size would make them harmless enough if it were not for an extraordinary power of multiplication. this power of growth and division is almost incredible. some of the species which have been carefully watched under the microscope have been found under favourable conditions to grow so rapidly as to divide every half hour, or even less. the number of offspring that would result in the course of twenty-four hours at this rate is of course easily computed. in one day each bacterium would produce over 16,500,000 descendants, and in two days about 281,500,000,000. it has been further calculated that these 281,500,000,000 would form about a solid pint of bacteria and weigh about a pound. at the end of the third day the total descendants would amount to 47,000,000,000,000, and would weigh about 16,000,000 pounds. of course these numbers have no significance, for they are never actual or even possible numbers. long before the offspring reach even into the millions their rate of multiplication is checked either by lack of food or by the accumulation of their own excreted products, which are injurious to them. but the figures do have interest since they show faintly what an unlimited power of multiplication these organisms have, and thus show us that in dealing with bacteria we are dealing with forces of almost infinite extent. this wonderful power of growth is chiefly due to the fact that bacteria feed upon food which is highly organized and already in condition for absorption. most plants must manufacture their own foods out of simpler substances, like carbonic dioxide (co2) and water, but bacteria, as a rule, feed upon complex organic material already prepared by the previous life of plants or animals. for this reason they can grow faster than other plants. not being obliged to make their own foods like most plants, nor to search for it like animals, but living in its midst, their rapidity of growth and multiplication is limited only by their power to seize and assimilate this food. as they grow in such masses of food, they cause certain chemical changes to take place in it, changes doubtless directly connected with their use of the material as food. recognising that they do cause chemical changes in food material, and remembering this marvellous power of growth, we are prepared to believe them capable of producing changes wherever they get a foothold and begin to grow. their power of feeding upon complex organic food and producing chemical changes therein, together with their marvellous power of assimilating this material as food, make them agents in nature of extreme importance. differences between different species of bacteria. while bacteria are thus very simple in form, there are a few other slight variations in detail which assist in distinguishing them. the rods are sometimes very blunt at the ends, almost as if cut square across, while in other species they are more rounded and occasionally slightly tapering. sometimes they are surrounded by a thin layer of some gelatinous substance, which forms what is called a capsule (fig. 10). this capsule may connect them and serve as a cement, to prevent the separate elements of a chain from falling apart. sometimes such a gelatinous secretion will unite great masses of bacteria into clusters, which may float on the surface of the liquid in which they grow or may sink to the bottom. such masses are called zoogloea, and their general appearance serves as one of the characters for distinguishing different species of bacteria (fig. 10, a and b). when growing in solid media, such as a nutritious liquid made stiff with gelatine, the different species have different methods of spreading from their central point of origin. a single bacterium in the midst of such a stiffened mass will feed upon it and produce descendants rapidly; but these descendants, not being able to move through the gelatine, will remain clustered together in a mass, which the bacteriologist calls a colony. but their method of clustering, due to different methods of growth, is by no means always alike, and these colonies show great differences in general appearance. the differences appear to be constant, however, for the same species of bacteria, and hence the shape and appearance of the colony enable bacteriologists to discern different species (fig. ii). all these points of difference are of practical use to the bacteriologist in distinguishing species. spore formation. in addition to their power of reproduction by simple division, many species of bacteria have a second method by means of spores. spores are special rounded or oval bits of bacteria protoplasm capable of resisting adverse conditions which would destroy the ordinary bacteria. they arise among bacteria in two different methods. endogenous spores.--these spores arise inside of the rods or the spiral forms (fig. 12). they first appear as slight granular masses, or as dark points which become gradually distinct from the rest of the rod. eventually there is thus formed inside the rod a clear, highly refractive, spherical or oval spore, which may even be of a greater diameter than the rod producing it, thus causing it to swell out and become spindle formed [fig. 12 c]. these spores may form in the middle or at the ends of the rods (fig. 12). they may use up all the protoplasm of the rod in their formation, or they may use only a small part of it, the rod which forms them continuing its activities in spite of the formation of the spores within it. they are always clear and highly refractive from containing little water, and they do not so readily absorb staining material as the ordinary rods. they appear to be covered with a layer of some substance which resists the stain, and which also enables them to resist various external agencies. this protective covering, together with their small amount of water, enables them to resist almost any amount of drying, a high degree of heat, and many other adverse conditions. commonly the spores break out of the rod, and the rod producing them dies, although sometimes the rod may continue its activity even after the spores have been produced. arthrogenous spores (?).--certain species of bacteria do not produce spores as just described, but may give rise to bodies that are sometimes called arthrospores. these bodies are formed as short segments of rods. a long rod may sometimes break up into several short rounded elements, which are clear and appear to have a somewhat increased power of resisting adverse conditions. the same may happen among the spherical forms, which only in rare instances form endogenous spores. among the spheres which form a chain of streptococci some may occasionally be slightly different from the rest. they are a little larger, and have been thought to have an increased resisting power like that of true spores (fig. 13 b). it is quite doubtful, however, whether it is proper to regard these bodies as spores. there is no good evidence that they have any special resisting power to heat like endogenous spores, and bacteriologists in general are inclined to regard them simply as resting cells. the term arthrospores has been given to them to indicate that they are formed as joints or segments, and this term may be a convenient one to retain although the bodies in question are not true spores. still a different method of spore formation occurs in a few peculiar bacteria. in this case (fig. 14) the protoplasm in the large thread breaks into many minute spherical bodies, which finally find exit. the spores thus formed may not be all alike, differences in size being noticed. this method of spore formation occurs only in a few special forms of bacteria. the matter of spore formation serves as one of the points for distinguishing species. some species do not form spores, at least under any of the conditions in which they have been studied. others form them readily in almost any condition, and others again only under special conditions which are adverse to their life. the method of spore formation is always uniform for any single species. whatever be the method of the formation of the spore, its purpose in the life of the bacterium is always the same. it serves as a means of keeping the species alive under conditions of adversity. its power of resisting heat or drying enables it to live where the ordinary active forms would be speedily killed. some of these spores are capable of resisting a heat of 180 degrees c. (360 degrees f.) for a short time, and boiling water they can resist for a long time. such spores when subsequently placed under favourable conditions will germinate and start bacterial activity anew. motion. some species of bacteria have the power of active motion, and may be seen darting rapidly to and fro in the liquid in which they are growing. this motion is produced by flagella which protrude from the body. these flagella (fig. 15) arise from a membrane surrounding the bacterium, but have an intimate connection with the protoplasmic content. their distribution is different in different species of bacteria. some species have a single flagellum at one end (fig. 15 a). others have one at each end (fig. 15 b). others, again, have, at least just before dividing, a bunch at one or both ends (fig. 15 c and d), while others, again, have many flagella distributed all over the body in dense profusion (fig. 15 e). these flagella keep up a lashing to and fro in the liquid, and the lashing serves to propel the bacteria through the liquid. internal structure. it is hardly possible to say much about the structure of the bacteria beyond the description of their external forms. with all the variations in detail mentioned, they are extraordinarily simple, and about all that can be seen is their external shape. of course, they have some internal structure, but we know very little in regard to it. some microscopists have described certain appearances which they think indicate internal structure. fig. 16 shows some of these appearances. the matter is as yet very obscure, however. the bacteria appear to have a membranous covering which sometimes is of a cellulose nature. within it is protoplasm which shows various uncertain appearances. some microscopists have thought they could find a nucleus, and have regarded bacteria as cells with inclosed nucleii (figs. 10 a and 15 f). others have regarded the whole bacterium as a nucleus without any protoplasm, while others, again, have concluded that the discerned internal structure is nothing except an appearance presented by the physical arrangement of the protoplasm. while we may believe that they have some internal structure, we must recognise that as yet microscopists have not been able to make it out. in short, the bacteria after two centuries of study appear to us about as they did at first. they must still be described as minute spheres, rods, or spirals, with no further discernible structure, sometimes motile and sometimes stationary, sometimes producing spores and sometimes not, and multiplying universally by binary fission. with all the development of the modern microscope we can hardly say more than this. our advance in knowledge of bacteria is connected almost wholly with their methods of growth and the effects they produce in nature. animals or plants? there has been in the past not a little question as to whether bacteria should be rightly classed with plants or with animals. they certainly have characters which ally them with both. their very common power of active independent motion and their common habit of living upon complex bodies for foods are animal characters, and have lent force to the suggestion that they are true animals. but their general form, their method of growth and formation of threads, and their method of spore formation are quite plantlike. their general form is very similar to a group of low green plants known as oscillaria. fig. 17 shows a group of these oscillariae, and the similarity of this to some of the thread-like bacteria is decided. the oscillariae are, however, true plants, and are of a green colour. bacteria are therefore today looked upon as a low type of plant which has no chlorophyll, [footnote: chlorophyll is the green colouring matter of plants.] but is related to oscillariae. the absence of the chlorophyll has forced them to adopt new relations to food, and compels them to feed upon complex foods instead of the simple ones, which form the food of green plants. we may have no hesitation, then, in calling them plants. it is interesting to notice that with this idea their place in the organic world is reduced to a small one systematically. they do not form a class by themselves, but are simply a subclass, or even a family, and a family closely related to several other common plants. but the absence of chlorophyll and the resulting peculiar life has brought about a curious anomaly. whereas their closest allies are known only to botanists, and are of no interest outside of their systematic relations, the bacteria are familiar to every one, and are demanding the life attention of hundreds of investigators. it is their absence of chlorophyll and their consequent dependence upon complex foods which has produced this anomaly. classification of bacteria. while it has generally been recognised that bacteria are plants, any further classification has proved a matter of great difficulty, and bacteriologists find it extremely difficult to devise means of distinguishing species. their extreme simplicity makes it no easy matter to find points by which any species can be recognised. but in spite of their similarity, there is no doubt that many different species exist. bacteria which appear to be almost identical, under the microscope prove to have entirely different properties, and must therefore be regarded as distinct species. but how to distinguish them has been a puzzle. microscopists have come to look upon the differences in shape, multiplication, and formation of spores as furnishing data sufficient to enable them to divide the bacteria into genera. the genus bacillus, for instance, is the name given to all rod-shaped bacteria which form endogenous spores, etc. but to distinguish smaller subdivisions it has been found necessary to fall back upon other characters, such as the shape of the colony produced in solid gelatine, the power to produce disease, or to oxidize nitrites, etc. thus at present the different species are distinguished rather by their physiological than their morphological characters. this is an unsatisfactory basis of classification, and has produced much confusion in the attempts to classify bacteria. the problem of determining the species of bacteria is to-day a very difficult one, and with our best methods is still unsatisfactorily solved. a few species of marked character are well known, and their powers of action so well understood that they can be readily recognised; but of the great host of bacteria studied, the large majority have been so slightly experimented upon that their characters are not known, and it is impossible, therefore, to distinguish many of them apart. we find that each bacteriologist working in any special line commonly keeps a list of the bacteria which he finds, with such data in regard to them as he has collected. such a list is of value to him, but commonly of little value to other bacteriologists from the insufficiency of the data. thus it happens that a large part of the different species of bacteria described in literature today have been found and studied by one investigator alone. by him they have been described and perhaps named. quite likely the same species may have been found by two or three other bacteriologists, but owing to the difficulty of comparing results and the incompleteness of the descriptions the identity of the species is not discovered, and they are probably described again under different names. the same process may be repeated over and over again, until the same species of bacterium will come to be known by several different names, as it has been studied by different observers. variation of bacteria. this matter is made even more confusing by the fact that any species of bacterium may show more or less variation. at one time in the history of bacteriology, a period lasting for many years, it was the prevalent opinion that there was no constancy among bacteria, but that the same species might assume almost any of the various forms and shapes, and possess various properties. bacteria were regarded by some as stages in the life history of higher plants. this question as to whether bacteria remain constant in character for any considerable length of time has ever been a prominent one with bacteriologists, and even to-day we hardly know what the final answer will be. it has been demonstrated beyond peradventure that some species may change their physiological characters. disease bacteria, for instance, under certain conditions lose their powers of developing disease. species which sour milk, or others which turn gelatine green, may lose their characters. now, since it is upon just such physiological characters as these that we must depend in order to separate different species of bacteria from each other, it will be seen that great confusion and uncertainty will result in our attempts to define species. further, it has been proved that there is sometimes more or less of a metamorphosis in the life history of certain species of bacteria. the same species may form a short rod, or a long thread, or break up into spherical spores, and thus either a short rod, or a thread, or a spherical form may belong to the same species. other species may be motile at one time and stationary at another, while at a third period it is a simple mass of spherical spores. a spherical form, when it lengthens before dividing, appears as a short rod, and a short rod form after dividing may be so short as to appear like a spherical organism. with all these reasons for confusion, it is not to be wondered at that no satisfactory classification of bacteria has been reached, or that different bacteriologists do not agree as to what constitutes a species, or whether two forms are identical or not. but with all the confusion there is slowly being obtained something like system. in spite of the fact that species may vary and show different properties under different conditions, the fundamental constancy of species is everywhere recognised to-day as a fact. the members of the same species may show different properties under different conditions, but it is believed that under identical conditions the properties will be constant. it is no more possible to convert one species into another than it is among the higher orders of plants. it is believed that bacteria do form a group of plants by themselves, and are not to be regarded as stages in the history of higher plants. it is believed that, together with a considerable amount of variability and an occasional somewhat long life history with successive stages, there is also an essential constancy. a systematic classification has been made which is becoming more or less satisfactory. we are constantly learning more and more of the characters, so that they can be recognised in different places by different observers. it is the conviction of all who work with bacteria that, in spite of the difficulties, it is only a matter of time when we shall have a classification and description of bacteria so complete as to characterize the different species accurately. even with our present incomplete knowledge of what characterizes a species, it is necessary to use some names. bacteria are commonly given a generic name based upon their microscopic appearance. there are only a few of these names. micrococcus, streptococcus, staphylococcus, sarcina, bacterium, bacillus, spirillum, are all the names in common use applying to the ordinary bacteria. there are a few others less commonly used. to this generic name a specific name is commonly added, based upon some physiological character. for example, bacillus typhosus is the name given to the bacillus which causes typhoid fever. such names are of great use when the species is a common and well-known one, but of doubtful value for less-known species it frequently happens that a bacteriologist makes a study of the bacteria found in a certain locality, and obtains thus a long list of species hitherto unknown. in these cases it is common simply to number these species rather than name them. this method is frequently advisable, since the bacteriologist can seldom hunt up all bacteriological literature with sufficient accuracy to determine whether some other bacteriologist may not have found the same species in an entirely different locality. one bacteriologist, for example, finds some seventy different species of bacteria in different cheeses. he studies them enough for his own purposes, but not sufficiently to determine whether some other person may not have found the same species perhaps in milk or water. he therefore simply numbers them--a method which conveys no suggestion as to whether they may be new species or not. this method avoids the giving of separate names to the same species found by different observers, and it is hoped that gradually accumulating knowledge will in time group together the forms which are really identical, but which have been described by different observers. where bacteria are found. there are no other plants or animals so universally found in nature as the bacteria. it is this universal presence, together with their great powers of multiplication, which renders them of so much importance in nature. they exist almost everywhere on the surface of the earth. they are in the soil, especially at its surface. they do not extend to very great depths of soil, however, few existing below four feet of soil. at the surface they are very abundant, especially if the soil is moist and full of organic material. the number may range from a few hundred to one hundred millions per gramme. [footnote: one gramme is fifteen grains.] the soil bacteria vary also in species, some two-score different species having been described as common in soil. they are in all bodies of water, both at the surface and below it. they are found at considerable depths in the ocean. all bodies of fresh water contain them, and all sediments in such bodies of water are filled with bacteria. they are in streams of running water in even greater quantity than in standing water. this is simply because running streams are being constantly supplied with water which has been washing the surface of the country and thus carrying off all surface accumulations. lakes or reservoirs, however, by standing quiet allow the bacteria to settle to the bottom, and the water thus gets somewhat purified. they are in the air, especially in regions of habitation. their numbers are greatest near the surface of the ground, and decrease in the upper strata of air. anything which tends to raise dust increases the number of bacteria in the air greatly, and the dust and emanations from the clothes of people crowded in a close room fill the air with bacteria in very great numbers. they are found in excessive abundance in every bit of decaying matter wherever it may be. manure heaps, dead bodies of animals, decaying trees, filth and slime and muck everywhere are filled with them, for it is in such places that they find their best nourishment. the bodies of animals contain them in the mouth, stomach, and intestine in great numbers, and this is, of course, equally true of man. on the surface of the body they cling in great quantity; attached to the clothes, under the finger nails, among the hairs, in every possible crevice or hiding place in the skin, and in all secretions. they do not, however, occur in the tissues of a healthy individual, either in the blood, muscle, gland, or any other organ. secretions, such as milk, urine, etc., always contain them, however, since the bacteria do exist in the ducts of the glands which conduct the secretions to the exterior, and thus, while the bacteria are never in the healthy gland itself, they always succeed in contaminating the secretion as it passes to the exterior. not only higher animals, but the lower animals also have their bodies more or less covered with bacteria. flies have them on their feet, bees among their hairs, etc. in short, wherever on the face of nature there is a lodging place for dust there will be found bacteria. in most of these localities they are dormant, or at least growing only a little. the bacteria clinging to the dry hair can grow but little, if at all, and those in pure water multiply very little. when dried as dust they are entirely dormant. but each individual bacterium or spore has the potential power of multiplication already noticed, and as soon as it by accident falls upon a place where there is food and moisture it will begin to multiply. everywhere in nature, then, exists this group of organisms with its almost inconceivable power of multiplication, but a power held in check by lack of food. furnish them with food and their potential powers become actual. such food is provided by the dead bodies of animals or plants, or by animal secretions, or from various other sources. the bacteria which are fortunate enough to get furnished with such food material continue to feed upon it until the food supply is exhausted or their growth is checked in some other way. they may be regarded, therefore, as a constant and universal power usually held in check. with their universal presence and their powers of producing chemical changes in food material, they are ever ready to produce changes in the face of nature, and to these changes we will now turn. chapter ii. miscellaneous use of bacteria in the arts. the foods upon which bacteria live are in endless variety, almost every product of animal or vegetable life serving to supply their needs. some species appear to require somewhat definite kinds of food, and have therefore rather narrow conditions of life, but the majority may live upon a great variety of organic compounds. as they consume the material which serves them as food they produce chemical changes therein. these changes are largely of a nature that the chemist knows as decomposition changes. by this is meant that the bacteria, seizing hold of ingredients which constitute their food, break them to pieces chemically. the molecule of the original food matter is split into simpler molecules, and the food is thus changed in its chemical nature. as a result, the compounds which appear in the decomposing solution are commonly simpler than the original food molecules. such products are in general called decomposition products, or sometimes cleavage products. sometimes, however, the bacteria have, in addition to their power of pulling their food to pieces, a further power of building other compounds out of the fragments, thus building up as well as pulling down. but, however they do it, bacteria when growing in any food material have the power of giving rise to numerous products which did not exist in the food mass before. because of their extraordinary powers of reproduction they are capable of producing these changes very rapidly and can give rise in a short time to large amounts of the peculiar products of their growth. it is to these powers of producing chemical changes in their food that bacteria owe all their importance in the world. their power of chemically destroying the food products is in itself of no little importance, but the products which arise as the result of this series of chemical changes are of an importance in the world which we are only just beginning to appreciate. in our attempt to outline the agency which bacteria play in our industries and in natural processes as well, we shall notice that they are sometimes of value simply for their power of producing decomposition; but their greatest value lies in the fact that they are important agents because of the products of their life. we may notice, in the first place, that in the arts there are several industries which may properly be classed together as maceration industries, all of which are based upon the decomposition powers of bacteria. hardly any animal or vegetable substance is able to resist their softening influence, and the artisan relies upon this power in several different directions. benefits derived from powers of decomposition. linen.--linen consists of certain woody fibres of the stem of the flax. the flax stem is not made up entirely of the valuable fibres, but largely of more brittle wood fibres, which are of no use. the valuable fibres are, however, closely united with the wood and with each other in such an intimate fashion that it is impossible to separate them by any mechanical means. the whole cellular substance of the stem is bound together by some cementing materials which hold it in a compact mass, probably a salt of calcium and pectinic acid. the art of preparing flax is a process of getting rid of the worthless wood fibres and preserving the valuable, longer, tougher, and more valuable fibres, which are then made into linen. but to separate them it is necessary first to soften the whole tissue. this is always done through the aid of bacteria. the flax stems, after proper preparation, are exposed to the action of moisture and heat, which soon develops a rapid bacterial growth. sometimes this is done by simply exposing the flax to the dew and rain and allowing it to lie thus exposed for some time. by another process the stems are completely immersed in water and allowed to remain for ten to fourteen days. by a third process the water in which the flax is immersed is heated from 75 degrees to 90 degrees f., with the addition of certain chemicals, for some fifty to sixty hours. in all cases the effect is the same. the moisture and the heat cause a growth of bacteria which proceeds with more or less rapidity according to the temperature and other conditions. a putrefactive fermentation is thus set up which softens the gummy substance holding the fibres together. the process is known as "retting," and after it is completed the fibres are easily isolated from each other. a purely mechanical process now easily separates the valuable fibres from the wood fibres. the whole process is a typical fermentation. a disagreeable odour arises from the fermenting flax, and the liquid after the fermentation is filled with products which make valuable manure. the process has not been scientifically studied until very recently. the bacillus which produces the "retting" is known now, however, and it has been shown that the "retting" is a process of decomposition of the pectin cement. no method of separating the linen fibres in the flax from the wood fibres has yet been devised which dispenses with the aid of bacteria. jute and hemp.--almost exactly the same use is made of bacterial action in the manufacture of jute und hemp. the commercial aspect of the jute industry has grown to be a large one, involving many millions of dollars. like linen, jute is a fibre of the inner bark of a plant, and is mixed in the bark with a mass of other useless fibrous material. as in the case of linen, a fermentation by bacteria is depended upon as a means of softening the material so that the fibres can be disassociated. the process is called "retting," as in the linen manufacture. the details of the process are somewhat different. the jute is commonly fermented in tanks of stagnant water, although sometimes it is allowed to soak in river water for a sufficient length of time to produce the softening. after the fermentation is thus started the jute fibre is separated from the wood, and is of a sufficient flexibility and toughness to be woven into sacking, carpets, curtains, table covers, and other coarse cloth. practically the same method is used in separating the tough fibres of the hemp. the hemp plant contains some long flexible fibres with others of no value, and bacterial fermentation is relied upon to soften the tissues so that they may be separated. cocoanut fibre, a somewhat similar material is obtained from the husk of the cocoanut by the same means. the unripened husk is allowed to steep and ferment in water for a long time, six months or a year being required. by this time the husk has become so softened that it can be beaten until the fibres separate and can be removed. they are subsequently made into a number of coarse articles, especially valuable for their toughness. door mats, brushes, ships' fenders, etc., are illustrations of the sort of articles made from them. in each of these processes the fermentation must have a tendency to soften the desired fibres as well as the connecting substance. putrefaction attacks all kinds of vegetable tissue, and if this "retting" continues too long the desired fibre is decidedly injured by the softening effect of the fermentation. it is quite probable that, even as commonly carried on, the fermentation has some slight injurious effect upon the fibre, and that if some purely mechanical means could be devised for separating the fibre from the wood it would produce a better material. but such mechanical means has not been devised, and at present a putrefactive fermentation appears to be the only practical method of separating the fibres. sponges.--a somewhat similar use is made of bacteria in the commercial preparation of sponges. the sponge of commerce is simply the fibrous skeleton of a marine animal. when it is alive this skeleton is completely filled with the softer parts of the animal, and to fit the sponge for use this softer organic material must be got rid of. it is easily accomplished by rotting. the fresh sponges are allowed to stand in the warm sun and very rapidly decay. bacteria make their way into the sponge and thoroughly decompose the soft tissues. after a short putrefaction of this sort the softened organic matter can be easily washed out of the skeleton and leave the clean fibre ready for market. leather preparation.--the tanning of leather is a purely chemical process, and in some processes the whole operation of preparing the leather is a chemical one. in others, however, especially in america, bacteria are brought into action at one stage. the dried hide which comes to the tannery must first have the hair removed together with the outer skin. the hide for this purpose must be moistened and softened. in some tanneries this is done by steeping it in chemicals. in others, however, it is put into water and slightly heated until fermentation arises. the fermentation softens it so that the outer skin can be easily removed with a knife, and the removal of hair is accomplished at the same time. bacterial putrefaction in the tannery is thus an assistance in preparing the skin for the tanning proper. even in the subsequent tanning a bacterial fermentation appears to play a part, but little is yet known in regard to it. maceration of skeletons.--the making of skeletons for museums and anatomical instruction in general is no very great industry, and yet it is one of importance. in the making of skeletons the process of maceration is commonly used as an aid. the maceration consists simply in allowing the skeleton to soak in water for a day or two after cleaning away the bulk of the muscles. the putrefaction that arises softens the connective tissues so much that the bones may be readily cleaned of flesh. citric acid.--bacterial fermentation is employed also in the ordinary preparation of citric acid. the acid is made chiefly from the juice of the lemon. the juice is pressed from the fruit and then allowed to ferment. the fermentation aids in separating a mucilaginous mass and making it thus possible to obtain the citric acid in a purer condition. the action is probably similar to the maceration processes described above, although it has not as yet been studied by bacteriologists. benefits derived from the products of bacterial life. while bacteria thus play a part in our industries simply from their power of producing decomposition, it is primarily because of the products of their action that they are of value. wherever bacteria seize hold of organic matter and feed upon it, there are certain to be developed new chemical compounds, resulting largely from decomposition, but partly also from constructive processes. these new compounds are of great variety. different species of bacteria do not by any means produce the same compounds even when growing in and decomposing the same food material. moreover, the same species of bacteria may give rise to different products when growing in different food materials. some of the compounds produced by such processes are poisonous, others are harmless. some are gaseous, others are liquids. some have peculiar odours, as may be recognised from the smell arising from a bit of decaying meat. others have peculiar tastes, as may be realized in the gamy taste of meat which is in the incipient stages of putrefaction. by purely empirical means mankind has learned methods of encouraging the development of some of these products, and is to-day making practical use of this power, possessed by bacteria, of furnishing desired chemical compounds. industries involving the investment of hundreds of millions of dollars are founded upon the products of bacterial life, and they have a far more important relation to our everyday life than is commonly imagined. in many cases the artisan who is dependent upon this action of microscopic life is unaware of the fact. his processes are those which experience has taught produce desired results, but, nevertheless, his dependence upon bacteria is none the less fundamental. bacteria in the fermentative industries. we may notice, first, several miscellaneous instances of the application of bacteria to various fermentative industries where their aid is of more or less value to man. in some of the examples to be mentioned the influence of bacteria is profound and fundamental, while in others it is only incidental. the fermentative industries of civilization are gigantic in extent, and have come to be an important factor in modern civilized life. the large part of the fermentation is based upon the growth of a class of microscopic plants which we call yeasts. bacteria and yeasts are both microscopic plants, and perhaps somewhat closely related to each other. the botanist finds a difference between them, based upon their method of multiplication, and therefore places them in different classes (fig. 2, page 19). in their general power of producing chemical changes in their food products, yeasts agree closely with bacteria, though the kinds of chemical changes are different. the whole of the great fermentative industries, in which are invested hundreds of millions of dollars, is based upon chemical decompositions produced by microscopic plants. in the great part of commercial fermentations alcohol is the product desired, and alcohol, though it is sometimes produced by bacteria, is in commercial quantities produced only by yeasts. hence it is that, although the fermentations produced by bacteria are more common in nature than those produced by yeasts and give rise to a much larger number of decomposition products, still their commercial aspect is decidedly less important than that of yeasts. nevertheless, bacteria are not without their importance in the ordinary fermentative processes. although they are of no importance as aids in the common fermentative processes, they are not infrequently the cause of much trouble. in the fermentation of malt to produce beer, or grape juice to produce wine, it is the desire of the brewer and vintner to have this fermentation produced by pure yeasts, unmixed with bacteria. if the yeast is pure the fermentation is uniform and successful. but the brewer and vintner have long known that the fermentation is frequently interfered with by irregularities. the troubles which arise have long been known, but the bacteriologist has finally discovered their cause, and in general their remedy. the cause of the chief troubles which arise in the fermentation is the presence of contaminating bacteria among the yeasts. these bacteria have been more or less carefully studied by bacteriologists, and their effect upon the beer or wine determined. some of them produce acid and render the products sour; others make them bitter; others, again, produce a slimy material which makes the wine or beer "ropy." something like a score of bacteria species have been found liable to occur in the fermenting material and destroy the value of the product of both the wine maker and the beer brewer. the species of bacteria which infect and injure wine are different from those which infect and injure beer. they are ever present as possibilities in the great alcoholic fermentations. they are dangers which must be guarded against. in former years the troubles from these sources were much greater than they are at present. since it has been demonstrated that the different imperfections in the fermentative process are due to bacterial impurities, commonly in the yeasts which are used to produce the fermentation, methods of avoiding them are readily devised. to-day the vintner has ready command of processes for avoiding the troubles which arise from bacteria, and the brewer is always provided with a microscope to show him the presence or absence of the contaminating bacteria. while, then, the alcoholic fermentations are not dependent upon bacteria, the proper management of these fermentations requires a knowledge of their habits and characters. there are certain other fermentative processes of more or less importance in their commercial aspects, which are directly dependent upon bacterial action, some of them we should unhesitatingly look upon as fermentations, while others would hardly be thought of as belonging to the fermentation industries. vinegar. the commercial importance of the manufacture of vinegar, though large, does not, of course, compare in extent with that of the alcoholic fermentations. vinegar is a weak solution of acetic acid, together with various other ingredients which have come from the materials furnishing the acid. in the manufacture of vinegar, alcohol is always used as the source of the acetic acid. the production of acetic acid from alcohol is a simple oxidation. the equation c2h6o + o2 = c2h4o2 + h2o shows the chemical change that occurs. this oxidation can be brought about by purely chemical means. while alcohol will not readily unite with oxygen under common conditions, if the alcohol is allowed to pass over a bit of platinum sponge the union readily occurs and acetic acid results. this method of acetic-acid production is possible experimentally, but is impracticable on any large scale. in the ordinary manufacture of vinegar the oxidation is a true fermentation, and brought about by the growth of bacteria. in the commercial manufacture of vinegar several different weak alcoholic solutions are used. the most common of these are fermented malt, weak wine, cider, and sometimes a weak solution of spirit to which is added sugar and malt. if these solutions are allowed to stand for a time in contact with air, they slowly turn sour by the gradual conversion of the alcohol into acetic acid. at the close of the process practically all of the alcohol has disappeared. ordinarily, however, not all of it has been converted into acetic acid, for the oxidation does not all stop at this step. as the oxidation goes on, some of the acid is oxidized into carbonic dioxide, which is, of course, dissipated at once into the air, and if the process is allowed to continue unchecked for a long enough period much of the acetic acid will be lost in this way. the oxidation of the alcohol in all commercial production of vinegar is brought about by the growth of bacteria in the liquid. when the vinegar production is going on properly, there is formed on the top of the liquid a dense felted mass known as the "mother of vinegar." this mass proves to be made of bacteria which have the power of absorbing oxygen from the air, or, at all events, of causing the alcohol to unite with oxygen. it was at first thought that a single species of bacterium was thus the cause of the oxidation of alcohol, and this was named mycoderma aceti. but further study has shown that several have the power, and that even in the commercial manufacture of vinegar several species play a part (fig. 18), although the different species are not yet very thoroughly studied. each appears to act best under different conditions. some of them act slowly, and others rapidly, the slowgrowing species appearing to produce the larger amount of acid in the end. after the amount of acetic acid reaches a certain percentage, the bacteria are unable to produce more, even though there be alcohol still left unoxidized. a percentage as high as fourteen per cent, commonly destroys all their power of growth. the production of the acid is wholly dependent upon the growth of the bacteria, and the secret of the successful vinegar manufacture is the skilful manipulation of these bacteria so as to keep them in the purest condition and to give them the best opportunity for growth. one method of vinegar manufacture which is quite rapid is carried on in a slightly different manner. a tall cylindrical chamber is filled with wood shavings, and a weak solution of alcohol is allowed to trickle slowly through it. the liquid after passing over the shavings comes out after a number of hours well charged with acetic acid. this process at first sight appears to be a purely chemical one, and reminds us of the oxidation which occurs when alcohol is allowed to pass over a platinum sponge. it has been claimed, indeed, that this is a chemical oxidation in which bacteria play no part. but this appears to be an error. it is always found necessary in this method to start the process by pouring upon the shavings some warm vinegar. unless in this way the shavings become charged with the vinegar-holding bacteria the alcohol will not undergo oxidation during its passage over them, and after the bacteria thus introduced have grown enough to coat the shavings thoroughly the acetic-acid production is much more rapid than at first. if vinegar is allowed to trickle slowly down a suspended string, so that its bacteria may distribute themselves through the string, and then alcohol be allowed to trickle over it in the same way, the oxidation takes place and acetic acid is formed. from the accumulation of such facts it has come to be recognised that all processes for the commercial manufacture of vinegar depend upon the action of bacteria. while the oxidation of alcohol into acetic acid may take place by purely chemical means, these processes are not practical on a large scale, and vinegar manufacturers everywhere depend upon bacteria as their agents in producing the oxidation. these bacteria, several species in all, feed upon the nitrogenous matter in the fermenting mass and produce the desired change in the alcohol. this vinegar fermentation is subject to certain irregularities, and the vinegar manufacturers can not always depend upon its occurring in a satisfactory manner. just as in brewing, so here, contaminating bacteria sometimes find their way into the fermenting mass and interfere with its normal course. in particular, the flavour of the vinegar is liable to suffer from such causes. as yet our vinegar manufacturers have not applied to acetic fermentation the same principle which has been so successful in brewing--namely, the use, as a starter of the fermentation, of a pure culture of the proper species of bacteria. this has been done experimentally and proves to be feasible. in practice, however, vinegar makers find that simpler methods of obtaining a starter--by means of which they procure a culture nearly though not absolutely pure--are perfectly satisfactory. it is uncertain whether really pure cultures will ever be used in this industry. lactic acid. the manufacture of lactic acid is an industry of less extent than that of acetic acid, and yet it is one which has some considerable commercial importance. lactic acid is used in no large quantity, although it is of some value as a medicine and in the arts. for its production we are wholly dependent upon bacteria. it is this acid which, as we shall see, is produced in the ordinary souring of milk, and a large number of species of bacteria are capable of producing the acid from milk sugar. any sample of sour milk may therefore always be depended upon to contain plenty of lactic organisms. in its manufacture for commercial purposes milk is sometimes used as a source, but more commonly other substances. sometimes a mixture of cane sugar and tartaric acid is used. to start the fermentation the mixture is inoculated with a mass of sour milk or decaying cheese, or both, such a mixture always containing lactic organisms. to be sure, it also contains many other bacteria which have different effects, but the acid producers are always so abundant and grow so vigorously that the lactic fermentation occurs in spite of all other bacteria. here also there is a possibility of an improvement in the process by the use of pure cultures of lactic organisms. up to the present, however, there has been no application of such methods. the commercial aspects of the industry are not upon a sufficiently large scale to call for much in this direction. at the present time the only method we have for the manufacture of lactic acid is dependent upon bacteria. chemical processes for its manufacture are known, but not employed commercially. there are several different kinds of lactic acid. they differ from each other in the relations of the atoms within their molecule, and in their relation to polarized light, some forms rotating the plane of polarized light to the right, others to the left, while others are inactive in this respect. all the types are produced by fermentation processes, different species of bacteria having powers of producing the different types. butyric acid. butyric acid is another acid for which we are chiefly dependent upon bacteria. this acid is of no very great importance, and its manufacture can hardly be called an industry; still it is to a certain extent made, and is an article of commerce. it is an acid that can be manufactured by chemical means, but, as in the case of the last two acids, its commercial manufacture is based upon bacterial action. quite a number of species of bacteria can produce butyric acid, and they produce it from a variety of different sources. butyric acid is a common ingredient in old milk and in butter, and its formation by bacteria was historically one of the first bacterial fermentations to be clearly understood. it can be produced also in various sugar and starchy solutions. glycerine may also undergo a butyric fermentation. the presence of this acid is occasionally troublesome, since it is one of the factors in the rancidity of butter and other similar materials. indigo preparation. the preparation of indigo from the indigo plant is a fermentative process brought about by a specific bacterium. the leaves of the plant are immersed in water in a large vat, and a rapid fermentation arises. as a result of the fermentation the part of the plant which is the basis of the indigo is separated from the leaves and dissolved in the water; and as a second feature of the fermentation the soluble material is changed in its chemical nature into indigo proper. as this change occurs the characteristic blue colour is developed, and the material is rendered insoluble in water. it therefore makes its appearance as a blue mass separated from the water, and is then removed as indigo. of the nature of the process we as yet know very little. that it is a fermentation is certain, and it has been proved that it is produced by a definite species of bacterium which occurs on the indigo leaves. if the sterilized leaves are placed in sterile water no fermentation occurs and no indigo is formed. if, however, some of the specific bacteria are added to the mass the fermentation soon begins and the blue colour of the indigo makes its appearance. it is plain, therefore, that indigo is a product of bacterial fermentation, and commonly due to a single definite species of bacterium. of the details of the formation, however, we as yet know little, and no practical application of the facts have yet been made. bacteria in tobacco curing. a fermentative process of quite a different nature, but of immense commercial value, is found in the preparation of tobacco. the process by which tobacco is prepared is a long and somewhat complicated one, consisting of a number of different stages. the tobacco, after being first dried in a careful manner, is subsequently allowed to absorb moisture from the atmosphere, and is then placed in large heaps to undergo a further change. this process appears to be a fermentation, for the temperature of the mass rises rapidly, and every indication of a fermentative action is seen. the tobacco in these heaps is changed occasionally, the heap being thrown down and built up again in such a way that the portion which was first at the bottom comes to the top, and in this way all parts of the heap may become equally affected by the process. after this process the tobacco is sent to the different manufacturers, who finish the process of curing. the further treatment it receives varies widely according to the desired product, whether for smoking or for snuff, etc. in all cases, however, fermentations play a prominent part. sometimes the leaves are directly inoculated with fermenting material. in the preparation of snuff the details of the process are more complicated than in the preparation of smoking tobacco. the tobacco, after being ground and mixed with certain ingredients, is allowed to undergo a fermentation which lasts for weeks, and indeed for months. in the different methods of preparing snuff the fermentations take place in different ways, and sometimes the tobacco is subjected to two or three different fermentative actions. the result of the whole is the slow preparation of the commercial product. it is during the final fermentative processes that the peculiar colour and flavour of the snuff are developed, and it is during the fermentation of the leaves of the smoking tobacco--either the original fermentation or the subsequent ones-that the special flavours and aromas of tobacco are produced. it can not be claimed for a moment that these changes by which the tobacco is cured and finally brought to a marketable condition are due wholly to bacteria. there is no question that chemical and physical phenomena play an important part in them. nevertheless, from the moment when the tobacco is cut in the fields until the time it is ready for market the curing is very intimately associated with bacteria and fermentative organisms in general. some of these processes are wholly brought about by bacterial life; in others the micro-organisms aid the process, though they perhaps can not be regarded as the sole agents. at the outset the tobacco producer has to contend with a number of micro-organisms which may produce diseases in his tobacco. during the drying process, if the temperature or the amount of moisture or the access of air is not kept in a proper condition, various troubles arise and various diseases make their appearance, which either injure or ruin the value of the product. these appear to be produced by micro-organisms of different sorts. during the fermentation which follows the drying the producer has to contend with micro-organisms that are troublesome to him; for unless the phenomena are properly regulated the fermentation that occurs produces effects upon the tobacco which ruin its character. from the time the tobacco is cut until the final stage in the curing the persons engaged in preparing it for market must be on a constant watch to prevent the growth within it of undesirable organisms. the preparation of tobacco is for this reason a delicate operation, and one that will be very likely to fail unless the greatest care is taken. in the several fermentative processes which occur in the preparation there is no question that micro-organisms aid the tobacco producer and manufacturer. bacteria produce the first fermentation that follows the drying, and it is these organisms too, in large measure, that give rise to all the subsequent fermentations, although seemingly in some cases purely chemical processes materially aid. now the special quality of the tobacco is in part dependent upon the peculiar type of fermentation which occurs in one or another of these fermenting actions. it is the fermentation that gives rise to the peculiar flavour and to the aroma of the different grades of tobacco. inasmuch as the various flavours which characterize tobacco of different grades are developed, at least to a large extent, during the fermentation processes, it is a natural supposition that the different qualities of the tobacco, so far as concerns flavour, are due to the different types of fermentation. the number of species of bacteria which are found upon the tobacco leaves in the various stages of its preparation is quite large, and from what we have already learned it is inevitable that the different kinds of bacteria will produce different results in the fermenting process. it would seem natural, therefore, to assume that the different flavours of different grades may not unlikely be due to the fact that the tobacco in the different cases has been fermented under the influence of different kinds of bacteria. nor is this simply a matter of inference. to a certain extent experimental evidence has borne out the conclusion, and has given at least a slight indication of practical results in the future. acting upon the suggestion that the difference between the high grades of tobacco and the poorer grades is due to the character of the bacteria that produce the fermentation, certain bacteriologists have attempted to obtain from a high quality of tobacco the species of bacteria which are infesting it. these bacteria have then been cultivated by bacteriological methods and used in experiments for the fermentation of tobacco. if it is true that the flavour of high grade tobacco is in large measure, or even in part, due to the action of the peculiar microbes from the soil where it grows, it ought to be possible to produce similar flavours in the leaves of tobacco grown in other localities, if the fermentation of the leaves is carried on by means of the pure cultures of bacteria obtained from the high grade tobacco. not very much has been done or is known in this connection as yet. two bacteriologists have experimented independently in fermenting tobacco leaves by the action of pure cultures of bacteria obtained from such sources. each of them reports successful experiments. each claims that they have been able to improve the quality of tobacco by inoculating the leaves with a pure culture of bacteria obtained from tobacco having high quality in flavour. in addition to this, several other bacteriologists have carried on experiments sufficient to indicate that the flavours of the tobacco and the character of the ripening may be decidedly changed by the use of different species of micro-organisms in the fermentations that go on during the curing processes. in regard to the whole matter, however, we must recognise that as yet we have very little knowledge. the subject has been under investigation for only a short time; and, while considerable information has been derived, this information is not thoroughly understood, and our knowledge in regard to the matter is as yet in rather a chaotic condition. it seems certain, however, that the quality of tobacco is in large measure dependent upon the character of the fermentations that occur at different stages of the curing. it seems certain also that these fermentations are wholly or chiefly produced by microorganisms, and that the character of the fermentation is in large measure dependent upon the species of micro-organisms that produce it. if these are facts, it would seem not improbable that a further study may produce practical results for this great industry. the study of yeasts and the methods of keeping yeast from contaminations has revolutionised the brewing industry. perhaps in this other fermentative industry, which is of such great commercial extent, the use of pure cultures of bacteria may in the future produce as great revolutions in methods as it has in the industry of the alcoholic fermentation. it must not, however, be inferred that the differences in grades of tobacco grown in different parts of the world are due solely to variations in the curing processes and to the types of fermentation. there are differences in the texture of the leaves, differences in the chemical composition of the tobaccoes, which are due undoubtedly to the soils and the climatic conditions in which they grow, and these, of course, will never be affected by changing the character of the ferment active processes. it is, however, probable that in so far as the flavours that distinguish the high and low grades of tobacco are due to the character of the fermentative processes, they may be in the future, at least to a large extent, controlled by the use of pure cultures in curing processes. seemingly, then, there is as great a future in the development of this fermentative industry as there has been in the past in the development of the fermentative industry associated with brewing and vinting. opium. opium for smoking purposes is commonly allowed to undergo a curing process which lasts several months. this appears to be somewhat similar to the curing of tobacco. apparently it is a fermentation due to the growth of microorganisms. the organisms in question are not, however, bacteria in this case, but a species of allied fungus. the plant is a mould, and it is claimed that inoculation of the opium with cultures of this mould hastens the curing. troublesome fermentations. before leaving this branch of the subject it is necessary to notice some of the troublesome fermentations which are ever interfering with our industries, requiring special methods, or, indeed, sometimes developing special industries to meet them. as agents of decomposition, bacteria will of course be a trouble whenever they get into material which it is desired to preserve. since they are abundant everywhere, it is necessary to count upon their attacking with certainty any fermentable substance which is exposed to air and water. hence they are frequently the cause of much trouble. in the fermentative industries they occasionally cause an improper sort of fermentation to occur unless care is taken to prevent undesired species of bacteria from being present. in vinegar making, improper species of bacteria obtaining access to the solution give rise to undesirable flavours, greatly injuring the product. in tobacco curing it is very common for the wrong species of bacteria to gain access to the tobacco at some stage of the curing and by their growth give rise to various troubles. it is the ubiquitous presence of bacteria which makes it impossible to preserve fruits, meats, or vegetables for any length of time without special methods. this fact in itself has caused the development of one of our most important industries. canning meats or fruits consists in nothing more than bringing them into a condition in which they will be preserved from attack of these micro-organisms. the method is extremely simple in theory. it is nothing more than heating the material to be preserved to a high temperature and then sealing it hermetically while it is still hot. the heat kills all the bacteria which may chance to be lodged in it, and the hermetical sealing prevents other bacteria from obtaining access. inasmuch as all organic decomposition is produced by bacterial growth, such sterilized and sealed material will be preserved indefinitely when the operation is performed carefully enough. the methods of accomplishing this with sufficient care are somewhat varied in different industries, but they are all fundamentally the same. it is an interesting fact that this method of preserving meats was devised in the last century, before the relation of micro-organisms to fermentation and putrefaction was really suspected. for a long time it had been in practical use while scientists were still disputing whether putrefaction could be avoided by preventing the access of bacteria. the industry has, however, developed wonderfully within the last few years, since the principles underlying it have been understood. this understanding has led to better methods of destroying bacterial life and to proper sealing, and these have of course led to greater success in the preservation, until to-day the canning industries are among those which involve capital reckoned in the millions. occasionally bacteria are of some value in food products. the gamy flavour of meats is nothing more than incipient decomposition. sauer kraut is a food mass intentionally allowed to ferment and sour. the value of bacteria in producing butter and cheese flavours is noticed elsewhere. but commonly our aim must be to prevent the growth of bacteria in foods. foods must be dried or cooked or kept on ice, or some other means adopted for preventing bacterial growth in them. it is their presence that forces us to keep our ice box, thus founding the ice business, as well as that of the manufacture of refrigerators. it is their presence, again, that forces us to smoke hams, to salt mackerel, to dry fish or other meats, to keep pork in brine, and to introduce numerous other details in the methods of food preparation and preservation. chapter iii. relation of bacteria to the dairy industry. dairying is one of the most primitive of our industries. from the very earliest period, ever since man began to keep domestic cattle, he has been familiar with dairying. during these many centuries certain methods of procedure have been developed which produce desired results. these methods, however, have been devised simply from the accumulation of experience, with very little knowledge as to the reasons underlying them. the methods of past centuries are, however, ceasing to be satisfactory. the advance of our civilization during the last half century has seen a marked expansion in the extent of the dairy industry. with this expansion has appeared the necessity for new methods, and dairymen have for years been looking for them. the last few years have been teaching us that the new methods are to be found along the line of the application of the discoveries of modern bacteriology. we have been learning that the dairyman is more closely related to bacteria and their activities than almost any other class of persons. modern dairying, apart from the matter of keeping the cow, consists largely in trying to prevent bacteria from growing in milk or in stimulating their growth in cream, butter, and cheese. these chief products of the dairy will be considered separately. sources of bacteria in milk. the first fact that claims our attention is, that milk at the time it is secreted from the udder of the healthy cow contains no bacteria. although bacteria are almost ubiquitous, they are not found in the circulating fluids of healthy animals, and are not secreted by their glands. milk when first secreted by the milk gland is therefore free from bacteria. it has taken a long time to demonstrate this fact, but it has been finally satisfactorily proved. secondly, it has been demonstrated that practically all of the normal changes which occur in milk after its secretion are caused by the growth of bacteria. this, too, was long denied, and for quite a number of years after putrefactions and fermentations were generally acknowledged to be caused by the growth of microorganisms, the changes which occurred in milk were excepted from the rule. the uniformity with which milk will sour, and the difficulty, or seeming impossibility, of preventing this change, led to the belief that the souring of milk was a normal change characteristic of milk, just as clotting is characteristic of blood. this was, however, eventually disproved, and it was finally demonstrated that, beyond a few physical changes connected with evaporation and a slight oxidation of the fat, milk, if kept free from bacteria, will undergo no change. if bacteria are not present, it will remain sweet indefinitely. but it is impossible to draw milk from the cow in such a manner that it will be free from bacteria except by the use of precautions absolutely impracticable in ordinary dairying. as milk is commonly drawn, it is sure to be contaminated by bacteria, and by the time it has entered the milk pail it contains frequently as many as half a million, or even a million, bacteria in every cubic inch of the milk. this seems almost incredible, but it has been demonstrated in many cases and is beyond question. since these bacteria are not in the secreted milk, they must come from some external sources, and these sources are the following: the first in importance is the cow herself; for while her milk when secreted is sterile, and while there are no bacteria in her blood, nevertheless the cow is the most prolific source of bacterial contamination. in the first place, the milk ducts are full of them. after each milking a little milk is always left in the duct, and this furnishes an ideal place for bacteria to grow. some bacteria from the air or elsewhere are sure to get into these ducts after the milking, and they begin at once to multiply rapidly. by the next milking they become very abundant in the ducts, and the first milk drawn washes most of them at once into the milk pail, where they can continue their growth in the milk. again, the exterior of the cow's body contains them in abundance. every hair, every particle of dirt, every bit of dried manure, is a lurking place for millions of bacteria. the hind quarters of a cow are commonly in a condition of much filth, for the farmer rarely grooms his cow, and during the milking, by her movements, by the switching of her tail, and by the rubbing she gets from the milker, no inconsiderable amount of this dirt and filth is brushed off and falls into the milk pail the farmer understands this source of dirt and usually feels it necessary to strain the milk after the milking. but the straining it receives through a coarse cloth, while it will remove the coarser particles of dirt, has no effect upon the bacteria, for these pass through any strainer unimpeded. again, the milk vessels themselves contain bacteria, for they are never washed absolutely clean. after the most thorough washing which the milk pail receives from the kitchen, there will always be left many bacteria clinging in the cracks of the tin or in the wood, ready to begin to grow as soon as the milk once more fills the pail the milker himself contributes to the supply, for he goes to the milking with unclean hands, unclean clothes, and not a few bacteria get from him to his milk pail. lastly, we find the air of the milking stall furnishing its quota of milk bacteria. this source of bacteria is, how ever, not so great as was formerly believed. that the air may contain many bacteria in its dust is certain, and doubtless these fall in some quantity into the milk, especially if the cattle are allowed to feed upon dusty hay before and during the milking. but unless the air is thus full of dust this source of bacteria is not very great, and compared with the bacteria from the other sources the air bacteria are unimportant. the milk thus gets filled with bacteria, and since it furnishes an excellent food these bacteria begin at once to grow. the milk when drawn is warm and at a temperature which especially stimulates bacterial growth. they multiply with great rapidity, and in the course of a few hours increase perhaps a thousandfold. the numbers which may be found after twenty-four hours are sometimes inconceivable; market milk may contain as many as five hundred millions per cubic inch; and while this is a decidedly extreme number, milk that is a day old will almost always contain many millions in each cubic inch, the number depending upon the age of the milk and its temperature. during this growth the bacteria have, of course, not been without their effect. recognising as we do that bacteria are agents for chemical change, we are prepared to see the milk undergoing some modifications during this rapid multiplication of bacteria. the changes which these bacteria produce in the milk and its products are numerous, and decidedly affect its value. they are both advantageous and disadvantageous to the dairyman. they are nuisances so far as concerns the milk producer, but allies of the butter and cheese maker. the effect of bacteria on milk. the first and most universal change effected in milk is its souring. so universal is this phenomenon that it is generally regarded as an inevitable change which can not be avoided, and, as already pointed out, has in the past been regarded as a normal property of milk. to-day, however, the phenomenon is well understood. it is due to the action of certain of the milk bacteria upon the milk sugar which converts it into lactic acid, and this acid gives the sour taste and curdles the milk. after this acid is produced in small quantity its presence proves deleterious to the growth of the bacteria, and further bacterial growth is checked. after souring, therefore, the milk for some time does not ordinarily undergo any further changes. milk souring has been commonly regarded as a single phenomenon, alike in all cases. when it was first studied by bacteriologists it was thought to be due in all cases to a single species of micro-organism which was discovered to be commonly present and named bacillus acidi lactici (fig. 19). this bacterium has certainly the power of souring milk rapidly, and is found to be very common in dairies in europe. as soon as bacteriologists turned their attention more closely to the subject it was found that the spontaneous souring of milk was not always caused by the same species of bacterium. instead of finding this bacillus acidi lactici always present, they found that quite a number of different species of bacteria have the power of souring milk, and are found in different specimens of soured milk. the number of species of bacteria which have been found to sour milk has increased until something over a hundred are known to have this power. these different species do not affect the milk in the same way. all produce some acid, but they differ in the kind and the amount of acid, and especially in the other changes which are effected at the same time that the milk is soured, so that the resulting soured milk is quite variable. in spite of this variety, however, the most recent work tends to show that the majority of cases of spontaneous souring of milk are produced by bacteria which, though somewhat variable, probably constitute a single species, and are identical with the bacillus acidi lactici (fig. 19). this species, found common in the dairies of europe, according to recent investigations occurs in this country as well. we may say, then, that while there are many species of bacteria infesting the dairy which can sour the milk, there is one which is more common and more universally found than others, and this is the ordinary cause of milk souring. when we study more carefully the effect upon the milk of the different species of bacteria found in the dairy, we find that there is a great variety of changes which they produce when they are allowed to grow in milk. the dairyman experiences many troubles with his milk. it sometimes curdles without becoming acid. sometimes it becomes bitter, or acquires an unpleasant "tainted" taste, or, again, a "soapy" taste. occasionally a dairyman finds his milk becoming slimy, instead of souring and curdling in the normal fashion. at such times, after a number of hours, the milk becomes so slimy that it can be drawn into long threads. such an infection proves very troublesome, for many a time it persists in spite of all attempts made to remedy it. again, in other cases the milk will turn blue, acquiring about the time it becomes sour a beautiful sky-blue colour. or it may become red, or occasionally yellow. all of these troubles the dairyman owes to the presence in his milk of unusual species of bacteria which grow there abundantly. bacteriologists have been able to make out satisfactorily the connection of all these infections with different species of the bacteria. a large number of species have been found to curdle milk without rendering it acid, several render it bitter, and a number produce a "tainted" and one a "soapy" taste. a score or more have been found which have the power of rendering the milk slimy. two different species at least have the power of turning the milk to sky-blue colour; two or three produce red pigments (fig. 20), and one or two have been found which produce a yellow colour. in short, it has been determined beyond question that all these infections, which are more or less troublesome to dairymen, are due to the growth of unusual bacteria in the milk. these various infections are all troublesome, and indeed it may be said that, so far as concerns the milk producer and the milk consumer, bacteria are from beginning to end a source of trouble. it is the desire of the milk producer to avoid them as far as possible--a desire which is shared also by everyone who has anything to do with milk as milk. having recognised that the various troubles, which occasionally occur even in the better class of dairies, are due to bacteria, the dairyman is, at least in a measure, prepared to avoid them. the avoiding of these troubles is moderately easy as soon as dairymen recognise the source from which the infectious organisms come, and also the fact that low temperatures will in all cases remedy the evil to a large extent. with this knowledge in hand the avoidance of all these troubles is only a question of care in handling the dairy. it must be recognised that most of these troublesome bacteria come from some unusual sources of infection. by unusual sources are meant those which the exercise of care will avoid. it is true that the souring bacteria appear to be so universally distributed that they can not be avoided by any ordinary means. but all other troublesome bacteria appear to be within control. the milkman must remember that the sources of the troubles which are liable to arise in his milk are in some form of filth: either filth on the cow, or dust in the hay which is scattered through the barn, or dirt on cows' udders, or some other unusual and avoidable source. these sources, from what we have already noticed, will always furnish the milk with bacteria; but under common conditions, and when the cow is kept in conditions of ordinary cleanliness, and frequently even when not cleanly, will only furnish bacteria that produce the universal souring. recognising this, the dairyman at once learns that his remedies for the troublesome infections are cleanliness and low temperatures. if he is careful to keep his milk vessels scrupulously clean; if he will keep his cow as cleanly as he does his horse; and if he will use care in and around the barn and dairy, and then apply low temperatures to the milk, he need never be disturbed by slimy or tainted milk, or any of these other troubles; or he can remove such infections speedily should they once appear. pure sweet milk is only a question of sufficient care. but care means labour and expense. as long as we demand cheap milk, so long will we be supplied with milk procured under conditions of filth. but when we learn that cheap milk is poor milk, and when we are willing to pay a little more for it, then only may we expect the use of greater care in the handling of the milk, resulting in a purer product. bacteriology has therefore taught us that the whole question of the milk supply in our communities is one of avoiding the too rapid growth of bacteria. these organisms are uniformly a nuisance to the milkman. to avoid their evil influence have been designed all the methods of caring for the dairy and the barn, all the methods of distributing milk in ice cars. moreover, all the special devices connected with the great industry of milk supply have for their foundation the attempt to avoid, in the first place, the presence of too great a number of bacteria, and. in the second place, the growth of these bacteria. bacteria in butter making. cream ripening.--passing from milk to butter, we find a somewhat different story, inasmuch as here bacteria are direct allies to the dairyman rather than his enemies. without being aware of it, butter makers have for years been making use of bacteria in their butter making and have been profiting by the products which the bacteria have furnished them. cream, as it is obtained from milk, will always contain bacteria in large quantity, and these bacteria will grow as readily in the cream as they will in the milk. the butter maker seldom churns his cream when it is freshly obtained from the milk. there are, it is true, some places where sweet cream butter is made and is in demand, but in the majority of butter-consuming countries a different quality of butter is desired, and the cream is subjected to a process known as "ripening" or "souring" before it is churned. in ripening, the cream is simply allowed to stand in a vat for a period varying from twelve hours to two or three days, according to circumstances. during this period certain changes take place therein. the bacteria which were in the cream originally, get an opportunity to grow, and by the time the ripening is complete they become extremely numerous. as a result, the character of the cream changes just as the milk is changed under similar circumstances. it becomes somewhat soured; it becomes slightly curdled, and acquires a peculiarly pleasant taste and an aroma which was not present in the original fresh cream. after this ripening the cream is churned. it is during the ripening that the bacteria produce their effect, for after the churning they are of less importance. part of them collect in the butter, part of them are washed off from the butter in the buttermilk and the subsequent processes. most of the bacteria that are left in the butter soon die, not finding there a favourable condition for growth; some of them, however, live and grow for some time and are prominent agents in the changes by which butter becomes rancid. the butter maker is concerned with the ripening rather than with later processes. the object of the ripening of cream is to render it in a better condition for butter making. the butter maker has learned by longexperience that ripened cream churns more rapidly than sweet cream, and that he obtains a larger yield of butter therefrom. the great object of the ripening, however, is to develop in the butter the peculiar flavour and aroma which is characteristic of the highest product. sweet cream butter lacks flavour and aroma, having indeed a taste almost identically the same as cream. butter, however, that is made from ripened cream has a peculiar delicate flavour and aroma which is well known to lovers of butter, and which is developed during the ripening process. bacteriologists have been able to explain with a considerable degree of accuracy the object of this ripening. the process is really a fermentation comparable to the fermentation that takes place in a brewer's malt. the growth of bacteria during the ripening produces chemical changes of a somewhat complicated character, and concerns each of the ingredients of the milk. the lactic-acid organisms affect the milk sugar and produce lactic acid; others act upon the fat, producing slight changes therein; while others act upon the casein and the albumens of the milk. as a result, various biproducts of decomposition arise, and it is these biproducts of decomposition that make the difference between the ripened and the unripened cream. they render it sour and curdle it, and they also produce the flavours and aromas that characterize it. products of decomposition are generally looked upon as undesirable for food, and this is equally true of these products that arise in cream if the decomposition is allowed to continue long enough. if the ripening, instead of being stopped at the end of a day or two, is allowed to continue several days, the cream becomes decayed and the butter made therefrom is decidedly offensive. but under the conditions of ordinary ripening, when the process is stopped at the right moment, the decomposition products are pleasant rather than unpleasant, and the flavours and aromas which they impart to the cream and to the subsequent butter are those that are desired. it is these decomposition products that give the peculiar character to a high quality of butter, and this peculiar quality is a matter that determines the price which the butter maker can obtain for his product. but, unfortunately, the butter maker is not always able to depend upon the ripening. while commonly it progresses in a satisfactory manner, sometimes, for no reason that he can assign, the ripening does not progress normally. instead of developing the pleasant aroma and flavour of the properly ripened cream, the cream develops unpleasant tastes. it may be bitter or somewhat tainted, and just as sure as these flavours develop in the cream, so sure does the quality of the butter suffer. moreover, it has been learned by experience that some creameries are incapable of obtaining an equally good ripening of their cream. while some of them will obtain favourable results, others, with equal care, will obtain a far less favourable flavour and aroma in their butter. the reason for all this has been explained by modern bacteriology. in the milk, and consequently in the cream, there are always found many bacteria, but these are not always of the same kinds. there are scores, and probably hundreds, of species of bacteria common in and around our barns and dairies, and the bacteria that are abundant and that grow in different lots of cream will not be always the same. it makes a decided difference in the character of the ripening, and in the consequent flavours and aromas, whether one or another species of bacteria has been growing in the cream. some species are found to produce good results with desired flavours, while others, under identical conditions, produce decidedly poor results with undesired flavours. if the butter maker obtains cream which is filled with a large number of bacteria capable of producing good flavours, then the ripening of his cream will be satisfactory and his butter will be of high quality. if, however, it chances that his cream contains only the species which produce unpleasant flavours, then the character of the ripening will be decidedly inferior and the butter will be of a poorer grade. fortunately the majority of the kinds of bacteria liable to get into the cream from ordinary sources are such as produce either good effects upon the cream or do not materially influence the flavour or aroma. hence it is that the ripening of cream will commonly produce good results. bacteriologists have learned that there are some species of bacteria more or less common around our barns which produce undesirable effects upon flavour, and should these become especially abundant in the cream, then the character of the ripening and the quality of the subsequent butter will suffer. these malign species of bacteria, however, are not very common in properly kept barns and dairies. hence the process that is so widely used, of simply allowing cream to ripen under the influence of any bacteria that happen to be in it, ordinarily produces good results. but our butter makers sometimes find, at the times when the cattle change from winter to summer or from summer to winter feed, that the ripening is abnormal. the reason appears to be that the cream has become infested with an abundance of malign species. the ripening that they produce is therefore an undesirable one, and the quality of the butter is sure to suffer. so long as butter was made only in private dairies it was a matter of comparatively little importance if there was an occasional falling off in quality of this sort. when it was made a few pounds at a time, and only once or twice a week, it was not a very serious matter if a few churnings of butter did suffer in quality. but to-day the butter-making industries are becoming more and more concentrated into large creameries, and it is a matter of a good deal more importance to discover some means by which a uniformly high quality can be insured. if a creamery which makes five hundred pounds of butter per day suffers from such an injurious ripening, the quality of its butter will fall off to such an extent as to command a lower price, and the creamery suffers materially. perhaps the continuation of such a trouble for two or three weeks would make a difference between financial success and failure in the creamery. with our concentration of the buttermaking industries it is becoming thus desirable to discover some means of regulating this process more accurately. the remedy of these occasional ill effects in cream ripening has not been within the reach of the butter maker. the butter maker must make butter with the cream that is furnished him, and if that cream is already impregnated with malign species of bacteria he is helpless. it is true that much can be done to remedy these difficulties by the exercise of especial care in the barns of the patrons of the creamery. if the barns, the cows, the dairies, the milk vessels, etc., are all kept in condition of strict cleanliness, if especial care is taken particularly at the seasons of the year when trouble is likely to arise, and if some attention is paid to the kind of food which the cattle eat, as a rule the cream will not become infected with injurious bacteria. it may be taken as a demonstrated fact that these malign bacteria come from sources of filth, and the careful avoidance of all such sources of filth will in a very large measure prevent their occurrence in the cream. such measures as these have been found to be practicable in many creameries. creameries which make the highest priced and the most uniform quality of butter are those in which the greatest care is taken in the barns and dairies to insure cleanliness and in the handling of the milk and cream. with such attention a large portion of the trouble which arises in the creameries from malign bacteria may be avoided. but these methods furnish no sure remedy against evils of improper species of bacteria in cream ripening, and do not furnish any sure means of obtaining uniform flavour in butter. even under the very best conditions the flavour of the butter will vary with the season of the year. butter made in the winter is inferior to that made in the summer months; and while this is doubtless due in part to the different food which the cattle have and to the character of the cream resulting therefrom, these differences in the flavour of the butter are also in part dependent upon the different species of bacteria which are present in the ripening of cream at different seasons. the species of bacteria in june cream are different from those that are commonly present in january cream, and this is certainly a factor in determining the difference between winter and summer butter. use of artificial bacteria cultures for cream ripening. bacteriologists have been for some time endeavouring to aid butter makers in this direction by furnishing them with the bacteria needful for the best results in cream ripening. the method of doing this is extremely simple in principle, but proves to be somewhat difficult in practice. it is only necessary to obtain the species of bacteria that produce the highest results, and then to furnish these in pure culture and in large quantity to the butter makers, to enable them to inoculate their cream with the species of bacteria which will produce the results that they desire. for this purpose bacteriologists have been for several years searching for the proper species of bacteria to produce the best results, and there have been put upon the market for sale several distinct "pure cultures" for this purpose. these have been obtained by different bacteriologists and dairymen in the northern european countries and also in the united states. these pure cultures are furnished to the dairymen in various forms, but they always consist of great quantities of certain kinds of bacteria which experience has found to be advantageous for the purpose of cream ripening. there have hitherto appeared a number of difficulties in the way of reaching complete success in these directions. the most prominent arises in devising a method of using pure cultures in the creamery. the cream which the butter makers desire to ripen is, as we have seen, already impregnated with bacteria, and would ripen in a fashion of its own even if no pure culture of bacteria were added thereto. pure cultures can not therefore be used as simply as can yeast in bread dough. it is plain that the simple addition of a pure culture to a mass of cream would not produce the desired effects, because the cream would be ripened then, not by the pure culture alone, but by the pure culture plus all of the bacteria that were originally present. it would, of course, be something of a question as to whether under these conditions the results would be favourable, and it would seem that this method would not furnish any means of getting rid of bad tastes and flavours which have come from the presence of malign species of bacteria. it is plainly desirable to get rid of the cream bacteria before the pure culture is added. this can be readily done by heating it to a temperature of 69 degrees c. (155 degrees f.) for a short time, this temperature being sufficient to destroy most of the bacteria. the subsequent addition of the pure culture of cream-ripening bacteria will cause the cream to ripen under the influence of the added culture alone. this method proves to be successful, and in the butter making countries in europe it is becoming rapidly adopted. in this country, however, this process has not as yet become very popular, inasmuch as the heating of the cream is a matter of considerable expense and trouble, and our butter makers have not been very ready to adopt it. for this reason, and also for the purpose of familiarizing butter makers with the use of pure cultures, it has been attempted to produce somewhat similar though less uniform results by the use of pure cultures in cream without previous healing. in the use of pure cultures in this way, the butter maker is directed to add to his cream a large amount of a prepared culture of certain species of bacteria, upon the principle that the addition of such a large number of bacteria to the cream, even though the cream is already inoculated with certain bacteria, will produce a ripening of the cream chiefly influenced by the artificially added culture. the culture thus added, being present in very much greater quantity than the other "wild" species, will have a much greater effect than any of them. this method, of course, cannot insure uniformity. while it may work satisfactorily in many cases, it is very evident that in others, when the cream is already filled with a large number of malign species of bacteria, such an artificial culture would not produce the desired results. this appears to be not only the theoretical but the actual experience. the addition of such pure cultures in many cases produces favourable results, but it does not always do so, and the result is not uniform. while the use of pure cultures in this way is an advantage over the method of simply allowing the cream to ripen normally without such additions, it is a method that is decidedly inferior to that which first pasteurizes the cream and subsequently adds a starter. there is still another method of adding bacteria to cream to insure a more advantageous ripening, which is frequently used, and, being simpler, is in many cases a decided advantage. this method is by the use of what is called a natural starter. a natural starter consists simply of a lot of cream which has been taken from the most favourable source possible--that is, from the cleanest and best dairy, or from the herd producing the best quality of cream--and allowing this cream to stand in a warm place for a couple of days until it becomes sour. the cream will by that time be filled with large numbers of bacteria, and this is then put as a starter into the vat of cream to be ripened. of course, in the use of this method the butter maker has no control over the kinds of bacteria that will grow in the starter, but it is found, practically, that if the cream is taken from a good source the results are extremely favourable, and there is produced in this way almost always an improvement in the butter. the use of pure cultures is still quite new, particularly in this country. in the european butter-making countries they have been used for a longer period and have become very much better known. what the future may develop along this line it is difficult to say; but it seems at least probable that as the difficulties in the details are mastered the time will come when starters will be used by our butter makers for their cream ripening, just as yeast is used by housewives for raising bread, or by brewers for fermenting malt. these starters will probably in time be furnished by bacteriologists. bacteriology, in other words, is offering in the near future to our butter makers a method of controlling the ripening of the cream in such a way as to insure the obtaining of a high and uniform quality of butter, so far, at least, as concerns flavour and aroma. bacteria in cheese. cheese ripening.--the third great product of the dairy industry is cheese, and in connection with this product the dairyman is even more dependent upon bacteria than he is in the production of butter. in the manufacture of cheese the casein of the milk is separated from the other products by the use of rennet, and is collected in large masses and pressed, forming the fresh cheese. this cheese is then set aside for several weeks, and sometimes for months, to undergo a process that is known as ripening. during the ripening there are developed in the cheese the peculiar flavours which are characteristic of the completed product. the taste of freshly made cheese is extremely unlike that of the ripened product. while butter made from unripened cream has a pleasant flavour, and one which is in many places particularly enjoyed, there is nowhere a demand for unripened cheese, for the freshly made cheese has a taste that scarce any one regards as pleasant. indeed, the whole value of the cheese is dependent upon the flavour of the product, and this flavour is developed during the ripening. the cheese maker finds in the ripening of his cheese the most difficult part of his manufacture. it is indeed a process over which he has very little control. even when all conditions seem to be correct, when cheese is made in the most careful manner, it not infrequently occurs that the ripening takes place in a manner that is entirely abnormal, and the resulting cheese becomes worthless. the cheese maker has been at an entire loss to understand these irregularities, nor has he possessed any means of removing them. the abnormal ripening that occurs takes on various types. sometimes the cheese will become extraordinarily porous, filled with large holes which cause the cheese to swell out of proper shape and become worthless. at other times various spots of red or blue appear in the manufactured cheese; while again unpleasant tastes and flavours develop which render the product of no value. sometimes a considerable portion of the product of the cheese factory undergoes such irregular ripening, and the product for a long time will thus be worthless. if some means could be discovered of removing these irregularities it would be a great boon to the cheese manufacturer; and very many attempts have been made in one way or another to furnish the cheese maker with some details in the manufacture which will enable him in a measure to control the ripening. the ripening of the cheese has been subjected to a large amount of study on the part of bacteriologists who have been interested in dairy products. that the ripening of cheese is the result of bacterial growth therein appears to be probable from a priori grounds. like the ripening of cream, it is a process that occurs somewhat slowly. it is a chemical change which is accompanied by the destruction of proteid matter; it takes place best at certain temperatures, and temperatures which we know are favourable to the growth of micro-organisms, all of which phenomena suggest to us the action of bacteria. moreover, the flavours and the tastes that arise have a decided resemblance in many cases to the decomposition products of bacteria, strikingly so in limburger cheese. when we come to study the matter of cheese ripening carefully we learn beyond question that this a priori conclusion is correct. the ripening of any cheese is dependent upon several different factors. the method of preparation, the amount of water left in the curd, the temperature of ripening, and other miscellaneous factors connected with the mechanical process of cheese manufacture, affect its character. but, in addition to all these factors, there is undoubtedly another one, and that is the number and the character of the bacteria that chance to be in the curd when the cheese is made. while it is found that cheeses which are treated by different processes will ripen in a different manner, it is also found that two cheeses which have been made under similar conditions and treated in identically the same way may also ripen in a different manner, so that the resulting flavour will vary. the variations between cheeses thus made may be slight or they may be considerable, but variations certainly do occur. every one knows the great difference in flavours of different cheeses, and these flavours are due in considerable measure to factors other than the simple mechanical process of making the cheese. the general similarity of the whole process to a bacterial fermentation leads us to believe at the outset that some of the differences in character are due to different kinds of bacteria that multiply in the cheese and produce decomposition therein. when the matter comes to be studied by bacteriology, the demonstration of this position becomes easy. that the ripening of cheese is due to growth of bacteria is very easily proved by manufacturing cheeses from milk which is deprived of bacteria. for instance, cheeses have been made from milk that has been either sterilized or pasteurized--which processes destroy most of the bacteria therein--and, treated otherwise in a normal manner, are set aside to ripen. these cheeses do not ripen, but remain for months with practically the same taste that they had originally. in other experiments the cheese has been treated with a small amount of disinfective, which is sufficient to prevent bacteria from growing, and again ripening is found to be absolutely prevented. furthermore, if the cheese under ordinary conditions is studied during the ripening process, it is found that bacteria are growing during the whole time. these facts all taken together plainly prove that the ripening of cheese is a fermentation due to bacteria. it will be noticed, however, that the conditions in the cheese are not favourable for very rapid bacterial growth. it is true that there is plenty of food in the cheese for bacterial life, but the cheese is not very moist; it is extremely dense, being subjected in all cases to more or less pressure. the penetration of oxygen into the centre of the mass must be extremely slight. the density, the lack of a great amount of moisture, and the lack of oxygen furnish conditions in which bacteria will not grow very rapidly. the conditions are far less favourable than those of ripening cream, and the bacteria do not grow with anything like the rapidity that they grow in cream. indeed, the growth of these organisms during the ripening is extremely slow compared to the possibilities of bacterial growth that we have already noticed. nevertheless, the bacteria do multiply in the cheese, and as the ripening goes on they become more and more abundant, although the number fluctuates, rising and falling under different conditions. when the attempt is made to determine the relation of the different kinds of ripening to different kinds of bacteria, it has thus far met with extremely little success. that different flavours are due to the ripening produced by different kinds of bacteria would appear to be almost certain when we remember, as we have already noticed, the different kinds of decomposition produced by different species of bacteria. it would seem, moreover, that it ought not to be very difficult to separate from the ripened cheese the bacteria which are present, and thus obtain the kind of bacteria necessary to produce the desired ripening. but for some reason this does not prove to be so easy in practice as it seems to be in theory. many different species of bacteria have been separated from cheeses. one bacteriologist, studying several cheeses, separated about eighty different species therefrom, and others have found perhaps as many more from different sources. moreover, experiments have been made with a considerable number of these different kinds of bacteria to determine whether they are capable of producing normal ripening. these experiments consist of making cheese out of milk that has been deprived of its bacteria, and which has been inoculated with large quantities of the species in question. hitherto these experiments have not been very satisfactory. in some cases the cheese appears to ripen scarcely at all; in other cases the ripening occurs, but the resulting cheese is of a peculiar character, entirely unlike the cheese that it is desired to imitate. there have been one or two experiments in recent times that give a little more promise of success than the earlier ones, for a few species of bacteria have been used in ripening with what the authors have thought to be promising success. the cheese made from the milk artificially inoculated with these species ripens in a satisfactory manner and gives some of the character desired, though up to the present time in no case has the typical normal ripening been produced in any of these experiments. but these experiments have demonstrated beyond question that the abnormal ripening which is common in cheese factories is due to the presence of undesirable species of bacteria in the milk. many of the experiments in making cheeses by means of artificial cultures of bacteria have resulted in decidedly abnormal cheeses. many of the cheeses thus manufactured have shown imperfections in ripening which are identical with those actually occurring in the cheese factory. several different species of bacteria have been found which, when artificially used thus for ripening cheese, will give rise to the porosity and the abnormal swelling of the cheese already referred to (fig. 24). others produced bad tastes and flavours, and enough has been done in this line to demonstrate beyond peradventure that the abnormal ripening of cheese is due primarily to the growth of improper species therein. quite a long list of species of bacteria which produce abnormal ripening have been isolated from cheeses, and have been studied and experimented with by bacteriologists. as a result of this study of abnormal ripening, there has been suggested a method of partially controlling these--remedying them. the method consists simply in testing the fermenting qualities of the milk used. a small sample of milk from different dairies is allowed to stand in the cheese factory by itself until it undergoes its normal souring. if the fermentation or souring that thus occurs is of a normal character, the milk is regarded as proper for cheese making. but if the fermentation that occurs in any particular sample of milk is unusual; if an extraordinary amount of gas bubbles are produced, or if unpleasant smells and tastes arise, the sample is regarded as unfavourable for cheese making, and as likely to produce abnormal ripening in the cheeses. milk from this source would therefore be excluded from the milk that is to be used in cheese making. this, of course, is a tentative and an unsatisfactory method of controlling the ripening, and yet it is one of some practical value to cheese makers. it is the only method that has yet been suggested of controlling the ripening. our bacteriologists, of course, are quite confident that in the future more practical results will be obtained along this line than in the past. if it is true that cheeses are ripened by bacteria; if it is true that different qualities in the cheese are due to the growth of different species of bacteria during the ripening, it would seem to be possible to obtain the proper kind of bacteria and to furnish them to the cheese maker for artificially inoculating his cheese, just as it has been possible to furnish artificially cultivated yeasts to the brewer, and as it has become possible to furnish artificially cultivated bacteria to the butter maker. we must, however, recognise this to be a matter for the future. up to the present time no practical results along the lines of bacteria have been obtained which our cheese manufacturers can make use of in the way of controlling with any accuracy this process of cheese ripening. thus it will be seen that in this last dairy product bacteria play even a more important part than in any of the others. the food value of cheese is dependent upon the casein which is present. the market price, however, is controlled entirely by the flavour, and this flavour is a product of bacterial growth. upon the action of bacteria, then, the cheese maker is absolutely dependent; and when our bacteriologists are able in the future to investigate this matter further, it seems to be at least possible that they may obtain some means of enabling the cheese maker to control the ripening accurately. not only so, but recognising the great variety in the flavours of cheese, and recognising that different kinds of bacteria undoubtedly produce different kinds of decomposition products, it seems to be at least possible that a time will come when the cheese maker will be able to produce at-will any particularly desired flavour in his cheese by the addition to it of particular species of bacteria, or particular mixtures of species of bacteria which have been discovered to produce the desired effects. chapter iv. bacteria in natural processes.--agriculture. thus far, in considering the relations of bacteria to mankind, we have taken into account only the arts and manufactures, and have found bacteria playing no unimportant part in many of the industries of our modern civilized life. so important are they that there is no one who is not directly affected by them. there is hardly a moment in our life when we are not using some of the direct or indirect products of bacterial action. we turn now, however, to the consideration of a matter of even more fundamental importance; for when we come to study bacteria in nature, we find that there are certain natural processes connected with the life of animals and plants that are fundamentally based upon their powers. living nature appears limitless, for life processes have been going on in the world through countless centuries with seemingly unimpaired vigour. at the very bottom we find this never-ending exhibition of vital power dependent upon certain activities of micro-organisms. so thoroughly is this true that, as we shall find after a short consideration, the continuance of life upon the surface of the world would be impossible if bacterial action were checked for any considerable length of time. the life of the globe is, in short, dependent upon these micro-organisms. bacteria as scavengers. in the first place, we may notice the value of these organisms simply as scavengers, keeping the surface of the earth in the proper condition for the growth of animals and plants. a large tree in the forest dies and falls to the ground. for a while the tree trunk lies there a massive structure, but in the course of months a slow change takes place in it. the bark becomes softened and falls from the wood. the wood also becomes more or less softened; it is preyed upon then by insect life; its density decreases more and more, until finally it crumbles into a soft, brownish, powdery mass, and eventually the whole sinks into the soil, is overgrown by mosses and other vegetation, and the tree trunk has disappeared from view. in the same way the body of the dead animal undergoes the process of the softening of its tissues by decay. the softer parts of the body rapidly dissipate, and even the bones themselves eventually are covered with the soil and disintegrated, until in time they, too, disappear from any visible existence. this whole process is one of decay, and the result is that the solid mass of the body of the tree or of the animal has been decomposed. what has become of it? the answer holds the secret of nature's eternal freshness. part of it has dissipated into the air in the form of gases and water vapour; part of it has changed its composition and has become incorporated into the soil, the final result being that the body of the plant or animal disappears as such, and its substance is converted into gaseous form, which is dissipated in the air or into simple compounds which sink into the earth. this whole process of decay of organic life is one in which bacteria play the most important part. in the case of the decomposition of the woody matter of the tree trunk, the process is begun by the agency of moulds, for this group of organisms alone appears to be capable of attacking such hard woody structure. the later part of the decay, however, is largely carried on by bacterial life. in the decomposition of the animal tissues, bacteria alone are the agents. thus the process by which organic matter is dissipated into the air or incorporated into the soil is one which is primarily presided over by bacterial life. viewing this matter in a purely mechanical light, the importance of bacteria in thus acting as scavengers can hardly be overestimated. if we think for a moment of the condition of the world were there no such decomposing agents to rid the earth's surface of the dead bodies of animals and plants, we shall see that long since the earth would have been uninhabitable. if the dead bodies of plants and animals of past ages simply accumulated on the surface of the ground without any forces to reduce them into simple compounds for dissipation, by their very bulk they would have long since completely covered the surface of the earth so as to afford no possible room for further growth of plants and animals. in a purely mechanical way, then, bacteria as decomposition agents are necessary to keep the surface of the earth fresh and unencumbered so that life can continue. bacteria as agents in nature's food cycle. but the matter by no means ends here. when we come to think of it, it is a matter of considerable surprise that the surface of the earth has been able to continue producing animals and plants for the many millions of years during which life has been in existence. plants and animals both require food, animals depending wholly upon plants therefor. plants, however, equally with animals, require food, and although they obtain a considerable portion of their food from the air, yet no inconsiderable part of it is obtained from the soil. the question is forced upon us, therefore, as to why the soil has not long since become exhausted of food. how could the soil continue to support plants year after year for millions of years, and yet remain as fertile as ever? the explanation of this phenomenon is in the simple fact that the processes of nature are such that the same food is used over and over again, first by the plant, then by the animal, and then again by the plant, and there is no necessity for any end of the process so long as the sun furnishes energy to keep the circulation continuous. one phase of this transference of food from animal to plant and from plant to animal is familiar to nearly every one. it is a well-known fact that animals in their respiration consume oxygen, but exhale it again in combination with carbon as carbonic dioxide. on the other hand, plants in their life consume the carbonic dioxide and exhale the oxygen again as free oxygen. thus each of these kingdoms makes use of the excreted product of the other, and this process can go on indefinitely, the animals furnishing our atmosphere with plenty of carbonic acid for plant life, and the plants excreting into the atmosphere at the same time an abundant sufficiency of oxygen for animal life. the oxygen thus passes in an endless round from animal to plant and from plant to animal. a similar cycle is true of all the other foods of animal and plant life, though in regard to the others the operation is more complex and more members are required to complete the chain. the transference of matter through a series of changes by which it is brought from a condition in which it is proper food for plants back again into a condition when it is once more a proper food for plants, is one of the interesting discoveries of modern science, and one in which, as we shall see, bacteria play a most important part. this food cycle is illustrated roughly by the accompanying diagram; but in order to understand it, an explanation of the various steps in this cycle is necessary. it will be noticed that at the bottom of the circle represented in fig. 25, at a, are given various ingredients which are found in the soil and which form plant foods. plant foods, as may be seen there, are obtained partly from the air as carbonic dioxide and water; but another portion comes from the soil. among the soil ingredients the most prominent are nitrates, which are the forms of nitrogen compounds most easily made use of by plants as a source of this important element. it should be stated also that there are other compounds in the soil which furnish plants with part of their food--compounds containing potassium, phosphorus, and some other elements. for simplicity's sake, however, these will be left out of consideration. beginning at the bottom of the cycle (fig. 25 a), plant life seizes the gases from the air and these foods from the soil, and by means of the energy furnished it by the sun's rays builds these simple chemical compounds into more complex ones. this gives us the second step, as shown in fig. 25 b, the products of plant life. these products of plant life consist of such materials as sugar, starches, fats, and proteids, all of which have been manufactured by the plant from the ingredients furnished it from the soil and air, and through the agency of the sun's rays. these products of plant life now form foods for the animal kingdom. starches, fats, and proteids are animal foods, and upon such complex bodies alone can the animal kingdom be fed. animal life, standing high up in the circle, is not capable of extracting its nutriment from the soil, but must take the more complex foods which have been manufactured by plant life. these complex foods enter now into the animal and take their place in the animal body. by the animal activities, some of the foods are at once decomposed into carbonic acid and water, which, being dissipated into the air, are brought back at once into the condition in which they can serve again as plant food. this part of the food is thus brought back again to the bottom of the circle (fig. 25, dotted lines). but while it is true that animals do thus reduce some of their foods to the simple condition of carbonic acid and water, this is not true of most of the foods which contain nitrogen. the nitrogenous foods are as necessary for the life as the carbon foods, and animals do not reduce their nitrogenous foods to the condition in which plants can prey upon them. while plants furnish them with nitrogenous food, they can not give it back to the plants. part of the nitrogenous foods animals build into new albumins (fig. 25 c); but a part of them they reduce at once into a somewhat simpler condition known as urea. urea is the form in which the nitrogen is commonly excreted from the animal body. but urea is not a plant food; for ordinary plants are entirely unable to make use of it. part of the nitrogen eaten by the animal is stored up in its body, and thus the body of the animal, after it has died, contains these nitrogen compounds of high complexity. but plants are not able to use these compounds. a plant can not be fed upon muscle tissue, nor upon fats, nor bones, for these are compounds so complex that the simple plant is unable to use them at all. so far, then, in the food cycle the compounds taken from the soil have been built up into compounds of greater and greater complexity; they have reached the top of this circle, and no part of them, except part of the carbon and oxygen, has become reduced again to plant food. in order that this material should again become capable of entering into the life of plants so as to go over the circle again, it is necessary for it to be once more reduced from its highly complex condition into a simpler one. now come into play these decomposition agencies which we have been studying under the head of scavengers. it will be noticed that the next step in the food cycle is taken by the decomposition bacteria. these organisms, existing, as we have already seen, in the air, in the soil, in the water, and always ready to seize hold of any organic substance that may furnish them with food, feed upon the products of animal life, whether they are such products as muscle tissue, or fat, or sugar, or whether they are the excreted products of animal life, such as urea, and produce therein the chemical decomposition changes already noticed. as a result of this chemical decomposition, the complex bodies are broken into simpler and simpler compounds, and the final result is a very thorough destruction of the animal body or the material excreted by animal life, and its reduction into forms simple enough for plants to use again as foods. thus the bacteria come in as a necessary link to connect the animal body, or the excretion from the animal body, with the soil again, and therefore with that part of the circle in which the material can once more serve as plant food. but in the decomposition that thus occurs through the agency of the putrefactive bacteria it very commonly happens that some of the food material is broken down into compounds too simple for use as plant food. as will be seen by a glance at the diagram (fig. 25 d), a portion of the cleavage products resulting from the destruction of these animal foods takes the form of carbonic-acid gas and water. these ingredients are at once in condition for plant life, as shown by the dotted lines. they pass off into the air, and the green leaves of vegetation everywhere again seize them, assimilate them, and use them as food. thus it is that the carbon and the oxygen have completed the cycle, and have come back again to the position in the circle where they started. in regard to the nitrogen portion of the food, however, it very commonly happens that the products which arise as the result of the decomposition processes are not yet in proper condition for plant food. they are reduced into a condition actually too simple for the use of plants. as a result of these putrefactive changes, the nitrogen products of animal life are broken frequently into compounds as simple as ammonia (nh3), or into compounds which the chemists speak of as nitrites (fig. 25 at d). now these compounds are not ordinarily within the reach of plant life. the luxuriant vegetation of the globe extracts its nitrogen from the soil in a form more complex than either of the compounds here mentioned; for, as we have seen, it is nitrates chiefly that furnish plants with their nitrogen food factor. but nitrates contain considerable oxygen. ammonia, which is one of the products of putrefactive decomposition, contains no oxygen, and nitrites, another factor, contains less oxygen than nitrates. these bodies are thus too simple for plants to make use of as a source of nitrogen. the chemical destruction of the food material which results from the action of the putrefactive bacteria is too thorough, and the nitrogen foods are not yet in condition to be used by plants. now comes in the agency of still another class of micro-organisms, the existence of which has been demonstrated to us during the last few years. in the soil everywhere, especially in fertile soil, is a class of bacteria which has received the name of nitrifying bacteria (fig. 26). these organisms grow in the soil and feed upon the soil ingredients. in the course of their life they have somewhat the same action upon the simple nitrogen cleavage products just mentioned as we have already noticed the vinegarproducing species have upon alcohol, viz., the bringing about a union with oxygen. there are apparently several different kinds of nitrifying bacteria with different powers. some of them cause an oxidation of the nitrogen products by means of which the ammonia is united with oxygen and built up into a series of products finally resulting in nitrates (fig. 26). by the action of other species still higher nitrogen compounds, including the nitrites, are further oxidized and built up into the form of nitrates. thus these nitrifying organisms form the last link in the chain that binds the animal kingdom to the vegetable kingdom (fig. 25 at 4). for after the nitrifying organisms have oxidized nitrogen cleavage products, the results of the oxidation in the form of nitrates or nitric acid are left in the soil, and may now be seized upon by the roots of plants, and begin once more their journey around the food cycle. in this way it will be seen that while plants, by building up compounds, form the connecting link between the soil and animal life, bacteria in the other half of the cycle, by reducing them again, give us the connecting link between animal life and the soil. the food cycle would be as incomplete without the agency of bacterial life as it would be without the agency of plant life. but even yet the food cycle is not complete. some of the processes of decomposition appear to cause a portion of the nitrogen to fly out of the circle at a tangent. in the process of decomposition which is going on through the agency of micro-organisms, a considerable part of the nitrogen is dissipated into the air in the form of free nitrogen. when a bit of meat decays, part of the meat is, indeed, converted into ammonia or other nitrogen compounds, but if the putrefaction is allowed to go on, in the end a considerable portion of it will be broken into still simpler forms, and the nitrogen will finally be dissipated into the air in the form of free nitrogen. this dissipation of free nitrogen into the air is going on in the world wherever putrefaction takes place. wherever decomposition of nitrogen products occurs some free nitrogen is eliminated. now, this part of the nitrogen has passed beyond the reach of plants, for plants can not extract free nitrogen from the air. in the diagram this is represented as a portion of the material which, through the agency of the decomposition bacteria, has been thrown out of the cycle at a tangent (fig. 25 e). it will, of course, be plain from this that the store of nitrogen food must be constantly diminishing. the soil may have been originally supplied with a given quantity of nitrogen compound, but if the decomposition products are causing considerable quantities of this nitrogen to be dissipated in the air, it plainly follows that the total amount of nitrogen food upon which the animal and vegetable kingdoms can depend is becoming constantly reduced by such dissipation. there are still other methods by which nitrogen is being lost from the food cycle. first, we may notice that the ordinary processes of vegetation result in a gradual draining of the soil and a throwing of its nitrogen into the ocean. the body of any animal or any plant that chances to fall into a brook or river is eventually carried to the sea, and the products of its decomposition pass into the ocean and are, of course, lost to the soil. now, while this gradual extraction of nitrogen from the soil by drainage is a slow one, it is nevertheless a sure one. it is far more rapid in these years of civilized life than in former times, since the products of the soil are given to the city, and then are thrown into its sewage our cities, then, with our present system of disposing of sewage, are draining from the soil the nitrogen compounds and throwing them away. in yet another direction must it be noticed that our nitrogen compounds are being lost to plant life--viz., by the use of various nitrogen compounds to form explosives. gunpowder, nitro-glycerine, dynamite, in fact, nearly all the explosives that are used the world over for all sorts of purposes, are nitrogen compounds. when they are exploded the nitrogen of the compound is dissipated into the air in the form of gas, much of it in the form of free nitrogen. the basis from which explosive compounds are made contains nitrogen in the form in which it can be used by plants. saltpetre, for example, is equally good as a fertilizer and as a basis for gunpowder. the products of the explosion are gases no longer capable of use by plants, and thus every explosion of nitrogen compounds aids in this gradual dissipation of nitrogen products, taking them from the store of plant foods and throwing them away. all of these agencies contribute to reduce the amount of material circulating in the food cycle of nature, and thus seem to tend inevitably in the end toward a termination of the processes of life; for as soon as the soil becomes exhausted of its nitrogen compounds, so soon will plant life cease from lack of nutrition, and the disappearance of animal life will follow rapidly. it is this loss of nitrogen in large measure that is forcing our agriculturists to purchase fertilizers. the last fifteen years have shown us, however, that here again we may look upon our friends, the bacteria, as agents for counteracting this dissipating tendency in the general processes of nature. bacterial life in at least two different ways appears to have the function of reclaiming from the atmosphere more or less of this dissipated free nitrogen. in the first place, it has been found in the last few years that soil entirely free from all common plants, but containing certain kinds of bacteria, if allowed to stand in contact with the air, will slowly but surely gain in the amount of nitrogen compounds that it contains. these nitrogen compounds are plainly manufactured by the bacteria in the soil; for unless the bacteria are present they do not accumulate, and they do accumulate inevitably if the bacteria are present in the proper quantity and the proper species. it appears that, as a rule, this fixation of nitrogen is not performed by any one species of microorganisms, but by two or three of them acting together. certain combinations of bacteria have been found which, when inoculated in the soil, will bring about this fixation of nitrogen, but no one of the species is capable of producing this result alone. we do not know to what extent these organisms are distributed in the soil, nor how widely this nitrogen fixation through bacterial life is going on. it is only within a short time that it has been demonstrated to exist, but we must look upon bacteria in the soil as one of the factors in reclaiming from the atmosphere the dissipated free nitrogen. the second method by which bacteria aid in the reclaiming of this lost nitrogen is by a combined action of certain species of bacteria and some of the higher plants. ordinary green plants, as already noted, are unable to make use of the free nitrogen of the atmosphere it was found, however, some fifteen years ago that some species of plants, chiefly the great family of legumes, which contains the pea plant, the bean, the clover, etc, are able, when growing in soil that is poor in nitrogen, to obtain nitrogen from some source other than the soil in which they grow. a pea plant in soil that contains no nitrogen products and watered with water that contains no nitrogen, will, after sprouting and growing for a length of time, be found to have accumulated a considerable quantity of fixed nitrogen in its tissues the only source of this nitrogen has been evidently from the air which bathes the leaves of the plant or permeates the soil and bathes its roots this fact was at first disputed, but subsequently demonstrated to be true, and was found later to be associated with the combined action of these legumes and certain soil bacteria. when a legume thus gains nitrogen from the air, it develops upon its roots little bunches known as root nodules or root tubercles. the nodules are sometimes the size of the head of a pm, and sometimes much larger than this, occasionally reaching the size of a large pea, or even larger. upon microscopic examination they are found to be little nests of bacteria in some way the soil organisms (fig 27) make their way into the roots of the sprouting plant, and finding there congenial environment, develop in considerable quantities and produce root tubercles in the root. now, by some entirely unknown process, the legume and the bacteria growing together succeed in extracting the nitrogen from the atmosphere which permeates the soil, and fixing this nitrogen in the tubercles and the roots in the form of nitrogen compounds. the result is that, after a proper period of growth, the amount of fixed nitrogen in the plant is found to have very decidedly increased (fig 25 e). this, of course, furnishes a starting point for the reclaiming of the lost atmospheric nitrogen. the legume continues to live its usual life, perhaps increasing the store of nitrogen in its roots and stems and leaves during the whole of its normal growth. subsequently, after having finished its ordinary life, the plant will die, and then the roots and stems and leaves, falling upon the ground and becoming buried, will be seized upon by the decomposition bacteria already mentioned. the nitrogen which has thus become fixed in their tissues will undergo the destructive changes already described. this will result eventually in the production of nitrates. thus some of the lost nitrogen is restored again to the soil in the form of nitrates, and may now start on its route once more around the cycle of food. it will be seen, then, that the food cycle is a complete one. beginning with the mineral ingredients in the soil, the food matter may start on its circulation from the soil to the plant, from the plant to the animal, from the animal to the bacterium and from the bacterium through a series of other bacteria back again to the soil in the condition in which it started. if, perchance, in this progress around the circle some of the nitrogen is thrown off at a tangent, this, too, is brought back again to the circle through the agency of bacterial life. and so the food material of animals and plants continues in this never-ceasing circulation. it is the sunlight that furnishes the energy for the motion. it is the sunlight that forces the food around the circle and keeps up the endless change; and so long as, the sun continues to shine upon the earth there seems to be no reason why the process should ever cease. it is this repeated circulation that has made the continuation of life possible for the millions and millions of years of the earth's history. it is this continued circulation that makes life possible still, and it is only this fact that the food is thus capable of ever circulating from animal to plant and from plant to animal that makes it possible for the living world to continue its existence. but, ah we have seen, one half of this great circle of food change is dependent upon bacterial life. without the bacterial life the animal body and the animal excretion could never be brought back again within the reach of the plant; and thus, were it not for the action of these microorganisms the food cycle would be incomplete and life could not continue indefinitely upon the surface of the earth. at the very foundation, the continuation of the present condition of nature and the existence of life during the past history of the world has been fundamentally based upon the ubiquitous presence of bacteria and upon their continual action in connection with both destructive and constructive processes. relation of bacteria to agriculture. we have already noticed that bacteria play an important part in some of the agricultural industries, particularly in the dairy. from the consideration of the matters just discussed, it is manifest that these organisms must have an even more intimate relation to the farmer's occupation. at the foundation, farming consists in the cultivation of plants and animals, and we have already seen how essential are the bacteria in the continuance of animal and plant life. but aside from these theoretical considerations, a little study shows that in a very practical manner the farmer is ever making use of bacteria, as a rule, quite unconsciously, but none the less positively. sprouting of seeds. even in the sprouting of seeds after they are sown in the soil bacterial life has its influence. when seeds are placed m moist soil they germinate under the influence of heat. the rich albuminous material in the seeds furnishes excellent food, and inasmuch as bacteria abound in the soil, it is inevitable that they should grow in and feed upon the seed. if the moisture is excessive and the heat considerable, they very frequently grow so rapidly in the seed as to destroy its life as a seedling. the seed rots in the ground as a result. this does not commonly occur, however, in ordinary soil. but even here bacteria do grow in the seed, though not so abundantly as to produce any injury. indeed, it has been claimed that their presence in the seed in small quantities is a necessity for the proper sprouting of the seed. it has been claimed that their growth tends to soften the food material in the seed, so that the young seedling can more readily absorb it for its own food, and that without such a softening the seed remains too hard for the plant to use. this may well be doubted, however, for seeds can apparently sprout well enough without the aid of bacteria. but, nevertheless, bacteria do grow in the seed during its germination, and thus do aid the plant in the softening of the food material. we can not regard them as essential to seed germination. it may well be claimed that they ordinarily play at least an incidental part in this fundamental life process, although it is uncertain whether the growth of seedlings is to any considerable extent aided thereby. the silo. in the management of a silo the farmer has undoubtedly another great bacteriological problem. in the attempt to preserve his summer-grown food for the winter use of his animals, he is hindered by the activity of common bacteria. if the food is kept moist, it is sure to undergo decomposition and be ruined in a short time as animal food. the farmer finds it necessary, therefore, to dry some kinds of foods, like hay. while he can thus preserve some foods, others can not be so treated. much of the rank growth of the farm, like cornstalks, is good food while it is fresh, but is of little value when dried. the farmer has from experience and observation discovered a method of managing bacterial growth which enables him to avoid their ordinary evil effects. this is by the use of the silo. the silo is a large, heavily built box, which is open only at the top. in the silo the green food is packed tightly, and when full all access of air is excluded, except at its surface. under these conditions the food remains moist, but nevertheless does not undergo its ordinary fermentations and putrefactions, and may be preserved for months without being ruined. the food in such a silo may be taken out months after it is packed, and will still be found to be in good condition for food. it is true that it has changed its character somewhat, but it is not decayed, and is eagerly eaten by cattle. we are yet very ignorant of the nature of the changes which occur m the food while in the silo. the food is not preserved from fermentation. when the siloxis packed slowly, a very decided fermentation occurs by which the mass is raised to a high temperature (140 degrees f. to 160 degrees f.). this heating is produced by certain species of bacteria which grow readily even at this high temperature. the fermentation uses up the air in the silo to a certain extent and produces a settling of the material which still further excludes air. the first fermentation soon ceases, and afterward only slow changes occur. certain acidproducing bacteria after a little begin to grow slowly, and in time the silage is rendered somewhat sour by the production of acetic acid. but the exclusion of air, the close packing, and the small amount of moisture appear to prevent the growth of the common putrefactive bacteria, and the silage remains good for a long time. in other methods of filling the silo, the food is very quickly packed and densely crowded together so as to exclude as much air as possible from the beginning. under these conditions the lack of moisture and air prevents fermentative action very largely. only certain acid-producing organisms grow, and these very slowly. the essential result in either case is that the common putrefactive bacteria are prevented from growing, probably by lack of sufficient oxygen and moisture, and thus the decay is prevented. the closely packed food offers just the same unfavourable condition for the growth of common putrefactive bacteria that we have already seen offered by the hard-pressed cheese, and the bacteria growth is in the same way held in check. our knowledge of the matter is as yet very slight, but we do know enough to understand that the successful management of a silo is dependent upon the manipulation of bacteria. the fertility of the soil. the farmer's sole duty is to extract food from the soil. this he does either directly by raising crops, or indirectly by raising animals which feed upon the products of the soil. in either case the fertility of the soil is the fundamental factor in his success. this fertility is a gift to him from the bacteria. even in the first formation of soil he is in a measure dependent upon bacteria. soil, as is well known, is produced in large part by the crumbling of the rocks into powder. this crumbling we generally call weathering, and regard it as due to the effect of moisture and cold upon the rocks, together with the oxidizing action of the air. doubtless this is true, and the weathering action is largely a physical and chemical one. nevertheless, in this fundamental process of rock disintegration bacterial action plays a part, though perhaps a small one. some species of bacteria, as we have seen, can live upon very simple foods, finding in free nitrogen and carbonates sufficiently highly complex material for their life. these organisms appear to grow on the bare surface of rocks, assimilating nitrogen from the air, and carbon from some widely diffused carbonates or from the co2 in the air. their secreted products of an acid nature help to soften the rocks, and thus aid in performing the first step in weathering. the soil is not, however, all made up of disintegrated rocks. it contains, besides, various ingredients which combine to make it fertile. among these are various sulphates which form important parts of plant foods. these sulphates appear to be formed, in part, at least, by bacterial agency. the decomposition of proteids gives rise, among other things, to hydrogen sulphide (h2s). this gas, which is of common occurrence in the atmosphere, is oxidized by bacterial growth into sulphuric acid, and this is the basis of part of the soil sulphates. the deposition of iron phosphates and iron silicates is probably also in a measure aided by bacterial action. all of these processes are factors in the formation of soil. beyond much question the rock disintegration which occurs everywhere in nature is chiefly the result of physical and chemical changes, but there is reason for believing that the physical and chemical processes are, to a slight extent at least, assisted by bacterial life. a more important factor of soil fertility is its nitrogen content, without which it is completely barren. the origin of these nitrogen ingredients has been more or less of a puzzle. fertile soil everywhere contains nitrates and other nitrogen compounds, and in certain parts of the world there are large accumulations of these compounds, like the nitrate beds of chili. that they have come ultimately from the free atmospheric nitrogen seems certain, and various attempts have been made to explain a method of this nitrogen fixation. it has been suggested that electrical discharges in the air may form nitric acid, which would readily then unite with soil ingredients to form nitrates. there is little reason, however, for believing this to be a very important factor but in the soil bacteria we find undoubtedly an efficient agency m this nitrogen fixation. as already seen, the bacteria are able to seize the free atmospheric nitrogen, converting it into nitrite and nitrates. we have also learned that they can act in connection with legumes and some other plants, enabling them to fix atmospheric nitrogen and store it m their roots. by these two means the nitrogen ingredient in the soil is prevented from becoming exhausted by the processes of dissipation constantly going on. further, by some such agency must we imagine the original nitrogen soil ingredient to have been derived. such an organic agency is the only one yet discerned which appears to have been efficient in furnishing virgin soil with its nitrates, and we must therefore look upon bacteria as essential to the original fertility of the soil. but in another direction still does the farmer depend directly upon bacteria the most important factor in the fertility of the soil is the part of it called humus. this humus is very complex, and never alike in different soils it contains nitrogen compounds in abundance, together with sulphates, phosphates, sugar, and many other substances. it is this which makes the garden soil different from sand, or the rich soil different from the sterile soil. if the soil is cultivated year after year, its food ingredients are slowly but surely exhausted. something is taken from the humus each year, and unless this be replaced the soil ceases to be able to support life. to keep up a constant yield from the soil the farmer understands that he must apply fertilizers more or less constantly. this application of fertilizers is simply feeding the crops. some of these fertilizers the farmer purchases, and knows little or nothing as to their origin. the most common method of feeding the crops is, however, by the use of ordinary barnyard manure. the reason why this material contains plant food we can understand, since it is made of the undigested part of food, together with all the urea and other excretions of animals, and contains, therefore, besides various minerals, all of the nitrogenous waste of animal life. these secretions are not at first fit for plant food. the farmer has learned by experience that such excretions, before they are of any use on his fields, must undergo a process of slow change, which is sometimes called ripening. fresh manure is sometimes used on the fields, but it is only made use of by the plants after the ripening process has occurred. fresh animal excretions are of little or no value as a fertilizer. the farmer, therefore, commonly allows it to remain in heaps for some time, and it undergoes a slow change, which gradually converts it into a condition in which it can be used by plants. this ripening is readily explained by the facts already considered the fresh animal secretions consist of various highly complex compounds of nitrogen, and the ripening is a process of their decomposition. the proteids are broken to pieces, and their nitrogen elements reduced to the form of nitrates, leucin, etc, or even to ammonia or free nitrogen. further, a second process occurs, the process of oxidation of these nitrogen compounds already noticed, and the ammonia and nitrites resulting from the decomposition are built into nitrates. in short, in this ripening manure the processes noticed in the first part of this chapter are taking place, by which the complex nitrogenous bodies are first reduced and then oxidized to form plant food. the ripening of manure is both an analytical and a synthetical process. by the analysis, proteids and other bodies are broken into very simple compounds, some of them, indeed, being dissipated into the air, but other portions are retained and then oxidized, and these latter become the real fertilizing materials. through the agency of bacteria the compost heap thus becomes the great source of plant food to the farmer. into this compost heap he throws garbage, straw, vegetable and animal substances in general, or any organic refuse which may be at hand. the various bacteria seize it all, and cause the decomposition which converts it into plant food again. the rotting of the compost heap is thus a gigantic cultivation of bacteria. this knowledge of the ripening process is further teaching the farmer how to prevent waste. in the ordinary decomposition of the compost heap not an inconsiderable portion of the nitrogen is lost in the air by dissipation as ammonia or free nitrogen. even his nitrates may be thus lost by bacterial action. this portion is lost to the farmer completely, and he can only hope to replace it either by purchasing nitrates in the form of commercial fertilizers, or by reclaiming it from the air by the use of the bacterial agencies already noticed. with the knowledge now at his command he is learning to prevent this waste. in the decomposition one large factor of loss is the ammonia, which, being a gas, is readily dissipated into the air. knowing this common result of bacterial action, the scientist has told the farmer that, by adding certain common chemicals to his decomposing manure heap, chemicals which will readily unite with ammonia, he may retain most of the nitrogen in this heap in the form of ammonia salts, which, once formed, no longer show a tendency to dissipate into the air. ordinary gypsum, or superphosphates, or plaster will readily unite with ammonia, and these added to the manure heap largely counteract the tendency of the nitrogen to waste, thus enabling the farmer to put back into his soil most of the nitrogen which was extracted from it by his crops and then used by his stock. his vegetable crops raise the nitrates into proteids. his animals feed upon the proteids, and perform his work or furnish him with milk. then his bacteria stock take the excreted or refuse nitrogen, and in his manure heap turn it back again into nitrates ready to begin the circle once more. this might go on almost indefinitely were it not for two facts, the farmer sends nitrogenous material off his farm in the milk or grains or other nitrogenous products, which he sells, and the decomposition processes, as we have seen, dissipate some of the nitrogen into the air as free nitrogen. to meet this emergency and loss the farmer has another method of enriching the soil, again depending upon bacteria. this is the socalled green manuring. here certain plants which seize nitrogen from the air are cultivated upon the field to be fertilized, and, instead of harvesting a crop, it is ploughed into the soil. or perhaps the tops may be harvested, the rest being ploughed into the soil. the vegetable material thus ploughed in lies over a season and enriches the soil. here the bacteria of the soil come into play in several directions. first, if the crop sowed be a legume, the soil bacteria assist it to seize the nitrogen from the air. the only plants which are of use in this green manuring are those which can, through the agency of bacteria, obtain nitrogen from the air and store it in their roots. second, after the crop is ploughed into the soil various decomposing bacteria seize upon it, pulling the compounds to pieces. the carbon is largely dissipated into the air as carbonic dioxide, where the next generation of plants can get hold of it. the minerals and the nitrogen remain in the soil. the nitrogenous portions go through the same series of decomposition and synthetical changes already described, and thus eventually the nitrogen seized from the air by the combined action of the legumes and the bacteria is converted into nitrates, and will serve for food for the next set of plants grown on the same soil. here is thus a practical method of using the nitrogen assimilation powers of bacteria, and reclaiming nitrogen from the air to replace that which has been lost. thus it is that the farmer's nitrogen problem of the fertile soil appears to resolve itself into a proper handling of bacteria. these organisms have stocked his soil in the first place. they convert all of his compost heap wastes into simple bodies, some of which are changed into plant foods, while others are at the same time lost. lastly, they may be made to reclaim this lost nitrogen, and the fanner, so soon as he has requisite knowledge of these facts, will be able to keep within his control the supply of this important element. the continued fertility of the soil is thus a gift from the bacteria. bacteria as sources of trouble to the farmer. while the topics already considered comprise the most important factors in agricultural bacteriology, the farmer's relations to bacteria do not end here. these organisms come incidentally into his life in many ways. they are not always his aids as they are in most of the instances thus far cited. they produce disease in his cattle, as will be noticed in the next chapter. bacteria are agents of decomposition, and they are just as likely to decompose material which the farmer wishes to preserve as they are to decompose material which the farmer desires to undergo the process of decay. they are as ready to attack his fruits and vegetables as to ripen his cream. the skin of fruits and vegetables is a moderately good protection of the interior from the attack of bacteria; but if the skin be broken in any place, bacteria get in and cause decay, and to prevent it the farmer uses a cold cellar. the bacteria prevent the farmer from preserving meats for any length of time unless he checks their growth in some way. they get into the eggs of his fowls and ruin them. their troublesome nature in the dairy in preventing the keeping of milk has already been noticed. if he plants his seeds in very moist, damp weather, the soil bacteria cause too rapid a decomposition of the seeds and they rot in the ground instead of sprouting. they produce disagreeable odours, and are the cause of most of the peculiar smells, good and bad, around the barn. they attack the organic matter which gets into his well or brook or pond, decomposing it, filling the water with disagreeable and perhaps poisonous products which render it unfit to drink. they not only aid in the decay of the fallen tree in his forests; but in the same way attack the timber which he wishes to preserve, especially if it is kept in a moist condition. thus they contribute largely to the gradual destruction of wooden structures. it is therefore the presence of these organisms which forces him to dry his hay, to smoke his hams, to corn his beef, to keep his fruits and vegetables cool and prevent skin bruises, to ice his dairy, to protect his timber from rain, to use stone instead of wooden foundations for buildings, etc. in general, when the farmer desires to get rid of any organic refuse, he depends upon bacteria, for they are his sole agents (aside from fire) for the final destruction of organic matter. when he wishes to convert waste organic refuse into fertilizing material, he uses the bacteria of his compost heap. on the other hand, whenever he desires to preserve organic material, the bacteria are the enemies against which he must carefully guard. thus the farmer's life from year's end to year's end is in most intimate association with bacteria. upon them he depends to insure the continued fertility of his soil and the constant continued production of good crops. upon them he depends to turn into plant food all the organic refuse from his house or from his barn. upon them he depends to replenish his stock of nitrogen. it is these organisms which furnish his dairy with its butter flavours and with the taste of its cheese. but, on the other hand, against them he must be constantly alert. all his food products must be protected from their ravages. a successful farmer's life, then, largely resolves itself into a skilful management of bacterial activity. to aid them in destroying or decomposing everything which he does not desire to preserve, and to prevent their destroying the organic material which he wishes to keep for future use, is the object of a considerable portion of farm labour; and the most successful farmer to-day, and we believe the most successful farmer of the future, is the one who most intelligently and skilfully manipulates these gigantic forces furnished him by the growth of his microscopical allies. relation of bacteria to coal. another one of nature's processes in which bacteria have played an important part is in the formation of coal. it is unnecessary to emphasize the importance of coal in modern civilization. aside from its use as fuel, upon which civilization is dependent, coal is a source of an endless variety of valuable products. it is the source of our illuminating gas, and ammonia is one of the products of the gas manufacture. from the coal also comes coal tar, the material from which such a long series of valuable materials, as aniline colours, carbolic acid, etc, is derived. the list of products which we owe to coal is very long, and the value of this material is hardly to be overrated. in the preparation of these ingredients from coal bacteria do not play any part. most of them are derived by means of distillation. but when asked for the agents which have given us the coal of the coal beds, we shall find that here, too, we owe a great debt to bacteria. coal, as is well known, has come from the accumulation of the luxuriant vegetable growth of the past geological ages. it has therefore been directly furnished us by the vegetation of the green plants of the past, and, in general, it represents so much carbonic dioxide which these plants have extracted from the atmosphere. but while the green plants have been the active agents in producing this assimilation, bacteria have played an important part in coal manufacture in two different directions. the first appears to be in furnishing these plants with nitrogen. without a store of fixed nitrogen in the soil these carboniferous plants could not have grown. this matter has already been considered. we have no very absolute knowledge as to the agency of bacteria in furnishing nitrogen for this vegetation in past ages, but there is every reason to believe that in the past, as in the present, the chief source of organic nitrogen has been from the atmosphere and derived from the atmosphere through the agency of bacteria. in the absence of any other known factor we may be pretty safe in the assumption that bacteria played an important part in this nitrogen fixation, and that bacteria must therefore be regarded as the agents which have furnished us the nitrogen stored in the coal. but in a later stage of coal formation bacteria have contributed more directly to the formation of coal. coal is not simply accumulated vegetation. the coal of our coal beds is very different in its chemical composition from the wood of the trees. it contains a much higher percentage of carbon and a lower percentage of hydrogen and oxygen than ordinary vegetable substances. the conversion of the vegetation of the carboniferous ages into coal was accompanied by a gradual loss of hydrogen and a consequent increase in the percentage of carbon. it is this change that has added to the density of the substance and makes the greater value of coal as fuel. there is little doubt now as to the method by which this woody material of the past has been converted into coal. the same process appears to be going on in a similar manner to-day in the peat beds of various northern countries. the fallen vegetation, trees, trunks, branches, and leaves, accumulate in masses, and, when the conditions of moisture and temperature are right, begin to undergo a fermentation. ordinarily this action of bacteria, as already noticed, produces an almost complete though slow oxidation of the carbon, and results in the total decay of the vegetable matter. but if the vegetable mass be covered by water and mud under proper conditions of moisture and temperature, a different kind of fermentation arises which does not produce such complete decay. the covering of water prevents the access of oxygen to the fermenting mass, an oxidation of the carbon is largely prevented, and the vegetable matter slowly changes its character. under the influence of this slow fermentation, aided, probably by pressure, the mass becomes more and more solid and condensed, its woody character becomes less and less distinct, and there is a gradual loss of the hydrogen and the oxygen. doubtless there is a loss of carbon also, for there is an evolution of marsh gas which contains carbon. but, in this slow fermentation taking place under the water in peat bogs and marshes the carbon loss is relatively small; the woody material does not become completely oxidized, as it does in free operations of decay. the loss of hydrogen and oxygen from the mass is greater than that of carbon, and the percentage of carbon therefore increases. this is not the ordinary kind of fermentation that goes on in vegetable accumulations. it requires special conditions and possibly special kinds of fermenting organisms. peat is not formed in all climates. in warm regions, or where the woody matter is freely exposed to the air, the fermentation of vegetable matter is more complete, and it is entirely destroyed by oxidation. it is only in colder regions and when covered with water that the destruction of the organic matter stops short of decay. but such incomplete fermentation is still going on in many parts of the world, and by its means vegetable accumulations are being converted into peat. this formation of peat appears to be a first step in the formation of denser coal. by a continuation of the same processes the mass becomes still more dense and solid. as we pass from the top to the bottom of such an accumulation of peat, we find it becoming denser and denser, and at the bottom it is commonly of a hard consistence, brownish in colour, and with only slight traces of the original woody structure. such material is called lignite. it contains a higher percentage of carbon than peat, but a lower percentage than coal, and is plainly a step in coal formation. but the process goes on, the hydrogen and oxygen loss continuing until there is finally produced true coal. if this is the correct understanding of the formation of coal, we see that we have plainly a process in which bacterial life has had a large and important share. we are, of course, densely ignorant of the exact processes going on. we know nothing positively as to the kind of microorganisms which produce this slow, peculiar fermentation. as yet, the fermentation going on in the formation of the peat has not been studied by the bacteriologists, and we do not know from direct experiment that it is a matter of bacterial action. it has been commonly regarded as simply a slow chemical change, but its general similarity to other fermentative processes is so great that we can have little hesitation in attributing it to micro-organisms, and doubtless to some forms of plants allied to bacteria. there is no reason for doubting that bacteria existed in the geological ages with essentially the same powers as they now possess, and to some forms of bacteria which grow in the absence of oxygen can we probably attribute the slow change which has produced coal. here, then, is another great source of wealth in nature for which we are dependent upon bacteria. while, of course, water and pressure were very essential factors in the deposition of coal, it was a peculiar kind of fermentation occurring in the vegetation that brought about the chemical changes in it which resulted in its transformation into coal. the vegetation of the carboniferous age was dependent upon the nitrogen fixed by the bacteria, and to these organisms also do we owe the fact that this vegetation was stored for us in the rocks. chapter v. parasitic bacteria and their relation to disease. perhaps the most universally known fact in regard to bacteria is that they are the cause of disease. it is this fact that has made them objects of such wide interest. this is the side of the subject that first attracted attention, has been most studied, and in regard to which there has been the greatest accumulation of evidence. so persistently has the relation of bacteria to disease been discussed and emphasized that the majority of readers are hardly able to disassociate the two. to most people the very word bacteria is almost equivalent to disease, and the thought of swallowing microbes in drinking water or milk is decidedly repugnant and alarming. in the public mind it is only necessary to demonstrate that an article holds bacteria to throw it under condemnation. we have already seen that bacteria are to be regarded as agents for good, and that from their fundamental relation to plant life they must be looked upon as our friends rather than as our enemies. it is true that there is another side to the story which relates to the parasitic species. these parasitic forms may do us direct or indirect injury. but the species of bacteria which are capable of doing us any injury, the pathogenic bacteria, are really very few compared to the great host of species which are harmless. a small number of species, perhaps a score or two, are pathogenic, while a much larger number, amounting to hundreds and perhaps thousands of species, are perfectly harmless. this latter class do no injury even though swallowed by man in thousands. they are not parasitic, and are unable to grow in the body of man. their presence is entirely consistent with the most perfect health, and, indeed, there are some reasons for believing that they are sometimes directly beneficial to health. it is entirely unjust to condemn all bacteria because a few chance to produce mischief. bacteria in general are agents for good rather than ill. there are, however, some species which cause mankind much trouble by interfering in one way or another with the normal processes of life. these pathogenic bacteria, or disease germs, do not all act alike, but bring about injury to man in a number of different ways. we may recognise two different classes among them, which, however, we shall see are connected by intermediate types. these two classes are, first, the pathogenic bacteria, which are not strictly parasitic but live free in nature; and, second, those which live as true parasites in the bodies of man or other animals. to understand the real relation of these two classes, we must first notice the method by which bacteria in general produce disease. method by which bacteria produce disease. since it was first clearly recognised that certain species of bacteria have the power of producing disease, the question as to how they do so has ever been a prominent one even if they do grow in the body, why should their presence give rise to the symptoms characterizing disease? various answers to this question have been given in the past it has been suggested that in their growth they consume the food of the body and thus exhaust it, that they produce an oxidation of the body tissues, or that they produce a reduction of these tissues, or that they mechanically interfere with the circulation none of these suggestions have proved of much value another view was early advanced, and has stood the test of time. this claim is that the bacteria while growing in the body produce poisons, and these poisons then have a direct action on the body we have already noticed that bacteria during their growth in any medium produce a large number of biproducts of decomposition. we noticed also that among these biproducts there are some which have a poisonous nature; so poisonous are they that when inoculated into the body of an animal they may produce poisoning and death. we have only to suppose that the pathogenic bacteria, when growing as parasites in man, produce such poisons, and we have at once an explanation of the method by which they give rise to disease. this explanation of germ disease is more than simple theory. it has been in many cases clearly demonstrated. it has been found that the bacteria which cause diphtheria, tetanus, typhoid, tuberculosis, and many other diseases, produce, even when growing in common culture media, poisons which are of a very violent nature. these poisons when inoculated into the bodies of animals give rise to much the same symptoms as the bacteria do themselves when growing as parasites in the animals. the chief difference in the results from inoculating an animal with the poison and with the living bacteria is in the rapidity of the action. when the poison is injected the poisoning symptoms are almost immediately seen, but when the living bacteria are inoculated the effect is only seen after several days or longer, not, in short, until the inoculated bacteria have had time enough to grow in the body and produce the poison in quantity. it has not by any means been shown that all pathogenic germs produce their effect in this way, but it has been proved to be the real method in quite a number of cases, and is extremely probable in others. while some bacteria perhaps produce results by a different method, we must recognise the production of poisons as at all events the common direct cause of the symptoms of disease. this explanation will enable us more clearly to understand the relation of different bacteria to disease. pathogenic germs which are not strictly parasitic recognising that bacteria may produce poisons, we readily see that it is not always necessary that they should be parasites in order to produce trouble. in their ordinary growth in nature such bacteria will produce no trouble the poisons will be produced in decaying material but will seldom be taken into the human body. these poisons, produced in the first stages of putrefaction, are oxidized by further stages of decomposition into harmless products. but should it happen that some of these bacteria obtained a chance to grow vigorously for a while in organic products that are subsequently swallowed as man's food, it is plain that evil results might follow. if such food is swallowed by man after the bacteria have produced their poisonous bodies, it will tend to produce an immediate poisoning of his system. the effect may be sudden and severe if considerable quantity of the poisonous material is swallowed, or slight but protracted if small quantities are repeatedly consumed in food. such instances are not uncommon. well-known examples are cases of ice-cream poisoning, poisoning from eating cheese or from drinking milk, or in not a few instances from eating fish or meats within which bacteria have had opportunity for growth. in all these cases the poison is swallowed in quantity sufficient to give rise quickly to severe symptoms, sometimes resulting fatally, and at other times passing off as soon as the body succeeds in throwing off the poisons. in other cases still, however, the amount of poison swallowed may be very slight, too slight to produce much effect unless the same be consumed repeatedly. all such trouble may be attributed to fermented or partly decayed food. it is difficult to distinguish such instances from others produced in a slightly different way, as follows: it may happen that the bacteria which grow in food products continue to grow in the food even after it is swallowed and has passed into the stomach or intestines. this appears particularly true of milk bacteria. under these conditions the bacteria are not in any proper sense parasitic, since they are simply living in and feeding upon the same food which they consume outside the body, and are not feeding upon the tissues of man. the poisons which they produce will continue to be developed as long as the bacteria continue to grow, whether in a milk pail or a human stomach. if now the poisons are absorbed by the body, they may produce a mild or severe disease which will be more or less lasting, continuing perhaps as long as the same food and the same bacteria are supplied to the individual. the most important disease of this class appears to be the dreaded cholera infantum, so common among infants who feed upon cow's milk in warm weather. it is easy to understand the nature of this disease when we remember the great number of bacteria in milk, especially in hot weather, and when we remember that the delicate organism of the infant will be thrown at once into disorder by slight amounts of poison which would have no appreciable effect upon the stronger adult. we can easily understand, further, how the disease readily yields to treatment if care is taken to sterilize the milk given to the patient. we do not know to-day the extent of the troubles which are produced by bacteria of this sort. they will, of course, be chiefly connected with our food products, and commonly, though not always, will affect the digestive functions. it is probable that many of the cases of summer diarrhoea are produced by some such cause, and if they could be traced to their source would be found to be produced by bacterial poisons swallowed with food or drink, or by similar poisons produced by bacteria growing in such food after it is swallowed by the individual. in hot weather, when bacteria are so abundant everywhere and growing so rapidly, it is impossible to avoid such dangers completely without exercising over all food a guard which would be decidedly oppressive. it is well to bear in mind, however, that the most common and most dangerous source of such poisons is milk or its products, and for this reason one should hesitate to drink milk in hot weather unless it is either quite fresh or has been boiled to destroy its bacteria. pathogenic bacteria which are true parasites. this class of pathogenic bacteria includes those which actually invade the body and feed upon its tissues instead of living simply upon swallowed food. it is difficult, however, to draw any sharp line separating the two classes. the bacteria which cause diphtheria (fig. 28), for instance, do not really invade the body. they grow in the throat, attached to its walls, and are confined to this external location or to the superficial tissues. this bacillus is, in short, only found in the mouth and throat, and is practically confined to the so-called false membranes. it never enters any of the tissues of the body, although attached to the mucous membrane. it grows vigorously in this membrane, and there secretes or in some way produces extremely violent poisons. these poisons are then absorbed by the body and give rise to the general symptoms of the disease. much the same is true of the bacillus which causes tetanus or lockjaw (fig. 29). this bacillus is commonly inoculated into the flesh of the victim by a wound made with some object which has been lying upon the earth where the bacillus lives. the bacillus grows readily after being inoculated, but it is localized at the point of the wound, without invading the tissue to any extent. it produces, however, during its growth several poisons which have been separated and studied. among them are some of the most violent poisons of which we have any knowledge. while the bacillus grows in the tissues around the wound it secretes these poisons, which are then absorbed by the body generally. their poisoning effects produce the violent symptoms of the disease. of much the same nature is asiatic cholera. this is caused by a bacillus which is able to grow rapidly in the intestines, feeding perhaps in part on the food in the intestines and perhaps in part upon the body secretions. to a slight extent also it appears to be able to invade the tissues of the body, for the bacilli are found in the walls of the intestines. but it is not a proper parasite, and the fatal disease it produces is the result of the absorption of the poisons secreted in the intestines. it is but a step from this to the true parasites. typhoid fever, for example, is a disease produced by bacteria which grow in the intestines, but which also invade the tissues more extensively than the cholera germs (fig. 30). they do not invade the body generally, however, but become somewhat localized in special glands like the liver, the spleen, etc. even here they do not appear to find a very favourable condition, for they do not grow extensively in these places. they are likely to be found in the spleen in small groups or centres, but not generally distributed through it. wherever they grow they produce poison, which has been called typhotoxine, and it is this poison chiefly which gives rise to the fever. quite a considerable number of the pathogenic germs are, like the typhoid bacillus, more or less confined to special places. instead of distributing themselves through the body after they find entrance, they are restricted to special organs. the most common example of a parasite of this sort is the tuberculosis bacillus, the cause of consumption, scrofula, white swelling, lupus, etc. (fig. 31). although this bacillus is very common and is able to attack almost any organ in the body, it is usually very restricted in growth. it may become localized in a small gland, a single joint, a small spot in the lungs, or in the glands of the mesentery, the other parts of the body remaining free from infection. not infrequently the whole trouble is thus confined to such a small locality that nothing serious results. but in other instances the bacilli may after a time slowly or rapidly distribute themselves from these centres, attacking more and more of the body until perhaps fatal results follow in the end. this disease is therefore commonly of very slow progress. again, we have still other parasites which are not thus confined, but which, as soon as they enter the body, produce a general infection, attacking the blood and perhaps nearly all tissues simultaneously. the most typical example of this sort is anthrax or malignant pustule, a disease fortunately rare in man (fig. 32). here the bacilli multiply in the blood, and very soon a general and fatal infection of the whole body arises, resulting from the abundance of the bacilli everywhere. some of the obscure diseases known as blood poisoning appear to be of the same general nature, these diseases resulting from a very general invasion of the whole body by certain pathogenic bacteria. in general, then, we see that the so-called germ diseases result from the action upon the body of poisons produced by bacterial growth. differences in the nature of these poisons produce differences in the character of the disease, and differences in the parasitic powers of the different species of bacteria produce wide differences in the course of the diseases and their relation to external phenomena. what diseases are due to bacteria? it is, of course, an extremely important matter to determine to what extent human diseases are caused by bacteria. it is not easy, nor indeed possible, to do this to-day with accuracy. it is no easy matter to prove that any particular disease is caused by bacteria. to do this it is necessary to find some particular bacterium present in all cases of the disease; to find some method of getting it to grow outside the body in culture media; to demonstrate its absence in healthy animals, or healthy human individuals if it be a human disease; and, finally, to reproduce the disease in healthy animals by inoculating them with the bacterium. all of these steps of proof present difficulties, but especially the last one. in the study of animals it is comparatively easy to reproduce a disease by inoculation. but experiments upon man are commonly impossible, and in the case of human diseases it is frequently very difficult or impossible to obtain the final test of the matter. after finding a specific bacterium associated with a disease, it is usually possible to experiment with it further upon animals only. but some human diseases do not attack animals, and in the case of diseases that may be given to animals it is frequently uncertain whether the disease produced in the animal by such inoculation is identical with the human disease in question, owing to the difference of symptoms in the different animals. as a consequence, the proof of the germ nature of different diseases varies all the way from absolute demonstration to mere suspicion. to give a complete and correct list of the diseases caused by bacteria, or to give a list of the bacteria species pathogenic to man, is therefore at present impossible. the difficulty of giving such a list is rendered greater from the fact that we have in recent years learned that the same species of pathogenic bacterium may produce different results under different conditions. when the subject of germ disease was first studied and the connection between bacteria and disease was first demonstrated, it was thought that each particular species of pathogenic bacteria produced a single definite disease; and conversely, each germ disease was supposed to have its own definite species of bacterium as its cause. recent study has shown, however, that this is not wholly true. it is true that some diseases do have such a definite relation to definite bacteria. the anthrax germ, for example, will always produce anthrax, no matter where or how it is inoculated into the body. so, also, in quite a number of other cases distinct specific bacteria are associated with distinct diseases. but, on the other hand, there are some pathogenic bacteria which are not so definite in their action, and produce different results in accordance with circumstances, the effect varying both with the organ attacked and with the condition of the individual. for instance, a considerable number of different types of blood poisoning, septicaemia, pyaemia, gangrene, inflammation of wounds, or formation of pus from slight skin wounds--indeed, a host of miscellaneous troubles, ranging all the way from a slight pus formation to a violent and severe blood poisoning--all appear to be caused by bacteria, and it is impossible to make out any definite species associated with the different types of these troubles. there are three common forms of so-called pus cocci, and these are found almost indiscriminately with various types of inflammatory troubles. moreover, these species of bacteria are found with almost absolute constancy in and around the body, even in health. they are on the clothing, on the skin, in the mouth and alimentary canal. here they exist, commonly doing no harm. they have, however, the power of doing injury if by chance they get into wounds. but their power of doing injury varies both with the condition of the individual and with variations in the bacteria themselves. if the individual is in a good condition of health these bacteria have little power of injuring him even when they do get into such wounds, while at times of feeble vitality they may do much more injury, and take the occasion of any little cut or bruise to enter under the skin and give rise to inflammation and pus. some people will develop slight abscesses or slight inflammations whenever the skin is bruised, while with others such bruises or cuts heal at once without trouble. both are doubtless subject to the same chance of infection, but the one resists, while the other does not. in common parlance, we say that such a tendency to abscesses indicates a bad condition of the blood--a phrase which means nothing. further, we find that the same species of bacterium may have varying powers of producing disease at different times. some species are universal inhabitants of the alimentary canal and are ordinarily harmless, while under other conditions of unknown character they invade the tissues and give rise to a serious and perhaps fatal disease. we may thus recognise some bacteria which may be compared to foreign invaders, while others are domestic enemies. the former, like the typhoid bacillus, always produce trouble when they succeed in entering the body and finding a foothold. the latter, like the normal intestinal bacilli, are always present but commonly harmless, only under special conditions becoming troublesome. all this shows that there are other factors in determining the course of a disease, or even the existence of a disease, than the simple presence of a peculiar species of pathogenic bacterium. from the facts just stated it will be evident that any list of germ diseases will be rather uncertain. still, the studies of the last twenty years or more have disclosed some definite relations of bacteria and disease, and a list of the diseases more or less definitely associated with distinct species of bacteria is of interest. such a list, including only well-known diseases, is as follows: name of disease. name of bacterium producing the disease. anthrax (malignant pustule). bacillus anthracis. cholera. spirillum cholera: asiaticae croupous pneumonia. micrococcus pneumonia crouposa. diphtheria. bacillus diphtheria. glanders. bacillus mallei. gonorrhoea. micrococcus gonorrhaeae influenza. bacillus of influenza. leprosy. bacillus leprae. relapsing fever. spirillum obermeieri. tetanus (lockjaw). bacillus tetani. tuberculosis (including consumption, scrofula, etc.) bacillus tuberculosis. typhoid fever. bacillus typhi abdominalis. various wound infections, including septicaemia, pyaemia, acute abscesses, ulcers, erysipelas, etc., are produced by a few forms of micrococci, resembling each other in many points but differing slightly. they are found almost indiscriminately in any of these wound infections, and none of them appears to have any definite relation to any special form of disease unless it be the micrococcus of erysipelas. the common pus micrococci are grouped under three species, staphylococcus pyogenes aureus, staphylococcus pyogenes, and streptococcus pyogenes. these three are the most common, but others are occasionally found. in addition to these, which may be regarded as demonstrated, the following diseases are with more or less certainty regarded as caused by distinct specific bacteria: bronchitis, endocarditis, measles, whooping-cough, peritonitis, pneumonia, syphilis. still another list might be given of diseases whose general nature indicates that they are caused by bacteria, but in connection with which no distinct bacterium has yet been found. as might be expected also, a larger list of animal diseases has been demonstrated to be caused by these organisms. in addition, quite a number of species of bacteria have been found in such material as faeces, putrefying blood, etc., which have been shown by experiment to be capable of producing diseases in animals, but in regard to which we have no evidence that they ever do produce actual disease under any normal conditions. these may contribute, perhaps, to the troubles arising from poisonous foods, but can not be regarded as disease germs proper. variability of pathogenic powers. as has already been stated, our ideas of the relation of bacteria to disease have undergone quite a change since they were first formulated, and we recognise other factors influencing disease besides the actual presence of the bacterium. these we may briefly consider under two heads, viz., variation in the bacterium, and variation in the susceptibility of the individual. the first will require only a brief consideration. that the same species of pathogenic bacteria at different times varies in its powers to produce disease has long been known. various conditions are known to affect thus the virulence of bacteria. the bacillus which is supposed to give rise to pneumonia loses its power to produce the disease after having been cultivated for a short time in ordinary culture media in the laboratory. this is easily understood upon the suggestion that it is a parasitic bacillus and does not thrive except under parasitic conditions. its pathogenic powers can sometimes be restored by passing it again through some susceptible animal. one of the most violent pathogenic bacteria is that which produces anthrax, but this loses its pathogenic powers if it is cultivated for a considerable period at a high temperature. the micrococcus which causes fowl cholera loses its power if it be cultivated in common culture media, care being taken to allow several days to elapse between the successive inoculations into new culture flasks. most pathogenic bacteria can in some way be so treated as to suffer a diminution or complete loss of their powers of producing a fatal disease. on the other hand, other conditions will cause an increase in the virulence of a pathogenic germ. the virus which produces hydrophobia is increased in violence if it is inoculated into a rabbit and subsequently taken from the rabbit for further inoculation. the fowl cholera micrococcus, which has been weakened as just mentioned, may be restored to its original violence by inoculating it into a small bird, like a sparrow, and inoculating a second bird from this. a few such inoculations will make it as active as ever. these variations doubtless exist among the species in nature as well as in artificial cultures. the bacteria which produce the various wound infections and abscesses, etc., appear to vary under normal conditions from a type capable of producing violent and fatal blood poisoning to a type producing only a simple abscess, or even to a type that is entirely innocuous. it is this factor, doubtless, which in a large measure determines the severity of any epidemic of a bacterial contagious disease. susceptibility of the individual. the very great modification of our early views has affected our ideas as to the power which individuals have of resisting the invasion of pathogenic bacteria. it has from the first been understood that some individuals are more susceptible to disease than others, and in attempting to determine the significance of this fact many valuable and interesting discoveries have been made. after the exposure to the disease there follows a period of some length in which there are no discernible effects. this is followed by the onset of the disease and its development to a crisis, and, if this be passed, by a recovery. the general course of a germ disease is divided into three stages: the stage of incubation, the development of the disease, and the recovery. the susceptibility of the body to a disease may be best considered under the three heads of invasion, resistance, recovery. means of invasion.--in order that a germ disease should arise in an individual, it is first necessary that the special bacterium which causes the disease should get into the body. there are several channels through which bacteria can thus find entrance; these are through the mouth, through the nose, through the skin, and occasionally through excretory ducts. those which come through the mouth come with the food or drink which we swallow; those which enter through the nose must be traced to the air; and those which enter through the skin come in most cases through contact with some infected object, such as direct contact with the body of an infected person or his clothing or some objects he has handled, etc. occasionally, perhaps, the bacteria may get into the skin from the air, but this is certainly uncommon and confined to a few diseases. there are here two facts of the utmost importance for every one to understand: first, that the chance of disease bacteria being carried to us through the air is very slight and confined to a few diseases, such as smallpox, tuberculosis, scarlet fever; etc., and, secondly, that the uninjured skin and the uninjured mucous membrane also is almost a sure protection against the invasion of the bacteria. if the skin is whole, without bruises or cuts, bacteria can seldom, if ever, find passage through it. these two facts are of the utmost importance, since of all sources of infection we have the least power to guard against infection through the air, and since of all means 'of entrance we can guard the skin with the greatest difficulty. we can easily render food free from pathogenic bacteria by heating it. the material we drink can similarly be rendered harmless, but we can not by any known means avoid breathing air, nor is there any known method of disinfecting the air, and it is impossible for those who have anything to do with sick persons to avoid entirely having contact either with the patient or with infected clothing or utensils. from the facts here given it will be seen that the individual's susceptibility to disease produced by parasitic bacteria will depend upon his habits of cleanliness, his care in handling infectious material, or care in cleansing the hands after such handling, upon his habit of eating food cooked or raw, and upon the condition of his skin and mucous membranes, since any kind of bruises will increase susceptibility. slight ailments, such as colds, which inflame the mucous membrane, will decrease its resisting power and render the individual more susceptible to the entrance of any pathogenic germs should they happen to be present. sores in the mouth or decayed teeth may in the same way be prominent factors in the individual's susceptibility. thus quite a number of purely physical factors may contribute to an individual's susceptibility. resisting power of the body.--even after the bacteria get into the body it is by no means certain that they will give rise to disease, for they have now a battle to fight before they can be sure of holding their own. it is now, indeed, that the actual conflict between the powers of the body and these microscopic invaders begins. after they have found entrance into the body the bacteria have arrayed against them strong resisting forces of the human organism, endeavouring to destroy and expel them. many of them are rapidly killed, and sometimes they are all destroyed without being able to gain a foothold. in such cases, of course, no trouble results. in other cases the body fails to overcome the powers of the invaders and they eventually multiply rapidly. in this struggle the success of the invaders is not necessarily a matter of numbers. they are simply struggling to gain a position in the body, where they can feed and grow. a few individuals may be entirely sufficient to seize such a foothold, and then these by multiplying may soon become indefinitely numerous. to protect itself, therefore, the human body must destroy every individual bacterium, or at least render them all incapable of growth. their marvellous reproductive powers give the bacteria an advantage in the battle. on the other hand, it takes time even for these rapidly multiplying beings to become sufficiently numerous to do injury. there is thus an interval after their penetration into the body when these invaders are weak in numbers. during this interval--the period of incubation--the body may organize a resistance sufficient to expel them. we do not as yet thoroughly understand the forces which the human organism is able to array against these invading foes. some of its methods of defence are, however, already intelligible to us, and we know enough, at all events, to give us an idea of the intensity of the conflict that is going on, and of the vigorous and powerful forces which the human organism is able to bring against its invading enemies. in the first place, we notice that a majority of bacteria are utterly unable to grow in the human body even if they do find entrance. there are known to bacteriologists to-day many hundreds, even thousands of species, but the vast majority of these find in the human tissues conditions so hostile to their life that they are utterly unable to grow therein. human flesh or human blood will furnish excellent food for them if the individual be dead, but living human flesh and blood in some way exerts a repressing influence upon them which is fatal to the growth of a vast majority of species. some few species, however, are not thus destroyed by the hostile agencies of the tissues of the animal, but are capable of growing and multiplying in the living body. these alone are what constitute the pathogenic bacteria, since, of course, these are the only bacteria which can produce disease by growing in the tissues of an animal. the fact that the vast majority of bacteria can not grow in the living organism shows clearly enough that there are some conditions existing in the living tissue hostile to bacterial life. there can be little doubt, moreover, that it is these same hostile conditions, which enable the body to resist the attack of the pathogenic species in cases where resistance is successfully made. what are the forces arrayed against these invaders? the essential nature of the battle appears to be a production of poisons and counter poisons. it appears to be an undoubted fact that the first step in repelling these bacteria is to flood them with certain poisons which check their growth. in the blood and lymph of man and other animals there are present certain products which have a direct deleterious influence upon the growth of micro-organisms. the existence of these poisons is undoubted, many an experiment having directly attested to their presence in the blood of animals. of their nature we know very little, but of their repressing influence upon bacterial growth we are sure. they have been named alexines, and they are produced in the living tissue, although as to the method of their production we are in ignorance. by the aid of these poisons the body is able to prevent the growth of the vast majority of bacteria which get into its tissues. ordinary micro-organisms are killed at once, for these alexines act as antiseptics, and common bacteria can no more grow in the living body than they could in a solution containing other poisons thus the body has a perfect protection against the majority of bacteria. the great host of species which are found in water, milk, air, in our mouths or clinging to our skin, and which are almost omnipresent in nature, are capable of growing well enough in ordinary lifeless organic foods, but just as soon as they succeed in finding entrance into living human tissue their growth is checked at once by these antiseptic agents which are poured upon them. such bacteria are therefore not pathogenic germs, and not sources of trouble to human health. there are, on the other hand, a few species of bacteria which may be able to retain their lodgment in the body m spite of this attempt of the individual to get rid of them. these, of course, constitute the pathogenic species, or so called "disease germs". only such species as can overcome this first resistance can be disease germs, for they alone can retain their foothold in the body. but how do these species overcome the poisons, which kill the other harmless bacteria? they, as well as the harmless forms, find these alexines injurious to their growth, but in some way they are able to counteract the poisons. in this general discussion of poisons we are dealing with a subject which is somewhat obscure, but apparently the pathogenic bacteria are able to overcome the alexines of the body by producing in their turn certain other products which neutralize the alexines, thus annulling their action. these pathogenic bacteria, when they get into the body, give rise at once to a group of bodies which have been named lysines. these lysines are as mysterious to us as the alexines, but they neutralize the effect of the alexines and thus overcome the resistance the body offers to bacterial growth the invaders can now multiply rapidly enough to get a lasting foothold in the body and then soon produce the abnormal symptoms which we call disease pathogenic bacteria thus differ from the non-pathogenic bacteria primarily in this power of secreting products which can neutralize the ordinary effects of the alexines, and so overcome the body's normal resistance to their parasitic life. even if the bacteria do thus overcome the alexines the battle is not yet over, for the individual has another method of defence which is now brought into activity to check the growth of the invading organisms. this second method of resistance is by means of a series of active cells found in the blood, known as white blood-corpuscles (fig. 33 a, b). they are minute bits of protoplasm present in the blood and lymph in large quantities. they are active cells, capable of locomotion and able to crawl out of the blood-vessels not infrequently they are found to take into their bodies small objects with which they come in contact. one of their duties is thus to engulf minute irritating bodies which may be in the tissues, and to carry them away for excretion. they thus act as scavengers these corpuscles certainly have some agency in warding off the attacks of pathogenic bacteria very commonly they collect in great numbers in the region of the body where invading bacteria are found. such invading bacteria exist upon them a strong attraction, and the corpuscles leave the blood-vessels and sometimes form a solid phalanx completely surrounding the invading germs. their collection at these points may make itself seen externally by the phenomenon we call inflammation. there is no question that the corpuscles engage in conflict with the bacteria when they thus surround them. there has been not a little dispute, however, as to the method by which they carry on the conflict. it has been held by some that the corpuscles actually take the bacteria into their bodies, swallow them, as it were, and subsequently digest them (fig. 33 c, d, e). this idea gave rise to the theory of phagocytosis, and the corpuscles were consequently named phagocytes. the study of several years has, however, made it probable that this is not the ordinary method by which the corpuscles destroy the bacteria. according to our present knowledge the method is a chemical one. these cells, when they thus collect in quantities around the invaders, appear to secrete from their own bodies certain injurious products which act upon the bacteria much as do the alexines already mentioned. these new bodies have a decidedly injurious effect upon the multiplying bacteria; they rapidly check their growth, and, acting in union with the alexines, may perhaps entirely destroy them. after the bacteria are thus killed, the white blood-corpuscles may load themselves with their dead bodies and carry them away (fig. 33 d, e). sometimes they pass back into the blood stream and carry the bacteria to various parts of the body for elimination. not infrequently the white corpuscles die in the contest, and then may accumulate in the form of pus and make their way through the skin to be discharged directly. the battle between these phagocytes and the bacteria goes on vigorously. if in the end the phagocytes prove too strong for the invaders, the bacteria are gradually all destroyed, and the attack is repelled. under these circumstances the individual commonly knows nothing--of the matter. this conflict has taken place entirely without any consciousness on his part, and he may not even know that he has been exposed to the attack of the bacteria. in other cases the bacteria prove too strong for the phagocytes. they multiply too rapidly, and sometimes they produce secretions which actually drive the phagocytes away. commonly, as already noticed, the corpuscles are attracted to the point of invasion, but in some cases, when a particularly deadly and vigorous species of bacteria invades the body, the secretions produced by them are so powerful as actually to drive the corpuscles away. under these circumstances the invading hosts have a chance to multiply unimpeded, to distribute themselves over the body, and the disease rapidly follows as the result of their poisoning action on the body tissues. it is plain, then, that the human body is not helpless in the presence of the bacteria of disease, but that it is supplied with powerful resistant forces. it must not be supposed, however, that the outline of the action of these forces just given is anything like a complete account of the matter; nor must it be inferred that the resistance is in all respects exactly as outlined. the subject has only recently been an object of investigation, and we are as yet in the dark in regard to many of the facts. the future may require us to modify to some extent even the brief outline which has been given. but while we recognise this uncertainty in the details, we may be assured of the general facts. the living body has some very efficacious resistant forces which prevent most bacteria from growing within its tissues, and which in large measure may be relied upon to drive out the true pathogenic bacteria. these resistant forces are in part associated with the productions of body poisons, and are in part associated with the active powers of special cells which have been called phagocytes. the origin of the poisons and the exact method of action of the phagocytes we may well leave to the future to explain. these resisting powers of the body will vary with conditions. it is evident that they are natural powers, and they will doubtless vary with the general condition of vigour of the individual. robust health, a body whose powers are strong, well nourished, and vigorous, will plainly furnish the conditions for the greatest resistance to bacterial diseases. one whose bodily activities are weakened by poor nutrition can offer less resistance. the question whether one shall suffer from a germ disease is not simply the question whether he shall be exposed, or even the question whether the bacteria shall find entrance into his body. it is equally dependent upon whether he has the bodily vigour to produce alexines in proper quantity, or to summon the phagocytes in sufficient abundance and vigour to ward off the attack. we may do much to prevent disease by sanitation, which aids in protecting the individual from attack; but we must not forget that the other half of the battle is of equal importance, and hence we must do all we can to strengthen the resisting forces of the organism. recovery from germ diseases. these resisting forces are not always sufficient to drive off the invaders. the organisms may retain their hold in the body for a time and eventually break down the resistance. after this they may multiply unimpeded and take entire possession of the body. as they become more numerous their poisonous products increase and begin to produce direct poisoning effects on the body. the incubation period is over and the disease comes on. the disease now runs its course. it becomes commonly more and more severe until a crisis is reached. then, unless the poisoning is so severe that death occurs, the effects pass away and recovery takes place. but why should not a germ disease be always fatal? if the bacteria thus take possession of the body and can grow there, why do they not always continue to multiply until they produce sufficient poison to destroy the life of the individual? such fatal results do, of course, occur, but in by far the larger proportion of cases recovery finally takes place. plainly, the body must have another set of resisting forces which is concerned in the final recovery. although weakened by the poisoning and suffering from the disease, it does not yield the battle, but somewhat slowly organizes a new attack upon the invaders. for a time the multiplying bacteria have an unimpeded course and grow rapidly; but finally their further increase is checked, their vigour impaired, and after this they diminish in numbers and are finally expelled from the body entirely. of the nature of this new resistance but little is yet known. we notice, in the first place, that commonly after such a recovery the individual has decidedly increased resistance to the disease. this increased resistance may be very lasting, and may be so considerable as to give almost complete immunity from the disease for many years, or for life. one attack of scarlet fever gives the individual great immunity for the future. on the other hand, the resistance thus derived may be very temporary, as in the case of diphtheria. but a certain amount of resistance appears to be always acquired. this power of resisting the activities of the parasites seems to be increased during the progress of the disease, and, if it becomes sufficient, it finally drives off the bacteria before they have produced death. after this, recovery takes place. to what this newly acquired resisting power is due is by no means clear to bacteriologists, although certain factors are already known. it appears beyond question that in the case of certain diseases the cells of the body after a time produce substances which serve as antidotes to the poisons produced by the bacteria during their growth in the body-antitoxines. in the case of diphtheria, for instance, the germs growing in the throat produce poisons which are absorbed by the body and give rise to the symptoms of the disease; but after a time the body cells react, and themselves produce a counter toxic body which neutralizes the poisonous effect of the diphtheria poison. this substance has been isolated from the blood of animals that have recovered from an attack of diphtheria, and has been called diphtheria antitoxine. but even with this knowledge the recovery is not fully explained. this antitoxine neutralizes the effects of the diphtheria toxine, and then the body develops strength to drive off the bacteria which have obtained lodgment in the throat. how they accomplish this latter achievement we do not know as yet. the antitoxme developed simply neutralizes the effects of the toxine. some other force must be at work to get rid of the bacteria, a force which can only exert itself after the poisoning effect of the poison is neutralized. in these cases, then, the recovery is due, first, to the development in the body of the natural antidotes to the toxic poisons, and, second, to some other unknown force which drives off the parasites. these facts are certainly surprising. if one had been asked to suggest the least likely theory to explain recovery from disease, he could hardly have found one more unlikely than that the body cells developed during the disease an antidote to the poison which the disease bacteria were producing. nevertheless, it is beyond question that such antidotes are formed during the course of the germ diseases. it has not yet been shown in all diseases, and it would be entirely too much to claim that this is the method of recovery in all cases. we may say, however, in regard to bacterial diseases in general, that after the bacteria enter the body at some weak point they have first a battle to fight with the resisting powers of the body, which appear to be partly biological and partly chemical. these resisting powers are in many cases entirely sufficient to prevent the bacteria from obtaining a foothold. if the invading host overcome the resisting powers, then they begin to multiply rapidly, and take possession of the body or some part of it. they continue to grow until either the individual dies or something occurs to check their growth. after the individual develops the renewed powers of checking their growth, recovery takes place, and the individual is then, because of these renewed powers of resistance, immune from a second attack of the disease for a variable length of time. this, in the merest outline, represents the relation of bacterial parasites to the human body but while this is a fair general expression of the matter, it must be recognised that different diseases differ much in their relations, and no general outline will apply to all they differ in their method of attack and in the point of attack. not only do they produce different kinds of poisons giving rise to different symptoms of poisoning; not only do they produce different results in different animals; not only do the different pathogenic species differ much in their power to develop serious disease, but the different species are very particular as to what species of animal they attack. some of them can live as parasites in man alone; some can live as parasites upon man and the mouse and a few other animals; some can live in various animals but not in man; some appear to be able to live in the field mouse, but not in the common mouse; some live in the horse; some in birds, but not in warm-blooded mammals; while others, again, can live almost equally well in the tissues of a long list of animals. those which can live as parasites upon man are, of course, especially related to human disease, and are of particular interest to the physician, while those which live in animals are in a similar way of interest to veterinarians. thus we see that parasitic bacteria show the widest variations. they differ in point of attack, in method of attack, and in the part of the body which they seize upon as a nucleus for growth. they differ in violence and in the character of the poisons they produce, as well as in their power of overcoming the resisting powers of the body. they differ at different times in their powers of producing disease. in short, they show such a large number of different methods of action that no general statements can be made which will apply universally, and no one method of guarding against them or in driving them off can be hoped to apply to any extended list of diseases. diseases caused by other organisms than bacteria. although the purpose of this work is to deal primarily with the bacterial world, it would hardly be fitting to leave the subject without some reference to diseases caused by organisms which do not belong to the group of bacteria. while most of the so-called germ diseases are caused by the bacteria which we have been studying in the previous chapters, there are some whose inciting cause is to be found among organisms belonging to other groups. some of these are plants of a higher organization than bacteria, but others are undoubtedly microscopic animals. their life habits are somewhat different from those of bacteria, and hence the course of the diseases is commonly different. of the diseases thus produced by microscopic animals or by higher plants, one or two are of importance enough to deserve special mention here. malaria.--the most important of these diseases is malaria in its various forms, and known under various names--chills and fever, autumnal fever, etc. this disease, so common almost everywhere, has been studied by physicians and scientists for a long time, and many have been the causes assigned to it. at one time it was thought to be the result of the growth of a bacterium, and a distinct bacillus was described as producing it. it has finally been shown, however, to be caused by a microscopic organism belonging to the group of unicellular animals, and somewhat closely related to the well-known amoeba. this organism is shown in fig. 34. the whole history of the malarial organism is not yet known. the following statements comprise the most important facts known in regard to it, and its relation to the disease in man. undoubtedly the malarial germ has some home outside the human body, but it is not yet very definitely known what this external home is; nor do we know from what source the human parasite is derived. it appears probable that water serves in some cases as its means of transference to man, and air in other cases. from some external source it gains access to man and finds its way into the blood. here it attacks the red blood-corpuscles, each malarial organism making its way into a single one (fig. 340). here it now grows, increasing in size at the expense of the substance of the corpuscle. as it becomes larger it becomes granular, and soon shows a tendency to separate into a number of irregular masses. finally it breaks up into many minute bodies called spores. these bodies break out of the corpuscle and for a time live a free life in the blood. after a time they make their way into other red blood-corpuscles, develop into new malarial amoeboid parasites, and repeat the growth and sporulation. this process can apparently be repeated many times without check. these organisms are thus to be regarded as parasites of the red corpuscles. it is, of course, easy to believe that an extensive parasitism and destruction of the corpuscles would be disastrous to the health of the individual, and the severity of the disease will depend upon the extent of the parasitism. corresponding to this life history of the organism, the disease malaria is commonly characterized by a decided intermittency, periods of chill and fever alternating with periods of intermission in which these symptoms are abated. the paroxysms of the disease, characterized by the chill, occur at the time that the spores are escaping from the blood-corpuscles and floating in the blood. after they have again found their way into a blood-corpuscle the fever diminishes, and during their growth in the corpuscle until the next sporulation the individual has a rest from the more severe symptoms. there appears to be more than one variety of the malarial organism, the different types differing in the length of time it takes for their growth and sporulation. there is one variety, the most common one, which requires two days for its growth, thus giving rise to the paroxysm of the disease about once in fortyeight hours; another variety appears to require three days for its growth; while still another variety appears to be decidedly irregular in its period of growth and sporulation. these facts readily explain some of the variations in the disease. certain other irregularities appear to be due to a different cause. more than one brood of parasites may be in the blood of the individual at the same time, one producing sporulation at one time and another at a different time. such a simultaneous growth of two independent broods may plainly produce almost any kind of modification in the regularity of the disease. the malarial organism appears to be very sensitive to quinine, a very small quantity being sufficient to kill it. upon this point depends the value of quinine as a medicine. if the drug be present in the blood at the time when the spores are set free from the blood-corpuscle, they are rapidly killed by it before they have a chance to enter another corpuscle. during their growth in the corpuscle they are far less sensitive to quinine than when they exist in the free condition as spores, and at this time the drug has little effect. the malarial organism is an animal, and can not be cultivated in the laboratory by any artificial method yet devised. its whole history is therefore not known. it doubtless has some home outside the blood of animals, and very likely it may pass through other stages of a metamorphosis in the bodies of other animals. most parasitic animals have two or more hosts upon which they live, alternating from one to the other, and that such is the case with the malarial parasite is at least probable. but as yet bacteriologists have been unable to discover anything very definite in regard to the matter. until we can learn something in regard to its life outside the blood of man we can do little in the way of devising methods to avoid it. malaria differs from most germ diseases in the fact that the organisms which produce it are not eliminated from the body in any way. in most germ diseases the germs are discharged from the patient by secretions or excretions of some kind, and from these excretions may readily find their way into other individuals. the malarial organism is not discharged from the body in any way, and hence is not contagious. if the parasite does pass part of its history in some other animal than man, there must be some means by which it passes from man to its other host. it has been suggested that some of the insects which feed upon human blood may serve as the second host and become inoculated when feeding upon such blood. this has been demonstrated with startling success in regard to the mosquito (anopheles), some investigators going so far as to say that this is the only way in which the disease can be communicated. several other microscopic animals occur as parasites upon man, and some of them are so definitely associated with certain diseases as to lead to the belief that they are the cause of these diseases. the only one of very common occurrence is a species known as amaeba coli, which is found in cases of dysentery. in a certain type of dysentery this organism is so universally found that there is little doubt that it is in some very intimate way associated with the cause of the disease. definite proof of the matter is, however, as yet wanting. on the side of plants, we find that several plants of a higher organization than bacteria may become parasitic upon the body of man and produce various types of disease. these plants belong mostly to the same group as the moulds, and they are especially apt to attack the skin. they grow in the skin, particularly under the hair, and may send their threadlike branches into some of the subdermal tissues. this produces irritation and inflammation of the skin, resulting in trouble, and making sores difficult to heal. so long as the plant continues to grow, the sores, of course, can not be healed, and when the organisms get into the skin under the hair it is frequently difficult to destroy them. among the diseases thus caused are ringworm, thrush, alopecia, etc. chapter vi. methods of combating parasitic bacteria. the chief advantage of knowing the cause of disease is that it gives us a vantage ground from which we may hope to find means of avoiding its evils. the study of medicine in the past history of the world has been almost purely empirical, with a very little of scientific basis. great hopes are now entertained that these new facts will place this matter upon a more strictly scientific foundation. certainly in the past twenty-five years, since bacteriology has been studied, more has been done to solve problems connected with disease than ever before. this new knowledge has been particularly directed toward means of avoiding disease. bacteriology has thus far borne fruit largely in the line of preventive medicine, although to a certain extent also along the line of curative medicine. this chapter will be devoted to considering how the study of bacteriology has contributed directly and indirectly to our power of combating disease. preventive medicine. in the study of medicine in the past centuries the only aim has been to discover methods of curing disease; at the present time a large and increasing amount of study is devoted to the methods of preventing disease. preventive medicine is a development of the last few years, and is based almost wholly upon our knowledge of bacteria. this subject is yearly becoming of more importance. forewarned is forearmed, and it has been found that to know the cause of a disease is a long step toward avoiding it. as some of our contagious and epidemic diseases have been studied in the light of bacteriological knowledge, it has been found possible to determine not only their cause, but also how infection is brought about, and consequently how contagion may be avoided. some of the results which have grown up so slowly as to be hardly appreciated are really great triumphs. for instance, the study of bacteriology first led us to suspect, and then demonstrated, that tuberculosis is a contagious disease, and from the time that this was thus proved there has been a slow, but, it is hoped, a sure decline in this disease. bacteriological study has shown that the source of cholera infection in cases of raging epidemics is, in large part at least, our drinking water; and since this has been known, although cholera has twice invaded europe, and has been widely distributed, it has not obtained any strong foothold or given rise to any serious epidemic except in a few cases where its ravages can be traced to recognised carelessness. it is very significant to compare the history of the cholera epidemics of the past few years with those of earlier dates. in the epidemics of earlier years the cholera swept ruthlessly through communities without check. in the last few years, although it has repeatedly knocked at the doors of many european cities, it has been commonly confined to isolated cases, except in a few instances where these facts concerning the relation to drinking water were ignored. the study of preventive medicine is yet in its infancy, but it has already accomplished much. it has developed modern systems of sanitation, has guided us in the building of hospitals, given rules for the management of the sick-room which largely prevent contagion from patient to nurse; it has told us what diseases are contagious, and in what way; it has told us what sources of contagion should be suspected and guarded against, and has thus done very much to prevent the spread of disease. its value is seen in the fact that there has been a constant decrease in the death rate since modern ideas of sanitation began to have any influence, and in the fact that our general epidemics are less severe than in former years, as well as in the fact that more people escape the diseases which were in former times almost universal. the study of preventive medicine takes into view several factors, all connected with the method and means of contagion. they are the following: the source of infectious material.--t has been learned that for most diseases the infectious material comes from individuals suffering with the disease, and that except in a few cases, like malaria, we must always look to individuals suffering from disease for all sources of contagion. it is found that pathogenic bacteria are in all these cases eliminated from the patient in some way, either from the alimentary canal or from skin secretions or otherwise, and that any nurse with common sense can have no difficulty in determining in what way the infectious material is eliminated from her patients. when this fact is known and taken into consideration it is a comparatively easy matter to devise valuable precautions against distribution of such material. it is thus of no small importance to remember that the simple presence of bacteria in food or drink is of no significance unless these bacteria have come from some source of disease infection. the method of distribution.--the bacteria must next get from the original source of the disease to the new susceptible individual. bacteria have no independent powers of distribution unless they be immersed in liquids, and therefore their passage from individual to individual must be a passive one. they are readily transferred, however, by a number of different means, and the study of these means is aiding much in checking contagion study along this line has shown that the means by which bacteria are carried are several. first we may notice food as a distributor. food may become contaminated by infectious material in many ways; for example, by contact with sewage, or with polluted water, or even with eating utensils which have been used by patients. water is also likely to be contaminated with infectious material, and is a fertile source for distributing typhoid and cholera. milk may become contaminated in a variety of ways, and be a source of distributing the bacteria which produce typhoid fever, tuberculosis, diphtheria, scarlet fever, and a few other less common diseases. again, infected clothing, bedding, or eating utensils may be taken from a patient and be used by another individual without proper cleansing. direct contact, or contact with infected animals, furnishes another method. insects sometimes carry the bacteria from person to person, and in some diseases (tuberculosis, and perhaps scarlet fever and smallpox) we must look to the air as a distributor of the infectious material. knowledge of these facts is helping to account for multitudes of mysterious cases of infection, especially when we combine them with the known sources of contagious matter. means of invasion.--bacteriology has shown us that different species of parasitic bacteria have different means of entering the body, and that each must enter the proper place in order to get a foothold. after we learn that typhoid infectious material must enter the mouth in order to produce the disease; that tuberculosis may find entrance through the nose in breathing, while types of blood poisoning enter only through wounds or broken skin, we learn at once fundamental facts as to the proper methods of meeting these dangers. we learn that with some diseases care exercised to prevent the swallowing of infectious material is sufficient to prevent contagion, while with others this is entirely insufficient. when all these facts are understood it is almost always perfectly possible to avoid contagion; and as these facts become more and more widely known direct contagion is sure to become less frequent. above all, it is telling us what becomes of the pathogenic bacteria after being eliminated from the body of the patient; how they may exist for a long time still active; how they may lurk in filth or water dormant but alive, or how they may even multiply there. preventive medicine is telling us how to destroy those thus lying in wait for a chance of infection, by discovering disinfectants and telling us especially where and when to use them. it has already taught us how to crush out certain forms of epidemics by the proper means of destroying bacteria, and is lessening the dangers from contagious diseases. in short, the study of bacteriology has brought us into a condition where we are no longer helpless in the presence of a raging epidemic. we no longer sit in helpless dismay, as did our ancestors, when an epidemic enters a community, but, knowing their causes and sources, set about at once to remove them. as a result, severe epidemics are becoming comparatively short-lived. bacteria in surgery. in no line of preventive medicine has bacteriology been of so much value and so striking in its results as in surgery. ever since surgery has been practised surgeons have had two difficulties to contend with. the first has been the shock resulting from the operation. this is dependent upon the extent of the operation, and must always be a part of a surgical operation. the second has been secondary effects following the operation. after the operation, even though it was successful, there were almost sure to arise secondary complications known as surgical fever, inflammation, blood poisoning, gangrene, etc., which frequently resulted fatally. these secondary complications were commonly much more serious than the shock of the operation, and it used to be the common occurrence for the patient to recover entirely from the shock, but yield to the fevers which followed. they appeared to be entirely unavoidable, and were indeed regarded as necessary parts of the healing of the wound. too frequently it appeared that the greater the care taken with the patient the more likely he was to suffer from some of these troubles. the soldier who was treated on the battlefield and nursed in an improvised field hospital would frequently recover, while the soldier who had the fortune to be taken into the regular hospital, where greater care was possible, succumbed to hospital gangrene. all these facts were clearly recognised, but the surgeon, through ignorance of their cause, was helpless in the presence of these inflammatory troubles, and felt it always necessary to take them into consideration. the demonstration that putrefaction and decay were caused by bacteria, and the early proof that the silkworm disease was produced by a micro-organism, led to the suggestion that the inflammatory diseases accompanying wounds were similarly caused. there are many striking similarities between these troubles and putrefaction, and the suggestion was an obvious one. at first, however, and for quite a number of years, it was impossible to demonstrate the theory by finding the distinct species of microorganisms which produced the troubles. we have already seen that there are several different species of bacteria which are associated with this general class of diseases, but that no specific one has any particular relation to a definite type of inflammation. this fact made discoveries in this connection a slow matter from the microscopical standpoint. but long before this demonstration was finally reached the theory had received practical application in the form of what has developed into antiseptic or aseptic surgery. antiseptic surgery is based simply upon the attempt to prevent the entrance of bacteria into the surgical wound. it is assumed that if these organisms are kept from the wound the healing will take place without the secondary fevers and inflammations which occur if they do get a chance to grow in the wound. the theory met with decided opposition at first, but accumulating facts demonstrated its value, and to-day its methods have been adopted everywhere in the civilized world. as the evidence has been accumulating, surgeons have learned many important facts, foremost among which is a knowledge of the common sources from which the infection of wounds occurs. at first it was thought that the air was the great source of infection, but the air bacteria have been found to be usually harmless. it has appeared that the more common sources are the surgeon's instruments, or his hands, or the clothing or sponges which are allowed to come in contact with the wounds. it has also appeared that the bacteria which produce this class of troubles are common species, existing everywhere and universally present around the body, clinging to the clothing or skin, and always on hand to enter the wound if occasion offers. they are always present, but commonly harmless. they are not foreign invaders like the more violent pathogenic species, such as those of asiatic cholera, but may be compared to domestic enemies at hand. it is these ever-present bacteria which the surgeon must guard against. the methods by which he does this need not detain us here. they consist essentially in bacteriological cleanliness. the operation is performed with sterilized instruments under most exacting conditions of cleanliness. the result has been a complete revolution in surgery. as the methods have become better understood and more thoroughly adopted, the instances of secondary troubles following surgical wounds have become less and less frequent until they have practically disappeared in all simple cases. to-day the surgeon recognises that when inflammatory troubles of this sort follow simple surgical wounds it is a testimony to his carelessness. the skilful surgeon has learned that with the precautions which he is able to take to-day he has to fear only the direct effect of the shock of the wound and its subsequent direct influence; but secondary surgical fevers, blood poisoning, and surgical gangrene need not be taken into consideration at all. indeed, the modern surgeon hardly knows what surgical gangrene is, and bacteriologists have had practically no chance to study it. secondary infections have largely disappeared, and the surgeon is concerned simply with the effect of the wound itself, and the power of the body to withstand the shock and subsequently heal the wound. with these secondary troubles no longer to disturb him, the surgeon has become more and more bold. operations formerly not dreamed of are now performed without hesitation. in former years an operation which opened the abdominal cavity was not thought possible, or at least it was so nearly certain to result fatally that it was resorted to only on the last extremity; while to-day such operations are hardly regarded as serious. even brain surgery is becoming more and more common. possibly our surgeons are passing too far to the other extreme, and, feeling their power of performing so many operations without inconvenience or danger, they are using the knife in cases where it would be better to leave nature to herself for her own healing. but, be this as it may, it is impossible to estimate the amount of suffering prevented and the number of lives saved by the mastery of the secondary inflammatory troubles which used to follow surgical wounds. preventive medicine, then, has for its object the prevention rather than the cure of disease. by showing the causes of disease and telling us where and how they are contracted, it is telling us how they may to a large extent be avoided. unlike practical medicine, this subject is one which has a direct relation to the general public. while it may be best that the knowledge of curative methods be confined largely to the medical profession, it is eminently desirable that a knowledge of all the facts bearing upon preventive medicine should be distributed as widely as possible. one person can not satisfactorily apply his knowledge of preventive medicine, if his neighbour is ignorant of or careless of the facts. we can not hope to achieve the possibilities lying along this line until there is a very wide distribution of knowledge. every epidemic that sweeps through our communities is a testimony to the crying need of education in regard to such simple facts as the source of infectious material, the methods of its distribution, and the means of rendering it harmless. prevention in inoculation. it has long been recognised that in most cases recovery from one attack of a contagious disease renders an individual more or less immune against a second attack. it is unusual for an individual to have the same contagious disease twice. this belief is certainly based upon fact, although the immunity thus acquired is subject to wide variations. there are some diseases in which there is little reason for thinking that any immunity is acquired, as in the case of tuberculosis, while there are others in which the immunity is very great and very lasting, as in the case of scarlet fever. moreover, the immunity differs with individuals. while some persons appear to acquire a lasting immunity by recovery from a single attack, others will yield to a second attack very readily. but in spite of this the fact of such acquired immunity is beyond question. apparently all infectious diseases from which a real recovery takes place are followed by a certain amount of protection from a second attack; but with some diseases the immunity is very fleeting, while with others it is more lasting. diseases which produce a general infection of the whole system are, as a rule, more likely to give rise to a lasting immunity than those which affect only small parts. tuberculosis, which, as already noticed, is commonly quite localized in the body, has little power of conveying immunity, while a disease like scarlet fever, which affects the whole system, conveys a more lasting protection. such immunity has long been known, and in the earlier years was sometimes voluntarily acquired; even to-day we find some individuals making use of the principle. it appears that a mild attack of such diseases produces immunity equally well with a severe attack, and acting upon this fact mothers have not infrequently intentionally exposed their children to certain diseases at seasons when they are mild, in order to have the disease "over with" and their children protected in the future. even the more severe diseases have at times been thus voluntarily acquired. in china it has sometimes been the custom thus to acquire smallpox. such methods are decidedly heroic, and of course to be heartily condemned. but the principle that a mild type of the disease conveys protection has been made use of in a more logical and defensible way. the first instance of this principle was in vaccination against smallpox, now practised for more than a century. cowpox is doubtless closely related to smallpox, and an attack of the former conveys a certain amount of protection against the latter. it was easy, therefore, to inoculate man with some of the infectious material from cowpox, and thus give him some protection against the more serious smallpox. this was a purely empirical discovery, and vaccination was practised long before the principle underlying it was understood, and long before the germ nature of disease was recognised. the principle was revived again, however, by pasteur, and this time with a logical thought as to its value. while working upon anthrax among animals, he learned that here, as in other diseases, recovery, when it occurred, conveyed immunity. this led him to ask if it were not possible to devise a method of giving to animals a mild form of the disease and thus protect them from the more severe type. the problem of giving a mild type of this extraordinarily severe disease was not an easy one. it could not be done, of course, by inoculating the animals with a small number of the bacteria, for their power of multiplication would soon make them indefinitely numerous. it was necessary in some way to diminish their violence. pasteur succeeded in doing this by causing them to grow in culture fluids for a time at a high temperature. this treatment diminished their violence so much that they could be inoculated into cattle, where they produced only the mildest type of indisposition, from which the animals speedily recovered. but even this mild type of the disease was triumphantly demonstrated to protect the animals from the most severe form of anthrax. the discovery was naturally hailed as a most remarkable one, and one which promised great things in the future. if it was thus possible, by direct laboratory methods, to find a means of inoculating against a serious disease like anthrax, why could not the same principle be applied to human diseases? the enthusiasts began at once to look forward to a time when all diseases should be thus conquered. but the principle has not borne the fruit at first expected. there is little doubt that it might be applied to quite a number of human diseases if a serious attempt should be made. but several objections arise against its wide application. in the first place, the inoculation thus necessary is really a serious matter. even vaccination, as is well known, sometimes, through faulty methods, results fatally, and it is a very serious thing to experiment upon human beings with anything so powerful for ill as pathogenic bacteria. the seriousness of the disease smallpox, its extraordinary contagiousness, and the comparatively mild results of vaccination, have made us willing to undergo vaccination at times of epidemics to avoid the somewhat great probability of taking the disease. but mankind is unwilling to undergo such an operation, even though mild, for the purpose of avoiding other less severe diseases, or diseases which are less likely to be taken. we are unwilling to be inoculated against mild diseases, or against the more severe ones which are uncommon. for instance, a method has been devised for rendering animals immune against lockjaw, which would probably apply equally well to man. but mankind in general will never adopt it, since the danger from lockjaw is so small. inoculation must then be reserved for diseases which are so severe and so common, or which occur in periodical epidemics of so great severity, as to make people in general willing to submit to inoculation as a protection. a further objection arises from the fact that the immunity acquired is not necessarily lasting. the cattle inoculated against anthrax retain their protective powers for only a few months. how long similar immunity might be retained in other cases we can not say, but plainly this fact would effectually prevent this method of protecting mankind from being used except in special cases. it is out of the question to think of constant and repeated inoculations against various diseases. as a result, the principle of inoculation as an aid in preventive medicine has not proved of very much value. the only other human disease in which it has been attempted seriously is asiatic cholera. this disease in times of epidemics is so severe and the chance of infection is so great as to justify such inoculation. several bacteriologists have in the last few years been trying to discover a harmless method of inoculating against this disease. apparently they have succeeded, for experiments in india, the home of the cholera, have been as successful as could be anticipated. bacteriological science has now in its possession a means of inoculation against cholera which is perhaps as efficacious as vaccination is against smallpox. whether it will ever be used to any extent is doubtful, since, as already pointed out, we are in a position to avoid cholera epidemics by other means. if we can protect our communities by guarding the water supply, it is not likely that the method of inoculation will ever be widely used. another instance of the application of preventive inoculation has been made, but one based upon a different principle. hydrophobia is certainly one of the most horrible of diseases, although comparatively rare. its rarity would effectually prevent mankind from submitting to a general inoculation against it, but its severity would make one who had been exposed to it by the bite of a rabid animal ready to submit to almost any treatment that promised to ward off the disease. in the attempt to discover a means of inoculating against this disease it was necessary, therefore, to find a method that could be applied after the time of exposure--i.e., after the individual had been bitten by the rabid animal. fortunately, the disease has a long period of incubation, and one that has proved long enough for the purpose. a method of inoculation against this disease has been devised by pasteur, which can be applied after the individual has been bitten by the rabid animal. apparently, however, this preventive inoculation is dependent upon a different principle from vaccination or inoculation against anthrax. it does not appear to give rise to a mild form of the disease, thus protecting the individual, but rather to an acquired tolerance of the chemical poisons produced by the disease. it is a well-known physiological fact that the body can become accustomed to tolerate poisons if inured to them by successively larger and larger doses. it is by this power, apparently, that the inoculation against hydrophobia produces its effect. material containing the hydrophobia poison (taken from the spinal cord of a rabbit dead with the disease) is injected into the individual after he has been bitten by a rabid animal. the poisonous material in the first injection is very weak, but is followed later by a more powerful inoculation. the result is that after a short time the individual has acquired the power of resisting the hydrophobia poisons. before the incubation period of the original infectious matter from the bite of the rabid animal has passed, the inoculated individual has so thoroughly acquired a tolerance of the poison that he successfully resists the attack of the infection. this method of inoculation thus neutralizes the effects of the disease by anticipating them. the method of treatment of hydrophobia met with extraordinarily violent opposition. for several years it was regarded as a mistake. but the constantly accumulating statistics from the pasteur institute have been so overwhelmingly on one side as to quiet opposition and bring about a general conviction that the method is a success. the method of preventive inoculation has not been extensively applied to human diseases in addition to those mentioned. in a few cases a similar method has been used to guard against diphtheria. among animals, experiment has shown that such methods can quite easily be obtained, and doubtless the same would be true of mankind if it was thought practical or feasible to apply them. but, for reasons mentioned, this feature of preventive medicine will always remain rather unimportant, and will be confined to a few of the more violent diseases. it may be well to raise the question as to why a single attack with recovery conveys immunity. this question is really a part of the one already discussed as to the method by which the body cures disease. we have seen that this is in part due to the development of chemical substances which either neutralize the poisons or act as germicide upon the bacteria, or both, and perhaps due in part to an active destruction of bacteria by cellular activity (phagocytosis). there is little reason to doubt that it is the same set of activities which renders the animal immune. the forces which drive off the invading bacteria in one case are still present to prevent a second attack of the same species of bacterium. the length of time during which these forces are active and sufficient to cope with any new invaders determines the length of time during which the immunity lasts. until, therefore, we can answer with more exactness just how cure is brought about in case of disease, we shall be unable to explain the method of immunity. limits of preventive medicine. with all the advance in preventive medicine we can not hope to avoid disease entirely. we are discovering that the sources of disease are on all sides of us, and so omnipresent that to avoid them completely is impossible. if we were to apply to our lives all the safeguards which bacteriology has taught us should be applied in order to avoid the different diseases, we would surround ourselves with conditions which would make life intolerable. it would be oppressive enough for us to eat no food except when it is hot, to drink no water except when boiled, and to drink no milk except after sterilization; but these would not satisfy the necessary conditions for avoiding disease. to meet all dangers, we should handle nothing which has not been sterilized, or should follow the handling by immediately sterilizing the hands; we should wear only disinfected clothes, we should never put our fingers in our mouths or touch our food with them; we should cease to ride in public conveyances, and, indeed, should cease to breathe common air. absolute prevention of the chance of infection is impossible. the most that preventive medicine can hope for is to point out the most common and prolific sources of infection, and thus enable civilized man to avoid some of his most common troubles. it becomes a question, therefore, where we will best draw the line in the employment of safeguards. shall we drink none except sterilized milk, and no water unless boiled? or shall we put these occasional sources of danger in the same category with bicycle and railroad accidents, dangers which can be avoided by not using the bicycle or riding on the rail, but in regard to which the remedy is too oppressive for application? indeed, when viewed in a broad philosophical light it may not be the best course for mankind to shun all dangers. strength in the organism comes from the use rather than the disuse of our powers. it is certain that the general health and vigour of mankind is to be developed by meeting rather than by shunning dangers. resistance to disease means bodily vigour, and this is to be developed in mankind by the application of the principle of natural selection. in accordance with this principle, disease will gradually remove the individuals of weak resisting powers, leaving those of greater vigour. parasitic bacteria are thus a means of preventing the continued life of the weaker members of the community, and so tend to strengthen mankind. by preventive medicine many a weak individual who would otherwise succumb earlier in the struggle is enabled to live a few years longer. whatever be our humanitarian feeling for the individual, we can not fail to admit that this survival of the weak is of no benefit to the race so far as the development of physical nature is concerned. indeed, if we were to take into consideration simply the physical nature of man we should be obliged to recommend a system such as the ancient spartans developed, of exposing to death all weakly individuals, that only the strong might live to become the fathers of future generations. in this light, of course, parasitic diseases would be an assistance rather than a detriment to the human race. of course such principles will never again be dominant among men, and our conscience tells us to do all we can to help the weak. we shall doubtless do all possible to develop preventive medicine in order to guard the weak against parasitic organisms. but it is at all events well for us to remember that we can never hope to develop the strength of the human race by shunning evil, but rather by combating it, and the power of the human race to resist the invasions of these organisms will never be developed by the line of action which guards us from attack. here, as in other directions, the principles of modern humanity have, together with their undoubted favourable influence upon mankind, certain tendencies toward weakness. while we shall still do our utmost to develop preventive medicine in a proper way, it may be well for us to remember these facts when we come to the practical question of determining where to draw the limits of the application of methods for preventing infectious diseases. curative medicine. bacteriology has hitherto contributed less to curative than to preventive medicine. nevertheless, its contributions to curative medicine have not been unimportant, and there is promise of much more in the future. it is, of course, unsafe to make predictions for the future, but the accomplishments of the last few years give much hope as to further results. drugs. it was at first thought that a knowledge of the specific bacteria which cause a disease would give a ready means of finding specific drugs for the cure of such disease. if a definite species of bacterium causes a disease and we can cultivate the organism in the laboratory, it is easy to find some drugs which will be fatal to its growth, and these same drugs, it would seem, should be valuable as medicines in these diseases. this hope has, however, proved largely illusive. it is very easy to find some drug which proves fatal to the specific germs while growing in the culture media of the laboratory, but commonly these are of little or no use when applied as medicines. in the first place, such substances are usually very deadly poisons. corrosive sublimate is a substance which destroys all pathogenic germs with great rapidity, but it is a deadly poison, and can not be used as a drug in sufficient quantity to destroy the parasitic bacteria in the body without at the same time producing poisonous effects on the body itself. it is evident that for any drug to be of value in thus destroying bacteria it must have some specially strong action upon the bacteria. its germicide action on the bacteria should be so strong that a dose which would be fatal or very injurious to them would be too small to have a deleterious influence on the body of the individual. it has not proved an easy task to discover drugs which will have any value as germicides when used in quantities so small as to produce no injurious effect on the body. a second difficulty is in getting the drug to produce its effect at the right point. a few diseases, as we have noticed, are produced by bacteria which distribute themselves almost indiscriminately over the body; but the majority are somewhat definitely localized in special points. tuberculosis may attack a single gland or a single lobe of the lung. typhoid germ is localized in the intestines, liver, spleen, etc. even if it were possible to find some drug which would have a very specific effect upon the tuberculosis bacillus, it is plain that it would be a very questionable method of procedure to introduce this into the whole system simply that it might have an effect upon a very small isolated gland. sometimes such a bacterial affection may be localized in places where it can be specially treated, as in the case of an attack on a dermal gland, and in these cases some of the germicides have proved to be of much value. indeed, the use of various disinfectants connected with abscesses and superficial infections has proved of much value. to this extent, in disinfecting wounds and as a local application, the development of our knowledge of disinfectants has given no little aid to curative medicine. very little success, however, has resulted in the attempt to find specific drugs for specific diseases, and it is at least doubtful whether many such will ever be found. the nearest approach to it is quinine as a specific poison for malarial troubles. malarious diseases are not, however, produced by bacteria but by a microscopic organism of a very different nature, thought to be an animal rather than a plant. besides this there has been little or no success in discovering specifics in the form of drugs which can be given as medicines or inoculated with the hope of destroying special kinds of pathogenic bacteria without injury to the body. while it is unwise to make predictions as to future discoveries, there seems at present little hope for a development of curative medicine along these lines. vis medicatrix naturae. the study of bacterial diseases as they progress in the body has emphasized above all things the fact that diseases are eventually cured by a natural rather than by an artificial process. if a pathogenic bacterium succeeds in passing the outer safeguards and entering the body, and if it then succeeds in overcoming the forces of resistance which we have already noticed, it will begin to multiply and produce mischief. this multiplication now goes on for a time unchecked, and there is little reason to expect that we can ever do much toward checking it by means of drugs. but after a little, conditions arise which are hostile to the further growth of the parasite. these hostile conditions are produced perhaps in part by the secretions from the bacteria, for bacteria are unable to flourish in a medium containing much of their own secretions. the secretions which they produce are poisons to them as well as to the individual in which they grow, and after these have become quite abundant the further growth of the bacterium is checked and finally stopped. partly, also, must we conclude that these hostile conditions are produced by active vital powers in the body of the individual attacked. the individual, as we have seen, in some cases develops a quantity of some substance which neutralizes the bacterial poisons and thus prevents their having their maximum effect. thus relieved from the direct effects of the poisons, the resisting powers are recuperated and once more begin to produce a direct destruction of the bacteria. possibly the bacteria, being now weakened by the presence of their own products of growth, more readily yield to the resisting forces of the cell life of the body. possibly the resisting forces are decidedly increased by the reactive effect of the bacteria and their poisons. but, at all events, in cases where recovery from parasitic diseases occurs, the revived powers of resistance finally overcome the bacteria, destroy them or drive them off, and the body recovers. all this is, of course, a natural process. the recovery from a disease produced by the invasion of parasitic bacteria depends upon whether the body can resist the bacterial poisons long enough for the recuperation of its resisting powers. if these poisons are very violent and produced rapidly, death will probably occur before the resisting powers are strong enough to drive off the bacteria. in the case of some diseases the poisons are so violent that this practically always occurs, recovery being very exceptional. the poison produced by the tetanus bacillus is of this nature, and recovery from lockjaw is of the rarest occurrence. but in many other diseases the body is able to withstand the poison, and later to recover its resisting powers sufficiently to drive off the invaders. in all cases, however, the process is a natural one and dependent upon the vital activity of the body. it is based at the foundation, doubtless, upon the powers of the body cells, either the phagocytes or other active cells. the body has, in short, its own forces for repelling invasions, and upon these forces must we depend for the power to produce recovery. it is evident that all these facts give us very little encouragement that we shall ever be able to cure diseases directly by means of drugs to destroy bacteria, but, on the contrary, that we must ever depend upon the resisting powers of the body. they teach us, moreover, along what line we must look for the future development of curative medicine. it is evident that scientific medicine must turn its attention toward the strengthening and stimulating of the resisting and curative forces of the body. it must be the physician's aim to enable the body to resist the poisons as well as possible and to stimulate it to re-enforce its resistant forces. drugs have a place in medicine, of course, but this place is chiefly to stimulate the body to react against its invading hosts. they are, as a rule, not specific against definite diseases. we can not hope for much in the way of discovering special medicines adapted to special diseases. we must simply look upon them as means which the physician has in hand for stimulating the natural forces of the body, and these may doubtless vary with different individual natures. recognising this, we can see also the logic of the small dose as compared to the large dose. a small dose of a drug may serve as a stimulant for the lagging forces, while a larger dose would directly repress them or produce injurious secondary effects. as soon as we recognise that the aim of medicine is not to destroy the disease but rather to stimulate the resisting forces of the body, the whole logic of therapeutics assumes a new aspect. physicians have understood this, and, especially in recent years, have guided their practice by it. if a moderate dose of quinine will check malaria in a few days, it does not follow that twice the dose will do it in half the time or with twice the certainty. the larger doses of the past, intended to drive out the disease, have been everywhere replaced by smaller doses designed to stimulate the lagging body powers. the modern physician makes no attempt to cure typhoid fever, having long since learned his inability to do this, at least if the fever once gets a foothold; but he turns his attention to every conceivable means of increasing the body's strength to resist the typhoid poison, confident that if he can thus enable the patient to resist the poisoning effects of the typhotoxine his patient will in the end react against the disease and drive off the invading bacteria. the physician's duty is to watch and guard, but he must depend upon the vital powers of his patient to carry on alone the actual battle with the bacterial invaders. antitoxines. in very recent times, however, our bacteriologists have been pointing out to the world certain entirely new means of assisting the body to fight its battles with bacterial diseases. as already noticed, one of the primal forces in the recovery, from some diseases, at least, is the development in the body of a substance which acts as an antidote to the bacterial poison. so long as this antitoxine is not present the poisons produced by the disease will have their full effect to weaken the body and prevent the revival of its resisting powers to drive off the bacteria. plainly, if it is possible to obtain this antitoxine in quantity and then inoculate it into the body when the toxic poisons are present, we have a means for decidedly assisting the body in its efforts to drive off the parasites. such an antidote to the bacterial poison would not, indeed, produce a cure, but it would perhaps have the effect of annulling the action of the poisons, and would thus give the body a much greater chance to master the bacteria. it is upon this principle that is based the use of antitoxines in diphtheria and tetanus it will be clear that to obtain the antitoxine we must depend upon some natural method for its production. we do not know enough of the chemical nature of the antitoxines to manufacture them artificially. of course we can not deny the possibility of their artificial production, and certain very recent experiments indicate that perhaps they may be made by the agency of electricity. at present, however, we must use natural methods, and the one commonly adopted is simple. some animal is selected whose blood is harmless to man and that is subject to the disease to be treated. for diphtheria a horse is chosen. this animal is inoculated with small quantities of the diphtheria poison without the diphtheria bacillus. this poison is easily obtained by causing the diphtheria bacillus to grow in common media in the laboratory for a while, and the toxines develop in quantity; then, by proper filtration, the bacteria themselves can be removed, leaving a pure solution of the toxic poison. small quantities of this poison are inoculated into the horse at successive intervals. the effect on the horse is the same as if the animal had the disease. its cells react and produce a considerable quantity of the antitoxine which remains in solution in the blood of the animal. this is not theory, but demonstrated fact. the blood of a horse so treated is found to have the effect of neutralizing the diphtheria poison, although the blood of the horse before such treatment has no such effect. thus there is developed in the horse's blood a quantity of the antitoxine, and now it may be used by physicians where needed. if some of this horse's blood, properly treated, be inoculated into the body of a person who is suffering from diphtheria, its effect, provided the theory of antitoxines is true, will be to counteract in part, at least, the poisons which are being produced in the patient by the diphtheria bacillus. this does not cure the disease nor in itself drive off the bacilli, but it does protect the body from the poisons to such an extent as to enable it more readily to assert its own resisting powers. this method of using antitoxines as a help in curing disease is very recent, and we can not even guess what may come of it. it has apparently been successfully applied in diphtheria. it has also been used in tetanus with slight success. the same principle has been used in obtaining an antidote for the poison of snake bites, since it has appeared that in this kind of poisoning the body will develop an antidote to the poison if it gets a chance. horses have been treated in the same way as with the diphtheria poison, and in the same way they develop a substance which neutralizes the snake poison. other diseases are being studied to-day with the hope of similar results. how much further the principle will go we can not say, nor can we be very confident that the same principle will apply very widely. the parasitic diseases are so different in nature that we can hardly expect that a method which is satisfactory in meeting one of the diseases will be very likely to be adapted to another. vaccination has proved of value in smallpox, but is not of use in other human diseases. inoculation with weakened germs has proved of value in anthrax and fowl cholera, but will not apply to all diseases. each of these parasites must be fought by special methods, and we must not expect that a method that is of value in one case must necessarily be of use elsewhere. above all, we must remember that the antitoxines do not cure in themselves; they only guard the body from the weakening effects of the poisons until it can cure itself, and, unless the body has resisting powers, the antitoxine will fail to produce the desired results. one further point in the action of the antitoxines must be noticed. as we have seen, a recovery from an attack of most germ diseases renders the individual for a time immune against a second attack. this applies less, however, to a recovery after the artificial inoculation with antitoxine than when the individual recovers without such aid. if the individual recovers quite independently of the artificial antitoxine, he does so in part because he has developed the antitoxines for counteracting the poison by his own powers. his cellular activities have, in other words, been for a moment at least turned in the direction of production of antitoxines. it is to be expected, therefore, that after the recovery they will still have this power, and so long as they possess it the individual will have protection from a second attack. when, however, the recovery results from the artificial inoculation of antitoxine the body cells have not actively produced antitoxine. the neutralization of the poisons has been a passive one, and after recovery the body cells are no more engaged in producing antitoxine than before. the antitoxine which was inoculated is soon eliminated by secretion, and the body is left with practically the same liability to attack as before. its immunity is decidedly fleeting, since it was dependent not upon any activity on the part of the body, but upon an artificial inoculation of a material which is rapidly eliminated by secretion. conclusion. it is hoped that the outline which has been given of the bacterial life of nature may serve to give some adequate idea of these organisms and correct the erroneous impressions in regard to them which are widely prevalent. it will be seen that, as our friends, bacteria play a vastly more important part in nature than they do as our enemies. these plants are minute and extraordinarily simple, but, nevertheless, there exists a large number of different species. the number of described forms already runs far into the hundreds, and we do not yet appear to be approaching the end of them. they are everywhere in nature, and their numbers are vast beyond conception. their powers of multiplication are inconceivable, and their ability to produce profound chemical changes is therefore unlimited. this vast host of living beings thus constitutes a force or series of forces of tremendous significance. most of the vast multitude we must regard as our friends. upon them the farmer is dependent for the fertility of his soil and the possibility of continued life in his crops. upon them the dairyman is dependent for his flavours. upon them important fermentative industries are dependent, and their universal powers come into action upon a commercial scale in many a place where we have little thought of them in past years. we must look upon them as agents ever at work, by means of which the surface of nature is enabled to remain fresh and green. their power is fundamental, and their activities are necessary for the continuance of life. a small number of the vast host, a score or two of species, unfortunately for us, find their most favourable living place in the human body, and thus become human parasites. by their growth they develop poisons and produce disease. this small class of parasites are then decidedly our enemies. but, taken all together, we must regard the bacteria as friends and allies. without them we should not have our epidemics, but without them we should not exist. without them it might be that some individuals would live a little longer, if indeed we could live at all. it is true that bacteria, by producing disease, once in a while cause the premature death of an individual; once in a while, indeed, they may sweep off a hundred or a thousand individuals; but it is equally true that without them plant and animal life would be impossible on the face of the earth. outlines of dairy bacteriology a concise manual for the use of students in dairying by h. l. russell dean of the college of agriculture, university of wisconsin eighth edition thoroughly revised madison, wisconsin h. l. russell 1907 copyrighted 1905 by h. l. russell state journal printing company, printers and stereotypers, madison, wis. transcriber's note: for text: a word surrounded by a cedilla such as ~this~ signifies that the word is bolded in the text. a word surrounded by underscores like _this_ signifies the word is italics in the text. for numbers and equations, underscores before bracketed numbers in equations denote a subscript. minor typos have been corrected. preface. knowledge in dairying, like all other technical industries, has grown mainly out of experience. many facts have been learned by observation, but the _why_ of each is frequently shrouded in mystery. modern dairying is attempting to build its more accurate knowledge upon a broader and surer foundation, and in doing this is seeking to ascertain the cause of well-established processes. in this, bacteriology is playing an important rôle. indeed, it may be safely predicted that future progress in dairying will, to a large extent, depend upon bacteriological research. as fleischmann, the eminent german dairy scientist, says: "the gradual abolition of uncertainty surrounding dairy manufacture is the present important duty which lies before us, and its solution can only be effected by bacteriology." it is therefore natural that the subject of dairy bacteriology has come to occupy an important place in the curriculum of almost every dairy school. an exposition of its principles is now recognized as an integral part of dairy science, for modern dairy practice is rapidly adopting the methods that have been developed as the result of bacteriological study. the rapid development of the subject has necessitated a frequent revision of this work, and it is gratifying to the writer that the attempt which has been made to keep these outlines abreast of bacteriological advance has been appreciated by students of dairying. while the text is prepared more especially for the practical dairy operator who wishes to understand the principles and reasons underlying his art, numerous references to original investigations have been added to aid the dairy investigator who wishes to work up the subject more thoroughly. my acknowledgments are due to the following for the loan of illustrations: wisconsin agricultural experiment station; creamery package mfg. co., chicago, ill.; and a. h. reid, philadelphia, pa. h. l. russell. university of wisconsin. contents. chapter i. structure of the bacteria and conditions governing their development and distribution 1 chapter ii. methods of studying bacteria 13 chapter iii. contamination of milk 19 chapter iv. fermentations in milk and their treatment 62 chapter v. relation of disease-bacteria to milk 82 diseases transmissible from animal to man through diseased milk 84 diseases transmissible to man through infection of milk after withdrawal 94 chapter vi. preservation of milk for commercial purposes 102 chapter vii. bacteria and butter making 134 bacterial defects in butter 156 chapter viii. bacteria in cheese 160 influence of bacteria in normal cheese processes 160 influence of bacteria in abnormal cheese processes 182 chapter i. structure of the bacteria and conditions governing their development and distribution. before one can gain any intelligent conception of the manner in which bacteria affect dairying, it is first necessary to know something of the life history of these organisms in general, how they live, move and react toward their environment. ~nature of bacteria.~ toadstools, smuts, rusts and mildews are known to even the casual observer, because they are of evident size. their plant-like nature can be more readily understood from their general structure and habits of life. the bacteria, however, are so small, that under ordinary conditions, they only become evident to our unaided senses by the by-products of their activity. when leeuwenhoek (pronounced lave-en-hake) in 1675 first discovered these tiny, rapidly-moving organisms he thought they were animals. indeed, under a microscope, many of them bear a close resemblance to those minute worms found in vinegar that are known as "vinegar-eels." the idea that they belonged to the animal kingdom continued to hold ground until after the middle of the nineteenth century; but with the improvement in microscopes, a more thorough study of these tiny structures was made possible, and their vegetable nature demonstrated. the bacteria as a class are separated from the fungi mainly by their method of growth; from the lower algae by the absence of chlorophyll, the green coloring matter of vegetable organisms. ~structure of bacteria.~ so far as structure is concerned the bacteria stand on the lowest plane of vegetable life. the single individual is composed of but a single cell, the structure of which does not differ essentially from that of many of the higher types of plant life. it is composed of a protoplasmic body which is surrounded by a thin membrane that separates it from neighboring cells that are alike in form and size. ~form and size.~ when a plant is composed of a single cell but little difference in form is to be expected. while there are intermediate stages that grade insensibly into each other, the bacteria may be grouped into three main types, so far as form is concerned. these are spherical, elongated, and spiral, and to these different types are given the names, respectively, _coccus_, _bacillus_ and _spirillum_ (plural, _cocci_, _bacilli_, _spirilla_) (fig. 1). a ball, a short rod, and a corkscrew serve as convenient models to illustrate these different forms. [illustration: fig. 1. different forms of bacteria. _a_, _b_, _c_, represent different types as to form: _a_, coccus, _b_, bacillus, _c_, spirillum; _d_, diplococcus or twin coccus; _e_, staphylococcus or cluster coccus; _f_ and _g_, different forms of bacilli, _g_ shows internal endospores within cell; _h_ and _i_, bacilli with motile organs (cilia).] in size, the bacteria are the smallest organisms that are known to exist. relatively there is considerable difference in size between the different species, yet in absolute amount this is so slight as to require the highest powers of the microscope to detect it. as an average diameter, one thirty-thousandth of an inch may be taken. it is difficult to comprehend such minute measurements, but if a hundred individual germs could be placed side by side, their total thickness would not equal that of a single sheet of paper upon which this page is printed. ~manner of growth.~ as the cell increases in size as a result of growth, it elongates in one direction, and finally a new cell wall is formed, dividing the so-called mother-cell into two, equal-sized daughter-cells. this process of cell division, known as _fission_, is continued until growth ceases and is especially characteristic of bacteria. ~cell arrangement.~ if fission goes on in the same plane continually, it results in the formation of a cell-row. a coccus forming such a chain of cells is called _strepto-coccus_ (chain-coccus). if only two cells cohere, it is called a _diplo-coccus_ (twin-coccus). if the second cell division plane is formed at right angles to the first, a _cell surface_ or _tetrad_ is formed. if growth takes place in three dimensions of space, a _cell mass_ or _sarcina_ is produced. frequently, these cell aggregates cohere so tenaciously that this arrangement is of value in distinguishing different species. ~spores.~ some bacteria possess the property of forming _spores_ within the mother cell (called _endospores_, fig. 1g) that are analogous in function to the seeds of higher plants and spores of fungi. by means of these structures which are endowed with greater powers of resistance than the vegetating cell, the organism is able to protect itself from the effect of an unfavorable environment. many of the bacilli form endospores but the cocci do not. it is these spore forms that make it so difficult to thoroughly sterilize milk. ~movement.~ many bacteria are unable to move from place to place. they have, however, a vibrating movement known as the _brownian_ motion that is purely physical. many other kinds are endowed with powers of locomotion. motion is produced by means of fine thread-like processes of protoplasm known as _cilia_ (sing. _cilium_) that are developed on the outer surface of the cell. by means of the rapid vibration of these organs, the cell is propelled through the medium. nearly all cocci are immotile, while the bacilli may or may not be. these cilia are so delicate that it requires special treatment to demonstrate their presence. ~classification.~ in classifying or arranging the different members of any group of living objects, certain similarities and dissimilarities must be considered. these are usually those that pertain to the structure and form, as such are regarded as most constant. with the bacteria these differences are so slight that they alone do not suffice to distinguish distinctly one species from another. as far as these characters can be used, they are taken, but in addition, many characteristics of a physiological nature are added. the way that the organism grows in different kinds of cultures, the by-products produced in different media, and effect on the animal body when injected into the same are also used as data in distinguishing one species from another. ~conditions favoring bacterial growth.~ the bacteria, in common with all other living organisms are affected by external conditions, either favorably or unfavorably. certain conditions must prevail before development can occur. thus, the organism must be supplied with an adequate and suitable food supply and with moisture. the temperature must also range between certain limits, and finally, the oxygen requirements of the organism must be considered. ~food supply.~ most bacteria are capable of living on dead, inert, organic matter, such as meats, milk and vegetable material, in which case, they are known as _saprophytes_. in contradistinction to this class is a smaller group known as _parasites_, which derive their nourishment from the living tissues of animals or plants. the first group comprise by far the larger number of known organisms which are concerned for the most part in the decomposition of organic matter. the parasitic group includes those which are the cause of various communicable diseases. between these two groups there is no sharp line of division, and in some cases, certain species possess the faculty of growing either as parasites or saprophytes, in which case they are known as _facultative_ parasites or saprophytes. the great majority of bacteria of interest in dairying belong to the saprophytic class; only those species capable of infecting milk through the development of disease in the animal are parasites in the strict sense of the term. most disease-producing species, as diphtheria or typhoid fever, while parasitic in man lead a saprophytic method of life so far as their relation to milk is concerned. bacteria require for their growth, nitrogen, hydrogen, carbon, oxygen, together with a limited amount of mineral matter. the nitrogen and carbon are most available in the form of organic compounds, such as albuminous material. carbon in the form of carbohydrates, as sugar or starch, is most readily attacked by bacteria. inasmuch as the bacteria are plant-cells, they must imbibe their food from material in solution. they are capable of living on solid substances, but in such cases, the food elements must be rendered soluble, before they can be appropriated. if nutritive liquids are too highly concentrated, as in the case of syrups and condensed milk, bacteria cannot grow therein, although all the necessary ingredients may be present. generally, bacteria prefer a neutral or slightly alkaline medium, rather than one of acid reaction; but there are numerous exceptions to this general rule, especially among the bacteria found in milk. ~temperature.~ growth of bacteria can only occur within certain temperature limits, the extremes of which are designated as the _minimum_ and _maximum_. below and above these respective limits, life may be retained in the cell for a time, but actual cell-multiplication is stopped. somewhere between these two cardinal temperature points, and generally nearer the maximum limit is the most favorable temperature for growth, known as the _optimum_. the temperature zone of most dairy bacteria in which growth occurs ranges from 40°-45° f. to somewhat above blood-heat, 105°-110° f., the optimum being from 80°-95° f. many parasitic species, because of their adaptation to the bodies of warm-blooded animals, generally have a narrower range, and a higher optimum, usually approximating the blood heat (98°-99° f). the broader growth limits of bacteria in comparison with other kinds of life explain why these organisms are so widely distributed in nature. ~air supply.~ most bacteria require as do the green plants and animal life, the free oxygen of the air for their respiration. these are called _aerobic_. some species, however, and some yeasts as well possess the peculiar property of taking the oxygen which they need from organic compounds such as sugar, etc., and are therefore able to live and grow under conditions where the atmospheric air is excluded. these are known as _anaerobic_. while some species grow strictly under one condition or the other, and hence are _obligate_ aerobes or anaerobes, others possess the ability of growing under either condition and are known as _facultative_ or optional forms. the great majority of milk bacteria are either obligate or facultative aerobes. ~rate of growth.~ the rate of bacterial development is naturally very much affected by external conditions, food supply and temperature exerting the most influence. in the neighborhood of the freezing point but little growth occurs. the rate increases with a rise in temperature until at the _optimum_ point, which is generally near the blood heat or slightly below (90°-98° f.), a single cell will form two cells in 20 to 30 minutes. if temperature rises much above blood heat rate of growth is lessened and finally ceases. under ideal conditions, rapidity of growth is astounding, but this initially rapid rate of development cannot be maintained indefinitely, for growth is soon limited by the accumulation of by-products of cell activity. thus, milk sours rapidly at ordinary temperatures until the accumulation of acid checks its development. ~detrimental effect of external conditions.~ environmental influences of a detrimental character are constantly at work on bacteria, tending to repress their development or destroy them. these act much more readily on the vegetating cell than on the more resistant spore. a thorough knowledge of the effect of these antagonistic forces is essential, for it is often by their means that undesirable bacteria may be killed out. ~effect of cold.~ while it is true that chilling largely prevents fermentative action, and actual freezing stops all growth processes, still it does not follow that exposure to low temperatures will effectually destroy the vitality of bacteria, even in the vegetative condition. numerous non-spore-bearing species remain alive in ice for a prolonged period, and recent experiments with liquid air show that even a temperature of -310° f. for hours does not effectually kill all exposed cells. ~effect of heat.~ high temperatures, on the other hand, will destroy any form of life, whether in the vegetative or latent stage. the temperature at which the vitality of the cell is lost is known as the _thermal death point_. this limit is not only dependent upon the nature of the organism, but varies with the time of exposure and the condition in which the heat is applied. in a moist atmosphere the penetrating power of heat is great; consequently cell-death occurs at a lower temperature than in a dry atmosphere. an increase in time of exposure lowers the temperature point at which death occurs. for vegetating forms the thermal death point of most bacteria ranges from 130°-140° f. where the exposure is made for ten minutes which is the standard arbitrarily selected. in the spore stage resistance is greatly increased, some forms being able to withstand steam at 210°-212° f. from one to three hours. if dry heat is employed, 260°-300° f. for an hour is necessary to kill spores. where steam is confined under pressure, a temperature of 230°-240° f. for 15-20 minutes suffices to kill all spores. ~drying.~ spore-bearing bacteria like anthrax withstand drying with impunity; even tuberculous material, although not possessing spores retains its infectious properties for many months. most of the dairy bacteria do not produce spores, and yet in a dry condition, they retain their vitality unimpaired for considerable periods, if they are not subjected to other detrimental influences. ~light.~ bright sunlight exerts on many species a powerful disinfecting action, a few hours being sufficient to destroy all cells that are reached by the sun's rays. even diffused light has a similar effect, although naturally less marked. the active rays in this disinfecting action are those of the chemical or violet end of the spectrum, and not the heat or red rays. ~influence of chemical substances.~ a great many chemical substances exert a more or less powerful toxic action of various kinds of life. many of these are of great service in destroying or holding bacterial growth in check. those that are toxic and result in the death of the cell are known as _disinfectants_; those that merely inhibit, or retard growth are known as _antiseptics_. all disinfectants must of necessity be antiseptic in their action, but not all antiseptics are disinfectants even when used in strong doses. disinfectants have no place in dairy work, except to destroy disease bacteria, or preserve milk for analytical purposes. corrosive sublimate or potassium bichromate are most frequently used for these purposes. the so-called chemical preservatives used to "keep" milk depend for their effect on the inhibition of bacterial growth. with a substance so violently toxic as formaldehyde (known as formalin, freezene) antiseptic doses are likely to be exceeded. in this country most states prohibit the use of these substances in milk. their only function in the dairy should be to check fermentative or putrefactive processes outside of milk and so keep the air free from taints. ~products of growth.~ all bacteria in their development form certain more or less characteristic by-products. with most dairy bacteria, these products are formed from the decomposition of the medium in which the bacteria may happen to live. such changes are known, collectively, as fermentations, and are characterised by the production of a large amount of by-products, as a result of the development of a relatively small amount of cell-life. the souring of milk, the formation of butyric acid, the making of vinegar from cider, are all examples of fermentative changes. with many bacteria, especially those that affect proteid matter, foul-smelling gases are formed. these are known as putrefactive changes. all organic matter, under the action of various organisms, sooner or later undergoes decay, and in different stages of these processes, acids, alkalies, gases and numerous other products are formed. many of these changes in organic matter occur only when such material is brought in direct contact with the living bacterial cell. in other instances, soluble, non-vital ferments known as _enzyms_ are produced by the living cell, which are able to act on organic matter, in a medium free from live cells, or under conditions where the activity of the cell is wholly suspended. these enzyms are not confined to bacteria but are found throughout the animal and plant world, especially in those processes that are concerned in digestion. among the better known of these non-vital ferments are rennet, the milk-curdling enzym; diastase or ptyalin of the saliva, the starch-converting enzym; pepsin and trypsin, the digestive ferments of the animal body. enzyms of these types are frequently found among the bacteria and yeasts and it is by virtue of this characteristic that these organisms are able to break down such enormous quantities of organic matter. most of these enzyms react toward heat, cold and chemical poisons in a manner quite similar to the living cells. in one respect they are readily differentiated, and that is, that practically all of them are capable of producing their characteristic chemical transformations under anaesthetic conditions, as in a saturated ether or chloroform atmosphere. ~distribution of bacteria.~ as bacteria possess greater powers of resistance than most other forms of life, they are to be found more widely distributed than any other type. at the surface of the earth, where conditions permit of their growth, they are found everywhere, except in the healthy tissues of animals and plants. in the superficial soil layers, they exist in myriads, as here they have abundance of nourishment. at the depth of several feet however, they diminish rapidly in numbers, and in the deeper soil layers, from six to ten feet or more, they are not present, because of the unsuitable growth conditions. the bacteria are found in the air because of their development in the soil below. they are unable to grow even in a moist atmosphere, but are so readily dislodged by wind currents that over land areas the lower strata of the air always contain them. they are more numerous in summer than in winter; city air contains larger numbers than country air. wherever dried fecal matter is present, as in barns, the air contains many forms. water contains generally enough organic matter in solution, so that certain types of bacterial life find favorable growth conditions. water in contact with the soil surface takes up many impurities, and is of necessity rich in microbes. as the rain water percolates into the soil, it loses its germ content, so that the normal ground water, like the deeper soil layers, contains practically no bacterial life. springs therefore are relatively deficient in germ life, except as they become infected with soil organisms, as the water issues from the soil. water may serve to disseminate certain infectious diseases as typhoid fever and cholera among human beings, and a number of animal maladies. while the inner tissues of healthy animals are free from bacteria, the natural passages as the respiratory and digestive tracts, being in more direct contact with the exterior, become more readily infected. this is particularly true with reference to the intestinal tract, for in the undigested residue, bacterial activity is at a maximum. the result is that fecal matter contains enormous numbers of organisms so that the possibility of pollution of any food medium such as milk with such material is sure to introduce elements that seriously affect the quality of the product. chapter ii. methods of studying bacteria. ~necessity of bacterial masses for study.~ the bacteria are so extremely small that it is impossible to study individual germs separately without the aid of first-class microscopes. for this reason, but little advance was made in the knowledge of these lower forms of plant life, until the introduction of culture methods, whereby a single organism could be cultivated and the progeny of this cell increased to such an extent in a short course of time, that they would be visible to the unaided eye. this is done by growing the bacteria in masses on various kinds of food media that are prepared for the purpose, but inasmuch as bacteria are so universally distributed, it becomes an impossibility to cultivate any special form, unless the medium in which they are grown is first freed from all pre-existing forms of germ life. to accomplish this, it is necessary to subject the nutrient medium to some method of sterilization, such as heat or filtration, whereby all life is completely destroyed or eliminated. such material after it has been rendered germ-free is kept in sterilized glass tubes and flasks, and is protected from infection by cotton stoppers. ~culture media.~ for culture media, many different substances are employed. in fact, bacteria will grow on almost any organic substance whether it is solid or fluid, provided the other essential conditions of growth are furnished. the food substances that are used for culture purposes are divided into two classes; solids and liquids. solid media may be either permanently solid like potatoes, or they may retain their solid properties only at certain temperatures like gelatin or agar. the latter two are of utmost importance in bacteriological research, for their use, which was introduced by koch, permits the separation of the different forms that may happen to be in any mixture. gelatin is used advantageously because the majority of bacteria present wider differences due to growth upon this medium than upon any other. it remains solid at ordinary temperatures, becoming liquid at about 70° f. agar, a gelatinous product derived from a japanese sea-weed, has a much higher melting point, and can be successfully used, especially with those organisms whose optimum growth point is above the melting point of gelatin. besides these solid media, different liquid substances are extensively used, such as beef broth, milk, and infusions of various vegetable and animal tissues. skim-milk is of especial value in studying the milk bacteria and may be used in its natural condition, or a few drops of litmus solution may be added in order to detect any change in its chemical reaction due to the bacteria. [illustration: fig. 2. a gelatin plate culture showing appearance of different organisms in a sample of milk. each mass represents a bacterial growth (colony) derived from a single cell. different forms react differently toward the gelatin, some liquefying the same, others growing in a restricted mass. _a_, represents a colony of the ordinary bread mold; _b_, a liquefying bacterium; _c_, and _d_, solid forms.] ~methods of isolation.~ suppose for instance one wishes to isolate the different varieties of bacteria found in milk. the method of procedure is as follows: sterile gelatin in glass tubes is melted and cooled down so as to be barely warm. to this gelatin which is germ-free a drop of milk is added. the gelatin is then gently shaken so as to thoroughly distribute the milk particles, and poured out into a sterile flat glass dish and quickly covered. this is allowed to stand on a cool surface until the gelatin hardens. after the culture plate has been left for twenty-four to thirty-six hours at the proper temperature, tiny spots will begin to appear on the surface, or in the depth of the culture medium. these patches are called _colonies_ and are composed of an almost infinite number of individual germs, the result of the continued growth of a single organism that was in the drop of milk which was firmly held in place when the gelatin solidified. the number of these colonies represents approximately the number of germs that were present in the milk drop. if the plate is not too thickly sown with these germs, the colonies will continue to grow and increase in size, and as they do, minute differences will begin to appear. these differences may be in the color, the contour and the texture of the colony, or the manner in which it acts toward gelatin. in order to make sure that the seeding in not too copious so as to interfere with continued study, an _attenuation_ is usually made. this consists in taking a drop of the infected gelatin in the first tube, and transferring it to another tube of sterile media. usually this operation is repeated again so that these culture plates are made with different amounts of seed with the expectation that in at least one plate the seeding will not be so thick as to prevent further study. for transferring the culture a loop made of platinum wire is used. by passing this through a gas flame, it can be sufficiently sterilized. [illustration: fig. 3. profile view of gelatin plate culture; _b_, a liquefying form that dissolves the gelatin; _c_ and _d_, surface colonies that do not liquefy the gelatin.] to further study the peculiarities of different germs, the separate colonies are transferred to other sterile tubes of culture material and thus _pure cultures_ of the various germs are secured. these cultures then serve as a basis for continued study and must be planted and grown upon all the different kinds of media that are obtainable. in this way the slight variations in the growth of different forms are detected and the peculiar characteristics are determined, so that the student is able to recognize this form when he meets it again. these culture methods are of essential importance in bacteriology, as it is the only way in which it is possible to secure a quantity of germs of the same kind. ~the microscope in bacterial investigation.~ in order to verify the purity of the cultures, the microscope is in constant demand throughout all the different stages of the isolating process. for this purpose, it is essential that the instrument used shall be one of strong magnifying powers (600-800 diameters), combined with sharp definition. [illustration: fig. 4. pure cultures of different kinds of bacteria in gelatin tubes. _a_, growth slight in this medium; _b_, growth copious at and near surface. fine parallel filaments growing out into medium liquefying at surface; _c_, a rapid liquefying form; _d_, a gas-producing form that grows equally well in lower part of tube as at surface (facultative anaerobe); _e_, an obligate anaerobe, that develops only in absence of air.] the microscopical examination of any germ is quite as essential as the determination of culture characteristics; in fact, the two must go hand in hand. the examination reveals not only the form and size of the individual germs, but the manner in which they are united with each other, as well as any peculiarities of movement that they may possess. in carrying out the microscopical part of the work, not only is the organism examined in a living condition, but preparations are made by using solutions of anilin dyes as staining agents. these are of great service in bringing out almost imperceptible differences. the art of staining has been carried to the highest degree of perfection in bacteriology, especially in the detection of germs that are found in diseased tissues in the animal or human body. in studying the peculiarities of any special organism, not only is it necessary that these cultural and microscopical characters should be closely observed, but special experiments must be carried out along different lines, in order to determine any special properties that the germ may possess. thus, the ability of any form to act as a fermentative organism can be tested by fermentation experiments; the property of causing disease, studied by the inoculation of pure cultures into animals. a great many different methods have been devised for the purpose of studying special characteristics of different bacteria, but a full description of these would necessarily be so lengthy that in a work of this character they must be omitted. for details of this nature consult standard reference books on bacteriological technique. chapter iii. contamination of milk. no more important lesson is to be learned than that which relates to the ways in which milk is contaminated with germ life of various kinds; for if these sources of infection are thoroughly recognized they can in large measure be prevented, and so the troubles which they engender overcome. various organisms find in milk a congenial field for development. yeasts and some fungi are capable of growth, but more particularly the bacteria. ~milk a suitable bacterial food.~ the readiness with which milk undergoes fermentative changes indicates that it is well adapted to nourish bacterial life. not only does it contain all the necessary nutritive substances but they are diluted in proper proportions so as to render them available for bacterial as well as mammalian life. of the nitrogenous compounds, the albumen is in readily assimilable form. the casein, being insoluble, is not directly available, until it is acted upon by proteid-dissolving enzyms like trypsin which may be secreted by bacteria. the fat is relatively resistant to change, although a few forms are capable of decomposing it. milk sugar, however, is an admirable food for many species, acids and sometimes gases being generally produced. ~condition when secreted.~ when examined under normal conditions milk always reveals bacterial life, yet in the secreting cells of the udder of a healthy cow germ life is not found. only when the gland is diseased are bacteria found in any abundance. in the passage of the milk from the secreting cells to the outside it receives its first infection, so that when drawn from the animal it generally contains a considerable number of organisms. [illustration: fig. 5. microscopic appearance of milk showing relative size of fat globules and bacteria.] ~contamination of milk.~ from this time until it is consumed in one form or another, it is continually subjected to contamination. the major part of this infection occurs while the milk is on the farm and the degree of care which is exercised while the product is in the hands of the milk producer is the determining factor in the course of bacterial changes involved. this of course does not exclude the possibility of contamination in the factory, but usually milk is so thoroughly seeded by the time it reaches the factory that the infection which occurs here plays a relatively minor rôle to that which happens earlier. the great majority of the organisms in milk are in no wise dangerous to health, but many species are capable of producing various fermentative changes that injure the quality of the product for butter or cheese. to be able to control abnormal changes of an undesirable character one must know the sources of infection which permit of the introduction of these unwelcome intruders. ~sources of infection.~ the bacterial life that finds its way into milk while it is yet on the farm may be traced to several sources, which may be grouped under the following heads: unclean dairy utensils, fore milk, coat of animal, and general atmospheric surroundings. the relative importance of these various factors fluctuates in each individual instance. ~dairy utensils.~ of first importance are the vessels that are used during milking, and also all storage cans and other dairy utensils that come in contact with the milk after it is drawn. by unclean utensils, actually _visible_ dirt need not always be considered, although such material is often present in cracks and angles of pails and cans. unless cleansed with especial care, these are apt to be filled with foul and decomposing material that suffices to seed thoroughly the milk. tin utensils are best. where made with joints, they should be well flushed with solder so as to be easily cleaned (see fig. 6). in much of the cheaper tin ware on the market, the soldering of joints and seams is very imperfect, affording a place of refuge for bacteria and dirt. cans are often used when they are in a condition wholly unsuitable for sanitary handling of milk. when the tin coating becomes broken and the can is rusty, the quality of the milk is often profoundly affected. olson[1] of the wisconsin station has shown that the action of rennet is greatly impaired where milk comes in contact with a rusty iron surface. [illustration: fig. 6.] with the introduction of the form or hand separator a new milk utensil has been added to those previously in use and one which is very frequently not well cleaned. where water is run through the machine to rinse out the milk particles, gross bacterial contamination occurs and the use of the machine much increases the germ content of the milk. every time the separator is used it should be taken apart and thoroughly cleaned and dried before reassembling.[2] ~use of milk-cans for transporting factory by-products.~ the general custom of using the milk-cans to carry back to the farm the factory by-products (skim-milk or whey) has much in it that is to be deprecated. these by-products are generally rich in bacterial life, more especially where the closest scrutiny is not given to the daily cleaning of the vats and tanks. too frequently the cans are not cleaned immediately upon arrival at the farm, so that the conditions are favorable for rapid fermentation. many of the taints that bother factories are directly traceable to such a cause. a few dirty patrons will thus seriously infect the whole supply. the responsibility for this defect should, however, not be laid entirely upon the shoulders of the producer. the factory operator should see that the refuse material does not accumulate in the waste vats from day to day and is not transformed into a more or less putrid mass. a dirty whey tank is not an especially good object lesson to the patron to keep his cans clean. it is possible that abnormal fermentations or even contagious diseases may thus be disseminated. suppose there appears in a dairy an infectious milk trouble, such as bitter milk. this milk is taken to the factory and passes unnoticed into the general milk-supply. the skim-milk from the separator is of course infected with the germ, and if conditions favor its growth, the whole lot soon becomes tainted. if this waste product is returned to the different patrons in the same cans that are used for the fresh milk, the probabilities are strongly in favor of some of the cans being contaminated and thus infecting the milk supply of the patrons. if the organism is endowed with spores so that it can withstand unfavorable conditions, this taint may be spread from patron to patron simply through the infection of the vessels that are used in the transportation of the by-products. connell has reported just such a case in a canadian cheese factory where an outbreak of slimy milk was traced to infected whey vats. typhoid fever among people, foot and mouth disease and tuberculosis among stock are not infrequently spread in this way. in denmark, portions of germany and some states in america, compulsory heating of factory by-products is practiced to eliminate this danger.[3] ~pollution of cans from whey tanks.~ the danger is greater in cheese factories than in creameries, for whey usually represents a more advanced stage of fermentation than skim-milk. the higher temperature at which it is drawn facilitates more rapid bacterial growth, and the conditions under which it is stored in many factories contribute to the ease with which fermentative changes can go on in it. often this by-product is stored in wooden cisterns or tanks, situated below ground, where it becomes impossible to clean them out thoroughly. a custom that is almost universally followed in the swiss cheese factories, here in this country, as in switzerland, is fully as reprehensible as any dairy custom could well be. in fig. 7 the arrangement in vogue for the disposal of the whey is shown. the hot whey is run out through the trough from the factory into the large trough that is placed over the row of barrels, as seen in the foreground. each patron thus has allotted to him in his individual barrel his portion of the whey, which he is supposed to remove day by day. no attempt is made to clean out these receptacles, and the inevitable result is that they become filled with a foul, malodorous liquid, especially in summer. when such material is taken home in the same set of cans that is used to bring the fresh milk (twice a day as is the usual custom in swiss factories), it is no wonder that this industry is seriously handicapped by "gassy" fermentations that injure materially the quality of the product. not only is the above danger a very considerable one, but the quality of the factory by-product for feeding purposes, whether it is skim-milk or whey, is impaired through the development of fermentative changes. [illustration: fig. 7. swiss cheese factory (wisconsin), showing careless way in which whey is handled. each patron's share is placed in a barrel, from which it is removed by him. no attempt is made to cleanse these receptacles.] ~improved methods of disposal of by-products.~ the difficulties which attend the distribution of these factory by-products have led to different methods of solution. one is to use another separate set of receptacles to carry back these products to the farm. this method has been tried, and while it is deemed impracticable by many to handle two sets of vessels, yet some of the most progressive factories report excellent results where this method is in use. large barrels could be used for this purpose to economize in wagon space. another method that has met with wider acceptance, especially in creameries, is the custom of pasteurizing or scalding the skim-milk immediately after it is separated, so that it is returned to the farmer in a hot condition. in factories where the whole milk is pasteurized, further treatment of the by-product is not necessary. in most factories steam, generally exhaust, is used directly in the milk, and experience has shown that such milk, without any cooling, will keep sweet for a considerable number of hours longer than the untreated product. it is noteworthy that the most advanced and progressive factories are the ones that appreciate the value of this work, and although it involves some time and expense, experience has shown the utility of the process in that a better grade of milk is furnished by the patrons of factories which follow this practice.[4] the exclusion of all danger of animal or human disease is also possible in this way. ~cleaning dairy utensils.~ the thorough cleaning of all dairy apparatus that in any way comes in contact with the milk is one of the most fundamental and important problems in dairying. all such apparatus should be so constructed as to permit of easy cleaning. tinware, preferably of the pressed variety, gives the best surface for this purpose and is best suited for the handling of milk. milk vessels should never be allowed to become dry when dirty, for dried particles of milk residue are extremely difficult to remove. in cleaning dairy utensils they should first be rinsed in lukewarm instead of hot water, so as to remove organic matter without coagulating the milk. then wash thoroughly in hot water, using a good washing powder. the best washing powders possess considerable disinfecting action.[5] strong alkalies should not be used. after washing rinse thoroughly in clean hot water. if steam is available, as it always is in creameries, cans and pails should be turned over jet for a few moments. while a momentary exposure will not suffice to completely sterilize such a vessel, yet many bacteria are killed in even a short exposure, and the cans dry more thoroughly and quickly when heated by steam. not only should the greatest care be paid to the condition of the cans and milk-pails, but all dippers, strainers, and other utensils that come in contact with the milk must be kept equally clean. cloth strainers, unless attended to, are objectionable, for the fine mesh of the cloth retains so much moisture that they become a veritable hot-bed of bacterial life, unless they are daily boiled or steamed. the inability to thoroughly render vessels bacteria-free with the conveniences which are generally to be found on the farm has led in some cases to the custom of washing and sterilizing the milk cans at the factory. ~germ content of milk utensils.~ naturally the number of bacteria found in different milk utensils after they have received their regular cleaning will be subject to great fluctuations; but, nevertheless, such determinations are of value as giving a scientific foundation for practical methods of improvement. the following studies may serve to indicate the relative importance of the utensils as a factor in milk contamination. two cans were taken, one of which had been cleaned in the ordinary way, while the other was sterilized by steaming. before milking, the udder was thoroughly cleaned and special precautions taken to avoid raising of dust; the fore milk was rejected. milk drawn into these two cans showed the following germ content: no. bacteria hours before per cc. souring. steamed pail 165 28-1/2 ordinary pail 4265 23 harrison[6] has shown how great a variation is in the bacterial content in milk cans. the utensils were rinsed with 100 cc. of sterile water and numerical determinations of this rinsing water made. in poorly cleaned cans, the average germ content was 442,000; in cans washed in tepid water and then scalded--the best farm practice--54,000, and in cans carefully washed and then steamed for five minutes, 880. another method used by the writer is to wash the utensil with 100 cc. sterile wash water, using a sterile swab to remove dirt. then repeat the process twice or more with fresh rinsing waters, making plate cultures from each. the following data were obtained from three such determinations: no. bacteria in different washings. total no. i. ii. iii. bacteria. 7,800,000 1,450,000 49,000 9,299,000 283,000 43,400 35,000 361,400 1,685,000 105,000 61,400 1,851,400 ~infection of milk in udder cavity.~ a frequently neglected but considerable factor of infection is that which is attributable to the bacteria which are present in the udder and which are removed in large numbers during the milking process. an examination of the fore milk, i. e., the first few streams from each teat, and that which is subsequently withdrawn, generally reveals a very much larger number of organisms in the fore milk.[7] not infrequently will this part of the milk when drawn under as careful conditions as possible, contain several score thousand organisms per cc. if successive bacterial determinations are made at different periods of the milking, as shown in the following experiment, a marked diminution is to be noted after the first portion of the milk is removed: _bacterial content at different periods of milking._ fore 200th 2000th 4300th 6500th strippings. milk. cc. cc. cc. cc. expt. 1 6,500 1,700 475 220 75 5 expt. 2 8,100 1,650 400 240 50 10 by some observers it has been claimed that it is possible to secure absolutely sterile milk in the strippings but this is rarely so. it is quite probable that such reported results are due to the fact that too small quantities of milk were used in the examinations and so erroneous conclusions were drawn. this marked diminution in numbers indicates that the larger proportion of the organisms found in the fore milk are present in the lower portion of the udder and milk ducts. when consideration is given to the structure of the udder, it is readily apparent that infection will be greater here than above. [illustration: fig. 8. sectional view of udder showing teat with milk duct connecting exterior with the milk cistern. milk sinuses are mostly shown in cross section interspersed and below the secreting tissue (moore and ward).] the udder is composed of secreting tissue (_gland cells_) held in place by fibrous connective tissue. ramifying throughout this glandular structure are numerous channels (_milk sinuses_) that serve to convey the milk from the cells where it is produced into the _milk cistern_, a common receptacle just above the teats. this cavity is connected with the exterior through the milk duct in the teat, which is more or less tightly closed by the circular sphincter muscles, thus preventing the milk from flowing out. the mucous membranes of the milk duct and cistern are naturally moist. the habits of the animal render it impossible to prevent infection of the external opening at the end of the teat and there is no mechanical reason why bacteria cannot readily find their way along the moist lining membrane for some distance. if organisms are adapted to this kind of an environment, ideal conditions exist for their multiplication, as moisture, warmth and suitable food supply are present. the question arises how far up into this organ is penetration possible? within late years numerous observations have been made on the presence of organisms in the upper portion of the udder in contact with the secreting tissue.[8] these investigations prove that bacteria are distributed throughout the whole of the udder, although numerically they are much less abundant in the region of the milk-secreting tissue than in the lower portion. ward's conclusions are "that milk when secreted by the glands of a healthy udder is sterile. it may however, immediately become contaminated by the bacteria which are normally present in the smaller milk ducts of the udder." ~nature of bacteria in fore milk.~ generally speaking the number of different species found in the fore milk is not large, and of those which do appear many occur at only occasional intervals. moore[9] in the examination of 9 udders found 20 different forms, and of these only 3 species predominated, all of which proved to be micrococci. streptococci have also been quite frequently reported. freudenreich[10] found the most common types to be cocci, belonging to both the liquefying and non-liquefying class. peptonizing[11] and spore-bearing[12] species have also been reported as well as gas-producing[13] forms allied to the colon bacillus. such findings are, however, due in all probability to accidental invasion. most investigators report the absence of the distinctively lactic-acid group of organisms.[14] ~origin of bacteria in udder.~ there is no question but that many of the types of bacteria found in the udder gain access from the outside. those belonging to the spore-bearing, digesting and intestinal types have such a favorable opportunity for introduction from outside and are so unlikely to have come directly from the body of the animal, that the external source of infection is much more probable. whether this explanation answers the origin of the cocci that are so generally found in the upper portion of the udder is questionable. the statement is ordinarily made that the inner tissues of healthy organs are bacteria-free, but the studies of ford[15] seem to indicate that 70 per cent. of such organs, removed under aseptic conditions from guinea pigs, rabbits, dogs and cats contained living organisms. others have reported similar results in which cocci have been found[16] very similar to those occurring in the udder. these findings increase the probability that the origin of this type is from the blood. the persistence of certain species in the udder for months as noted by ward indicates possibility of growth of some forms at least. stocking[17] has shown where cows are not milked clean that the germ content of succeeding milkings is greatly increased. ~artificial introduction of bacteria into udder.~ if bacteria are capable of actually developing in the udder proper, it ought to be possible to easily demonstrate this by the artificial introduction of cultures. in a number of cases[18] such experiments have been made with various saprophytic forms, such as _b. prodigiosus_, lactic acid bacilli and others. in no case has it appeared evident that actual growth has occurred, although the introduced organism has been demonstrated in diminishing numbers for 5-6 days. even the common lactic acid germ and a yellow liquefying coccus isolated from the fore milk failed to persist for more than a few days when thus artificially introduced. this failure to colonize is indeed curious and needs explanation. is it due to unsuitable environmental conditions or attributable to the germicidal influence of the milk? various body fluids are known to possess the property of destroying bacteria and it is claimed by fokker[19] that this same property was found in freshly drawn milk. this peculiarity has also been investigated by freudenreich,[20] and hunziker[21] who find a similar property. no material increase in germ content takes place in milk for several hours when chilled to 40°-70° f.; on the other hand an actual, but usually not a marked decrease is observed for about 6 hours. this phenomenon varies with the milk of different cows. nothing is known as to the cause of this apparent germicidal action. the question is yet by no means satisfactorily settled, although the facts on which the hypothesis is based are not in controversy. if such a peculiarity belongs to milk, it is not at all improbable that it may serve to keep down the germ content in the udder. freudenreich[22] found that udders which were not examined for some time after death showed abundant growth, which fact he attributed to the loss of this germicidal property. the infection of the whole milk can be materially reduced by rejecting the fore milk, but it is questionable whether such rejection is worth while, except in the case of "sanitary" dairies where milk is produced with as low a germ content as possible. the intrinsic loss in butter fat in the fore milk is inconsiderable as the first few streams contain only about one-fifth the normal fat content. ~infection of milk after withdrawal from animal.~ the germ content of the milk, when it is being drawn from the animal is immediately increased upon contact with the atmosphere. these organisms are derived from the surrounding air and the utensils in which the milk is received and stored. the number of organisms which find their way into the milk depends largely upon the character of the surroundings. bacteria are so intimately associated with dirt, dust and filth of all kinds that wherever the latter are found, the former are sure to be present in abundance. the most important factors in the infection of the milk after withdrawal are the pollution which is directly traceable to the animal herself and the condition of the milk utensils. fortunately both of these sources of contamination are capable of being greatly minimized by more careful methods of handling. ~infection directly from the cow.~ it is a popular belief that the organisms found in milk are derived from the feed and water which the cow consumes, the same passing directly from the intestinal tract to the milk by the way of the blood circulation. such a view has no foundation in fact as bacteria absorbed into the circulation are practically all destroyed in the tissues by the action of the body fluids and cells.[23] while organisms cannot pass readily from the intestine to the udder, yet this must not be interpreted as indicating that no attention should be given to the bacterial character of the material consumed. the water supply given should be pure and wholesome and no decomposed or spoiled food should be used. the infection traceable directly to the cow is modified materially by the conditions under which the animal is kept and the character of the feed consumed. the nature of the fecal matter is in part dependent upon the character of the food. the more nitrogenous rations with which animals are now fed leads to the production of softer fecal discharges which is more likely to soil the coat of the animal unless care is taken. the same is true with animals kept on pasture in comparison with those fed dry fodder. stall-fed animals, however, are more likely to have their flanks fouled, unless special attention is paid to the removal of the manure. all dairy stalls should be provided with a manure drop which should be cleaned as frequently as circumstances will permit. [illustration: fig. 9. showing the bacterial contamination arising from hair. these hairs were allowed to fall on a sterile gelatin surface. the adherent bacteria developed readily in this medium, and the number of bacteria thus introduced into the milk from these hairs can be estimated by the number of developing colonies.] the animal herself contributes materially to the quota of germ life finding its way into the milk through the dislodgment of dust and filth particles adhering to its hairy coat. the nature of this coat is such as to favor the retention of these particles. unless care is taken the flanks and udder become polluted with fecal matter, which upon drying is displaced with every movement of the animal. every hair or dirt particle so dislodged and finding its way into the milk-pail adds its quota of organisms to the liquid. this can be readily demonstrated by placing cow's hairs collected with care on the moist surface of gelatin culture plates. almost invariably, bacteria will be found in considerable numbers adhering to such hairs as is indicated in fig. 9. dirt particles are even richer in germ life. not only is there the dislodgment of hairs, epithelial scales and masses of dirt and filth, but during the milking process, as at all other times, every motion of the animal is accompanied by a shower of _invisible_ particles more or less teeming with bacterial life. the amount of actual impurities found in milk is often considerable and when it is remembered that about one-half of fresh manure dissolves in milk,[24] and thus does not appear as sediment, the presence of this undissolved residue bespeaks filthy conditions as to milking. from actual tests made, it is computed that the city of berlin, germany consumes about 300 pounds of such dirt and filth daily. renk has laid down the following rule with reference to this insoluble dirt: if a sample of milk shows any evidence of impurity settling on a transparent bottom within two hours, it should be regarded as too dirty for use. while the number of organisms here introduced is at all times large, the character of the species is of even greater import. derived primarily from dirt and fecal matter, it is no wonder that such forms are able to produce very undesirable fermentative changes. ~influence of milker.~ the condition of the milker is not to be ignored in determining all possible factors of infection, for when clothed in the dust-laden garments that have been worn all day, a favorable opportunity for direct contamination is possible. the filthy practice of wetting the hands with milk just before milking is to be condemned. the milker's hands should be washed immediately before milking in clean water and dried. a pinch of vaseline on hands is sometimes used to obtain a firmer grasp and prevents the ready dislodgment of scales.[25] it must also be borne in mind that the milker may spread disease through the milk. in typhoid fever and diphtheria, the germs often remain in the system for weeks and thus make infection possible. stocking[26] has shown that the individual milker exerts a potent influence on the total germ content of milk, even where the procedure is quite the same. in sanitary dairies milkers are usually clad in white duck suits. ~milking by machinery.~ several mechanical devices have been invented for milking, some of which have been tested bacteriologically as to their efficiency. harrison[27] has examined the "thistle" machine but found a much higher germ content than with hand-drawn milk. the recent introduction of the burrel-lawrence-kennedy machine has led to numerous tests in which very satisfactory results have been obtained. if the rubber parts of the milker are thoroughly cleaned and kept in lime water solution, they remain nearly sterile. when milk is properly handled, the germ content may be greatly reduced. ~reduction in dirt and adherent bacteria.~ no factor of contamination is so susceptible of improvement as that which relates to the reduction in filth and dirt which gains access during and immediately subsequent to the milking. the care which is taken to keep the coat of the animal clean by grooming lessens very much the grosser portion of such contamination, but with a dry, hairy coat, fine scales and dust particles must of necessity be dislodged.[28] ordinarily the patron thinks all evidence of such dirt is removed if the milk is strained, but this process only lessens the difficulty; it does not overcome it. various methods are in use, the effectiveness of which is subject to considerable variation. some of these look to the elimination of the bacteria after they are once introduced into the milk; others to the prevention of infection in the first place. _1. straining the milk._ most of the visible, solid particles of filth, such as hairs, dirt particles, etc., can be removed by simple straining, the time-honored process of purification. as ordinarily carried out, this process often contributes to instead of diminishing the germ life in milk. the strainer cloths unless washed and thoroughly sterilized by boiling harbor multitudes of organisms from day to day and may thus actually add to the organisms present. various methods have been suggested for this simple process, but the most practical and efficient strainer is that made of fine wire gauze to which is added 3-4 layers of cheese cloth, the whole to set over the storage milk can. _2. filtration._ in europe especially, the system of cleaning milk by filtration through sand, gravel and other substances has been quite extensively used. these filters are built in sections and the milk passes from below upward. the filtering substance is washed in hot water immediately after use and then steamed and finally baked. while it is possible to remove the solid impurities in this way, the germ content cannot be greatly reduced.[29] cellulose filters have also been suggested[30] as an improvement over the sand filters. methods of filtration of this character have not been used under commercial conditions here in this country. _3. clarification in separator._ within recent years the custom has grown of clarifying milk or removing the visible dirt by passing the milk through a centrifugal separator the cream and skim milk being remixed after separation. this process naturally removes the solid impurities as dirt, hairs, epithelial scales and cells, also some of the casein, making what is known as centrifuge slime. this conglomerate mass is incomparably rich in germ life and the natural inference would be that the bacterial content of the milk would be greatly reduced by this procedure. eckles and barnes[31] noted a reduction of 37 to 56 per cent. of the bacteria but others have failed to observe such reductions.[32] this condition is explained by the more thorough breaking up of the bacterial masses in the process, thus apparently not reducing them in numbers. it is somewhat surprising that in spite of the elimination of much organic matter and bacteria, such clarified milk sours as rapidly as the untreated product.[33] the mechanical shock of separation ruptures the clusters of fat globules and so delays creaming and also lessens the consistency of cream derived from such milk. this practical disadvantage together with the increased expense of the operation and the failure to materially enhance the keeping quality of the product outweigh the advantage which might come from removal of solid impurities which can be largely accomplished on the farm by efficient straining. _4. washing the udder._ if a surface is moist, bacteria adherent to it cannot be dislodged by ordinary movements. thus the air over snow-covered mountains or oceans is relatively devoid of germ life. the method of moistening the udder is applied with success to the hairy coat of the animal thus subserving the double purpose of cleaning the animal and preventing in large measure the continual dislodgment of dust particles. after these parts have been well carded to remove loose hairs and dirt particles, the skin should be thoroughly moistened with clean water and then wiped. it has been urged that this procedure lessens the yield of milk but eckles[34] concludes from experiments that when the animal becomes accustomed to this treatment, no noticeable change in amount of milk or butter-fat is produced. the effectiveness of this method in reducing the actual amount of dirt and filth introduced into the milk as well as the great diminution in germ life is shown by the instructive experiments of fraser[35] who found that the actual amount of dirt dislodged from udders of apparently clean animals during the milking process was three and one-half times as much as when the cow's udders were washed. from udders visibly polluted one ounce of such filth was removed in 275 pounds of milk, while from cows whose udders had been washed, the same amount of dirt was distributed through 24,030 pounds. fraser observed as a result of 420 examinations that the average germ content of 4-inch culture dishes under clean but unwashed udders was 578, while under washed animals it was reduced to 192. from numerous tests made in the writer's laboratory, it is evident that the germ content of the milk in the pail is increased from 20,000-40,000 bacteria _per minute_ during the milking period. by far the larger part of this pollution can be easily prevented by cleaning and dampening the udder. _5. diminishing exposed surface of pail._ the entrance of organisms into the milk can be greatly reduced by lessening the area of the milk pail directly exposed to the dust shower. a number of so-called sanitary or hygienic milk pails have been devised for this purpose. in one case the pail is smaller at the top than bottom, but in most of them the common form is kept and the exposed area is lessened by means of a cover, the milk being received through a narrower opening. in some cases, strainers are also interposed so as to remove more effectually the coarse particles. it is necessary to have these covers and strainers constructed in such a way so they can be easily removed and cleaned. [illustration: fig. 10. sanitary milk pails designed to diminish the introduction of hairs, scales, dirt, etc., into milk.] stocking tested one of these pails (a, fig. 10) and found that 63 per cent of the dirt and 29 per cent. of the bacteria were prevented from passing into the milk. eckles examined one in which the germ content was found to be 3200 per cc. as against 43200 per cc. in a common open pail. this milk did not sour until it was 64 hours old in the first case while in the latter it curdled in 43 hours. ~air in barn.~ the atmosphere of the barn where the milking is done may frequently contribute considerable infection. germ life is incapable of development in the air, but in a dried condition, organisms may retain their vitality for long periods. anything which contributes to the production of dust in the stable and aids in the stirring up of the same increases the number of organisms to be found in the air (fig. 11). thus, the feeding of dry fodder and the bedding of animals with straw adds greatly to the germ life floating in the air. dust may vary much in its germ content depending upon its origin. fraser found the dust from corn meal to contain only about one-sixth to one-eighth as much germ life as that from hay or bran.[36] in time most of these dust particles settle to the floor, but where the herd is kept in the barn, the constant movement of the animals keeps these particles more or less in motion. much can be done by forethought to lessen the germ content of stables. feeding dry feed should not be done until after milking.[37] in some of the better sanitary dairies, it is customary to have a special milking room that is arranged with special reference to the elimination of all dust. in this way this source of infection may be quite obviated as the air of a clean, still room is relatively free from bacteria, especially if the floor is moistened. it has often been noted that the milk of stall-fed animals does not keep as well as that milked out of doors, a condition in part attributable to the lessened contamination. [illustration: fig. 11. effect of contaminated air. the number of spots indicate the colonies that have developed from the bacteria which fell in 30 seconds on the surface of the gelatin plate (3 inches in diameter). this exposure was made at time the cows were fed.] ~relative importance of different sources of infection.~ it is impossible to measure accurately the influence of the different sources of infection as these are continually subject to modification in each and every case. as a general rule, however, where milk is drawn and handled without any special care, the utensils and the animal contribute the larger proportion of dirt and bacteria that find their way into milk. where the manner of milking and handling is designed to exclude the largest number of organisms possible, the bacteria appearing in the fore milk make up the major portion remaining. by putting into practice the various suggestions that have been made with reference to diminishing the bacterial content of milk, it is possible to greatly reduce the number of organisms found therein, and at the same time materially improve the keeping quality of the milk. backhaus[38] estimates that the germ life in milk can be easily reduced to one-two thousandth of its original number by using care in milking. he reports a series of experiments covering two years in which milk was secured that averaged less than 10,000 bacteria per cc., while that secured under ordinary conditions averaged over 500,000. [illustration: fig. 12. bacterial content of milk handled in ordinary way. each spot represents a colony growing on gelatin plate. compare with fig. 13, where same quantity of milk is used in making culture. over 15,000 bacteria per cc. in this milk.] fig. 13 gives an illustration as to what care in milking will do in the way of eliminating bacteria. fig. 12 shows a gelatin plate seeded with the same quantity of milk that was used in making the culture indicated by fig. 13. the first plate was inoculated with milk drawn under good conditions, the germ content of which was found to be 15,500 bacteria per cc., while the sample secured under as nearly aseptic conditions as possible (fig. 13) contained only 330 organisms in the same volume. [illustration: fig. 13. bacterial content of milk drawn with care. diminished germ content is shown by smaller number of colonies (330 bacteria per cc.). compare this culture with that shown in fig. 12.] ~"sanitary" or "certified" milk.~ within recent years there has been more or less generally introduced into many cities, the custom of supplying high grade milk that has been handled in a way so as to diminish its germ content as much as possible. milk of this character is frequently known as "sanitary," "hygienic" or "certified," the last term being used in connection with a certification from veterinary authorities or boards of health as to the freedom of animals from contagious disease. frequently a numerical bacterial standard is exacted as a pre-requisite to the recommendation of the board of examining physicians. thus, the pediatric society of philadelphia requires all children's milk that receives its recommendation to have not more than 10,000 bacteria per cc. such a standard has its value in the scrupulous cleanliness that must prevail in order to secure these results. this in itself is practically a guarantee of the absence of those bacteria liable to produce trouble in children. the number of organisms found in such milks is surprisingly low when compared with ordinary milk. naturally, there is considerable fluctuation from day to day, and occasionally the germ content is increased to a high figure without any apparent reason. the average results though, show a greatly reduced number of organisms. de schweinitz[39] found in a washington dairy in 113 examinations extending throughout a year, an average of 6,485 bacteria per cc. the daily analyses made of the walker-gordon supply sold in philadelphia for an entire year, showed that the milk almost always contained less than 5,000 bacteria per cc. and on 120 days out of the year the germ content was 1,000 organisms per cc. or less. from a practical point of view, the improvement in quality of sanitary milk, in comparison with the ordinary product is seen in the enhanced keeping quality. during the paris exposition in 1900, milk and cream from several such dairies in the united states were shipped to paris, arriving in good condition after 15 to 18 days transit. when milk has been handled in such a way, it is evident that it is much better suited to serve as a food supply than where it has undergone the fermentative changes incident to the development of myriads of organisms. ~application of foregoing precautions to all milk producers.~ milk is so susceptible to bacterial changes that it is necessary to protect it from invasion, if its original purity is to be maintained, and yet, from a practical point of view, the use to which it is destined has much to do with the care necessary to take in handling. the effect of the bacterial contamination of milk depends largely upon the way in which the product is used. to the milk-man engaged in the distribution of milk for direct consumption, all bacterial life is more or less of a detriment, while to the butter-maker and cheese-maker some forms are a direct necessity. it is unnecessary and impracticable to require the same degree of care in handling milk destined to be worked up into factory products as is done, for instance, in sanitary milk supplies, but this fact should not be interpreted to mean that the care of milk for factories is a matter of small consequence. in fact no more important dairy problem exists, and the purer and better the quality of the raw material the better the product will be. particularly is this true with reference to cheese-making. dairymen have learned many lessons in the severe school of experience, but it is earnestly to be hoped that future conditions will not be summed up in the words of the eminent german dairy scientist, prof. fleischmann, when he says that "all the results of scientific investigation which have found such great practical application in the treatment of disease, in disinfection, and in the preservation of various products, are almost entirely ignored in milking." ~growth of bacteria in milk.~ milk is so well suited as a medium for the development of germ life that it might be expected that all microörganisms would develop rapidly therein, and yet, as a matter of fact, growth does not begin at once, even though the milk may be richly seeded. at ordinary temperatures, such as 70° f., no appreciable increase is to be noted for a period of 6-9 hours; at lower temperatures (54° f.) this period is prolonged to 30-40 hours or even longer. after this period has elapsed, active growth begins and continues more or less rapidly until after curdling. the cause of this suspended development is attributed to the germicidal properties inherent to the milk.[40] milk is of course seeded with a considerable variety of organisms at first. the liquefying and inert species are the most abundant, the distinctively lactic acid class occurring sparsely, if at all. as milk increases in age, germ growth begins to occur. more or less development of all types go on, but soon the lactic species gain the ascendency, owing to their being better suited to this environment; they soon outstrip all other species, with the result that normal curdling generally supervenes. the growth of this type is largely conditioned by the presence of the milk sugar. if the sugar is removed from milk by dialysis, the liquid undergoes putrefactive changes due to the fact that the putrefactive bacteria are able to grow if no acid is produced. ~relation of temperature to growth.~ when growth does once begin in milk, the temperature at which it is stored exerts the most profound effect on the rate of development. when milk is not artificially cooled, it retains its heat for some hours, and consequently the conditions become very favorable for the rapid multiplication of the contained organisms, as is shown in following results obtained by freudenreich[41]: _no. of bacteria per cc. in milk kept at different temperatures._ 77° f. 95° f. 5 hrs. after milking 10,000 30,000 8 " " " 25,000 12,000,000 12 " " " 46,000 35,280,000 26 " " " 5,700,000 50,000,000 [illustration: fig. 14. effect of cooling milk on the growth of bacteria.] conn[42] is inclined to regard temperature of more significance in determining the keeping quality than the original infection of the milk itself. milk which curdled in 18 hours at 98° f., did not curdle in 48 hours at 70°, and often did not change in two weeks, if the temperature was kept at 50° f. where kept for a considerable period at this low temperature, the milk becomes filled with bacteria of the undesirable putrefactive type, the lactic group being unable to form acid in any appreciable amounts. running well water can be used for cooling, if it is possible to secure it at a temperature of 48°-50° f. the use of ice, of course, gives better results, and in summer is greatly to be desired. the influence of these lowered temperatures makes it possible to ship milk long distances[43] by rail for city supplies, if the temperature is kept low during transit. ~mixing night and morning milk.~ not infrequently it happens when old milk is mixed with new, that the course of the fermentative changes is more rapid than would have been the case if the two milks had been kept apart. thus, adding the cooled night milk to the warm morning milk sometimes produces more rapid changes in both. the explanation for this often imperfectly understood phenomenon is that germ growth may have gone on in the cooled milk, and when this material is added to the warmer, but bacteria-poor, fresh milk, the temperature of the whole mass is raised to a point suitable for the more rapid growth of all bacteria than would have occurred if the older milk had been kept chilled. ~number of bacteria in milk.~ the number of organisms found in milk depends upon (1) the original amount of contamination, (2) the age of the milk, and (3) the temperature at which it has been held. these factors all fluctuate greatly in different cases; consequently, the germ life is subject to exceedingly wide variations. here in america, milk reaches the consumer with less bacteria than in europe, although it may often be older. this is due largely to the more wide-spread use of ice for chilling the milk _en route_ to market. examinations have been made of various supplies with the following results: sedgwick and batchelder found in 57 tests of boston milk from 30,000-4,220,000 per cc. jordan and heineman found 30% of samples of chicago milk to range from 100,000 to 1,000,000 while nearly one half were from 1-20,000,000 per cc. the germ content of city milks increase rapidly in the summer months. park[44] found 250,000 organisms per cc. in winter, about 1,000,000 in cool weather and 5,000,000 per cc. in hot summer weather. knox and bassett in baltimore report 1,500,000 in spring and nearly 4,500,000 in summer. eckles[45] studied milk under factory conditions. he finds from 1,000,000 to 5,000,000 per cc. in winter, and in summer from 15-30 millions. ~bacterial standards for city supplies.~ it would be very desirable to have a hygienic standard for city milk supplies, as there is a butter fat and milk-solid test, but the wide spread variation in germ content and the impracticability of utilizing ordinary bacterial determinations (on account of time required) makes the selection of such a standard difficult. some hold, as park, that such a standard is feasible. the new york city milk commission has set a standard of 30,000 bacteria per cc. for their certified milk and 100,000 per cc. for inspected milk. rochester, n. y. has attempted the enforcement of such a standard (limit, 100,000 per cc.) with good results it is claimed while boston has placed the legal limit at 500,000 per cc. quantitative standards would seem more applicable to "certified" or sanitary supplies than to general city supplies, where the wide range in conditions lead to such enormous variations that the bacterial standard seems too refined a method for practical routine inspection. ~other tests.~ any test to be of much service must be capable of being quickly applied. the writer believes for city milk inspectors that the acid test would serve a very useful purpose. this test measures the acidity of the milk. there is, of course, no close and direct relationship between the development of acidity and the growth of bacteria, yet in a general way one follows the other at normal temperatures. where the temperature is kept rather low, bacterial growth might go on without much acid development, but in the great majority of cases a high degree of acidity means either old milk, in which there has been a long period of incubation, or high temperature, where rapid bacterial growth has been possible. either of these conditions encourages germ growth and thus impairs the quality of the milk. the rapid determination of acidity may be made in an approximate manner so as to serve as a test at the weigh-can or intake. the test is best made by the use of the well known alkaline tablet which is composed of a solid alkali, and the indicator, phenolphthalein. the tablets are dissolved in water, one to each ounce used. a number of white cups are filled with the proper quantity of the solution necessary to neutralize say, 0.2 per cent. lactic acid. then, as the milk is delivered, the proper quantity is taken from each can to which is added the tablet solution. a retention of the pink color shows that there is not sufficient acid in the milk to neutralize the alkali used; a disappearance of color indicates an excess of acid. the standard selected is of course arbitrarily chosen. in our experience, 0.2 per cent. acidity (figured as lactic), has proven a satisfactory point. with carefully handled milk the acidity ought to be reduced to about 0.15 per cent. the acidity of the milk may be abnormally reduced if milk is kept in rusty cans, owing to action of acid on the metal. [illustration: fig. 15. apparatus used in making rapid acid test. a definite quantity of the alkali solution and indicator is placed in the white tea cup. to this is added the quantity of milk by means of the cartridge measure which would just be neutralized if the acidity was 0.2 per cent. a retention of the pink color shows a low acid milk; its disappearance, a high acid milk.] ~kinds of bacteria in milk.~ the number of bacteria in milk is not of so much consequence as the kinds present. with reference to the number of different species, the more dirt and foreign matter the milk contains, the larger the number of varieties found in the same. while milk may contain forms that are injurious to man, still the great majority of them have no apparent effect on human health. in their effect on milk, the case is much different. depending upon their action in milk, they may be grouped into three classes: 1. inert group, those producing no visible change in the milk. 2. sour milk forms, those breaking up the milk sugar with or without the formation of gas. 3. digesting or peptonizing group, those capable of rendering the casein of milk soluble and more or less completely digested. a surprisingly large number of bacteria that are found in milk belong to the first class. undoubtedly they affect the chemical characteristics of the milk somewhat, but not to the extent that it becomes physically perceptible. eckles[46] reports in a creamery supply from 20 to 55 per cent. of entire flora as included in this class. by far the most important group is that embraced under the second head. it includes not only the true lactic acid types in which no gas is formed, but those species capable of producing gases and various kinds of acids. these organisms are the distinctively milk bacteria, although they do not predominate when the milk is first drawn. their adaptation to this medium is normally shown, however, by this extremely rapid growth, in which they soon gain the ascendency over all other species present. it is to this lactic acid class that the favorable flavor-producing organisms belong which are concerned in butter-making. they are also indispensable in cheese-making. the third class represents those capable of producing a liquefied or digested condition on gelatin or in milk. they are usually the spore-bearing species which gain access from filth and dirt. their high powers of resistance due to spores makes it difficult to eradicate this type, although they are materially held in subjection by the lactic bacteria. the number of different kinds that have been found in milk is quite considerable, something over 200 species having been described more or less thoroughly. in all probability, however, many of these forms will be found to be identical when they are subjected to a more critical study. ~direct absorption of taints.~ a tainted condition in milk may result from the development of bacteria, acting upon various constituents of the milk, and transforming these in such a way as to produce by-products that impair the flavor or appearance of the liquid; or it may be produced by the milk being brought in contact with any odoriferous or aromatic substance, under conditions that permit of the direct absorption of such odors. this latter class of taints is entirely independent of bacterial action, and is largely attributable to the physical property which milk possesses of being able to absorb volatile odors, the fat in particular, having a great affinity for many of these substances. this direct absorption may occur before the milk is withdrawn from the animal, or afterwards if exposed to strong odors. it is not uncommon for the milk of animals advanced in lactation to have a more or less strongly marked odor and taste; sometimes this is apt to be bitter, at other times salty to the taste. it is a defect that is peculiar to individual animals and is liable to recur at approximately the same period in lactation. the peculiar "cowy" or "animal odor" of fresh milk is an inherent peculiarity that is due to the direct absorption of volatile elements from the animal herself. this condition is very much exaggerated when the animal consumes strong-flavored substances as garlic, leeks, turnips and cabbage. the volatile substances that give to these vegetables their characteristic odor are quickly diffused through the system, and if such foods are consumed some few hours before milking, the odor in the milk will be most pronounced. the intensity of such taints is diminished greatly and often wholly disappears, if the milking is not done for some hours (8-12) after such foods are consumed. this same principle applies in lesser degree to many green fodders that are more suitable as feed for animals, as silage, green rye, rape, etc. not infrequently, such fodders as these produce so strong a taint in milk as to render it useless for human use. troubles from such sources could be entirely obviated by feeding limited quantities of such material immediately after milking. under such conditions the taint produced is usually eliminated before the next milking. the milk of swill-fed cows is said to possess a peculiar taste, and the urine of animals fed on this food is said to be abnormally acid. brewers' grains and distillery slops when fed in excess also induce a similar condition in the milk. milk may also acquire other than volatile substances directly from the animal, as in cases where drugs, as belladonna, castor oil, sulfur, turpentine, jalap, croton oil, and many others have been used as medicine. such mineral poisons as arsenic have been known to appear eight hours after ingestion, and persist for a period of three weeks before being eliminated. ~absorption of odors after milking.~ if milk is brought in contact with strong odors after being drawn from the animal, it will absorb them readily, as in the barn, where frequently it is exposed to the odor of manure and other fermenting organic matter. it has long been a popular belief that milk evolves odors and cannot absorb them so long as it is warmer than the surrounding air, but from experimental evidence, the writer[47] has definitely shown that the direct absorption of odors takes place much more rapidly when the milk is warm than when cold, although under either condition, it absorbs volatile substances with considerable avidity. in this test fresh milk was exposed to an atmosphere impregnated with odors of various essential oils and other odor-bearing substances. under these conditions, the cooler milk was tainted very much less than the milk at body temperature even where the period of exposure was brief. it is therefore evident that an exposure in the cow barn where the volatile emanations from the animals themselves and their excreta taint the air will often result in the absorption of these odors by the milk to such an extent as to seriously affect the flavor. the custom of straining the milk in the barn has long been deprecated as inconsistent with proper dairy practice, and in the light of the above experiments, an additional reason is evident why this should not be done. even after milk is thoroughly cooled, it may absorb odors as seen where the same is stored in a refrigerator with certain fruits, meats, fish, etc. ~distinguishing bacterial from non-bacterial taints.~ in perfectly fresh milk, it is relatively easy to distinguish between taints caused by the growth of bacteria and those attributable to direct absorption. if the taint is evident at time of milking, it is in all probability due to character of feed consumed, or possibly to medicines. if, however, the intensity of the taint grows more pronounced as the milk becomes older, then it is probably due to living organisms, which require a certain period of incubation before their fermentative properties are most evident. moreover, if the difficulty is of bacterial origin, it can be frequently transferred to another lot of milk (heated or sterilized is preferable) by inoculating same with some of the original milk. not all abnormal fermentations are able though to compete with the lactic acid bacteria, and hence outbreaks of this sort soon die out by the re-establishment of more normal conditions. ~treatment of directly absorbed taints.~ much can be done to overcome taints of this nature by exercising greater care in regard to the feed of animals, and especially as to the time of feeding and milking. but with milk already tainted, it is often possible to materially improve its condition. thorough aeration has been frequently recommended, but most satisfactory results have been obtained where a combined process of aeration and pasteurization was resorted to. where the milk is used in making butter, the difficulty has been successfully met by washing the cream with twice its volume of hot water in which a little saltpeter has been dissolved (one teaspoonful per gallon), and then separating it again.[48] the treatment of abnormal conditions due to bacteria has been given already under the respective sources of infection, and is also still further amplified in following chapter. ~aeration.~ it is a common belief that aeration is of great aid in improving the quality of milk, yet, when closely studied, no material improvement can be determined, either where the milk is made into butter or sold as milk. dean in canada and storch in denmark have both experimented on the influence of aeration in butter making, but with negative results. marshall and doane failed to observe any material improvement in keeping quality, but it is true that odors are eliminated from the milk during aeration. the infection of the milk during aeration often more than counterbalances the reputed advantage. especially is this so if the aeration is carried out in an atmosphere that is not perfectly clean and pure. in practice aeration differs greatly. in some cases, air is forced into the milk; in others, the milk is allowed to distribute itself in a thin sheet over a broad surface and fall some distance so that it is brought intimately in contact with the air. this latter process is certainly much more effective if carried out under conditions which preclude infection. it must be remembered that aeration is frequently combined with cooling, in which case the reputed advantages may not be entirely attributable to the process of aeration. ~infection of milk in the factory.~ the problem of proper handling of milk is not entirely solved when the milk is delivered to the factory or creamery, although it might be said that the danger of infection is much greater while the milk is on the farm. in the factory, infection can be minimized because effective measures of cleanliness can be more easily applied. steam is available in most cases, so that all vats, cans, churns and pails can be thoroughly scalded. special emphasis should be given to the matter of cleaning pumps and pipes. the difficulty of keeping these utensils clean often leads to neglect and subsequent infection. in swiss cheese factories, the custom of using home-made rennet solutions is responsible for considerable factory infection. natural rennets are soaked in whey which is kept warm in order to extract the rennet ferment. this solution when used for curdling the milk often adds undesirable yeasts and other gas-generating organisms, which are later the cause of abnormal ferment action in the cheese (see page 186). the influence of the air on the germ content of the milk is, as a rule, overestimated. if the air is quiet, and free from dust, the amount of germ life in the same is not relatively large. in a creamery or factory, infection from this source ought to be much reduced, for the reason that the floors and wall are, as a rule, quite damp, and hence germ life cannot easily be dislodged. the majority of organisms found under such conditions come from the person of the operators and attendants. any infection can easily be prevented by having the ripening cream-vats covered with a canvas cloth. the clothing of the operator should be different from the ordinary wearing-apparel. if made of white duck, the presence of dirt is more quickly recognized, and greater care will therefore be taken than if ordinary clothes are worn. the surroundings of the factory have much to do with the danger of germ infection. many factories are poorly constructed and the drainage is poor, so that filth and slime collect about and especially under the factory. the emanations from these give the peculiar "factory odor" that indicates fermenting matter. not only are these odors absorbed directly, but germ life from the same is apt to find its way into the milk. connell[49] has recently reported a serious defect in cheese that was traced to germ infection from defective factory drains. the water supply of a factory is also a question of prime importance. when taken from a shallow well, especially if surface drainage from the factory is possible, the water may be contaminated to such an extent as to introduce undesirable bacteria in such numbers that the normal course of fermentation may be changed. the quality of the water, aside from flavor, can be best determined by making a curd test (p. 76) which is done by adding some of the water to boiled milk and incubating the same. if "gassy" fermentations occur, it signifies an abnormal condition. in deep wells, pumped as thoroughly as is generally the case with factory wells, the germ content should be very low, ranging from a few score to a few hundred bacteria per cc. at most. harrison[50] has recently traced an off-flavor in cheese in a canadian factory to an infection arising from the water-supply. he found the same germ in both water and cheese and by inoculating a culture into pasteurized milk succeeded in producing the undesirable flavor. the danger from ice is much less, for the reason that good dairy practice does not sanction using ice directly in contact with milk or cream. then, too, ice is largely purified in the process of freezing, although if secured from a polluted source, reliance should not be placed in the method of purification; for even freezing does not destroy all vegetating bacteria. footnotes: [1] olson. 24 rept. wis. expt. stat., 1907. [2] erf and melick bull. 131, kan. expt. stat., apr. 1905. [3] storch (40 rept. danish expt. stat., copenhagen, 1898) has devised a test whereby it can be determined whether this treatment has been carried out or not: milk contains a soluble enzym known as galactase which has the property of decomposing hydrogen peroxid. if milk is heated to 176° f. (80° c.) or above, this enzym is destroyed so that the above reaction no longer takes place. if potassium iodid and starch are added to unheated milk and the same treated with hydrogen peroxid, the decomposition of the latter agent releases oxygen which acts on the potassium salt, which in turn gives off free iodine that turns the starch blue. [4] mckay, n. y. prod. rev., mch. 22, 1899. [5] doane, bull. 79, md. expt. stat., jan. 1902. [6] harrison, 22 rept. ont. agr'l coll., 1896, p. 113. [7] moore and ward, bull. 158, cornell expt. stat., jan. 1899; ward, bull. 178, cornell expt. stat., jan. 1900. [8] harrison, 22 rept. ont. agr. coll., 1896, p. 108; moore, 12 rept. bur. animal ind., u. s. dept. ag., 1895-6, p. 261. [9] moore, bacteria in milk, n. y. dept. ag., 1902. [10] freudenreich, cent. f. bakt., ii abt., 10: 418, 1903. [11] harrison, 22 rept. ont. agr. coll., 1896, p. 108. [12] marshall, bull. 147, mich. expt. stat., p. 42. [13] moore and ward, bull. 158, cornell expt. stat., jan. 1899. [14] burr, r. h. cent. f. bakt., ii abt., 8: 236, 1902. freudenreich, l. c. p. 418. ward, bull. 178, cornell expt. stat., p. 277. bolley (cent. f. bakt., ii abt., 1: 795, 1895), in 30 experiments found 12 out of 16 species to belong to lactic class. harrison (trans. can. inst., 7: 474, 1902-3) records the lactic type as most commonly present. [15] ford, journ. of hyg., 1901, 1: 277. [16] freudenreich, l. c. p. 421. [17] stocking, bull. 42, storrs expt. stat., june, 1906. [18] dinwiddie, bull, 45 ark. expt. stat., p. 57. ward, journ. appld. mic. 1: 205, 1898. appel, milch zeit., no. 17, 1900. harrison and cumming, journ. appld. mic. 5: 2087. russell and hastings, 21 rept. wis. expt. stat., 158, 1904. [19] fokker, zeit. f. hyg., 9: 41, 1890. [20] freudenreich, ann. de microg., 3: 118, 1891. [21] hunziker, bull. 197, cornell expt. stat., dec. 1901. [22] freudenreich, cent. f. bakt., ii abt., 10: 417, 1903. [23] this general statement is in the main correct, although ford (journ. of hyg., 1: 277, 1901) claims to have found organisms sparingly present in healthy tissues. [24] backhaus, milch zeit., 26: 357, 1897. [25] freudenreich, die bakteriologie, p. 30. [26] stocking, bull. 42, storrs expt. stat., june 1906. [27] harrison, cent. f. bakt., ii abt., 5: 183, 1899. [28] drysdale, trans. high. and agr. soc. scotland. 5 series, 10: 166, 1898. [29] schuppan, (cent. f. bakt., 13: 155, 1893) claims to have found a reduction of 48 per cent. in the copenhagen filters while in the more extended work of dunbar and kister (milch zeit., pp. 753, 787, 1899) the bacterial content was higher in the filtered milk in 17 cases out of 22. [30] backhaus and cronheim, journ. f. landw., 45: 222, 1897. [31] eckles and barnes, bull. 159 iowa expt. stat., aug. 1901. [32] dunbar and kister, milch zeit., p. 753, 1899. harrison and streit, trans. can. inst., 7: 488, 1902-3. [33] doane, bull. 88 md. expt. stat., may 1903. [34] eckles, hoard's dairyman, july 8, 1898. [35] fraser, bull. 91, ill. expt. stat. [36] fraser, bull. 91, ill. expt. stat., dec. 1903. [37] stocking, bull. 42, storrs expt. stat., june, 1906. [38] backhaus. ber. landw. inst. univ. königsberg 2: 12, 1897. [39] de schweinitz, nat. med. rev., april, 1899. [40] conn, proc. soc. amer. bacteriologists, 1902. [41] freudenreich, ann. de microg., 2:115, 1890. [42] conn, bull. 26, storrs expt. stat. [43] new york city is supplied with milk that is shipped 350 miles. [44] park, n. y. univ. bull., 1: 85, 1901. [45] eckles, bull. 59, iowa expt. stat., aug. 1901. [46] eckles, bull. 59, iowa expt. stat., aug. 1901. [47] russell, 15 rept. wis. expt. stat. 1898, p. 104. [48] alvord, circ. no. 9, u. s. dept. agric. (div. of bot.). [49] connell, rept. of commissioner of agr., canada, 1897, part xvi, p. 15. [50] harrison, hoard's dairyman, march 4, 1898. chapter iv. fermentations in milk and their treatment. under the conditions in which milk is drawn, it is practically impossible to secure the same without bacterial contamination. the result of the introduction of these organisms often changes its character materially as most bacteria cause the production of more or less pronounced fermentative processes. under normal conditions, milk sours, i. e., develops lactic acid, but at times this more common fermentation may be replaced by other changes which are marked by the production of some other more or less undesirable flavor, odor or change in appearance. in referring to these changes, it is usually customary to designate them after the most prominent by-product formed, but it must be kept in mind that generally some other decomposition products are usually produced. whether the organisms producing this or that series of changes prevail or not depends upon the initial seeding, and the conditions under which the milk is kept. ordinarily, the lactic acid organisms grow so luxuriantly in the milk that they overpower all competitors and so determine the nature of the fermentation; but occasionally the milk becomes infected with other types of bacteria in relatively large numbers and the conditions may be especially suitable to the development of these forms, thereby modifying the course of the normal changes that occur. the kinds of bacteria that find it possible to develop in milk may be included under two heads: 1. those which cause no appreciable change in the milk, either in taste, odor or appearance. while these are frequently designated as the inert bacteria, it must not be supposed that they have absolutely no effect on milk. it is probably true in most cases that slight changes of a chemical nature are produced, but the nature of the changes do not permit of ready recognition. 2. this class embraces all those organisms which, as a result of their growth, are capable of producing evident changes. these transformations may be such as to affect the taste, as in the sour milk or in the bitter fermentations, or the odor, as in some of the fetid changes, or the appearance of the milk, as in the slimy and color changes later described. ~souring of milk.~ ordinarily if milk is allowed to stand for several days at ordinary temperatures it turns sour. this is due to the formation of lactic acid, which is produced by the decomposition of the milk-sugar. while this change is well nigh universal, it does not occur without a pre-existing cause, and that is the presence of certain living bacterial forms. these organisms develop in milk with great rapidity, and the decomposition changes that are noted in souring are due to the by-products of their development. the milk-sugar undergoes fermentation, the chief product being lactic acid, although various other by-products, as other organic acids (acetic, formic and succinic), different alcohols and gaseous products, as co_{2}, h, n and methane (ch_{4}) are produced in small amounts. in this fermentation, the acidity begins to be evident to the taste when it reaches about 0.3 per cent., calculated as lactic acid. as the formation of acid goes on, the casein is precipitated and incipient curdling or lobbering of the milk occurs. this begins to be apparent when the acidity is about 0.4 per cent., but the curd becomes more solid with increasing acidity. the rapidity of curdling is also dependent upon the temperature of the milk. thus milk which at ordinary temperatures might remain fluid often curdles when heated. the growth of the bacteria is continued until about 0.8 to 1.0 per cent. acid is formed, although the maximum amount fluctuates considerably with different lactic acid species. further formation then ceases even though all of the milk-sugar is not used up, because of the inability of the lactic bacteria to continue their growth in such acid solutions. as this acidity is really in the milk serum, cream never develops so much acid as milk, because a larger proportion of its volume is made up of butter-fat globules. this fact must be considered in the ripening of cream in butter-making where the per cent. of fat is subject to wide fluctuations. the formation of lactic acid is a characteristic that is possessed by a large number of bacteria, micrococci as well as bacilli being numerously represented. still the preponderance of evidence is in favor of the view that a few types are responsible for most of these changes. the most common type found in spontaneously soured milk changes the milk-sugar into lactic acid without the production of any gas. this type has been described by various workers on european as well as american milks, and is designated by conn as the _bact. lactis acidi_ type.[51] it is subject to considerable variation under different conditions. curiously enough if milk which has been drawn with special care is examined immediately after milking, the lactic organisms are not usually found. they are incapable of development in the udder itself, as shown by injections into the milk cistern. they abound, however, on hay, in dust, in the barn air, on the hairy coat of the animal, and from these sources easily gain access to the milk. in this medium they find an exceptionally favorable environment and soon begin a very rapid growth, so that by the time milk is consumed, either in the form of milk or milk products, they make up numerically the larger portion of the bacteria present. another widely disseminated, although numerically less prevalent, type is _b. lactis aerogenes_. this type forms gas in milk so that the soured milk is torn by the presence of gas bubbles. it also grows more luxuriantly in contact with the air. other types occur more or less sporadically, some of which are capable of liquefying the casein of milk while at the same time they also develop lactic acid. conn and aikman refer to the fact that over one hundred species capable of producing variable quantities of lactic acid are already known. it is fair to presume, however, that a careful comparative study of these would show that simply racial differences exist in many cases, and therefore, that they are not distinct species. as a group these bacteria are characterized by their inability to liquefy gelatin or develop spores. on account of this latter characteristic they are easily destroyed when milk is pasteurized. they live under aerobic or anaerobic conditions, many of them being able to grow in either environment, although, according to mcdonnell,[52] they are more virulent when air is not excluded. while growth of these lactic forms may go on in milk throughout a relatively wide range in temperature, appreciable quantities of acid are not produced except very slowly at temperatures below 50° f.[53] from the standpoint of frequency the most common abnormal changes that occur in milk are those in which gases of varying character are developed in connection with acids, from the milk sugar. other volatile products imparting bad flavors usually accompany gas production. these fermentations are of most serious import in the cheese industry, as they are especially prone to develop in the manufacture of milk into certain types of cheese. not often is their development so rapid that they appear in the milk while it is yet in the hands of the milk producer, but almost invariably the introduction of the causal organisms takes place while the milk is on the farm. numerous varieties of bacteria possess this property of producing gas (h and co_{2} are most common although n and methane (ch_{4}) are sometimes produced). the more common forms are those represented by _b. lactis aerogenes_ and the common fecal type, _b. coli commune_. the ordinary habitat of this type is dirt and intestinal filth. hence careless methods of milk handling invite this type of abnormal change in milk. it is a wide-spread belief that thunder storms cause milk to sour prematurely, but this idea has no scientific foundation. experiments[54] with the electric spark, ozone and loud detonations show no effect on acid development, but the atmospheric conditions usually incident to a thunder storm are such as permit of a more rapid growth of organisms. there is no reason to believe but that the phenomenon of souring is wholly related to the development of bacteria. sterile milks are never affected by the action of electric storms. ~"gassy" milks.~ where these gas bacteria abound, the amount of lactic acid is generally reduced, due to the splitting up of some of the sugar into gaseous products. this type of germ life does not seem to be able to develop well in the presence of the typical lactic acid non gas-forming bacteria. [illustration: fig. 16. cheese made from "gassy" milk.] ~"sweet curdling" and digesting fermentations.~ not infrequently milk, instead of undergoing spontaneous souring, curdles in a weakly acid or neutral condition, in which state it is said to have undergone "sweet curdling." the coagulation of the milk is caused by the action of enzyms of a rennet type that are formed by the growth of various species of bacteria. later the whey separates more or less perfectly from the curd, producing a "wheyed off" condition. generally the coagulum in these cases is soft and somewhat slimy. the curd usually diminishes in bulk, due to the gradual digestion or peptonization of the casein by proteid-dissolving enzyms (tryptic type) that are also produced by the bacteria causing the change. a large number of bacteria possess the property of affecting milk in this way. so far as known they are able to liquefy gelatin (also a peptonizing process) and form spores. the tyrothrix type of bacteria (so named by duclaux on account of the supposed relation to cheese ripening) belongs to this class. the hay and potato forms are also digesters. organisms of this type are generally associated with filth and manure, and find their way into the milk from the accumulations on the coat of the animal. conn[55] has separated the rennet enzym from bacterial cultures in a relatively pure condition, while fermi[56] has isolated the digestive ferment from several species. duclaux[57] has given to this digesting enzym the name _casease_ or cheese ferment. these isolated ferments when added to fresh milk possess the power of causing the characteristic curdling and subsequent digestion quite independent of cell development. the quantity of ferment produced by different species differs materially in some cases. in these digestive fermentations, the chemical transformations are profound, the complex proteid molecule being broken down into albumoses, peptones, amido-acids (tyrosin and leucin) and ammonia as well as fatty acids. not infrequently these fermentations gain the ascendency over the normal souring change, but under ordinary conditions they are held in abeyance, although this type of bacteria is always present to some extent in milk. when the lactic acid bacteria are destroyed, as in boiled, sterilized or pasteurized milk, these rennet-producing, digesting species develop. ~butyric acid fermentations.~ the formation of butyric acid in milk which may be recognized by the "rancid butter" odor is not infrequently seen in old, sour milk, and for a long time was thought to be a continuation of the lactic fermentation, but it is now believed that these organisms find more favorable conditions for growth, not so much on account of the lactic acid formed as in the absence of dissolved oxygen in the milk which is consumed by the sour-milk organisms. most of the butyric class of bacteria are spore-bearing, and hence they are frequently present in boiled or sterilized milk. the by-products formed in this series of changes are quite numerous. in most cases, butyric acid is prominent, but in addition to this, other organic acids, as lactic, succinic, and acetic, are produced, likewise different alcohols. concerning the chemical origin of butyric acid there is yet some doubt. duclaux[58] affirms that the fat, sugar and casein are all decomposed by various forms. in some cases, the reaction of the milk is alkaline, with other species it may be neutral or acid. this type of fermentation has not received the study it deserves. in milk these organisms are not of great importance, as this fermentation does not readily gain the ascendency over the lactic bacteria. ~ropy or slimy milk.~ the viscosity of milk is often markedly increased over that which it normally possesses. the intensity of this abnormal condition may vary much; in some cases the milk becoming viscous or slimy; in others stringing out into long threads, several feet in length, as in fig. 17. two sets of conditions are responsible for these ropy or slimy milks. the most common is where the milk is clotted or stringy when drawn, as in some forms of garget. this is generally due to the presence of viscid pus, and is often accompanied by a bloody discharge, such a condition representing an inflamed state of the udder. ropiness of this character is not usually communicable from one lot of milk to another. [illustration: fig. 17. ropy milk.] the communicable form of ropy milk only appears after the milk has been drawn from the udder for a day or so, and is caused by the development of various species of bacteria which find their way into the milk after it is drawn. these defects are liable to occur at any season of the year. their presence in a dairy is a source of much trouble, as the unsightly appearance of the milk precludes its use as food, although there is no evidence that these ropy fermentations are dangerous to health. there are undoubtedly a number of different species of bacteria that are capable of producing these viscid changes,[59] but it is quite probable that they are not of equal importance in infecting milk under natural conditions. in the majority of cases studied in this country,[60] the causal organism seems to be _b. lactis viscosus_, a form first found by adametz in surface waters.[61] this organism possesses the property of developing at low temperatures (45°-50° f.), and consequently it is often able in winter to supplant the lactic-acid forms. ward has found this germ repeatedly in water tanks where milk cans are cooled; and under these conditions it is easy to see how infection of the milk might occur. marshall[62] reports an outbreak which he traced to an external infection of the udder; in another case, the slime-forming organism was abundant in the barn dust. a defect of this character is often perpetuated in a dairy for some time, and may therefore become exceedingly troublesome. in one instance in the writer's experience, a milk dealer lost over $150 a month for several months from ropy cream. failure to properly sterilize cans, and particularly strainer cloths, is frequently responsible for a continuance of trouble of this sort. the slimy substance formed in milk comes from various constituents of the milk, and the chemical character of the slime produced also varies with different germs. in some cases the slimy material is merely the swollen outer cell membrane of the bacteria themselves as in the case of _b. lactis viscosus_; in others it is due to the decomposition of the proteids, but often the chief decomposition product appears to come from a viscous fermentation of the milk-sugar. an interesting case of a fermentation of this class being utilized in dairying is seen in the use of "lange wei" (long or stringy whey) which is employed as a starter in holland to control the gassy fermentations in edam cheese. this slimy change is due to the growth of _streptococcus hollandicus_.[63] ~alcoholic fermentations.~ although glucose or cane-sugar solutions are extremely prone to undergo alcoholic fermentation, milk sugar does not readily undergo this change. where such changes are produced it is due to yeasts. several outbreaks attributable to such a cause have been reported.[64] russell and hastings[65] have found these milk-sugar splitting yeasts particularly abundant in regions where swiss cheese is made, a condition made possible by the use of whey-soaked rennets in making such cheese. kephir and koumiss are liquors much used in the orient which are made from milk that has undergone alcoholic fermentation. koumiss was originally made from mare's milk but is now often made from cows' milk by adding cane sugar and yeast. in addition to the co_{2} developed, alcohol, lactic acid, and casein-dissolving ferments are formed. kephir is made by adding to milk kephir grains, which are a mass of yeast and bacterial cells. the yeasts produce alcohol and co_{2} while the bacteria change the casein of milk, rendering it more digestible. these beverages are frequently recommended to persons who seem to be unable to digest raw milk readily. the exact nature of the changes produced are not yet well understood.[66] ~bitter milk.~ the presence of bitter substances in milk may be ascribed to a variety of causes. a number of plants, such as lupines, ragweed and chicory, possess the property of affecting milk when the same are consumed by animals. at certain stages in lactation, a bitter salty taste is occasionally to be noted that is peculiar to individual animals. a considerable number of cases of bitter milk have, however, been traced to bacterial origin. for a number of years the bitter fermentation of milk was thought to be associated with the butyric fermentation, but weigmann[67] showed that the two conditions were not dependent upon each other. he found that the organism which produced the bitter taste acted upon the casein. conn[68] observed a coccus form in bitter cream that was able to impart a bitter flavor to milk. sometimes a bitter condition does not develop in the milk, but may appear later in the milk products, as in the case of a micrococcus which freudenreich[69] found in cheese. harrison[70] has traced a common bitter condition in canadian milk to a milk-sugar splitting yeast, _torula amara_ which not only grows rapidly in milk but produces an undesirable bitterness in cheddar cheese. cream ripened at low temperatures not infrequently develops a bitter flavor, showing that the optimum temperature for this type of fermentation is below the typical lactic acid change. milk that has been heated often develops a bitter condition. the explanation of this is that the bacteria producing the bitter substances usually possess endospores, and that while the boiling or sterilizing of milk easily kills the lactic acid germs, these forms on account of their greater resisting powers are not destroyed by the heat. ~soapy milk:~ a soapy flavor in milk was traced by weigmann and zirn[71] to a specific bacillus, _b. lactis saponacei_, that they found gained access to the milk in one case from the bedding and in another instance from hay. a similar outbreak has been reported in this country,[72] due to a germ acting on the casein and albumen. ~red milk.~ the most common trouble of this nature in milk is due to presence of blood, which is most frequently caused by some wound in the udder. the ingestion of certain plants as sedges and scouring rushes is also said to cause a bloody condition; madders impart a reddish tinge due to coloring matter absorbed. defects of this class can be readily distinguished from those due to germ growth because they are apparent at time of milking. where blood is actually present, the corpuscles settle out in a short time if left undisturbed. there are a number of chromogenic or color-producing bacteria that are able to grow in milk, but their action is so slow that generally they are not of much consequence. moreover their development is usually confined to the surface of the milk as it stands in a vessel. the most important is the well-known _b. prodigiosus_. another form found at times in milk possessing low acidity[73] is _b. lactis erythrogenes_. this species only develops the red color in the dark. in the light, it forms a yellow pigment. various other organisms have been reported at different times.[74] ~blue milk.~ blue milk has been known for many years, its communicable nature being established as long ago as 1838. it appears on the surface of milk first as isolated particles of bluish or grey color, which later become confluent, the blue color increasing in intensity as the acidity increases. the causal organism, _b. cyanogenes_, is very resistant toward drying,[75] thus accounting for its persistence. in mecklenberg an outbreak of this sort once continued for several years. it has frequently been observed in europe in the past, but is not now so often reported. occasional outbreaks have been reported in this country. ~other kinds of colored milk.~ two or three chromogenic forms producing still other colors have occasionally been found in milk. adametz[76] discovered in a sample of cooked milk a peculiar form (_bacillus synxanthus_) that produced a citron-yellow appearance which precipitated and finally rendered soluble the casein. adametz, conn, and list have described other species that confer tints of yellow on milk. some of these are bright lemon, others orange, and some amber in color. still other color-producing bacteria, such as those that produce violet or green changes in the milk, have been observed. in fact, almost any of the chromogenic bacteria are able to produce their color changes in milk as it is such an excellent food medium. under ordinary conditions, these do not gain access to milk in sufficient numbers so that they modify the appearance of it except in occasional instances. ~treatment of abnormal fermentations.~ if the taint is recognized as of bacterial origin (see p. 57) and is found in the mixed milk of the herd, it is necessary to ascertain, first, whether it is a general trouble, or restricted to one or more animals. this can sometimes be done by separating the milk of the different cows and noting whether any abnormal condition develops in the respective samples. ~fermentation tests.~ the most satisfactory way to detect the presence of the taints more often present is to make a fermentation test of one kind or another. these tests are most frequently used at the factory, to enable the maker to detect the presence of milk that is likely to prove unfit for use, especially in cheese making. they are based upon the principle that if milk is held at a moderately high temperature, the bacteria will develop rapidly. a number of different methods have been devised for this purpose. in walther's lacto-fermentator samples of milk are simply allowed to stand in bottles or glass jars until they sour. they are examined at intervals of several hours. if the curdled milk is homogeneous and has a pure acid smell, the milk is regarded as all right. if it floats in a turbid serum, is full of gas or ragged holes, it is abnormal. as generally carried out, no attempt is made to have these vessels sterile. gerber's test is a similar test that has been extensively employed in switzerland. sometimes a few drops of rennet are added to the milk so as to curdle the same, and thus permit of the more ready detection of the gas that is evolved. ~wisconsin curd test.~ the method of testing milk described below was devised at the wisconsin experiment station in 1895 by babcock, russell and decker.[77] it was used first in connection with experimental work on the influence of gas-generating bacteria in cheese making, but its applicability to the detection of all taints in milk produced by bacteria makes it a valuable test for abnormal fermentations in general. in the curd test a small pat of curd is made in a glass jar from each sample of milk. these tests may be made in any receptacle that has been cleaned in boiling water, and to keep the temperature more nearly uniform these jars should be immersed in warm water, as in a wash tub or some other receptacle. when the milk is about 95° f., about ten drops of rennet extract are added to each sample and mixed thoroughly with the milk. the jars should then remain undisturbed until the milk is completely curdled; then the curd is cut into small pieces with a case knife and stirred to expel the whey. the whey should be poured off at frequent intervals until the curd mats. if the sample be kept at blood heat (98° f.) for six to eight hours, it will be ready to examine. [illustration: fig. 18. improved bottles for making curd test. _a_, test bottle complete; _b_, bottle showing construction of cover; _s_, sieve to hold back the curd when bottle is inverted; _c_, outer cover with _(d h)_ drain holes to permit of removal of whey.] more convenient types of this test than the improvised apparatus just alluded to have been devised by different dairy manufacturers. generally, they consist of a special bottle having a full-sized top, thus permitting the easy removal of the curd. the one shown in fig. 18 is provided with a sieve of such construction that the bottles will drain thoroughly if inclined in an inverted position. ~interpretation of results of test.~ the curd from a good milk has a firm, solid texture, and should contain at most only a few small pin holes. it may have some large, irregular, "mechanical" holes where the curd particles have failed to cement, as is seen in fig. 19. if gas-producing bacteria are very prevalent in the milk, the conditions under which the test is made cause such a rapid growth of the same that the evidence of the abnormal fermentation may be readily seen in the spongy texture of the curd (fig. 20). if the undesirable organisms are not very abundant and the conditions not especially suited to their growth, the "pin holes" will be less frequent. [illustration: fig. 19. curd from a good milk. the large irregular holes are mechanical.] sometimes the curds show no evidence of gas, but their abnormal condition can be recognized by the "mushy" texture and the presence of "off" flavors that are rendered more apparent by keeping them in closed bottles. this condition is abnormal and is apt to produce quite as serious results as if gas was formed. ~overcoming taints by use of starters.~ another method of combatting abnormal fermentations that is often fruitful, is that which rests upon the inability of one kind of bacteria to grow in the same medium in competition with certain other species. some of the undesirable taints in factories can be controlled in large part by the introduction of starters made from certain organisms that are able to obtain the ascendency over the taint-producing germ. such a method is commonly followed when a lactic ferment, either a commercial pure culture, or a home-made starter, is added to milk to overcome the effect of gas-generating bacteria. [illustration: fig. 20. curd from a badly tainted milk. large ragged holes are mechanical; numerous small holes due to gas. this curd was a "floater."] a similar illustration is seen in the case of the "lange wei" (slimy whey), that is used in the manufacture of edam cheese to control the character of the fermentation of the milk. this same method is sometimes applied in dealing with certain abnormal fermentations that are apt to occur on the farm. it is particularly useful with those tainted milks known as "sweet curdling." the ferment organisms concerned in this change are unable to develop in the presence of lactic acid bacteria, so the addition of a clean sour milk as a starter restores the normal conditions by giving the ordinary milk bacteria the ascendency. ~chemical disinfection.~ in exceptional instances it may be necessary to employ chemical disinfectants to restore the normal conditions. of course with such diseases as tuberculosis, very stringent measures are required, as they are such a direct menace to human life, but with these abnormal or taint-producing fermentations, care and cleanliness, well directed, will usually overcome the trouble. if it becomes necessary to employ chemical substances as disinfecting agents, their use should always be preceded by a thorough cleansing with hot water so that the germicide may come in direct contact with the surface to be disinfected. it must be borne in mind that many chemicals act as deodorants, _i.e._, destroy the offensive odor, without destroying the cause of the trouble. _sulfur_ is often recommended as a disinfecting agent, but its use should be carefully controlled, otherwise the vapors have but little germicidal power. the common practice of burning a small quantity in a room or any closed space for a few moments has little or no effect upon germ life. the effect of sulfur vapor (so_{2}) alone upon germ life is relatively slight, but if this gas is produced in the presence of moisture, sulfurous acid (h_{2}so_{3}) is formed, which is much more efficient. to use this agent effectively, it must be burned in large quantities in a moist atmosphere (three lbs. to every 1,000 cubic feet of space), for at least twelve hours. after this operation, the space should be thoroughly aired. _formalin_, a watery solution of a gas known as formaldehyde, is a new disinfectant that recent experience has demonstrated to be very useful. it may be used as a gas where rooms are to be disinfected, or applied as a liquid where desired. it is much more powerful in its action than sulfur, and it has a great advantage over mercury and other strong disinfectants, as it is not so poisonous to man as it is to the lower forms of life. _bleaching powder or chloride of lime_ is often recommended where a chemical can be advantageously used. this substance is a good disinfectant as well as a deodorant, and if applied as a wash, in the proportion of four to six ounces of the powder to one gallon of water, it will destroy most forms of life. in many cases this agent is inapplicable on account of its odor. _corrosive sublimate_ (hgcl_{2}) for most purposes is a good disinfectant, but it is such an intense poison that its use is dangerous in places that are at all accessible to stock. for the disinfection of walls in stables and barns, common thin _white wash_ ca(oh)_{2} is admirably adapted if made from freshly-burned quick lime. it possesses strong germicidal powers, increases the amount of light in the barn, is a good absorbent of odors, and is exceedingly cheap. carbolic acid, creosote, and such products, while excellent disinfectants, cannot well be used on account of their odor, especially in factories. for gutters, drains, and waste pipes in factories, _vitriol salts_ (sulfates of copper, iron and zinc) are sometimes used. these are deodorants as well as disinfectants, and are not so objectionable to use on account of their odor. these suggestions as to the use of chemicals, however, only apply to extreme cases and should not be brought into requisition until a thorough application of hot water, soap, a little soda, and the scrubbing brush have failed to do their work. footnotes: [51] günther and thierfelder, arch. f. hyg., 25:164, 1895; leichmann, cent. f. bakt., 2:281, 1896; esten, 9 rept. storrs expt. stat., p. 44, 1896; dinwiddie, bull. 45, ark. expt. stat., may, 1897; kozai, zeit. f. hyg., 38:386, 1901; weigmann, hyg. milk congress, hamburg, 1903, p. 375. [52] mcdonnell, inaug. diss., kiel. 1899, p. 39. [53] kayser, cent. f. bakt. ii. abt. 1:436. [54] treadwell, science, 1894, 17:178. [55] conn, 5 rept. storrs expt. stat., 1892, p. 396. [56] fermi, arch. f. hyg., 1892, 14:1. [57] duclaux, le lait, p. 121. [58] duclaux, principes de laiterie, p. 67. [59] guillebeau (milch zeit., 1892, p. 808) has studied over a dozen different forms that possess this property. [60] ward, bull. 165, cornell expt. stat., mch., 1899; also bull. 195, ibid., nov., 1901. [61] adametz, landw. jahr., 1891, p. 185. [62] marshall, mich. expt. stat., bull. 140. [63] milch zeit., 1899, p. 982. [64] duclaux, principes de laiterie, p. 60. heinze and cohn, zeit. f. hyg., 46: 286, 1904. [65] bull. 128, wis. expt. stat., sept. 1905. [66] freudenreich, landw. jahr. d. schweiz, 1896, 10; 1. [67] weigmann, milch zeit., 1890, p. 881. [68] conn, 3 rept. storrs expt. stat., 1890, p. 158. [69] freudenreich, fühl. landw. ztg. 43: 361. [70] harrison, bull. 120 ont. agr'l. coll., may, 1902. [71] milch zeit. 22:569. [72] marshall, bull. 146, mich. expt. stat., p. 16. [73] grotenfelt, milch zeit., 1889, p. 263. [74] menge, cent. f. bakt., 6:596; keferstein, cent. f. bakt., 21:177. [75] heim, arb. a. d. kais. gesundheitsamte, 5:578. [76] adametz, milch zeit., 1890, p. 225. [77] 12 rept. wis. expt. stat., 1895, p. 148; also bull. 67, ibid., june, 1898. chapter v. relation of disease-bacteria to milk. practical experience with epidemic disease has abundantly demonstrated the fact that milk not infrequently serves as a vehicle for the dissemination of contagion. attention has been prominently called to this relation by ernest hart,[78] who in 1880 compiled statistical evidence showing the numerous outbreaks of various contagious diseases that had been associated with milk infection up to that time. since then, further compilations have been made by freeman,[79] and also by busey and kober,[80] who have collected the data with reference to outbreaks from 1880 to 1899. these statistics indicate the relative importance of milk as a factor in the dissemination of disease. the danger from this source is much intensified for the reason that milk, generally speaking, is consumed in a raw state; and also because a considerable number of disease-producing bacteria are able, not merely to exist, but actually thrive and grow in milk, even though the normal milk bacteria are also present. moreover the recognition of the presence of such pathogenic forms is complicated by the fact that often they do not alter the appearance of the milk sufficiently so that their presence can be detected by a physical examination. these facts which have been experimentally determined, coupled with the numerous clinical cases on record, make a strong case against milk serving as an agent in the dissemination of disease. ~origin of pathogenic bacteria in milk.~ disease-producing bacteria may be grouped with reference to their relation toward milk into two classes, depending upon the manner in which infection occurs: class i. disease-producing bacteria capable of being transmitted directly from a diseased animal to man through the medium of infected milk. class ii. bacteria pathogenic for man but not for cattle which are capable of thriving in milk after it is drawn from the animal. in the first group the disease produced by the specific organism must be common to both cattle and man. the organism must live a parasitic life in the animal, developing in the udder, and so infect the milk supply. it may, of course, happen that diseases toward which domestic animals alone are susceptible may be spread from one animal to another in this way without affecting human beings. in the second group, the bacterial species lives a saprophytic existence, growing in milk, if it happens to find its way therein. in such cases milk indirectly serves as an agent in the dissemination of disease, by giving conditions favorable to the growth of the disease germ. by far the most important of diseases that may be transmitted directly from animal to man through a diseased milk supply is tuberculosis, but in addition to this, foot and mouth disease (aphthous fever in children), anthrax and acute enteric troubles have also been traced to a similar source of infection. the most important specific diseases that have been disseminated through subsequent pollution of the milk are typhoid fever, diphtheria, scarlet fever and cholera, but, of course, the possibility exists that any disease germ capable of living and thriving in milk may be spread in this way. in addition to these diseases that are caused by the introduction of specific organisms (the causal organism of scarlet fever has not yet been definitely determined), there are a large number of more or less illy-defined troubles of an intestinal character that occur especially in infants and young children that are undoubtedly attributable to the activity of microörganisms that gain access to milk during and subsequent to the milking, and which produce changes in milk before or after its ingestion that result in the formation of toxic products. diseases transmissible from animal to man through diseased milk. ~tuberculosis.~ in view of the wide-spread distribution of this disease in both the human and the bovine race, the relation of the same to milk supplies is a question of great importance. it is now generally admitted that the different types of tubercular disease found in different kinds of animals and man are attributable to the development of the same organism, _bacillus tuberculosis_, although there are varieties of this organism found in different species of animals that are sufficiently distinct to permit of recognition. the question of prime importance is, whether the bovine type is transmissible to the human or not. artificial inoculation of cattle with tuberculous human sputum as well as pure cultures of this variety show that the human type is able to make but slight headway in cattle. this would indicate that the danger of cattle acquiring the infection from man would in all probability be very slight, but these experiments offer no answer as to the possibility of transmission from the bovine to the human. manifestly it is impossible to solve this problem by direct experiment upon man except by artificial inoculation, but comparative experiments upon animals throw some light on the question. theo. smith[81] and others[82] have made parallel experiments with animals such as guinea pigs, rabbits and pigeons, inoculated with both bovine and human cultures of this organism. the results obtained in the case of all animals tested show that the virulence of the two types was much different, but that the bovine cultures were much more severe. while of course this does not prove that transmission from bovine to human is possible, still the importance of the fact must not be overlooked. in a number of cases record of accidental infection from cattle to man has been noted.[83] these have occurred with persons engaged in making post-mortem examinations on tuberculous animals, and the tubercular nature of the wound was proven in some cases by excision and inoculation. in addition to data of this sort that is practically experimental in character, there are also strong clinical reasons for considering that infection of human beings may occur through the medium of milk. naturally such infection should produce intestinal tuberculosis, and it is noteworthy that this phase of the disease is quite common in children especially between the ages of two and five.[84] it is difficult to determine, though, whether primary infection occurred through the intestine, for, usually, other organs also become involved. in a considerable number of cases in which tubercular infection by the most common channel, inhalation, seems to be excluded, the evidence is strong that the disease was contracted through the medium of the milk, but it is always very difficult to exclude the possibility of pulmonary infection. tuberculosis as a bovine disease has increased rapidly during recent decades throughout many portions of the world. this has been most marked in dairy regions. its extremely insidious nature does not permit of an early recognition by physical means, and it was not until the introduction of the tuberculin test[85] in 1892, as a diagnostic aid that accurate knowledge of its distribution was possible. the quite general introduction of this test in many regions has revealed an alarmingly large percentage of animals as affected. in denmark in 1894 over forty per cent were diagnosed as tubercular. in some parts of germany almost as bad a condition has been revealed. slaughter-house statistics also show that the disease has increased rapidly since 1890. in this country the disease on the average is much less than in europe and is also very irregularly distributed. in herds where it gained a foothold some years ago, often the majority of animals are frequently infected; many herds, in fact the great majority, are wholly free from all taint. the disease has undoubtedly been most frequently introduced through the purchase of apparently healthy but incipiently affected animals. consequently in the older dairy regions where stock has been improved the most by breeding, more of the disease exists than among the western and southern cattle. [illustration fig. 21: front view of a tuberculous udder, showing extent of swelling in single quarter.] ~infectiousness of milk of reacting animals.~ where the disease appears in the udder the milk almost invariably contains the tubercle organism. under such conditions the appearance of the milk is not materially altered at first, but as the disease progresses the percentage of fat generally diminishes, and at times in the more advanced stages where the physical condition of the udder is changed (fig. 21), the milk may become "watery"; but the percentage of animals showing such udder lesions is not large, usually not more than a few per cent. (4 per cent. according to ostertag.) on the other hand, in the earlier phases of the disease, where its presence has been recognized solely by the aid of the tuberculin test, before there are any recognizable physical symptoms in any part of the animal, the milk is generally unaffected. between these extremes, however, is found a large proportion of cases, concerning which so definite data are not available. the results of investigators on this point are conflicting and further information is much desired. some have asserted so long as the udder itself shows no lesions that no tubercle bacilli would be present,[86] but the findings of a considerable number of investigators[87] indicate that even when the udder is apparently not diseased the milk may contain the specific organism as revealed by inoculation experiments upon animals. in some cases, however, it has been demonstrated by post-mortem examination that discoverable udder lesions existed that were not recognizable before autopsy was made. in the experimental evidence collected, a varying percentage of reacting animals were found that gave positive results; and this number was generally sufficient to indicate that the danger of using milk from reacting animals was considerable, even though apparently no disease could be found in the udder. the infectiousness of milk can also be proven by the frequent contraction of the disease in other animals, such as calves and pigs which may be fed on the skim milk. the very rapid increase of the disease among the swine of germany and denmark,[88] and the frequently reported cases of intestinal infection of young stock also attest the presence of the organism in milk. the tubercle bacillus is so markedly parasitic in its habits, that, under ordinary conditions, it is incapable of growing at normal air temperatures. there is, therefore, no danger of the germ developing in milk after it is drawn from the animal, unless the same is kept at practically blood heat. even though the milk of some reacting animals may not contain the dangerous organism at the time of making the test, it is quite impossible to foretell how long it will remain free. as the disease becomes more generalized, or if tuberculous lesions should develop in the udder, the milk may pass from a healthy to an infectious state. this fact makes it advisable to exclude from milk supplies intended for human use, all milk of animals that respond to the tuberculin test; or at least to treat it in a manner so as to render it safe. whether it is necessary to do this or not if the milk is made into butter or cheese is a somewhat different question. exclusion or treatment is rendered more imperative in milk supplies, because the danger is greater with children with whom milk is often a prominent constituent of their diet, and also for the reason that the child is more susceptible to intestinal infection than the adult. the danger of infection is much lessened in butter or cheese, because the processes of manufacture tend to diminish the number of organisms originally present in the milk, and inasmuch as no growth can ordinarily take place in these products the danger is minimized. moreover, the fact that these foods are consumed by the individual in smaller amounts than is generally the case where milk is used, and also to a greater extent by adults, lessens still further the danger of infection. notwithstanding this, numerous observers[89] especially in germany have succeeded in finding the tubercle bacillus in market butter, but this fact is not so surprising when it is remembered that a very large fraction of their cattle show the presence of the disease as indicated by the tuberculin test, a condition that does not obtain in any large section in this country. the observations on the presence of the tubercle bacillus in butter have been questioned somewhat of late[2] by the determination of the fact that butter may contain an organism that possesses the property of being stained in the same way as the tubercle organism. differentiation between the two forms is rendered more difficult by the fact that this tubercle-like organism is also capable of producing in animals lesions that stimulate those of tuberculosis, although a careful examination reveals definite differences. petri[90] has recently determined that both the true tubercle and the acid-resisting butter organism may be readily found in market butter. in the various milk products it has been experimentally determined that the true tubercle bacillus is able to retain its vitality in butter for a number of months and in cheese for nearly a year. ~treatment of milk from tuberculosis cows.~ while it has been shown that it is practically impossible to foretell whether the milk of any reacting animal actually contains tubercle bacilli or not, still the interests of public health demand that no milk from such stock be used for human food until it has been rendered safe by some satisfactory treatment. _1. heating._ by far the best treatment that can be given such milk is to heat it. the temperature at which this should be done depends upon the thermal death point of the tubercle bacillus, a question concerning which there has been considerable difference of opinion until very recently. according to the work of some of the earlier investigators, the tubercle bacillus in its vegetative stage is endowed with powers of resistance greater than those possessed by any other pathogenic organism. this work has not been substantiated by the most recent investigations on this subject. in determining the thermal death point of this organism, as of any other, not only must the temperature be considered, but the period of exposure as well, and where that exposure is made in milk, another factor must be considered, viz., the presence of conditions permitting of the formation of a "scalded layer," for as smith[91] first pointed out, the resistance of the tubercle organism toward heat is greatly increased under these conditions. if tuberculous milk is heated in a closed receptacle where this scalded membrane cannot be produced, the tubercle bacillus is killed at 140° f. in 15 to 20 minutes. these results which were first determined by smith, under laboratory conditions, and confirmed by russell and hastings,[92] where tuberculous milk was heated in commercial pasteurizers, have also been verified by hesse.[93] a great practical advantage which accrues from the treatment of milk at 140° f. is that the natural creaming is practically unaffected. of course, where a higher temperature is employed, the period of exposure may be materially lessened. if milk is momentarily heated to 176° f., it is certainly sufficient to destroy the tubercle bacillus. this is the plan practiced in denmark where all skim milk and whey must be heated to this temperature before it can be taken back to the farm, a plan which is designed to prevent the dissemination of tuberculosis and foot and mouth disease by means of the mixed creamery by-products. this course renders it possible to utilize with perfect safety, for milk supplies, the milk of herds reacting to the tuberculin test, and as butter of the best quality can be made from cream or milk heated to even high temperatures,[94] it thus becomes possible to prevent with slight expense what would otherwise entail a large loss. _2. dilution._ another method that has been suggested for the treatment of this suspected milk is dilution with a relatively large volume of perfectly healthy milk. it is a well known fact that to produce infection, it requires the simultaneous introduction of a number of organisms, and in the case of tuberculosis, especially that produced by ingestion, this number is thought to be considerable. gebhardt[95] found that the milk of tuberculous cows, which was virulent when injected by itself into animals, was innocuous when diluted with 40 to 100 times its volume of healthy milk. this fact is hardly to be relied upon in practice, unless the proportion of reacting to healthy cows is positively known. it has also been claimed in the centrifugal separation of cream from milk[96] that by far the larger number of tubercle bacilli were thrown out with the separator slime. moore[97] has shown that the tubercle bacilli in an artificially infected milk might be reduced in this way, so as to be no longer microscopically demonstrable, yet the purification was not complete enough to prevent the infection of animals inoculated with the milk. another way to exclude all possibility of tubercular infection in milk supplies is to reject all milk from reacting animals. this method is often followed where pasteurization or sterilization is not desired. in dairies where the keeping quality is dependent upon the exclusion of bacteria by stringent conditions as to milking and handling ("sanitary" or "hygienic" milk), the tuberculin test is frequently used as a basis to insure healthy milk. ~foot and mouth disease.~ the wide-spread extension of this disease throughout europe in recent years has given abundant opportunity to show that while it is distinctively an animal malady, it is also transmissible to man, although the disease is rarely fatal. the causal organism has not been determined with certainty, but it has been shown that the milk of affected animals possesses infectious properties[98] although appearing unchanged in earlier phases of the disease. hertwig showed the direct transmissibility of the disease to man by experiments made on himself and others. by ingesting milk from an affected animal, he was able to produce the symptoms of the disease, the mucous membrane of the mouth being covered with the small vesicles that characterize the malady. it has also been shown that the virus of the disease may be conveyed in butter.[99] this disease is practically unknown in this country, although widely spread in europe. there are a number of other bovine diseases such as anthrax,[100] lockjaw,[101] and hydrophobia[102] in which it has been shown that the virus of the disease is at times to be found in the milk supply, but often the milk becomes visibly affected, so that the danger of using the same is greatly minimized. there are also a number of inflammatory udder troubles known as garget or mammitis. in most of these, the physical appearance of the milk is so changed, and often pus is present to such a degree as to give a very disagreeable appearance to the milk. pus-forming bacteria (staphylococci and streptococci) are to be found associated with such troubles. a number of cases of gastric and intestinal catarrh have been reported as caused by such milks.[103] diseases transmissible to man through infection of milk after withdrawal. milk is so well adapted to the development of bacteria in general, that it is not surprising to find it a suitable medium for the growth of many pathogenic species even at ordinary temperatures. not infrequently, disease-producing bacteria are able to grow in raw milk in competition with the normal milk bacteria, so that even a slight contamination may suffice to produce infection. the diseases that are most frequently disseminated in this way are typhoid fever, diphtheria, scarlet fever and cholera, together with the various illy-defined intestinal troubles of a toxic character that occur in children, especially under the name of cholera infantum, summer complaint, etc. diseases of this class are not derived directly from animals because cattle are not susceptible to the same. ~modes of infection.~ in a variety of ways, however, the milk may be subject to contaminating influences after it is drawn from the animal, and so give opportunity for the development of disease-producing bacteria. the more important methods of infection are as follows: _1. infection directly from a pre-existing case of disease on premises._ quite frequently a person in the early stage of a diseased condition may continue at his usual vocation as helper in the barn or dairy, and so give opportunity for direct infection to occur. in the so-called cases of "walking typhoid," this danger is emphasized. it is noteworthy in typhoid fever that the bacilli frequently persist in the urine and in diphtheria they often remain in the throat until after convalescence. in some cases infection has been traced to storage of the milk in rooms in the house where it became polluted directly by the emanations of the patient.[104] among the dwellings of the lower classes where a single room has to be used in common this source of infection has been most frequently observed. _2. infection through the medium of another person._ not infrequently another individual may serve in the capacity of nurse or attendant to a sick person, and also assist in the handling of the milk, either in milking the animals or caring for the milk after it has been drawn. busey and kober report twenty-one outbreaks of typhoid fever in which dairy employees also acted in the capacity of nurses. _3. pollution of milk utensils._ the most frequent method of infection of cans, pails, etc., is in cleaning them with water that may be polluted with disease organisms. often wells may be contaminated with diseased matter of intestinal origin, as in typhoid fever, and the use of water at normal temperatures, or even in a lukewarm condition, give conditions permitting of infection. intentional adulteration of milk with water inadvertently taken from polluted sources has caused quite a number of typhoid outbreaks.[105] sedgwick and chapin[106] found in the springfield, mass., epidemic of typhoid that the milk cans were placed in a well to cool the milk, and it was subsequently shown that the well was polluted with typhoid fecal matter. _4. pollution of udder_ of animal _by wading in infected water_, or by washing same with contaminated water. this method of infection would only be likely to occur in case of typhoid. an outbreak at the university of virginia in 1893[107] was ascribed to the latter cause. _5. pollution of creamery by-products, skim-milk, etc._ where the milk supply of one patron becomes infected with pathogenic bacteria, it is possible that disease may be disseminated through the medium of the creamery, the infective agent remaining in the skim milk after separation and so polluting the mixed supply. this condition is more likely to prevail with typhoid because of the greater tolerance of this organism for acids such as would be found in raw milk. the outbreaks at brandon,[108] england, in 1893, castle island,[109] ireland, and marlboro,[110] mass., in 1894, were traced to such an origin. while most outbreaks of disease associated with a polluted milk supply originate in the use of the milk itself, yet infected milk may serve to cause disease even when used in other ways. several outbreaks of typhoid fever have been traced to the use of ice cream where there were strong reasons for believing that the milk used in the manufacture of the product was polluted.[111] hankin[112] details a case of an indian confection made largely from milk that caused a typhoid outbreak in a british regiment. although the evidence that milk may not infrequently serve as an agent in spreading disease is conclusive enough to satisfactorily prove the proposition, yet it should be borne in mind that the organism of any specific disease in question has rarely ever been found. the reasons for this are quite the same as those that govern the situation in the case of polluted waters, except that the difficulties of the problem are much greater in the case of milk than with water. the inability to readily separate the typhoid germ, for instance, from the colon bacillus, an organism frequently found in milk, presents technical difficulties not easily overcome. the most potent reason of failure to find disease bacteria is the fact that infection in any case must occur sometime previous to the appearance of the outbreak. not only is there the usual period of incubation, but it rarely happens that an outbreak is investigated until a number of cases have occurred. in this interim the original cause of infection may have ceased to be operative. ~typhoid fever.~ with reference to the diseases likely to to be disseminated through the medium of milk, infected after being drawn from the animal, typhoid fever is the most important. the reason for this is due (1) to the wide spread distribution of the disease; (2) to the fact that the typhoid bacillus is one that is capable of withstanding considerable amounts of acid, and consequently finds even in raw milk containing the normal lactic acid bacteria conditions favorable for its growth.[113] ability to grow under these conditions can be shown not only experimentally, but there is abundant clinical evidence that even a slight infection often causes extensive outbreaks, as in the stamford, conn., outbreak in 1895 where 386 cases developed in a few weeks, 97 per cent. of which occurred on the route of one milk-man. in this case the milk cans were thoroughly and properly cleaned, but were rinsed out with _cold_ water from a shallow well that was found to be polluted. the most common mode of pollution of milk with typhoid organisms is where the milk utensils are infected in one way or another.[114] second in importance is the carrying of infection by persons serving in the dual capacity of nurse and dairy attendant. ~cholera.~ this germ does not find milk so favorable a nutrient medium as the typhoid organism, because it is much more sensitive toward the action of acids. kitasato[115] found, however, that it could live in raw milk from one to four days, depending upon the amount of acid present. in boiled or sterilized milk it grows more freely, as the acid-producing forms are thereby eliminated. in butter it dies out in a few days (4 to 5). on account of the above relation not a large number of cholera outbreaks have been traced to milk, but simpson[116] records a very striking case in india where a number of sailors, upon reaching port, secured a quantity of milk. of the crew which consumed this, every one was taken ill, and four out of ten died, while those who did not partake escaped without any disease. it was later shown that the milk was adulterated with water taken from an open pool in a cholera infected district. ~diphtheria.~ milk occasionally, though not often, serves as a medium for the dissemination of diphtheria. swithinbank and newman[117] cites four cases in which the causal organism has been isolated from milk. it has been observed that growth occurs more rapidly in raw than in sterilized milk.[118] infection in this disease is more frequently attributable to direct infection from patient on account of the long persistence of this germ in the throat, or indirectly through the medium of an attendant. ~scarlet fever.~ although it is more difficult to study the relation of this disease to contaminated milk supplies, because the causal germ of scarlet fever is not yet known, yet the origin of a considerable number of epidemics has been traced to polluted milk supplies. milk doubtless is infected most frequently from persons in the earlier stages of the disease when the infectivity of the disease is greater. ~diarrhoeal diseases.~ milk not infrequently acquires the property of producing diseases of the digestive tract by reason of the development of various bacteria that form more or less poisonous by-products. these troubles occur most frequently during the summer months, especially with infants and children, as in cholera infantum and summer complaint. the higher mortality of bottle-fed infants[119] in comparison with those that are nursed directly is explicable on the theory that cows' milk is the carrier of the infection, because in many cases it is not consumed until there has been ample time for the development of organisms in it. where milk is pasteurized or boiled it is found that the mortality among children is greatly reduced. as a cause of sickness and death these diseases exceed in importance all other specific diseases previously referred to. these troubles have generally been explained as produced by bacteria of the putrefactive class which find their way into the milk through the introduction of filth and dirt at time of milking.[120] flügge[121] has demonstrated that certain peptonizing species possess toxic properties for animals. recent experimental inquiry[122] has demonstrated that the dysentery bacillus (shiga) probably bears a causal relation to some of these summer complaints. ~ptomaine poisoning.~ many cases of poisoning from food products are also reported with adults. these are due to the formation of various toxic products, generally ptomaines, that are produced as a result of infection of foods by different bacteria. one of these substances, _tyrotoxicon_, was isolated by vaughan[123] from cheese and various other products of milk, and found to possess the property of producing symptoms of poisoning similar to those that are noted in such cases. he attributes the production of this toxic effect to the decomposition of the elements in the milk induced by putrefactive forms of bacteria that develop where milk is improperly kept.[124] often outbreaks of this character[125] assume the proportions of an epidemic, where a large number of persons use the tainted food. footnotes: [78] hart, trans. int. med. cong., london, 1881, 4:491-544. [79] freeman, med. rec., march 28, 1896. [80] busey and kober, rept. health off. of dist. of col., washington, d. c., 1895, p. 299. these authors present in this report an elaborate article on morbific and infectious milk, giving a very complete bibliography of 180 numbers. they append to hart's list (which is published in full) additional outbreaks which have occurred since, together with full data as to extent of epidemic, circumstances governing the outbreak, as well as name of original reporter and reference. [81] smith, theo., journ. of expt. med., 1898, 3:451. [82] dinwiddie, bull. 57, ark. expt. stat., june, 1899; ravenel, univ. of penn. med. bull., sept. 1901. [83] ravenel, journ. of comp. med. & vet. arch., dec. 1897; hartzell, journ. amer. med. ass'n, april 16, 1898. [84] stille, brit. med. journ., aug. 19, 1899. [85] this test is made by injecting into the animal a small quantity of tuberculin, which is a sterilized glycerin extract of cultures of the tubercle bacillus. in a tuberculous animal, even in the very earliest phases of the disease, tuberculin causes a temporary fever that lasts for a few hours. by taking the temperature a number of times before and after injection it is possible to readily recognize any febrile condition. a positive diagnosis is made where the temperature after inoculation is at least 2.0° f. above the average normal, and where the reaction fever is continued for a period of some hours. [86] martin, brit. med. journ. 1895, 1:937; nocard, les tuberculoses animales, 1895. [87] c. o. jensen, milch kunde und milch hygiene, p. 69. [88] ostertag, milch zeit., 22:672. [89] obermüller, hyg. rund., 1897, p. 712; petri, arb. a. d. kais. ges. amte, 1898, 14: 1; hormann und morgenroth, hyg. rund., 1898, p. 217. [90] rabinowitsch, zeit. f. hyg., 1897, 26: 90. [91] th. smith. journ. of expt. med., 1899, 4:217. [92] russell and hastings, 18 rept. wis. expt. stat., 1901. [93] hesse, zeit. f. hyg., 1900, 34:346. [94] practically all of the finest butter made in denmark is made from cream that has been pasteurized at temperatures varying from 160°-185° f. [95] gebhardt, virch. arch., 1890, 119:12. [96] scheurlen, arb. a. d. k. ges. amte, 1891, 7:269; bang, milch zeit., 1893, p. 672. [97] moore, year book of u. s. dept. agr., 1895, p. 432. [98] weigel and noack, jahres. d. ges. med., 1890, p. 642; weissenberg, allg. med. cent. zeit., 1890, p. 1; baum, arch. f. thierheilkunde, 1892, 18:16. [99] schneider, münch, med. wochenschr., 1893, no. 27; fröhner, zeit f. fleisch u. milchhygiene, 1891, p. 55. [100] feser, deutsche zeit. f. thiermed., 1880, 6:166. [101] nocard, bull. gén., 1885, p. 54. [102] deutsche viertelsjahr. f. offentl. gesundheitspflege, 1890, 20:444. [103] zeit. f. fleisch und milch hygiene, 11:114. [104] e. roth, deutsche vierteljahresschr. f. offentl. gesundheitspfl., 1890, 22:238 [105] s. w. north, london practitioner, 1889, 43:393. [106] sedgwick and chapin, boston med. & surg. journ., 1893, 129:485. [107] dabney, phila. med. news, 1893, 63:630. [108] welphy, london lancet, 1894, 2:1085. [109] brit. med. journ., 1894, 1:815. [110] mass. bd. health rept., 1894, p. 765. [111] turner, london practitioner, 1892, 49:141; munro, brit. med. journ., 1894, 2:829. [112] hankin, brit. med. journ., 1894, 2:613. [113] heim (arb. a. d. kais. gesundheitsamte, 1889, 5:303) finds it capable of living from 20-30 days in milk. [114] schüder (zeit. f. hyg., 1902, 38:34) examined the statistics of 638 typhoid epidemics. he found 71 per cent. due to infected drinking water, 17 per cent. to infected milk, and 3.5 per cent. caused by other forms of food. [115] kitasato. arb. a. d. kais. gesundheitsamte, 1:470. [116] simpson, london practitioner, 1887, 39:144. [117] swithinbank and newman, bacteriology of milk, p. 341. [118] schottelius and ellerhorst. milch zeit., 1897, pp. 40 and 73. [119] baginsky, hyg. rund., 1895, p. 176. [120] gaffky, deutsch. med. wochen., 18:14. [121] flügge. zeit., f. hyg., 17:272, 1894. [122] duval and bassett, studies from the rockefeller inst. for med. research, 2:7, 1904. [123] zeit. f. physiol. chemie, 10:146; 9 intern. hyg. cong. (london), 1891, p. 118. [124] vaughan and perkins, arch. f. hyg., 27:308. [125] newton and wallace (phila. med. news, 1887, 50:570) report three outbreaks at long branch, n. j., two of which occurred in summer hotels. chapter vi. bacteria and milk supplies with especial reference to methods of preservation. to the milk dealer or distributor, bacteria are more or less of a detriment. none of the organisms that find their way into milk, nor the by-products formed by their growth, improve the quality of milk supplies. it is therefore especially desirable from the milk-dealer's point of view that these changes should be held in abeyance as much as possible. then too, the possibility that milk may serve as a medium for the dissemination of disease-breeding bacteria makes it advisable to protect this food supply from all possible infection from suspicious sources. in considering, therefore, the relation of bacteria to general milk supplies, the _economic_ and the _hygienic_ standpoints must be taken into consideration. ordinarily much more emphasis is laid upon the first requirement. if the supply presents no abnormal feature as to taste, odor and appearance, unfortunately but little attention is paid to the possibility of infection by disease germs. the methods of control which are applicable to general milk supplies are based on the following foundations: (1) the exclusion of all bacterial life, as far as practicable, at the time the milk is drawn, and the subsequent storage of the same at temperatures unfavorable for the growth of the organisms that do gain access; (2) the removal of the bacteria, wholly or in part, after they have once gained access. until within comparatively recent years, practically no attention was given to the character of milk supplies, except possibly as to the percentage of butter fat, and sometimes the milk solids which it contained. so long as the product could be placed in the hands of the consumer in such shape as not to be rejected by him as unfit for food, no further attention was likely to be given to its character. at present, however, much more emphasis is being given to the quality of milk, especially as to its germ content; and the milk dealer is beginning to recognize the necessity of a greater degree of control. this control must not merely concern the handling of the product after it reaches him, but should go back to the milk producer on the farm. here especially, it is necessary to inculcate those methods of cleanliness which will prevent in large measure the wholesale infection that ordinarily occurs. the two watch words which are of the utmost importance to the milk dealer are _cleanliness_ and _cold_. if the milk is properly drawn from the animal in a clean manner and is immediately and thoroughly chilled, the dealer has little to fear as to his product. whenever serious difficulties do arise, attributable to bacterial changes, it is because negligence has been permitted in one or both directions. the influence of cleanliness in diminishing the bacterial life in milk and that of low temperatures in repressing the growth of those forms which inevitably gain access has been fully dealt with in preceding chapters. it is of course not practicable to take all of these precautions to which reference has been made in the securing of large supplies of market milk for city use, but great improvement over existing conditions could be secured if the public would demand a better supervision of this important food article. boards of health in our larger cities are awakening to the importance of this question and are becoming increasingly active in the matter of better regulations and the enforcement of the same. new york city board of health has taken an advanced position in requiring that all milk sold in the city shall be chilled down to 45° f. immediately after milking and shall be transported to the city in refrigerator cars. reference has already been made to the application of the acid test (page 52) in the inspection of city milk supplies, and it is the opinion of the writer that the curd test (see page 76) could also be used with advantage in determining the sanitary character of milk. this test reveals the presence of bacteria usually associated with dirt and permits of the recognition of milks that have been carelessly handled. from personal knowledge of examinations made of the milk supplies in a number of wisconsin cities it appears that this test could be utilized with evident advantage. ~"sanitary" or "certified" milk supplies.~ in a number of the larger cities, the attempt has been made to improve the quality of the milk supplies by the installation of dairies in which is produced an especially high grade of milk. frequently the inspection of the dairy as well as the examination of the milk at stated intervals is under the control of milk commissions or medical societies and as it is customary to distribute the certificate of the examining board with the product, such milks are frequently known as "certified." in such dairies the tuberculin test is used at regular intervals, and the herd inspected frequently by competent veterinarians. the methods of control inaugurated as to clean milking and subsequent handling are such as to insure the diminution of the bacteria to the lowest possible point. the bacterial limit set by the pediatric society of philadelphia is 10,000 organisms per cc. often it is possible to improve very materially on this standard and not infrequently is the supply produced where it contains only a few thousand organisms per cc. where such a degree of care is exercised, naturally a considerably higher price must be paid for the product,[126] and it should be remembered that the development of such a system is only possible in relatively large centers where the dealer can cater to a selected high-class trade. moreover, it should also be borne in mind that such a method of control is only feasible in dairies that are under individual control. the impossibility of exercising adequate control with reference to the milking process and the care which should be given the milk immediately thereafter, when the same is produced on different farms under various auspices is evident. preservation of milk supplies. while much can be done to improve the quality of milk supplies by excluding a large proportion of the bacteria which normally gain access to the milk, and preventing the rapid growth of those that do find their way therein, yet for general municipal purposes, any practical method of preservation[127] that is applicable on a commercial scale must rest largely upon the destruction of bacteria that are present in the milk. the two possible methods by which bacteria can be destroyed after they have once gained access is (1) by the use of chemical preservatives; (2) by the aid of physical methods. ~chemical preservatives.~ numerous attempts have been made to find some chemical substance that could be added to milk which would preserve it without interfering with its nutritive properties, but as a general rule a substance that is toxic enough to destroy or inhibit the growth of bacterial life exerts a prejudicial effect on the tissues of the body. the use of chemicals, such as carbolic acid, mercury salts and mineral acids, that are able to entirely destroy all life, is of course excluded, except when milk is preserved for analytical purposes; but a number of milder substances are more or less extensively employed, although the statutes of practically all states forbid their use. the substances so used may be grouped in two classes: 1. those that unite chemically with certain by-products of bacterial growth to form inert substances. thus bicarbonate of soda neutralizes the acid in souring milk, although it does not destroy the lactic acid bacteria. 2. those that act directly upon the bacteria in milk, restraining or inhibiting their development. the substances most frequently utilized are salicylic acid, formaldehyde and boracic acid. these are nearly always sold to the milk handler, under some proprietary name, at prices greatly in excess of what the crude chemicals could be bought for in the open market. formaldehyde has been widely advertised of late, but its use is fraught with the greatest danger, for it practically renders insoluble all albuminous matter and its toxic effect is greatly increased in larger doses. these substances are generally used by milk handlers who know nothing of their poisonous action, and although it may be possible for adults to withstand their use in dilute form, without serious results, yet their addition to general milk supplies that may be used by children is little short of criminal. the sale of these preparations for use in milk finds its only outlet with those dairymen who are anxious to escape the exactions that must be met by all who attempt to handle milk in the best possible manner. farrington has suggested a simple means for the detection of preservalin (boracic acid).[128] when this substance is added to fresh milk, it increases the acidity of milk without affecting its taste. as normal milk tastes sour when it contains about 0.3 per cent lactic acid, a milk that tests as much or more than this without tasting sour has been probably treated with this antiseptic agent. ~physical methods of preservation.~ methods based upon the application of physical forces are less likely to injure the nutritive value of milk, and are consequently more effective, if of any value whatever. a number of methods have been tried more or less thoroughly in an experimental way that have not yet been reduced to a practical basis, as electricity, use of a vacuum, and increased pressure.[129] condensation has long been used with great success, but in this process the nature of the milk is materially changed. the keeping quality in condensed milk often depends upon the action of another principle, viz., the inhibition of bacterial growth by reason of the concentration of the medium. this condition is reached either by adding sugar and so increasing the soluble solids, or by driving off the water by evaporation, preferably in a vacuum pan. temperature changes are, however, of the most value in preserving milk, for by a variation in temperature all bacterial growth can be brought to a standstill, and under proper conditions thoroughly destroyed. ~use of low temperatures.~ the effect of chilling or rapid cooling on the keeping quality of milk is well known. when the temperature of milk is lowered to the neighborhood of 45° f., the development of bacterial life is so slow as to materially increase the period that milk remains sweet. within recent years, attempts have been made to preserve milk so that it could be shipped long distances by freezing the product, which in the form of milk-ice could be held for an indefinite period without change.[130] a modification of this process known as casse's system has been in use more or less extensively in copenhagen and in several places in germany. this consists of adding a small block of milk-ice (frozen milk) to large cans of milk (one part to about fifty of milk) which may or may not be pasteurized.[131] this reduces the temperature so that the milk remains sweet considerably longer. such a process might permit of the shipment of milk for long distances with safety but as a matter of fact, the system has not met with especial favor. [illustration: fig. 22. microscopic appearance of normal milk showing the fat-globules aggregated in clusters.] ~use of high temperatures.~ heat has long been used as a preserving agent. milk has been scalded or cooked to keep it from time immemorial. heat may be used at different temperatures, and when so applied exerts a varying effect, depending upon temperature employed. all methods of preservation by heat rest, however, upon the application of the heat under the following conditions: 1. a temperature above the maximum growing-point (105°-115° f.) and below the thermal death-point (130°-140° f.) will prevent further growth, and consequently fermentative action. 2. a temperature above the thermal death-point destroys bacteria, and thereby stops all changes. this temperature varies, however, with the condition of the bacteria, and for spores is much higher than for vegetative forms. attempts have been made to employ the first principle in shipping milk by rail, viz., prolonged heating above growing temperature, but when milk is so heated, its physical appearance is changed.[132] the methods of heating most satisfactorily used are known as sterilization and pasteurization, in which a degree of temperature is used approximating the boiling and scalding points respectively. [illustration: fig. 23. microscopic appearance of milk heated above 140° f., showing the homogeneous distribution of fat-globules. the physical change noted in comparison with fig. 22 causes the diminished consistency of pasteurized cream.] ~effect of heat on milk.~ when milk is subjected to the action of heat, a number of changes in its physical and chemical properties are to be noted. _1. diminished "body."_ when milk, but more especially cream, is heated to 140° f. or above, it becomes thinner in consistency or "body," a condition which is due to a change in the grouping of the fat globules. in normal milk, the butter fat for the most part is massed in microscopic clots as (fig. 22). when exposed to 140° f. or above for ten minutes these fat-globule clots break down, and the globules become homogeneously distributed (fig. 23). a _momentary_ exposure to heat as high as 158°-160° may be made without serious effect on the cream lime; but above this the cream rises so poorly and slowly that it gives the impression of thinner milk. _2. cooked taste._ if milk is heated for some minutes to 160° f., it acquires a cooked taste that becomes more pronounced as the temperature is further raised. milk so heated develops on its surface a pellicle or "skin." the cause of this change in taste is not well known. usually it has been explained as being produced by changes in the nitrogenous elements in the milk, particularly in the albumen. thoerner[133] has pointed out the coincidence that exists between the appearance of a cooked taste and the loss of certain gases that are expelled by heating. he finds that the milk heated in closed vessels from which the gas cannot escape has a much less pronounced cooked flavor than if heated in an open vessel. the so-called "skin" on the surface of heated milk is not formed when the milk is heated in a tightly-closed receptacle. by some[134] it is asserted that this layer is composed of albumen, but there is evidence to show that it is modified casein due to the rapid evaporation of the milk serum at the surface of the milk. _3. digestibility._ considerable difference of opinion has existed in the minds of medical men as to the relative digestibility of raw and heated milks. a considerable amount of experimental work has been done by making artificial digestion experiments with enzyms, also digestion experiments with animals, and in a few cases with children. the results obtained by different investigators are quite contradictory, although the preponderance of evidence seems to be in favor of the view that heating does impair the digestibility of milk, especially if the temperature attains the sterilizing point.[135] it has been observed that there is a noteworthy increase in amount of rickets,[136] scurvy and marasmus in children where highly-heated milks are employed. these objections do not obtain with reference to milk heated to moderate temperatures, as in pasteurization, although even this lower temperature lessens slightly its digestibility. the successful use of pasteurized milks in children's hospitals is evidence of its usefulness. _4. fermentative changes._ the normal souring change in milk is due to the predominance of the lactic acid bacteria, but as these organisms as a class do not possess spores, they are readily killed when heated above the thermal death-point of the developing cell. the destruction of the lactic forms leaves the spore-bearing types possessors of the field, and consequently the fermentative changes in heated milk are not those that usually occur, but are characterized by the curdling of the milk from the action of rennet enzyms. _5. action of rennet._ heating milk causes the soluble lime salts to be precipitated, and as the curdling of milk by rennet (in cheese-making) is dependent upon the presence of these salts, their absence in heated milks greatly retards the action of rennet. this renders it difficult to utilize heated milks in cheese-making unless the soluble lime salts are restored, which can be done by adding solutions of calcium chlorid. ~sterilization.~ as ordinarily used in dairying, sterilization means the application of heat at temperatures approximating, if not exceeding, 212° f. it does not necessarily imply that milk so treated is sterile, i. e., germ-free; for, on account of the resistance of spores, it is practically impossible to destroy entirely _all_ these hardy forms. if milk is heated at temperatures above the boiling point, as is done where steam pressure is utilized, it can be rendered practically germ-free. such methods are employed where it is designed to keep milk sweet for a long period of time. the treatment of milk by sterilization has not met with any general favor in this country, although it has been more widely introduced abroad. in most cases the process is carried out after the milk is bottled; and considerable ingenuity has been exercised in the construction of devices which will permit of the closure of the bottles after the sterilizing process has been completed. milks heated to so high a temperature have a more or less pronounced boiled or cooked taste, a condition that does not meet with general favor in this country. the apparatus suitable for this purpose must, of necessity, be so constructed as to withstand steam pressure, and consequently is considerably more expensive than that required for the simpler pasteurizing process. ~pasteurization.~ in this method the degree of heat used ranges from 140° to 185° f. and the application is made for only a limited length of time. the process was first extensively used by pasteur (from whom it derives its name) in combating various maladies of beer and wine. its importance as a means of increasing the keeping quality of milk was not generally recognized until a few years ago; but the method is now growing rapidly in favor as a means of preserving milk for commercial purposes. the method does not destroy all germ-life in milk; it affects only those organisms that are in a growing, vegetative condition; but if the milk is quickly cooled, it enhances the keeping quality very materially. it is unfortunate that this same term is used in connection with the heating of cream as a preparatory step to the use of pure cultures in cream-ripening in butter-making. the objects to be accomplished vary materially and the details of the two processes are also quite different. while pasteurizing can be performed on a small scale by the individual, the process can also be adapted to the commercial treatment of large quantities of milk. the apparatus necessary for this purpose is not nearly so expensive as that used in sterilizing, a factor of importance when other advantages are considered. in this country pasteurization has made considerable headway, not only in supplying a milk that is designed to serve as children's food, but even for general purposes. ~requirements essential in pasteurization.~ while considerable latitude with reference to pasteurizing limits is permitted, yet there are certain conditions which should be observed, and these, in a sense, fix the limits that should be employed. these may be designated as (1) the _physical_, and (2) the _biological_ requirements. ~physical requirements.~ _1. avoidance of scalded or cooked taste._ the english and american people are so averse to a scalded or cooked flavor in milk that it is practically impossible for a highly heated product to be sold in competition with ordinary raw milk. in pasteurization then, care must be taken not to exceed the temperature at which a permanently cooked flavor is developed. as previously observed, this point varies with the period of exposure. a momentary exposure to a temperature of about 170° f. may be made without any material alteration, but if the heat is maintained for a few minutes (ten minutes or over), a temperature of 158° to 160° f. is about the maximum that can be employed with safety. _2. normal creaming of the milk._ it is especially desirable that a sharp and definite cream line be evident on the milk soon after pasteurization. if this fails to appear, the natural inference of the consumer is that the milk is skimmed. if the milk be heated to a temperature sufficiently high to cause the fat-globule clusters to disintegrate (see figs. 22 and 23), the globules do not rise to the surface as readily as before and the cream line remains indistinct. where the exposure is made for a considerable period of time (10 minutes or more), the maximum temperature which can be used without producing this change is about 140° f.; if the exposure is made for a very brief time, a minute or less, the milk may be heated to 158°-160 f.° without injuring the creaming property. _3. no diminution in cream "body."_ coincident with this change which takes place in the creaming of the milk is the change in body or consistency which is noted where cream is pasteurized at too high a temperature. for the same reason as given under (2) cream heated above these temperatures is reduced in apparent thickness and appears to contain less butter-fat. of course the pasteurizing process does not change the fat content, but its "body" is apparently so affected. thus a 25 per cent. cream may seem to be no thicker or heavier than an 18 per cent. raw cream. this real reduction in consistency naturally affects the readiness with which the cream can be whipped. ~biological requirements.~ _1. enhanced keeping quality._ in commercial practice the essential biological requirement is expressed in the enhanced keeping quality of the pasteurized milk. this expresses in a practical way the reduction in germ life accomplished by the pasteurizing process. the improvement in keeping quality depends upon the temperature and time of exposure, but fully as much also on the way in which the pasteurized product is handled after heating. the lowest temperature which can be used with success to kill the active, vegetative bacteria is about 140° f., at which point it requires about ten minutes exposure. if this period is curtailed the temperature must be raised accordingly. an exposure to a temperature of 175° f. for a minute has approximately the same effect as the lower degree of heat for the longer time. the following bacteriological studies as to the effect which a variation in temperature exerts on bacterial life in milk are of importance as indicating the foundation for the selection of the proper limits. in the following table the exposures were made for a uniform period (20 minutes): _the bacterial content of milk heated at different temperatures._ number of bacteria per cc. in milk. 45° c. 50° c. 55° c. 60° c. 65° c. 70° c. unheated 113° f. 122° f. 131° f. 140° f. 149° f. 158° f. series i. 2,895,000 --- 1,260,000 798,000 32,000 5,770 3,900 series ii. 750,000 665,000 262,400 201,000 950 700 705 series iii. 1,350,000 1,100,000 260,000 215,000 575 610 650 series iv. 1,750,000 --- 87,360 --- 4,000 3,500 3,600 it appears from these results that the most marked decrease in temperature occurs at 140° f. (60° c.). it should also be observed that an increase in heat above this temperature did not materially diminish the number of organisms present, indicating that those forms remaining were in a spore or resistant condition. it was noted, however, that the developing colonies grew more slowly in the plates made from the highly heated milk, showing that their vitality was injured to a greater extent even though not killed. _2. destruction of disease bacteria._ while milk should be pasteurized so as to destroy all active, multiplying bacteria, it is particularly important to destroy any organisms of a disease nature that might find their way into the same. fortunately most of the bacteria capable of thriving in milk before or after it is drawn from the animal are not able to form spores and hence succumb to proper pasteurization. such is the case with the diphtheria, cholera and typhoid organisms. the organism that is invested with most interest in this connection is the tubercle bacillus. on account of its more or less frequent occurrence in milk and its reputed high powers of resistance, it may well be taken as a standard in pasteurizing. ~thermal death limits of tubercle bacillus.~ concerning the exact temperature at which this germ is destroyed there is considerable difference of opinion. part of this arises from the inherent difficulty in determining exactly when the organism is killed (due to its failure to grow readily on artificial media), and part from the lack of uniform conditions of exposure. the standards that previously have been most generally accepted are those of de man,[137] who found that thirty minutes exposure at 149° f., fifteen minutes at 155° f., or ten minutes at 167° f., sufficed to destroy this germ. more recently it has been demonstrated,[138] and these results confirmed,[139] that if tuberculous milk is heated in closed receptacles where the surface pellicle does not form, the vitality of this disease germ is destroyed at 140° f. in 10-15 minutes, while an exposure at 160° f. requires only about one minute.[140] if the conditions of heating are such that the surface of the milk is exposed to the air, the resistance of bacteria is greatly increased. when heated in open vessels smith found that the tubercle organism was not killed in some cases where the exposure was made for at least an hour. russell and hastings[141] have shown an instance where the thermal death-point of a micrococcus isolated from pasteurized milk was increased 12.5° f., by heating it under conditions that permitted of the formation of the scalded layer. it is therefore apparent that apparatus used for pasteurization should be constructed so as to avoid this defect. ~methods of treatment.~ two different systems of pasteurization have grown up in the treatment of milk. one of these has been developed from the hygienic or sanitary aspect of the problem and is used more particularly in the treatment of cream and relatively small milk supplies. the other system has been developed primarily from the commercial point of view where a large amount of milk must be treated in the minimum time. in the first method the milk is heated for a longer period of time, about fifteen minutes at a relatively low temperature from 140°-155° f.; in the other, the milk is exposed to the source of heat only while it is passing rapidly through the apparatus. naturally, the exposure under such conditions must be made at a considerably higher temperature, usually in the neighborhood of 160° f. the types of apparatus used in these respective processes naturally varies. where the heating is prolonged, the apparatus employed is built on the principle of a _tank_ or _reservoir_ in which a given volume of milk may be held at any given temperature for any given period of time. when the heat is applied for a much shorter period of time, the milk is passed in a continuous stream through the machine. naturally the capacity of a continuous-flow apparatus is much greater than a machine that operates on the intermittent principle; hence, for large supplies, as in city distribution, this system has a great advantage. the question as to relative efficiency is however one which should be given most careful consideration. ~pasteurizing apparatus.~ the problems to be solved in the pasteurization of milk and cream designed for direct consumption are so materially different from where the process is used in butter-making that the type of machinery for each purpose is quite different. the equipment necessary for the first purpose may be divided into two general classes: 1. apparatus of limited capacity designed for family use. 2. apparatus of sufficient capacity to pasteurize on a commercial scale. ~domestic pasteurizers.~ in pasteurizing milk for individual use, it is not desirable to treat at one time more than will be consumed in one day; hence an apparatus holding a few bottles will suffice. in this case the treatment can best be performed in the bottle itself, thereby lessening the danger of infection. several different types of pasteurizers are on the market; but special apparatus is by no means necessary for the purpose. the process can be efficiently performed by any one with the addition of an ordinary dairy thermometer to the common utensils found in the kitchen. fig. 24 indicates a simple contrivance that can be readily arranged for this purpose. the following suggestions indicate the different steps of the process: 1. use only fresh milk. 2. place milk in clean bottles or fruit cans, filling to a uniform level, closing bottles tightly with a cork or cover. if pint and quart cans are used at the same time, an inverted bowl will equalize the level. set these in a flat-bottomed tin pail and fill with warm water to same level as milk. an inverted pie tin punched with holes will serve as a stand on which to place the bottles during the heating process. 3. heat water in pail until the temperature of same reaches 155° to 160° f.; then remove from source of direct heat, cover with a cloth or tin cover, and allow the whole to stand for half an hour. in the preparation of milk for children, it is not advisable to use the low-temperature treatment (140° f.) that is recommended for commercial city delivery. [illustration: fig. 24. a home-made pasteurizer.] 4. remove bottles of milk and cool them as rapidly as possible without danger to bottles and store in a refrigerator. ~commercial pasteurizers.~ the two methods of pasteurization practiced commercially for the preservation of milk and cream have been developed because of the two types of machinery now in use. apparatus constructed on the reservoir or tank principle permits of the retention of the milk for any desired period of time. therefore, a lower temperature can be employed in the treatment. in those machines where the milk flows through the heater in a more or less continuous stream, the period of exposure is necessarily curtailed, thereby necessitating a higher temperature. ~reservoir pasteurizers.~ the simplest type of apparatus suitable for pasteurizing on this principle is where the milk is placed in shotgun cans and immersed in water heated by steam. ordinary tanks surrounded with water spaces can also be used successfully. the boyd cream ripening vat has also been tried. in this the milk is heated by a swinging coil immersed in the vat through which hot water circulates. in 1894 the writer[142] constructed a tank pasteurizer which consisted of a long, narrow vat surrounded by a steam-heated water chamber. both the milk and the water chambers were provided with mechanical agitators having a to-and-fro movement. [illustration: fig. 25. pott's pasteurizer.] another machine which has been quite generally introduced is the potts' rotating pasteurizer. this apparatus has a central milk chamber that is surrounded with an outer shell containing hot water. the whole machine revolves on a horizontal axis, and the cream or milk is thus thoroughly agitated during the heating process. ~continuous-flow pasteurizers.~ the demand for greater capacity than can be secured in the reservoir machines has led to the perfection of several kinds of apparatus where the milk is heated momentarily as it flows through the apparatus. most of these were primarily introduced for the treatment of cream for butter-making purposes, but they are frequently employed for the treatment of milk on a large scale in city milk trade. many of them are of european origin although of late years several have been devised in this country. the general principle of construction is much the same in most of them. the milk is spread out in a thin sheet, and is treated by passing it over a surface, heated either with steam directly or preferably with hot water. where steam is used directly, it is impossible to prevent the "scalding on" of the milk proteids to the heated surface. in some of these machines (thiel, kuehne, lawrence, de laval, and hochmuth), a ribbed surface is employed over which the milk flows, while the opposite surface is heated with hot water or steam. monrad, lefeldt and lentsch employ a centrifugal apparatus in which a thin layer of milk is heated in a revolving drum. in some types of apparatus, as in the miller machine, an american pasteurizer, the milk is forced in a thin sheet between two heated surfaces, thereby facilitating the heating process. in the farrington machine heated discs rotate in a reservoir through which the milk flows in a continuous stream. one of the most economical types of apparatus is the regenerator type (a german machine), in which the milk passes over the heating surface in a thin stream and then is carried back over the incoming cold milk so that the heated liquid is partially cooled by the inflowing fresh milk. in machines of this class it requires very much less steam to heat up the milk than in those in which the cold milk is heated wholly by the hot water. a number of machines have been constructed on the principle of a reservoir which is fed by a constantly flowing stream. in some kinds of apparatus of this type no attempt is made to prevent the mixing of the recently introduced milk with that which has been partially heated. the pattern for this reservoir type is fjord's heater, in which the milk is stirred by a stirrer. this apparatus was originally designed as a heater for milk before separation, but it has since been materially modified so that it is better adapted to the purposes of pasteurization. reid was the first to introduce this type of machine into america. ~objections to continuous flow pasteurizers.~ in all continuous flow pasteurizers certain defects are more or less evident. while they fulfill the important requirement of large capacity, an absolute essential where large volumes of milk are being handled, it does not of necessity follow that they conform to all the hygienic and physical requirements that should be kept in mind. the greatest difficulty is the shortened period of exposure. the period which the milk is actually heated is often not more than a minute or so. another serious defect is the inability to heat _all_ of the milk for a uniform period of time. at best, the milk is exposed for an extremely short time, but even then portions pass through the machine much more quickly than do the remainder. those portions in contact with the walls of the apparatus are retarded by friction and are materially delayed in their passage, while the particles in the center of the stream, however thin, flow through in the least possible time. the following simple method enables the factory operator to test the period of exposure in the machine: start the machine full of water, and after the same has become heated to the proper temperature, change the inflow to full-cream milk, continuing at the same rate. note the exact time of change and also when first evidence of milkiness begins to appear at outflow. if samples are taken from first appearance of milky condition and thereafter at different intervals for several minutes, it is possible, by determining the amount of butter-fat in the same, to calculate with exactness how long it takes for the milk to entirely replace the water. tests made by the writer[143] on the miller pasteurizer showed, when fed at the rate of 1,700 pounds per hour, the minimum period of exposure to be 15 seconds, and the maximum about 60-70 seconds, while about two-thirds of the milk passed the machine in 40-50 seconds. this manifest variation in the rate of flow of the milk through the machine is undoubtedly the reason why the results of this type of treatment are subject to so much variation. naturally, even a fatal temperature to bacterial life can be reduced to a point where actual destruction of even vegetating cells does not occur. ~bacterial efficiency of reservoir pasteurizers.~ the bacterial content of pasteurized milk and cream will depend somewhat on the number of organisms originally present in the same. naturally, if mixed milk brought to a creamery is pasteurized, the number of organisms remaining after treatment would be greater than if the raw material was fresh and produced on a single farm. an examination of milk and cream pasteurized on a commercial scale in the russell vat at the wisconsin dairy school showed that over 99.8 per cent of the bacterial life in raw milk or cream was destroyed by the heat employed, i. e., 155° f. for twenty minutes duration.[144] in nearly one-half of the samples of milk, the germ content in the pasteurized sample fell below 1,000 bacteria per cc., and the average of twenty-five samples contained 6,140 bacteria per cc. in cream the germ content was higher, averaging about 25,000 bacteria per cc. this milk was taken from the general creamery supply, which was high in organisms, containing on an average 3,675,000 bacteria per cc. de schweinitz[145] has reported the germ content of a supply furnished in washington which was treated at 158° to 160° f. for fifteen minutes. this supply came from a single source. figures reported were from 48-hour-old agar plates. undoubtedly these would have been higher if a longer period of incubation had been maintained. the average of 82 samples, taken for the period of one year, showed 325 bacteria per cc. [illustration: fig. 26. effect of pasteurizing on germ content of milk. black square represents bacteria of raw milk; small white square, those remaining after pasteurization.] ~bacterial efficiency of continuous-flow pasteurizers.~ a quantitative determination of the bacteria found in milk and cream when treated in machinery of this class almost always shows a degree of variation in results that is not to be noted in the discontinuous apparatus. [illustration: fig. 27. reid's continuous pasteurizer.] harding and rogers[146] have tested the efficiency of one of the danish type of continuous pasteurizers. these experiments were made at 158°, 176° and 185° f. they found the efficiency of the machine not wholly satisfactory at the lower temperatures. at 158° f. the average of fourteen tests gave 15,300 bacteria per cc., with a maximum to minimum range from 62,790 to 120. twenty-five examinations at 176° f. showed an average of only 117, with a range from 300 to 20. the results at 185° f. showed practically the same results as noted at 176° f. considerable trouble was experienced with the "scalding on" of the milk to the walls of the machine when milk of high acidity was used. jensen[147] details the results of 139 tests in 1899, made by the copenhagen health commission. in 66 samples from one hundred thousand to one million organisms per cc. were found, and in 22 cases from one to five millions. nineteen tests showed less than 10,000 per cc. in a series of tests conducted by the writer[148] on a miller pasteurizer in commercial operation, an average of 21 tests showed 12,350 bacteria remaining in the milk when the milk was pasteurized from 156°-164° f. the raw milk in these tests ran from 115,000 to about one million organisms per cc. a recently devised machine of this type (pasteur) has been tested by lehmann, who found that it was necessary to heat the milk as high as 176° to 185° f., in order to secure satisfactory results on the bacterial content of the cream. the writer tested reid's pasteurizer at 155° to 165° f. with the following results: in some cases as many as 40 per cent. of the bacteria survived, which number in some cases exceeded 2,000,000 bacteria per cc. ~pasteurizing details.~ while the pasteurizing process is exceedingly simple, yet, in order to secure the best results, certain conditions must be rigidly observed in the treatment before and after the heating process. it is important to select the best possible milk for pasteurizing, for if the milk has not been milked under clean conditions, it is likely to be rich in the spore-bearing bacteria. old milk, or milk that has not been kept at a low temperature, is much richer in germ-life than perfectly fresh or thoroughly chilled milk. the true standard for selecting milk for pasteurization should be to determine the actual number of bacterial _spores_ that are able to resist the heating process, but this method is impracticable under commercial conditions. the following method, while only approximate in its results, will be found helpful: assuming that the age or treatment of the milk bears a certain relation to the presence of spores, and that the acid increases in a general way with an increase in age or temperature, the amount of acid present may be taken as an approximate index of the suitability of the milk for pasteurizing purposes. biological tests were carried out in the author's laboratory[149] on milks having a high and low acid content, and it was shown that the milk with the least acid was, as a rule, the freest from spore-bearing bacteria. this acid determination can be made at the weigh-can by employing the farrington alkaline tablet which is used in cream-ripening. where milk is pasteurized under general creamery conditions, none should be used containing more than 0.2 per cent acidity. if only perfectly fresh milk is used, the amount of acid will generally be about 0.15 per cent with phenolphthalein as indicator. [illustration: fig. 28. diagram showing temperature changes in pasteurizing, and the relation of same to bacterial growth. shaded zone represents limits of bacterial growth, 50°-109° f. (10°-43° c.), the intensity of shading indicating rapidity of development. the solid black line shows temperature of milk during the process. the necessity for rapid cooling is evident as the milk falls in temperature to that of growing zone.] emphasis has already been laid on the selection of a proper limit of pasteurizing (p. 114). it should be kept constantly in mind that the thermal death-point of any organism depends not alone on the temperature used, but on the period of exposure. with the lower limits given, 140° f., it is necessary to expose the milk for not less than fifteen minutes. if a higher heat is employed (and the cooked flavor disregarded) the period of exposure may be curtailed. ~chilling the milk.~ it is very essential in pasteurizing that the heated milk be immediately chilled in order to prevent the germination of the resistant spores, for if germination once occurs, growth can go on at relatively low temperatures. the following experiments by marshall[150] are of interest as showing the influence of refrigeration on germination of spores: cultures of organisms that had been isolated from pasteurized milk were inoculated into bouillon. one set was left to grow at room temperature, another was pasteurized and allowed to stand at same temperature, while another heated set was kept in a refrigerator. the unheated cultures at room temperature showed evidence of growth in thirty trials in an average of 26 hours; 29 heated cultures at room temperature all developed in an average of 50 hours, while the heated cultures kept in refrigerator showed no growth in 45 days with but four exceptions. practically all of the rapid-process machines are provided with especially constructed cooling devices. in some of them, as in the miller and farrington, the cooling is effected by passing the milk through two separate coolers that are constructed in the same general way as the heater. with the first cooler, cold running water is employed, the temperature often being lowered in this way to 58° or 60° f. further lessening of the temperature is secured by an additional ice water or brine cooler which brings the temperature down to 40°-50° f. in the economical use of ice the ice itself should be applied as closely as possibly to the milk to be cooled, for the larger part of the chilling value of ice comes from the melting of the same. to convert a pound of ice at 32° f. into a pound of water at the same temperature, if we disregard radiation, would require as much heat as would suffice to raise 142 pounds of water one degree f., or one pound of water 142° f. the absorptive capacity of milk for heat (specific heat) is not quite the same as it is with water, being .847 for milk in comparison with 1.0 for water.[151] hot milk would therefore require somewhat less ice to cool it than would be required by any equal volume of water at the same temperature. ~bottling the product.~ if the milk has been properly pasteurized, it should, of course, be dispensed in sterilized bottles. glass bottles with plain pulp caps are best, and these should be thoroughly sterilized in steam before using. the bottling can best be done in a commercial bottling machine. care must be taken to thoroughly clean this apparatus after use each day. rubber valves in these machines suffer deterioration rapidly. [illustration: fig. 29. relative consistency of pasteurized cream before (a) and after (b) treatment with viscogen as shown by rate of flow down inclined glass plate.] ~restoration of "body" of pasteurized cream.~ the action of heat causes the tiny groupings of fat globules in normal milk (fig. 22) to break up, and with this change, which occurs in the neighborhood of 140° f., where the milk is heated for about 15 minutes and at about 160-165° f. where rapidly heated in a continuous stream, the consistency of the liquid is diminished, notwithstanding the fact that the fat-content remains unchanged. babcock and the writer[152] devised the following "cure" for this apparent defect. if a strong solution of cane sugar is added to freshly slacked lime and the mixture allowed to stand, a clear fluid can be decanted off. the addition of this alkaline liquid, which is called "viscogen," to pasteurized cream in proportions of about one part of sugar-lime solution to 100 to 150 of cream, restores the consistency of the cream, as it causes the fat globules to cluster together in small groups. the relative viscosity of creams can easily be determined by the following method (fig. 29): take a perfectly clean piece of glass (plate or picture glass is preferable, as it is less liable to be wavy). drop on one edge two or three drops of cream at intervals of an inch or so. then incline piece of glass at such an angle as to cause the cream to flow down surface of glass. the cream, having the heavier body or viscosity, will move more slowly. if several samples of each cream are taken, then the aggregate lengths of the different cream paths may be taken, thereby eliminating slight differences due to condition of glass. footnotes: [126] from 10 to 16 cents per quart is usually paid for such milks. [127] much improvement in quality could be made by more careful control of milk during shipment, especially as to refrigeration; also as to the care taken on the farms. the use of the ordinary milking machine (see page 37), would go far to reduce the germ content of milk. [128] farrington, journ. amer. chem. soc., sept., 1896. [129] hite, bull. 58, west va. expt. stat., 1899. [130] milch zeit., 1895, no. 9. [131] ibid., 1897, no. 33. [132] bernstein, milch zeit., 1894, pp. 184, 200. [133] thoerner, chem. zeit., 18:845. [134] snyder, chemistry of dairying, p. 59. [135] doane and price (bull. 77, md. expt. stat., aug. 1901) give quite a full resumé of the work on this subject in connection with rather extensive experiments made by them on feeding animals with raw, pasteurized and sterilized milks. [136] rickets is a disease in which the bones lack sufficient mineral matter to give them proper firmness. marasmus is a condition in which the ingested food seems to fail to nourish the body and gradual wasting away occurs. [137] de man, arch. f. hyg., 1893, 18:133. [138] th. smith, journ. of expt. med., 1899, 4:217. [139] russell and hastings, 17 rept. wis. expt. stat., 1900, p. 147. [140] russell and hastings, 21 rept. ibid., 1904. [141] russell and hastings, 18 rept. ibid., 1901. [142] russell, bull. 44, wis. expt. stat. [143] russell, 22 wis. expt. stat. rept., 1905, p. 232. [144] russell, 12 wis. expt. stat. rept., 1895, p. 160. [145] de schweinitz, nat. med. rev., 1899, no. 11. [146] harding and rogers. bull. 182, n. y. (geneva) expt. stat., dec., 1899. [147] jensen, milchkunde und milch hygiene, p. 132. [148] 22 wis. expt. stat. rept., 1905, p. 236. [149] shockley, thesis, univ. of wis., 1896. [150] marshall, mich. expt. stat., bull. 147, p. 47. [151] fleischmann, landw. versuchts stat., 17:251. [152] babcock and russell, bull. 54, wis. expt. stat., aug. 1896. chapter vii. bacteria and butter-making. in making butter from the butter fat in milk, it is necessary to concentrate the fat globules into cream, preliminary to the churning process. the cream may be raised by the gravity process or separated from the milk by centrifugal action. in either case the bacteria that are normally present in the milk differentiate themselves in varying numbers in the cream and the skim-milk. the cream always contains per cc. a great many more than the skim-milk, the reason for this being that the bacteria are caught and held in the masses of fat globules, which, on account of their lighter specific gravity, move toward the surface of the milk or toward the interior of the separator bowl. this filtering action of the fat globules is similar to what happens in muddy water upon standing. as the suspended particles fall to the bottom they carry with them a large number of the organisms that are in the liquid. ~various creaming methods.~ the creaming method has an important bearing on the kind as well as the number of the bacteria that are to be found in the cream. the difference in species is largely determined by the difference in ripening temperature, while the varying number is governed more by the age of the milk. _1. primitive gravity methods._ in the old shallow-pan process, the temperature of the milk is relatively high, as the milk is allowed to cool naturally. this comparatively high temperature favors especially the development of those forms whose optimum growing-point is near the air temperature. by this method the cream layer is exposed to the air for a longer time than with any other, and consequently the contamination from this source is greater. usually cream obtained by the shallow-pan process will contain a larger number of species and also have a higher acid content. _2. modern gravity methods._ in the cooley process, or any of the modern gravity methods where cold water or ice is used to lower the temperature, the conditions do not favor the growth of a large variety of species. the number of bacteria in the cream will depend largely upon the manner in which the milk is handled previous to setting. if care is used in milking, and the milk is kept so as to exclude outside contamination, the cream will be freer from bacteria than if carelessness prevails in handling the milk. only those forms will develop in abundance that are able to grow at the low temperature at which the milk is set. cream raised by this method is less frequently infected with undesirable forms than that which is creamed at a higher temperature. _3. centrifugal method._ separator cream should contain less germ-life than that which is secured in the old way. it should contain only those forms that have found their way into the milk during and subsequent to the milking, for the cream is ordinarily separated so soon that there is but little opportunity of infection, if care is taken in the handling. as a consequence, the number of species found therein is smaller. where milk is separated, it is always prudent to cool the cream so as to check growth, as the milk is generally heated before separating in order to skim efficiently. although cream is numerically much richer in bacteria than milk, yet the changes due to bacterial action are slower; hence milk sours more rapidly than cream. for this same reason, cream will sour sooner when it remains on the milk than it will if it is separated as soon as possible. this fact indicates the necessity of early creaming, so as to increase the keeping quality of the product, and is another argument in favor of the separator process. ~ripening of cream.~ if cream is allowed to remain at ordinary temperatures, it undergoes a series of fermentation changes that are exceedingly complex in character, the result of which is to produce in butter made from the same the characteristic flavor and aroma that are so well known in this article. we are so accustomed to the development of these flavors in butter that they are not generally recognized as being intimately associated with bacterial activity unless compared with butter made from perfectly fresh cream. sweet-cream butter lacks the aromatic principle that is prominent in the ripened product, and while the flavor is delicate, it is relatively unpronounced. in the primitive method of butter-making, where the butter was made on the farm, the ripening of cream became a necessity in order that sufficient material might be accumulated to make a churning. the ripening change occurred spontaneously without the exercise of any especial control. with the development of the creamery system came the necessity of exercising a control of this process, and therefore the modern butter-maker must understand the principles which are involved in this series of complex changes that largely give to his product its commercial value. in these ripening changes three different factors are to be taken into consideration: the development of acid, flavor and aroma. much confusion in the past has arisen from a failure to discriminate between these qualities. while all three are produced simultaneously in ordinary ripening, it does not necessarily follow that they are produced by the same cause. if the ripening changes are allowed to go too far, undesirable rather than beneficial decomposition products are produced. these greatly impair the value of butter, so that it becomes necessary to know just to what extent this process should be carried. in cream ripening there is a very marked bacterial growth, the extent of which is determined mainly by the temperature of the cream. conn and esten[153] find that the number of organisms may vary widely in unripened cream, but that the germ content of the ripened product is more uniform. when cream is ready for the churn, it often contains 500,000,000 organisms per cc., and frequently even a higher number. this represents a germ content that has no parallel in any natural material. the larger proportion of bacteria in cream as it is found in the creamery belong to the acid-producing class, but in the process of ripening, these forms seem to thrive still better, so that when it is ready for churning the germ content of the cream is practically made up of this type. ~effect on churning.~ in fresh cream the fat globules which are suspended in the milk serum are surrounded by a film of albuminous material which prevents them from coalescing readily. during the ripening changes, this enveloping substance is modified, probably by partial solution, so that the globules cohere when agitated, as in churning. the result is that ripened cream churns more easily, and as it is possible to cause a larger number of the smaller fat-globules to cohere to the butter granules, the yield is slightly larger--a point of considerable economic importance where large quantities of butter are made. ~development of acid.~ the result of this enormous bacterial multiplication is that acid is produced in cream, lactic being the principal acid so formed. other organic acids are undoubtedly formed as well as certain aromatic products. while the production of acid as a result of fermentative activity is usually accompanied with a development of flavor, the flavor is not directly produced by the formation of acid. if cream is treated in proper proportions with a commercial acid, as hydrochloric,[154] it assumes the same churning properties as found in normally ripened cream, but is devoid of the desired aromatic qualities. lactic acid[155] has also been used in a similar way but with no better results. the amount of acidity that should be developed under natural conditions so as to secure the optimum quality as to flavor and aroma is the most important question in cream ripening. concerning this there have been two somewhat divergent views as to what is best in practice, some holding that better results were obtained with cream ripened to a high degree of acidity than where a less amount was developed.[156] the present tendency seems to be to develop somewhat more than formerly, as it is thought that this secures more of the "high, quick" flavor wanted in the market. on the average, cream is ripened to about 0.5 to 0.65 per cent. acidity, a higher percentage than this giving a strong-flavored butter. in the determination of acidity, the most convenient method is to employ the farrington alkaline tablet, which permits of an accurate and rapid estimation of the acidity in the ripening cream. the amount of acidity to be produced must of necessity be governed by the amount of butter-fat present, for the formation of acid is confined to the serum of the cream; consequently, a rich cream would show less acid by titration than a thinner cream, and still contain really as much acid as the other. the importance of this factor is evident in gathered-cream factories. the rate of ripening is dependent upon the conditions that affect the rate of growth of bacterial life, such as time and temperature, number of organisms in cream and also the per cent of butter fat in the cream. some years ago it was customary to ripen cream at about 50° to 60° f., but more recently better results have been obtained, it is claimed, where the ripening temperature is increased and the period of ripening lessened. as high a temperature as 70° to 75° f. has been recommended. it should be said that this variation in practice may have a valid scientific foundation, for the temperature of the ripening cream is undoubtedly the most potent factor in determining what kind of bacteria will develop most luxuriantly. it is well known that those forms that are capable of producing bitter flavors are able to thrive better at a lower temperature than some of the desirable ripening species. the importance of this factor would be lessened where a pure culture was used in pasteurized cream, because here practically the selected organism alone controls the field. it is frequently asserted that better results are obtained by stirring the cream and so exposing it to the air as much as possible. experiments made at the ontario agricultural college, however, show practically no difference in the quality of the butter made by these two methods. the great majority of the bacteria in the cream belong to the facultative class, and are able to grow under conditions where they are not in direct contact with the air. ~flavor and aroma.~ the basis for the peculiar flavor or taste which ripened cream-butter possesses is due, in large part, to the formation of certain decomposition products formed by various bacteria. aroma is a quality often confounded with flavor, but this is produced by volatile products only, which appeal to the sense of smell rather than taste. generally a good flavor is accompanied by a desirable aroma, but the origin of the two qualities is not necessarily dependent on the same organisms. the quality of flavor and aroma in butter is, of course, also affected by other conditions, as, for instance, the presence or absence of salt, as well as the inherent qualities of the milk, that are controlled, to some extent at least, by the character of the feed which is consumed by the animal. the exact source of these desirable but evanescent qualities in butter is not yet satisfactorily determined. according to storch,[157] flavors are produced by the decomposition of the milk sugar and the absorption of the volatile flavors by the butter fat. conn[158] holds that the nitrogenous elements in cream serve as food for bacteria, and in the decomposition of which the desired aromatic substance is produced. the change is unquestionably a complex one, and cannot be explained as a single fermentation. there is no longer much doubt but that both acid-forming and casein-digesting species can take part in the production of proper flavors as well as desirable aromas. the researches of conn,[159] who has studied this question most exhaustively, indicate that both of these types of decomposition participate in the production of flavor and aroma. he has shown that both flavor and aroma production are independent of acid; that many good flavor-producing forms belong to that class which renders milk alkaline, or do not change the reaction at all. some of these species liquefied gelatin and would therefore belong to the casein-dissolving class. those species that produced bad flavors are also included in both fermentative types. conn has found a number of organisms that are favorable flavor-producers; in fact they were much more numerous than desirable aroma-yielding species. none of the favorable aroma forms according to his investigations were lactic-acid species,--a view which is also shared by weigmann.[160] mcdonnell[161] has found that the production of aroma in certain cases varies at different temperatures, the most pronounced being evolved near the optimum growing temperature, which, as a general rule, is too high for cream ripening. the majority of bacteria in ripening cream do not seem to exert any marked influence in butter. a considerable number of species are positively beneficial, inasmuch as they produce a good flavor or aroma. a more limited number are concerned in the production of undesirable ripening changes. this condition being true, it may seem strange that butter is as good as it is, because so frequently the requisite care is not given to the development of proper ripening. in all probability the chief reason why this is so is that those bacteria that find milk and cream pre-eminently suited to their development, e. g. the lactic-acid class, are either neutral or beneficial in their effect on butter. ~use of starters.~ experience has amply demonstrated that it is possible to control the nature of the fermentative changes that occur in ripening cream to such an extent as to materially improve the quality of the butter. this is frequently done by the addition of a "starter." while starters have been employed for many years for the purpose mentioned, it is only recently that their nature has been understood. a starter may be selected from widely divergent sources, but in all cases it is sure to contain a large number of bacteria, and the presumption is that they are of such a nature as to produce desirable fermentative changes in the cream. in the selection of these so-called natural starters, it follows that they must be chosen under such conditions as experience has shown to give favorable results. for this purpose, whole milk from a single animal is often used where the same is observed to sour with the production of no gas or other undesirable taint. a skim-milk starter from a mixed supply is recommended by many. butter milk is frequently employed, but in the opinion of butter experts is not as suitable as the others mentioned. it not infrequently happens that the practical operator may be misled in selecting a starter that is not desirable, or by continuing its use after it has become contaminated. in 1890[162] a new system of cream ripening was introduced in denmark by storch that possesses the merit of being a truly scientific and at the same time practical method. this consisted in the use of pure cultures of specific organisms that were selected on account of their ability to produce a desirable ripening change in cream. the introduction of these so-called culture starters has become universal in denmark, and in parts of germany. their use is also rapidly extending in this country, australia and new zealand. ~principles of pure-culture cream-ripening.~ in the proper use of pure cultures for ripening cream, it is necessary first to eliminate as far as possible the bacteria already present in cream before the culture starter is added. this result is accomplished by heating the cream to a temperature sufficiently high to destroy the vegetating organisms. the addition of a properly selected starter will then give the chosen organism such an impetus as will generally enable it to gain the ascendency over any other bacteria and so control the character of the ripening. the principle employed is quite like that practiced in raising grain. the farmer prepares his soil by plowing, in this way killing the weeds. then he sows his selected grain, which is merely a pure culture, and by the rapid growth of this, other forms are held in check. the attempt has been made to use these culture starters in raw sweet cream, but it can scarcely be expected that the most beneficial results will be attained in this way. this method has been justified on the basis of the following experiments. where cream is pasteurized and no starter is added, the spore-bearing forms frequently produce undesirable flavors. these can almost always be controlled if a culture starter is added, the obnoxious form being repressed by the presence of the added starter. this condition is interpreted as indicating that the addition of a starter to cream which already contains developing bacteria will prevent those originally present in the cream from growing.[163] this repressive action of one species on another is a well-known bacteriological fact, but it must be remembered that such an explanation is only applicable in those cases where the culture organism is better able to develop than those forms that already exist in the cream. if the culture organism is added to raw milk or cream which already contains a flora that is well suited to develop in this medium, it is quite doubtful whether it would gain the supremacy in the ripening cream. the above method of adding a culture to raw cream renders cream-ripening details less burdensome, but at the same time danish experience, which is entitled to most credence on this question, is opposed to this method. ~reputed advantages of culture starters.~ _1. flavor and aroma._ naturally the flavor produced by pure-culture ferments depends upon the character of the organism used. those which are most extensively used are able to produce a perfectly clean but mild flavor, and a delicate but not pronounced aroma. the "high, quick" flavor and aroma that is so much desired in the american market is not readily obtained by the use of cultures. it is quite problematical whether the use of any single species will give any more marked aroma than normally occurs in natural ripening. _2. uniformity of product._ culture starters produce a more uniform product because the type of fermentation is under more complete control, and herein is the greatest advantage to be derived from their use. even the best butter-maker at times will fail to secure uniform results if his starter is not perfectly satisfactory. _3. keeping quality of product._ butter made from pasteurized cream to which a pure-culture starter has been added will keep much better than the ordinary product, because the diversity of the bacterial flora is less and the milk is therefore not so likely to contain those organisms that produce an "off" condition. _4. elimination of taints._ many defective conditions in butter are attributable to the growth of undesirable bacteria in the cream that result in the formation of "off" flavors and taints. if cream is pasteurized, thereby destroying these organisms, then ripened with pure ferments, it is generally possible to eliminate the abnormal conditions.[164] taints may also be present in cream due to direct absorption from the cow or through exposure to foul odors.[165] troubles of this sort may thus be carried over to the butter. this is particularly true in regions where leeks and wild onions abound, as in some of the atlantic states. the heating of the cream tends to expel these volatile taints, so that a fairly good article of butter can be made from what would otherwise be a relatively worthless product. ~characteristics desired in culture starters.~ certain conditions as the following are desirable in starters made from pure cultures: 1. vigorous growth in milk at ordinary ripening temperatures. 2. ability to form acid so as to facilitate churning and increase the yield of butter. 3. able to produce a clean flavor and desirable aroma. 4. impart a good keeping quality to butter. 5. not easily modified in its flavor-producing qualities by artificial cultivation. these different conditions are difficult to attain, for the reason that some of them seem to be in part incompatible. weigmann[166] found that a good aroma was generally an evanescent property, and therefore opposed to good keeping quality. conn has shown that the functions of acid-formation, flavor and aroma production are not necessarily related, and therefore the chances of finding a single organism that possesses all the desirable attributes are not very good. in all probability no one germ possesses all of these desirable qualities, but natural ripening is the resultant of the action of several forms.[167] this idea has led to the attempt at mixing selected organisms that have been chosen on account of certain favorable characteristics which they might possess. the difficulty of maintaining such a composite culture in its correct proportions when it is propagated in the creamery is seemingly well nigh insuperable, as one organism is very apt to develop more or less rapidly than the other. a very satisfactory way in which these cultures are marketed is to mix the bacterial growth with some sterile, inert, dry substance. this is the method used in most of the danish cultures. in this country, some of the more prominent cultures employed are marketed in a liquid form. ~culture vs. home-made starters.~ one great advantage which has accrued from the use of culture or commercial starters has been that in emphasizing the need of closer control of the ripening process, greater attention has been paid to the carrying out of the details. in the hands of the better operators, the differences in flavor of butter made with a culture or a natural starter are not marked,[168] but in the hands of those who fail to make a good product under ordinary conditions, an improvement is often secured where a commercial culture is used. ~pasteurization as applied to butter-making.~ this process, as applied to butter making, is often confounded with the treatment of milk and cream for direct consumption. it is unfortunate that the same term is used in connection with the two methods, for they have but little in common except in the use of heat to destroy the germ life of the milk. in pasteurizing cream for butter-making, it is not necessary to observe the stringent precautions that are to be noted in the preservation of milk; for the addition of a rapidly developing starter controls at once the fermentative changes that subsequently occur. then again, the physical requirement as to the production of a cooked taste is not so stringent in butter-making. while a cooked taste is imparted to milk or even cream at about 158° f., it is possible to make butter that shows no permanent cooked taste from cream that has been raised as high as 185° or even 195° f. this is due to the fact that the fat does not readily take up those substances that give to scalded milk its peculiar flavor. unless care is taken in the manipulation of the heated cream, the grain or body of the butter may be injured. this tendency can be overcome if the ripened cream is chilled to 48° f. for about two hours before churning. it is also essential that the heated cream should be quickly and thoroughly chilled after being pasteurized. the danes, who were the first to employ pasteurization in butter-making, used, in the beginning, a temperature ranging from 158° to 167° f., but owing to the prevalence of such diseases as tuberculosis and foot-and-mouth disease, it became necessary to treat all of the skim milk that was returned from the creameries. for this purpose the skim milk is heated to a temperature of 176° f., it having been more recently determined that this degree of heat is sufficient to destroy the seeds of disease. with the use of this higher temperature the capacity of the pasteurizing apparatus is considerably reduced, but the higher temperature is rendered necessary by the prevailing conditions as to disease. when the system was first introduced in denmark, two methods of procedure were followed: the whole milk was heated to a sufficiently high temperature to thoroughly pasteurize it before it was separated, or it was separated first, and the cream pasteurized afterwards. in the latter case, it is necessary to heat the skim milk after separation to destroy the disease organisms, but this can be quickly done by the use of steam directly. much more care must be used in heating the cream in order to prevent injury to the grain of the butter. in spite of the extra trouble of heating the cream and skim milk separately, this method has practically supplanted the single heating. with the continual spread of tuberculosis in america the heating of skim milk separately is beginning to be introduced.[169] ~use of starters in pasteurized and unpasteurized cream.~ in order to secure the beneficial results presumably attributable to the use of a starter, natural as well as a pure culture, it should be employed in cream in which the bacteria have first been killed out by pasteurization. this is certainly the most logical and scientific method and is the way in which the process has been developed in denmark. here in this country, the use of pure cultures has been quite rapidly extended, but the system of heating the cream has been used in only a slight measure. the increased labor and expense incurred in pasteurizing the cream has naturally militated somewhat against the wide-spread use of the process, but doubtless the main factor has been the inability to secure as high a flavor where the cream was heated as in the unheated product. as the demands of the market change from a high, quick flavor to one that is somewhat milder but of better keeping quality, doubtless pasteurization of the cream will become more and more popular. that such a change is gradually occurring is already evident, although as yet only a small proportion of butter made in this country is now made in this way. where the cream is unheated, a considerable number of species will be found, and even the addition of a pure culture, if that culture is of the lactic acid-producing species, will to some extent control the type of fermentation that occurs. such would not be the case with a culture composed of the casein-digesting type of bacteria. only those forms could thus be used which are especially well suited to development in raw cream. for this reason the pure culture ferments that are generally employed in creamery practice are organisms of the lactic acid type, able to grow rapidly in cream and produce a pure cream flavor in the butter. ~purity of commercial starters.~ naturally the butter maker is forced to rely on the laboratory for his commercial starter, and the question will often arise as to the purity and vigor of the various ferments employed. as there is no way for the factory operator to ascertain the actual condition of the starter, except by using the same, the greatest care should be taken by the manufacturer to insure the absolute purity of the seed used. a bacteriological examination of the various cultures which have been placed on the market not infrequently reveals an impure condition. in several cases the writer has found a not inconsiderable number of liquefying bacteria mixed with the selected organism. molds not infrequently are found in cultures put up in the dry form. doubtless the effect of these accidental contaminations is considerably less in the case of a starter composed of a distinctively lactic acid-producing organism than with a form which is less capable of thriving vigorously in milk, and it should be said that these impurities can frequently be eliminated by continued propagation. the virility and vigor of the starter is also a fluctuating factor, dependent in part at least, upon the conditions under which the organism is grown. in some cases the germ is cultivated in solutions in which acid cannot be formed in abundance. where the conditions permit of the formation of acid, as would be the case if sugar was present with a lactic acid-producing species, the vitality of the culture is often impaired by the action of the gradually accumulating acid. some manufacturers attempt to minimize this deleterious condition by adding carbonate of lime which unites with the acid that is formed. ~propagation of starters for cream-ripening.~ the preparation and propagation of a starter for cream-ripening is a process involving considerable bacteriological knowledge, whether the starter is of domestic origin or prepared from a pure-culture ferment. in any event, it is necessary that the starter should be handled in a way so as to prevent the introduction of foreign bacteria as far as possible. it should be remembered at all times that the starter is a live thing and must be handled throughout its entire history in a way so as to retain its vitality and vigor unimpaired. the following points should be taken into consideration in growing the starter and transferring it from day to day: 1. if a commercial starter is used, see that it is fresh and that the seal has not been broken. if the culture is too old, the larger part of the organisms may have died out before it is transferred, in which case the effect of its addition to the sterilized milk would be of little value. when the commercial ferment is received, it should be stored in the refrigerator pending its use so as to retard as much as possible the changes that naturally go on in the culture liquid. be careful that the bottle is not exposed to the influence of direct sunlight for in a transparent medium the organisms may be readily killed by the disinfecting action of the sun's rays. 2. if a home-made starter is employed, use the greatest possible care in selecting the milk that is to be used as a basis for the starter. 3. for the propagation and perpetuation of the starter from day to day, it is necessary that the same should be grown in milk that is as germ-free as it is possible to secure it. for this purpose sterilize some fresh skim-milk in a covered can that has previously been well steamed. this can be done easily by setting cans containing skim-milk in a vat filled with water and heating the same to 180° f. or above for one-half hour or more. steam should not be introduced directly. this process destroys all but a few of the most resistant spore-bearing organisms. this will give a cooked flavor to the milk, but will not affect the cream to which the starter is added. dairy supply houses are now introducing the use of starter cans that are specially made for this purpose. 4. after the heated milk is cooled down to about 70° or 80° f., it can be inoculated with the desired culture. sometimes it is desirable to "build up" the starter by propagating it first in a smaller volume of milk, and then after this has developed, adding it to a larger amount. this method is of particular value where a large amount of starter is needed for the cream-ripening. 5. after the milk has been inoculated, it should be kept at a temperature that is suitable for the rapid development of the contained bacteria, 65°-75° f., which temperature should be kept as uniform as possible. this can best be done by setting the covered can in a vat filled with warm water. the starter cans are often arranged so that temperature can be controlled by circulating water. 6. the starter should not be too thoroughly curdled when it is needed for use, but should be well soured and only partially curdled for it is difficult to break up thoroughly the curd particles if the starter is completely curdled. if these curd masses are added to ripening cream, white specks may appear in the butter. 7. the vigor of the starter is in all probability stronger when the milk is on the point of curdling than it is after the curd has been formed some time. the continued formation of lactic acid kills many of the bacteria and thus weakens the fermentative action. it is therefore highly important that the acidity of the starter should be closely watched. 8. do not refrigerate the starter when it has reached the proper stage of development, as this retards the bacterial growth in the same manner as cold weather checks the growth of grain. it is preferable to dilute the starter, if it cannot be used when ready, with sufficient freshly sterilized sweet milk to hold the acidity at the proper point and thus keep the bacteria in the starter in a condition which will favor vigorous growth. 9. the starter should be propagated from day to day by adding a small quantity to a new lot of freshly prepared milk. for this purpose two propagating cans should be provided so that one starter may be in use while the other is being prepared. ~how long should a starter be propagated?~ no hard-and-fast rule can be given for this, for it depends largely upon how carefully the starter is handled during its propagation. if the starter is grown in sterilized milk kept in steamed vessels and is handled with sterile dippers, it is possible to maintain it in a state of relative purity for a considerable period of time; if, however, no especial care is given, it will soon become infected by the air, and the retention of its purity will depend more upon the ability of the contained organism to choke out foreign growths than upon any other factor. experience seems to indicate that pure-culture starters "run out" sooner than domestic starters. while it is possible, by bacteriological methods, to determine with accuracy the actual condition of a starter as to its germ content, still such methods are inapplicable in creamery practice. here the maker must rely largely upon the general appearance of the starter as determined by taste and smell. the supply houses that deal in cultures of this class generally expect to supply a new culture at least every month. ~bacteria in butter.~ as ripened cream is necessarily rich in bacteria, it follows that butter will also contain germ life in varying amounts, but as butter-fat is not well adapted for bacterial food, the number of germs in butter is usually less than in ripened cream. sweet-cream butter is naturally poorer in germ life than that made from ripened cream. grotenfelt reports in sweet-cream butter, the so-called "paris butter," only a few bacteria while in acid cream butter the germ content runs from scores to hundreds of thousands. ~effect of bacteria in wash water.~ an important factor in contamination may be the wash water that is used. much carelessness often prevails regarding the location and drainage of the creamery well, and if same becomes polluted with organic matter, bacterial growth goes on apace. melick[170] has made some interesting studies on using pasteurized and sterilized well waters for washing. he found a direct relation to exist between the bacterial content of the wash water and the keeping quality of the butter. some creameries have tried filtered water but under ordinary conditions a filter, unless it is tended to with great regularity, becomes a source of infection rather than otherwise. ~changes in germ content.~ the bacteria that are incorporated with the butter as it first "comes" undergo a slight increase for the first few days. the duration of this period of increase is dependent largely upon the condition of the butter. if the buttermilk is well worked out of the butter, the increase is slight and lasts for a few days only, while the presence of so nutritious a medium as buttermilk affords conditions much more favorable for the continued growth of the organisms. while there may be many varieties in butter when it is fresh, they are very soon reduced in kind as well as number. the lactic acid group of organisms disappear quite rapidly; the spore-bearing species remaining for a somewhat longer time. butter examined after it is several months old is often found to be almost free from germs. in the manufacture of butter there is much that is dependent upon the mechanical processes of churning, washing, salting and working the product. these processes do not involve any bacteriological principles other than those that are incident to cleanliness. the cream, if ripened properly, will contain such enormous numbers of favorable forms that the access of the few organisms that are derived from the churn, the air, or the water in washing will have little effect, unless the conditions are abnormal. bacterial defects in butter. ~rancid change in butter.~ fresh butter has a peculiar aroma that is very desirable and one that enhances the market price, if it can be retained; but this delicate flavor is more or less evanescent, soon disappearing, even in the best makes. while a good butter loses with age some of the peculiar aroma that it possesses when first made, yet a gilt-edged product should retain its good keeping qualities for some length of time. all butters, however, sooner or later undergo a change that renders them worthless for table use. this change is usually a rancidity that is observed in all stale products of this class. the cause of this rancid condition in butter was at first attributed to the formation of butyric acid, but it is now recognized that other changes also enter in.[171] light and especially air also exert a marked effect on the flavor of butter. where butter is kept in small packages it is much more prone to develop off flavors than when packed in large tubs. from the carefully executed experiments of jensen it appears that some of the molds as well as certain species of bacteria are able to incite these changes. these organisms are common in the air and water and it therefore readily follows that inoculation occurs. practically, rancidity is held in check by storing butter at low temperatures where germ growth is quite suspended. ~lack of flavor.~ often this may be due to improper handling of the cream in not allowing it to ripen far enough, but sometimes it is impossible to produce a high flavor. the lack of flavor in this case is due to the absence of the proper flavor-producing organisms. this condition can usually be overcome by the addition of a proper starter. ~putrid butter.~ this specific butter trouble has been observed in denmark, where it has been studied by jensen.[172] butter affected by it rapidly acquires a peculiar putrid odor that ruins it for table use. sometimes, this flavor may be developed in the cream previous to churning. jensen found the trouble to be due to several different putrefactive bacteria. one form which he called _bacillus foetidus lactis_, a close ally of the common feces bacillus, produced this rotten odor and taste in milk in a very short time. fortunately, this organism was easily killed by a comparatively low heat, so that pasteurization of the cream and use of a culture starter quickly eliminated the trouble, where it was tried. ~turnip-flavored butter.~ butter sometimes acquires a peculiar flavor recalling the order of turnips, rutabagas, and other root crops. often this trouble is due to feeding, there being in several of these crops, aromatic substances that pass directly into the milk, but in some instances the trouble arises from bacteria that are able to produce decomposition products,[173] the odor and taste of which strongly recalls these vegetables. ~"cowy" butter.~ frequently there is to be noted in milk a peculiar odor that resembles that of the cow stable. usually this defect in milk has been ascribed to the absorption of impure gases by the milk as it cools, although the gases and odors naturally present in fresh milk have this peculiar property that is demonstrable by certain methods of aeration. occasionally it is transmitted to butter, and recently pammel[174] has isolated from butter a bacillus that produced in milk the same peculiar odor so commonly present in stables. ~lardy and tallowy butter.~ the presence of this unpleasant taste in butter may be due to a variety of causes. in some instances, improper food seems to be the source of the trouble; then again, butter exposed to direct sunlight bleaches in color and develops a lardy flavor.[175] in addition to these, cases have been found in which the defect has been traced to the action of bacteria. storch[176] has described a lactic-acid form in a sample of tallowy butter that was able to produce this disagreeable odor. ~oily butter.~ jensen has isolated one of the causes of the dreaded oily butter that is reported quite frequently in denmark. the specific organism that he found belongs to the sour-milk bacteria. in twenty-four hours it curdles milk, the curd being solid like that of ordinary sour milk. there is produced, however, in addition to this, an unpleasant odor and taste resembling that of machine oil, a peculiarity that is transmitted directly to butter made from affected cream. ~bitter butter.~ now and then butter develops a bitter taste that may be due to a variety of different bacterial forms. in most cases, the bitter flavor in the butter is derived primarily from the bacteria present in the cream or milk. several of the fermentations of this character in milk are also to be found in butter. in addition to these defects produced by a biological cause, bitter flavors in butter are sometimes produced by the milk being impregnated with volatile, bitter substances derived from weeds. ~moldy butter.~ this defect is perhaps the most serious because most common. it is produced by the development of a number of different varieties of molds. the trouble appears most frequently in packed butter on the outside of the mass of butter in contact with the tub. mold spores are so widely disseminated that if proper conditions are given for their germination, they are almost sure to develop. in some cases the mold is due to the growth of the ordinary bread mold, _penicillium glaucum_; in other cases a black mold develops, due often to _cladosporium butyri_. not infrequently trouble of this character is associated with the use of parchment wrappers. the difficulty can easily be held in check by soaking the parchment linings and the tubs in a strong brine, or paraffining the inside of the tub. ~fishy butter.~ considerable trouble has been experienced in australian butter exported to europe in which a fishy flavor developed. it was noted that the production of this defect seemed to be dependent upon the storage temperature at which the butter was kept. when the butter was refrigerated at 15° f. no further difficulty was experienced. it is claimed that the cause of this condition is due to the formation of trimethylamine (herring brine odor) due to the growth of the mold fungus _oidium lactis_, developing in combination with the lactic-acid bacteria. a fishy taste is sometimes noted in canned butter. rogers[177] has determined that this flavor is caused by yeasts (_torula_) which produce fat-splitting enzyms capable of producing this undesirable change. footnotes: [153] conn and esten, cent. f. bakt., ii abt., 1901, 7:746. [154] tiemann, milch zeit., 23:701. [155] milch zeit., 1889, p. 7; 1894, p. 624; 1895, p. 383. [156] dean, ont. agr. coll., 1897, p. 66. [157] storch, nogle, unders. over floed. syrning, 1890. [158] conn, 6 storrs expt. stat., 1893, p. 66. [159] conn, 9 storrs expt. stat., 1896, p. 17. [160] weigmann, milch zeit., 1891, p. 793 [161] mcdonnell, ü. milchsäure bakterien (diss. kiel, 1899), p. 43. [162] storch, milch zeit., 1890, p. 304. [163] conn, 9 storrs expt. stat., 1896, p. 25. [164] milch zeit., 1891, p. 122; 1894, p. 284; 1895, p. 56; 1896, p. 163. [165] mckay, bull. 32, iowa expt. stat., p. 47 [166] weigmann, landw. woch. f. schl. hol., no. 2, 1890. [167] weigmann, cent. f. bakt., ii abt., 3:497, 1897. [168] at the national creamery buttermakers' association for 1901, 193 out of 240 exhibitors used starters. of those that employed starters, nearly one-half used commercial cultures. there was practically no difference in the average score of the two classes of starters, but those using starters ranked nearly two points higher in flavor than those that did not. [169] russell, bull. 143, wis. expt. stat., feb. 1907. [170] melick, bull. 138, kansas expt. stat., june 1906. [171] reinmann, cent. f. bakt., 1900, 6:131; jensen, landw. jahr. d. schweiz, 1901. [172] jensen, cent. f. bakt., 1891, 11:409. [173] jensen, milch zeit., 1892, 6, nos. 5 and 6. [174] pammel, bull. 21, iowa expt. stat., p. 803. [175] fischer, hyg. rund., 5:573. [176] storch, 18 rept. danish agric. expt. stat., 1890. [177] rogers bull. 57, b. a. i. u. s. dept agric., 1904. chapter viii. bacteria in cheese. the art of cheese-making, like all other phases of dairying, has been developed mainly as a result of empirical methods. within the last decade or so, the subject has received more attention from the scientific point of view and the underlying causes determined to some extent. since the subject has been investigated from the bacteriological point of view, much light has been thrown on the cause of many changes that were heretofore inexplicable. our knowledge, as yet, is quite meager, but enough has already been determined to indicate that the whole industry is largely based on the phenomena of ferment action, and that the application of bacteriological principles and ideas is sure to yield more than ordinary results, in explaining, in a rational way, the reasons underlying many of the processes to be observed in this industry. the problem of good milk is a vital one in any phase of dairy activity, but it is pre-eminently so in cheese-making, for the ability to make a first-class product depends to a large extent on the quality of the raw material. cheese contains so large a proportion of nitrogenous constituents that it is admirably suited, as a food medium, to the development of bacteria; much better, in fact, than butter. influence of bacteria in normal cheese processes. in the manufacture of cheddar cheese bacteria exert a marked influence in the initial stages of the process. to produce the proper texture that characterizes cheddar cheese, it is necessary to develop a certain amount of acid which acts upon the casein. this acidity is measured by the development of the lactic-acid bacteria that normally abound in the milk; or, as the cheese-maker expresses it, the milk is "ripened" to the proper point. the action of the rennet, which is added to precipitate the casein of the milk, is markedly affected by the amount of acid present, as well as the temperature. hence it is desirable to have a standard amount of acidity as well as a standard temperature for coagulation, so as to unify conditions. it frequently happens that the milk is abnormal with reference to its bacterial content, on account of the absence of the proper lactic bacteria, or the presence of forms capable of producing fermentative changes of an undesirable character. in such cases the maker attempts to overcome the effect of the unwelcome bacteria by adding a "starter;" or he must vary his method of manufacture to some extent to meet these new conditions. ~use of starters.~ a starter may be employed to hasten the ripening of milk that is extremely sweet, so as to curtail the time necessary to get the cheese to press; or it may be used to overcome the effect of abnormal conditions. the starter that is employed is generally one of domestic origin, and is usually taken from skim milk that has been allowed to ferment and sour under carefully controlled conditions. of course much depends upon the quality of the starter, and in a natural starter there is always the possibility that it may not be perfectly pure. within recent years the attempt has been made to control the effect of the starter more thoroughly by using pure cultures of some desirable lactic-acid form.[178] this has rendered the making of cheese not only more uniform, but has aided in repressing abnormal fermentations particularly those that are characterized by the production of gas. recently, pure cultures of adametz's _b. nobilis_, a digesting organism that is claimed to be the cause of the breaking down of the casein and also of the peculiar aroma of emmenthaler cheese, has been placed on the market under the name _tyrogen_. it is claimed that the use of this starter, which is added directly to the milk and also rubbed on the surface of the cheese, results in the improvement of the curds, assists in the development of the proper holes, imparts a favorable aroma and hastens ripening.[179] campbell[180] states that the discoloration of cheese in england, which is due to the formation of white spots that are produced by the bleaching of the coloring matter in the cheese, may be overcome by the use of lactic-acid starters. the use of stringy or slimy whey has been advocated in holland for some years as a means of overcoming the tendency toward gas formation in edam cheese which is made from practically sweet milk. this fermentation, the essential feature of which is produced by a culture of _streptococcus hollandicus_,[181] develops acid in a marked degree, thereby inhibiting the production of gas. the use of masses of moldy bread in directing the fermentation of roquefort cheese is another illustration of the empirical development of starters, although in this instance it is added after the curds have been prepared for the press. ~pasteurizing milk for cheese-making.~ if it were possible to use properly pasteurized milk in cheese-making, then practically all abnormal conditions could be controlled by the use of properly selected starters. numerous attempts have been made to perfect this system with reference to cheddar cheese, but so far they have been attended with imperfect success. the reason for this is that in pasteurizing milk, the soluble lime salts are precipitated by the action of heat, and under these conditions rennet extract does not curdle the casein in a normal manner. this condition can be restored, in part at least, by the addition of soluble lime salts, such as calcium chlorid; but in our experience, desirable results were not obtained where heated milks to which this calcium solution had been added were made into cheddar cheese. considerable experience has been gained in the use of heated milks in the manufacture of certain types of foreign cheese. klein[182] finds that brick cheese can be successfully made even where the milk is heated as high as 185° f. an increased weight is secured by the addition of the coagulated albumin and also increased moisture. ~bacteria in rennet.~ in the use of natural rennets, such as are frequently employed in the making of swiss cheese, considerable numbers of bacteria are added to the milk. although these rennets are preserved in salt, alcohol or boric acid, they are never free from bacteria. adametz[183] found ten different species and from 640,000 to 900,000 bacteria per cc. in natural rennets. freudenreich has shown that rennet extract solutions can be used in swiss cheese-making quite as well as natural rennets; but to secure the best results, a small quantity of pure lactic ferment must be added to simulate the conditions that prevail when natural rennets are soaked in whey, which, it must be remembered, is a fluid rich in bacterial life. where rennet extract or tablets are used, as is generally the case in cheddar making, the number of bacteria added is so infinitesimal as to be negligible. ~development of acid.~ in the manufacture of cheddar cheese, the development of acid exerts an important influence on the character of the product. this is brought about by holding the curds at temperatures favorable to the growth of the bacteria in the same. under these conditions the lactic-acid organisms, which usually predominate, develop very rapidly, producing thereby considerable quantities of acid which change materially the texture of the curds. the lactic acid acts upon the casein in solutions containing salt, causing it to dissolve to some extent, thus forming the initial compounds of digestion.[184] this solution of the casein is expressed physically by the "stringing" of the curds on a hot iron. this causes the curds to mat, producing a close, solid body, free from mechanical holes. still further, the development of this acid is necessary for the digestive activity of the pepsin in the rennet extract. in some varieties of cheese, as the swiss, acid is not developed and the character of the cheese is much different from that of cheddar. in all such varieties, a great deal more trouble is experienced from the production of "gassy" curds, because the development of the gas-producing bacteria is held in check by the rapid growth of the lactic acid-producing species. ~bacteria in green cheese.~ the conditions under which cheese is made permit of the development of bacteria throughout the entire process. the cooking or heating of curds to expel the excessive moisture is never so high as to be fatal to germ life; on the contrary, the acidity of the curd and whey is continually increased by the development of bacteria in the same. the body of green cheese fresh from the press is, to a considerable extent, dependent upon the acid produced in the curds. if the curds are put to press in a relatively sweet condition the texture is open and porous. the curd particles do not mat closely together and "mechanical holes," rough and irregular in outline, occur. very often, at relatively high temperatures, such cheese begin to "huff," soon after being taken from the press, a condition due to the development of gas, produced by gas-generating bacteria acting on the sugar in the curd. this gas finds its way readily into these ragged holes, greatly distending them, as in fig. 30. [illustration: fig. 30. _l_, a sweet curd cheese direct from the press. "mechanical" holes due to lack of acid development; _p_, same cheese four days later, mechanical holes distended by development of gas.] ~physical changes in ripening cheese.~ when a green cheese is taken from the press, the curd is tough, firm, but elastic. it has no value as a food product for immediate use, because it lacks a desirable flavor and is not readily digestible. it is nothing but precipitated casein and fat. in a short time, a deep-seated change occurs. physically this change is demonstrated in the modification that the curd undergoes. gradually it breaks down and becomes plastic, the elastic, tough curd being changed into a softened mass. this change in texture of the cheese is also accompanied by a marked change in flavor. the green cheese has no distinctively cheese flavor, but in course of time, with the gradual change of texture, the peculiar flavor incident to ripe cheese is developed. the characteristic texture and flavor are susceptible of considerable modification that is induced not only by variation in methods of manufacture, but by the conditions under which the cheese are cured. the amount of moisture incorporated with the curd materially affects the physical appearance of the cheese, and the rate of change in the same. the ripening temperature, likewise the moisture content of the surrounding air, also exerts a marked influence on the physical properties of the cheese. to some extent the action of these forces is purely physical, as in the gradual loss by drying, but in other respects they are associated with chemical transformations. ~chemical changes in ripening cheese.~ coincident with the physical breaking down of the curd comes a change in the chemical nature of the casein. the hitherto insoluble casein is gradually transformed into soluble nitrogenous substances (_caseone_ of duclaux, or _caseogluten_ of weigmann). this chemical phenomenon is a breaking-down process that is analogous to the peptonization of proteids, although in addition to the peptones and albumoses characteristic of peptic digestion, amido-acids and ammonia are to be found. the quantity of these lower products increases with the age of the cheese. the chemical reaction of cheese is normally acid to phenolphthalein, although there is generally no free acid, as shown by congo red, the lactic acid being converted into salts as fast as formed. in very old cheese, undergoing putrefactive changes, especially on the outside, an alkaline reaction may be present, due to the formation of free ammonia. the changes that occur in a ripening cheese are for the most part confined to the proteids. according to most investigators the fat remains practically unchanged, although the researches of weigmann and backe[185] show that fatty acids are formed from the fat. in the green cheese considerable milk-sugar is present, but, as a result of the fermentation that occurs, this is rapidly converted into acid products. ~bacterial flora of cheese.~ it might naturally be expected that the green cheese, fresh from the press, would contain practically the same kind of bacteria that are in the milk, but a study of cheese shows a peculiar change in the character of the flora. in the first place, fresh cottage cheese, made by the coagulation of the casein through the action of acid, has a more diversified flora than cheese made with rennet, for the reason, as given by lafar,[186] that the fermentative process is farther advanced. when different varieties of cheese are made from milk in the same locality, the germ content of even the ripened product has a marked similarity, as is illustrated by adametz's work[187] on emmenthaler or swiss hard cheese, and schweitzer hauskäse, a soft variety. of the nine species of bacilli and cocci found in mature emmenthaler, eight of them were also present in ripened hauskäse. different investigators have studied the bacterial flora of various kinds of cheese, but as yet little comparative systematic work has been done. freudenreich[188] has determined the character and number of bacteria in emmenthaler cheese, and russell[189] the same for cheddar cheese. the same general law has also been noted in canadian[190] and english[191] cheese. at first a marked decrease in numbers is usually noted, lasting for a day or two. this is followed by an enormous increase, caused by the rapid growth of the lactic-acid type. the development may reach scores of millions and often over a hundred million organisms per gram. synchronous with this increase, the peptonizing and gas-producing bacteria gradually disappear. this rapid development, which lasts only for a few weeks, is followed by a general decline. in the ripening of cheese a question arises as to whether the process goes on throughout the entire mass of cheese, or whether it is more active at or near the surface. in the case of many of the soft cheese, such as brie and limburger, bacterial and mold development is exceedingly active on the exterior, and the enzyms secreted by these organisms diffuse toward the interior. that such a condition occurs in the hard type of cheese made with rennet is extremely improbable. most observers agree that in this type of cheese the ripening progresses throughout the entire mass, although adametz opposes this view and considers that in emmenthaler cheese the development of the specific aroma-producing organism occurs in the superficial layers. jensen has shown, however, that the greatest amount of soluble nitrogenous products are to be found in the innermost part of the cheese, a condition that is not reconcilable with the view that the most active ripening is on the exterior.[192] the course of development of bacteria in cheddar cheese is materially influenced by the ripening temperature. in cheese ripened at relatively low temperatures (50°-55° f.),[193] a high germ content is maintained for a much longer period of time than at higher temperatures. under these conditions the lactic-acid type continues in the ascendancy as usual. in cheese cured at high temperatures (80°-86° f.) the number of organisms is greatly diminished, and they fail to persist in appreciable numbers for as long a time as in cheese cured at temperatures more frequently employed. ~influence of temperature on curing.~ temperature exerts a most potent influence on the quality of the cheese, as determined not only by the rate of ripening but the nature of the process itself. much of the poor quality of cheese is attributable to the effect of improper curing conditions. probably in the initial stage of this industry cheese were allowed to ripen without any sort of control, with the inevitable result that during the summer months the temperature generally fluctuated so much as to impair seriously the quality. the effect of high temperatures (70° f. and above) is to produce a rapid curing, and, therefore, a short lived cheese; also a sharp, strong flavor, and generally a more or less open texture. unless the cheese is made from the best quality of milk, it is very apt to undergo abnormal fermentations, more especially those of a gassy character. [illustration: fig. 31. influence of curing temperature on texture of cheese. upper row ripened eight months at 60° f.; lower row at 40° f.] where cheese is ripened at low temperatures, ranging from 50° f. down to nearly the freezing temperatures, it is found that the quality is greatly improved.[194] such cheese are thoroughly broken down from a physical point of view even though they may not show such a high per cent of soluble nitrogenous products. they have an excellent texture, generally solid and firm, free from all tendency to openness; and, moreover, their flavor is clean and entirely devoid of the sharp, undesirable tang that so frequently appears in old cheese. the keeping quality of such cheese is much superior to the ordinary product. the introduction of this new system of cheese-curing promises much from a practical point of view, and undoubtedly a more complete study of the subject from a scientific point of view will aid materially in unraveling some of the problems as to flavor production. ~theories of cheese curing.~ within the last few years considerable study has been given the subject of cheese curing or ripening, in order to explain how this physical and chemical transformation is brought about. much of the misconception that has arisen relative to the cause of cheese ripening comes from a confusion of terms. in the ordinary use of the word, ripening or curing of cheese is intended to signify the sum total of all the changes that result in converting the green product as it comes from the press into the edible substance that is known as cured cheese. as previously shown, the most marked chemical transformation that occurs is that which has to do with the peptonization or breaking down of the casein. it is true that under ordinary conditions this decomposition process is also accompanied with the formation of certain flavor-producing substances, more or less aromatic in character; but it by no means follows that these two processes are necessarily due to the same cause. the majority of investigators have failed to consider these two questions of casein decomposition and flavor as independent, or at least as not necessarily related. they are undoubtedly closely bound together, but it will be shown later that the problems are quite different and possibly susceptible of more thorough understanding when considered separately. in the earlier theories of cheese ripening it was thought to be purely a chemical change, but, with the growth of bacteriological science, evidence was forthcoming that seemed to indicate that the activity of organisms entered into the problem. schaffer[195] showed that if milk was boiled and made into cheese, the casein failed to break down. adametz[196] added to green cheese various disinfectants, as creolin and thymol, and found that this practically stopped the curing process. from these experiments he drew the conclusion that bacteria must be the cause of the change, because these organisms were killed; but when it is considered that such treatment would also destroy the activity of enzyms as well as vital ferments, it is evident that these experiments were quite indecisive. a determination of the nature of the by-products found in maturing cheese indicates that the general character of the ripening change is a peptonization or digestion of the casein. until recently the most widely accepted views relating to the cause of this change have been those which ascribed the transformation to the activity of micro-organisms, although concerning the nature of these organisms there has been no unanimity of opinion. the overwhelming development of bacteria in all cheeses naturally gave support to this view; and such experiments as detailed above strengthened the idea that the casein transformation could not occur where these ferment organisms were destroyed. the very nature of the changes produced in the casein signified that to take part in this process any organism must possess the property of dissolving the proteid molecule, casein, and forming therefrom by-products that are most generally found in other digestive or peptonizing changes of this class. ~digestive bacterial theory.~ the first theory propounded was that of duclaux,[197] who in 1887 advanced the idea that this change was due to that type of bacteria which is able to liquefy gelatin, peptonize milk, and cause a hydrolytic change in proteids. to this widely-spread group that he found in cheese, he gave the generic name _tyrothrix_ (cheese hairs). according to him, these organisms do not function directly as ripening agents, but they secrete an enzym or unorganized ferment to which he applies the name _casease_. this ferment acts upon the casein of milk, converting it into a soluble product known as _caseone_. these organisms are found in normal milk, and if they function as casein transformers, one would naturally expect them to be present, at least frequently, if not predominating in the ripening cheese; but such is not the case. in typical cheddar or swiss cheese, they rapidly disappear (p. 168), although in the moister, softer varieties, they persist for considerable periods of time. according to freudenreich, even where these organisms are added in large numbers to the curd, they soon perish, an observation that is not regarded as correct by the later adherents to the digestive bacterial theory, as adametz and winkler. duclaux's experiments were made with liquid media for isolation purposes, and his work, therefore, cannot be regarded as satisfactory as that carried out with more modern technical methods. recently this theory has been revived by adametz,[198] who claims to have found in emmenthaler cheese a digesting species, one of the tyrothrix type, which is capable of peptonizing the casein and at the same time producing the characteristic flavor of this class of cheese. this organism, called by him _bacillus nobilis_, the edelpilz of emmenthaler cheese, has been subjected to comparative experiments, and in the cheese made with pure cultures of this germ better results are claimed to have been secured. sufficient experiments have not as yet been reported by other investigators to warrant the acceptance of the claims made relative to the effect of this organism. ~lactic-acid bacterial theory.~ it has already been shown that the lactic-acid bacteria seems to find in the green cheese the optimum conditions of development; that they increase enormously in numbers for a short period, and then finally decline. this marked development, coincident with the breaking down of the casein, has led to the view which has been so ably expounded by freudenreich[199] that this type of bacterial action is concerned in the ripening of cheese. this group of bacteria is, under ordinary conditions, unable to liquefy gelatin, or digest milk, or, in fact, to exert, under ordinary conditions, any proteolytic or peptonizing properties. this has been the stumbling-block to the acceptance of this hypothesis, as an explanation of the breaking down of the casein. freudenreich has recently carried on experiments which he believes solve the problem. by growing cultures of these organisms in milk, to which sterile, freshly precipitated chalk had been added, he was able to prolong the development of bacteria for a considerable period of time, and as a result finds that an appreciable part of the casein is digested; but this action is so slow compared with what normally occurs in a cheese, that exception may well be taken to this type of experiment alone. weigmann[200] inclines to the view that the lactic-acid bacteria are not the true cause of the peptonizing process, but that their development prepares the soil, as it were, for those forms that are more directly concerned in the peptonizing process. this they do by developing an acid substratum that renders possible the more luxuriant growth of the aroma-producing species. according to gorini,[201] certain of the tyrothrix forms function at high temperatures as lactic acid producing bacteria, while at lower temperatures they act as peptonizers. on this basis he seeks to reconcile the discrepancies that appear in the experiments of other investigators. ~digestive milk enzym theory.~ in 1897 babcock and the writer[202] showed that milk underwent digestive changes spontaneously when bacterial activity was suspended by the addition of such anaesthetics as ether, chloroform and benzol. the chemical nature of the by-products produced by this auto-digestion of milk resembles quite closely those found in ripened cheese, except that ammonia is not produced as is the case in old cheese. the cause of the decomposition of the casein, they found to be due to the action of a milk enzym which is inherent to the milk itself. this digestive ferment may be separated from fresh milk by concentrating centrifuge slime extracts by the usual physiological reagents. this ferment, called by them _galactase_, on account of its origin in milk, is a proteolytic enzym of the tryptic type. its activity is destroyed by strong chemicals such as formaldehyde, corrosive sublimate, also when heated to 175° f. or above. when such extracts are added to boiled milk, the digestive process is started anew, and the by-products produced are very similar to those noted in a normal cheese. jensen[203] has also shown that the addition of pancreatic extracts to cheese accelerated the formation of soluble nitrogenous products. the action of galactase in milk and cheese has been confirmed by freudenreich[204] and jensen,[205] as well as by american investigators, and this enzym is now generally accepted as one of the factors concerned in the decomposition of the casein. freudenreich believes it is able to change casein into albumose and peptones, but that the lactic-acid bacteria are chiefly responsible for the further decomposition of the nitrogen to amid form. failure before to recognize the presence of galactase in milk is attributable to the fact that all attempts to secure sterile milk had been made by heating the same, in which case galactase was necessarily destroyed. a brief exposure at 176° f. is sufficient to destroy its activity, and even an exposure at lower temperatures weakens its action considerably, especially if the reaction of the medium is acid. this undoubtedly explains the contradictory results obtained in the ripening of cheese from pasteurized milk, such cheese occasionally breaking down in an abnormal manner. the results mentioned on page 172, in which cheese failed to ripen when treated with disinfectants,--experiments which were supposed at that time to be the foundation of the bacterial theory of casein digestion--are now explicable on an entirely different basis. in these cases the casein was not peptonized, because these strong disinfectants destroyed the activity of the enzyms as well as the bacteria. another important factor in the breaking down of the casein is the _pepsin_ in the rennet extract. the digestive influence of this agent was first demonstrated for cheddar cheese by babcock, russell and vivian,[206] and simultaneously, although independently, by jensen[207] in emmenthaler cheese. in this digestive action, only albumoses and higher peptones are produced. the activity of pepsin does not become manifest until there is about 0.3 per cent. acid which is approximately the amount developed in the cheddar process. these two factors undoubtedly account for by far the larger proportion of the changes in the casein; and yet, the formation of ammonia in well ripened cheese is not accounted for by these factors. this by-product is the main end product of proteid digestion by the liquefying bacteria but their apparent infrequency in cheese makes it difficult to understand how they can function prominently in the change, unless the small quantity of digestive enzyms excreted by them in their growth in milk is capable of continuing its action until a cumulative effect is obtained. although much light has been thrown on this question by the researches of the last few years, the matter is far from being satisfactorily settled at the present time and the subject needs much more critical work. if liquefying bacteria abound in the milk, doubtless they exert some action, but the rôle of bacteria is doubtless much greater in the production of flavor than in the decomposition of the curd. ~conditions determining quality.~ in determining the quality of cheese, several factors are to be taken into consideration. first and foremost is the flavor, which determines more than anything else the value of the product. this should be mild and pleasant, although with age the intensity of the same generally increases but at no time should it have any bitter, sour, or otherwise undesirable taste or aroma. texture registers more accurately the physical nature of the ripening. the cheese should not be curdy and harsh, but should yield quite readily to pressure under the thumb, becoming on manipulation waxy and plastic instead of crumbly or mealy. body refers to the openness or closeness of the curd particles, a close, compact mass being most desirable. the color of cheese should be even, not wavy, streaked or bleached. for a cheese to possess all of these characteristics in an optimum degree is to be perfect in every respect--a condition that is rarely reached. so many factors influence this condition that the problem of making a perfect cheese becomes exceedingly difficult. not only must the quality of the milk--the raw material to be used in the manufacture--be perfectly satisfactory, but the factory management while the curds are in the vat demands great skill and careful attention; and finally, the long period of curing in which variation in temperature or moisture conditions may seriously affect the quality,--all of these stages, more or less critical, must be successfully gone through, before the product reaches its highest state of development. it is of course true that many phases of this complex series of processes have no direct relation to bacteria, yet it frequently happens that the result attained is influenced at some preceding stage by the action of bacteria in one way or another. thus the influence of the acidity developed in the curds is felt throughout the whole life of the cheese, an over-development of lactic-acid bacteria producing a sour condition that leaves its impress not only on flavor but texture. an insufficient development of acid fails to soften the curd-particles so as to permit of close matting, the consequence being that the body of the cheese remains loose and open, a condition favorable to the development of gas-generating organisms. ~production of flavor.~ the importance of flavor as determining the quality of cheese makes it imperative that the nature of the substances that confer on cheese its peculiar aromatic qualities and taste be thoroughly understood. it is to be regretted that the results obtained so far are not more satisfactory, for improvement in technique is hardly to be expected until the reason for the process is thoroughly understood. the view that is most generally accepted is that this most important phase of cheese curing is dependent upon bacterial activity, but the organisms that are concerned in this process have not as yet been satisfactorily determined. in a number of cases, different species of bacteria have been separated from milk and cheese that have the power of producing aromatic compounds that resemble, in some cases, the peculiar flavors and odors that characterize some of the foreign kinds of cheese; but an introduction of these into curd has not resulted in the production of the peculiar variety, even though the methods of manufacture and curing were closely followed. the similarity in germ content in different varieties of cheese made in the same locality has perhaps a bearing on this question of flavor as related to bacteria. of the nine different species of bacteria found in emmenthaler cheese by adametz, eight of them were also present in ripened hauskäse. if specific flavors are solely the result of specific bacterial action, it might naturally be expected that the character of the flora would differ. some suggestive experiments were made by babcock and russell on the question of flavor as related to bacterial growth, by changing the nature of the environment in cheese by washing the curds on the racks with warm water. in this way the sugar and most of the ash were removed. under such conditions the character of the bacterial flora was materially modified. while the liquefying type of bacteria was very sparse in normal cheddar, they developed luxuriantly in the washed cheese. the flavor at the same time was markedly affected. the control cheddar was of good quality, while that made from the washed curds was decidedly off, and in the course of ripening became vile. it may be these two results are simply coincidences, but other data[208] bear out the view that the flavor was to some extent related to the nature of the bacteria developing in the cheese. this was strengthened materially by adding different sugars to washed curds, in which case it was found that the flavor was much improved, while the more normal lactic-acid type of bacteria again became predominant. ~ripening of moldy cheese.~ in a number of foreign cheeses, the peculiar flavor obtained is in part due to the action of various fungi which grow in the cheese, and there produce certain by-products that flavor the cheese. among the most important of these are the roquefort cheese of france, stilton of england, and gorgonzola of italy. roquefort cheese is made from goat's or cow's milk, and in order to introduce the desired mold, which is the ordinary bread-mold, _penicillium glaucum_, carefully-prepared moldy bread-crumbs are added to the curd. at ordinary temperatures this organism develops too rapidly, so that the cheese to ripen properly must be kept at a low temperature. the town of roquefort is situated in a limestone country, in a region full of caves, and it is in these natural caves that most of the ripening is done. these caverns are always very moist and have a temperature ranging from 35° to 44° f., so that the growth of the fungus is retarded considerably. the spread of the mold throughout the ripening mass is also assisted in a mechanical way. the partially-matured cheese are run through a machine that pricks them full of small holes. these slender canals allow the mold organism to penetrate the whole mass more thoroughly, the moldy straw matting upon which the ripening cheese are placed helping to furnish an abundant seeding of the desired germ. when new factories are constructed it is of advantage to introduce this necessary germ in quantities, and the practice is sometimes followed of rubbing the walls and cellars of the new location with material taken from the old established factory. in this custom, developed in purely an empirical manner, is to be seen a striking illustration of a bacteriological process crudely carried out. in the stilton cheese, one of the highly prized moldy cheeses of england, the desired mold fungus is introduced into the green cheese by exchanging plugs taken with a cheese trier from a ripe stilton. ~ripening of soft cheese.~ the type of ripening which takes place in the soft cheeses is materially different from that which occurs in the hard type. the peptonizing action does not go on uniformly throughout the cheese, but is hastened by the development of molds and bacteria on the outside that exert a solvent action on the casein. for this reason, soft cheeses are usually made up in small sizes, so that this action may be hastened. the organisms that take part in this process are those that are able to form enzyms (similar in their action to trypsin, galactase, etc.), and these soluble ferments gradually diffuse from the outside through the cheese. most of these peptonizing bacteria are hindered in their growth by the presence of lactic acid, so that in many cases the appearance of the digesting organisms on the surface is delayed until the acidity of the mass is reduced to the proper point by the development of other organisms, principally molds, which prefer an acid substratum for their growth. in brie cheese a blue coating of mold develops on the surface. in the course of a few weeks, a white felting appears which later changes to red. this slimy coat below the mold layer is made up of diverse species of bacteria and fungi that are able to grow after the acid is reduced by the blue mold. the organisms in the red slimy coat act upon the casein, producing an alkaline reaction that is unfavorable to the growth of the blue mold. two sets of organisms are, therefore essential in the ripening process, one preparing the soil for the ferment that later produces the requisite ripening changes. as ordinarily carried on, the process is an empirical one, and if the red coat does not develop as expected, the maker resorts to all kinds of devices to bring out the desired ferment. the appearance of the right form is dependent, however, upon the proper reaction of the cheese, and if this is not suitable, the wished-for growth will not appear. influence of bacteria in abnormal cheese processes. the reason why cheese is more subject to abnormal fermentation than butter is because its high nitrogen content favors the continued development of bacteria for some time after it is made. it must be borne in mind, in considering the more important of these changes, that not all defective conditions in cheese are attributable to the influence of living organisms. troubles frequently arise from errors in manufacturing details, as too prolonged cooking of curds, too high heating, or the development of insufficient or too much acid. then again, the production of undesirable flavors or impairment in texture may arise from imperfect curing conditions. our knowledge regarding the exact nature of these indefinite faults is as yet too inadequate to enable many of these undesirable conditions to be traced to their proper source; but in many cases the taints observed in a factory are due to the abnormal development of certain bacteria, capable of evolving unpleasant or even putrid odors. most of them are seeded in the milk before it comes to the factory and are due to careless manipulation of the milk while it is still on the farm. others gain access to the milk in the factory, owing to unclean conditions of one sort or another. sometimes the cheese-maker is able to overcome these taints by vigorous treatment, but often they pass on into the cheese, only to detract from the market value of the product. most frequently these "off" flavors appear in cheese that are cured at too high temperatures, say above 65° f. ~"gassy" fermentations in cheese.~ one of the worst and at the same time most common troubles in cheese-making is where the cheese undergoes a fermentation marked by the evolution of gas. the presence of gas is recognized by the appearance either of spherical or lens-shaped holes of various sizes in the green cheese; often they appear in the curd before it is put to press. usually in this condition the curds look as if they had been punctured with a pin, and are known as "pin holey" curds. where the gas holes are larger, they are known as "swiss holes" from their resemblance to the normal holes in the swiss product. if the development of gas is abundant, these holes are restricted in size. often the formation of gas may be so intense as to cause the curds to float on the surface of the whey before they are removed. such curds are known as "floaters" or "bloaters." if "gassy" curds are put to press, the abnormal fermentation may continue. the further production of gas causes the green cheese to "huff" or swell, until it may be considerably distorted as in fig. 33. in such cases the texture of the cheese is greatly injured, and the flavor is generally impaired. [illustration: fig. 33. cheese made from gassy milk.] such abnormal changes may occur at any season of the year, but the trouble is most common in summer, especially in the latter part. this defect is less likely to occur in cheese that is well cheddared than in sweet curd cheese. when acidity is produced, these gassy fermentations are checked, and in good cheddar the body is so close and firm as not readily to permit of gaseous changes. in swiss cheese, which is essentially a sweet curd cheese, these fermentations are very troublesome. where large holes are formed in abundance (blähen), the trouble reaches its maximum. if the gas holes are very numerous and therefore small it is called a "nissler." sometimes the normal "eyes" are even wanting when it is said to be "blind" or a "gläsler." [illustration: fig. 34. block swiss cheese showing "gassy" fermentation.] one method of procedure which is likely to cause trouble in swiss factories is often produced by the use of sour, fermented whey in which to soak the natural rennets. freudenreich and steinegger[209] have shown that a much more uniform quality of cheese can be made with rennet extract if it is prepared with a starter made from a pure lactic ferment. the cause of the difficulty has long been charged to various sources, such as a lack of aeration, improper feeding, retention of animal gases, etc., but in all these cases it was nothing more than a surmise. very often the milk does not betray any visible symptom of fermentation when received, and the trouble is not to be recognized until the process of cheese-making is well advanced. studies from a biological standpoint have, however, thrown much light on this troublesome problem; and it is now known that the formation of gas, either in the curd or after it has been put to press, is due entirely to the breaking down of certain elements, such as the sugar of milk, due to the influence of various living germs. this trouble is, then, a type fermentation, and is, therefore, much more widely distributed than it would be if it was caused by a single specific organism. these gas-producing organisms are to be found, sparingly at least, in almost all milks, but are normally held in check by the ordinary lactic species. among them are a large number of the bacteria, although yeasts and allied germs are often present and are likewise able to set up fermentative changes of this sort. in these cases the milk-sugar is decomposed in such a way as to give off co_{2} and h, and in some cases, alcohol. russell and hastings[210] found a lactose-splitting yeast in a severe outbreak of gassy cheese in a swiss factory. in this case the gas did not develop until the cheese were a few weeks old. in severe cases the cheese actually cracked to pieces. according to guillebeau, a close relation exists between those germs that are able to produce an infectious inflammation (mastitis) in the udder of the cow and some forms capable of gas evolution. if pure cultures of these gas-producing bacteria are added to perfectly sweet milk, it is possible to artificially produce the conditions in cheese that so frequently appear in practice. ~treatment of "pin-holey" curds.~ when this type of fermentation appears during the manufacture of the cheese, the maker can control it in part within certain limits. these methods of treatment are, as a rule, purely mechanical, as when the curds are piled and turned, and subsequently ground in a curd mill. after the gas has been forced out, the curds are then put to press and the whole mats into a compact mass. another method of treatment based upon bacteriological principles is the addition of a starter to induce the formation of acid. where acid is developed as a result of the growth of the lactic-acid bacteria, the gas-producing species do not readily thrive. another reason why acid aids in repressing the development of gas is that the curd particles are partially softened or digested by the action of the acid. this causes them to mat together more closely, and there is not left in the cheese the irregular mechanical openings in which the developing gas may find lodgment. another method that is also useful with these curds is to employ salt. this represses gaseous fermentations, and the use of more salt than usual in making the cheese will very often restrain the production of gas. tendency to form gas in edam cheese is controlled by the addition of a starter prepared from slimy whey (lange wei) which is caused by the development of an acid-forming organism. some have recommended the custom of washing the curds to remove the whey and the gas-producing bacteria contained therein. care must be taken not to carry this too far, for the removal of the sugar permits taint-producing organisms to thrive.[211] the temperature at which the cheese is cured also materially affects the development of gas. at high curing temperatures, gas-producing organisms develop rapidly; therefore more trouble is experienced in summer than at other seasons. if milks which are prone to undergo "gassy" development are excluded from the general supply, it would be possible to eliminate the source of the entire trouble. to aid in the early recognition of such milks that are not apparently affected when brought to the factory, fermentation or curd tests (p. 76) are of great value. the use of this test in the hands of the factory operator often enables him to detect the exact source of the trouble, which may frequently be confined to the milk delivered by a single patron. ~"fruity" or "sweet" flavor.~ not infrequently the product of a factory may acquire during the process of ripening what is known as a "sweet" or "fruity" flavor. this flavor resembles the odor of fermented fruit or the bouquet of certain kinds of wine. it has been noted in widely different sections of the country and its presence bears no relation to the other qualities of the cheese. the cause of this trouble has recently been traced[212] to the presence of various kinds of yeasts. ordinarily yeasts are rarely present in good cheese, but in cheese affected with this trouble they abound. the addition of starters made from yeast cultures resulted in the production of the undesirable condition. ~mottled cheese.~ the color of cheese is sometimes cut to that extent that the cheese presents a wavy or mottled appearance. this condition is apt to appear if the ripening temperature is somewhat high, or larger quantities of rennet used than usual. the cause of the defect is obscure, but it has been demonstrated that the same is communicable if a starter is made by grating some of this mottled cheese into milk. the bacteriology of the trouble has not yet been worked out, but the defect is undoubtedly due to an organism that is able to grow in the ripening cheese. it has been claimed that the use of a pure lactic ferment as a starter enables one to overcome this defect. ~bitter cheese.~ bitter flavors are sometimes developed in cheese especially where the ripening process is carried on at a low temperature in the presence of an excess of moisture for a considerable length of time. guillebeau[213] isolated several forms from emmenthaler cheese which he connected with udder inflammation that were able to produce a bitter substance in cheese. von freudenreich[214] has described a new form _micrococcus casei amari_ (micrococcus of bitter cheese) that was found in a sample of bitter cheese. this germ is closely related to conn's micrococcus of bitter milk. it develops lactic acid rapidly, coagulating the milk and producing an intensely bitter taste in the course of one to three days. when milk infected with this organism is made into cheese, there is formed in a few days a decomposition product that imparts a marked bitter flavor to the cheese. harrison[215] has recently found a yeast that grows in the milk and also in the cheese which produces an undesirable bitter change. it is peculiar that some of the organisms that are able to produce bitter products in milk do not retain this property when the milk is worked up into cheese. ~putrid or rotten cheese.~ sometimes cheese undergoes a putrefactive decomposition in which the texture is profoundly modified and various foul smelling gases are evolved. these often begin on the exterior as small circumscribed spots that slowly extend into the cheese, changing the casein into a soft slimy mass. then, again, the interior of the cheese undergoes this slimy decomposition. the soft varieties are more prone toward this fermentation than the hard, although the firm cheeses are by no means exempt from the trouble. the "verlaufen" or "running" of limburger cheese is a fermentation allied to this. it is where the inside of the cheese breaks down into a soft semi-fluid mass. in severe cases, the rind may even be ruptured, in which case the whole interior of the cheese flows out as a thick slimy mass, having sometimes a putrid odor. the conditions favoring this putrid decomposition are usually associated with an excess of moisture, and an abnormally low ripening temperature. ~rusty spot.~ this name is applied to the development of small yellowish-red or orange spots that are formed sometimes throughout the whole mass of cheddar cheese. a close inspection shows the colored points to be located along the edges of the curd particles. according to harding,[216] this trouble is most common in spring and fall. the cause of the difficulty has been traced by connell[217] to the development of a chromogenic bacterium, _bacillus rudensis_. the organism can be most readily isolated on a potato surface rather than with the usual isolating media, agar or gelatin. ~other pigment changes.~ occasionally, with the hard type of cheese, but more frequently with the softer foreign varieties, various abnormal conditions arise that are marked by the production of different pigments in or on the cheese. more frequently these are merely superficial and affect only the outer layers of the cheese. generally they are attributable to the development of certain chromogenic organisms (bacteria, molds and yeasts), although occasionally due to other causes, as in the case of a blue discoloration sometimes noted in foreign cheese made in copper kettles.[218] de vries[219] has described a blue condition that is found in edam cheese. it appears first as a small blue spot on the inside, increasing rapidly in size until the whole mass is affected. this defect he was able to show was produced by a pigment-forming organism, _b. cyaneo-fuscus_. by the use of slimy whey (lange wei) this abnormal change was controlled. ~moldy cheese.~ with many varieties of cheese, especially some of the foreign types, the presence of mold on the exterior is not regarded as detrimental; in fact a limited development is much desired. in hard rennet cheese as cheddar or swiss, the market demands a product free from mold, although it should be said that this condition is imposed by the desire to secure a good-looking cheese rather than any injury in flavor that the mold causes. mold spores are so widely distributed that, if proper temperature and moisture conditions prevail, these spores will always develop. at temperatures in the neighborhood of 40° f. and below, mold growth is exceedingly slow, and often fructification does not occur, the only evidence of the mold being the white, felt-like covering that is made up of the vegetating filaments. the use of paraffin has been suggested as a means of overcoming this growth, the cheese being dipped at an early stage into melted paraffin. recent experiments have shown that "off" flavors sometimes develop where cheese are paraffined directly from the press. if paraffin is too hard, it has a tendency to crack and separate from the rind, thus allowing molds to develop beneath the paraffin coat, where the conditions are ideal as to moisture, for evaporation is excluded and the air consequently saturated. the use of formalin (2% solution) has been suggested as a wash for the outside of the cheese. this substance or sulfur is also applied in a gaseous form. double bandaging is also resorted to as a means of making the cheese more presentable through the removal of the outer bandage. the nature of these molds has not been thoroughly studied as yet. the ordinary blue-green bread mold, _penicillium glaucum_, is most frequently found, but there are numerous other forms that appear, especially at low temperatures. ~poisonous cheese.~ cases of acute poisoning arising from the ingestion of cheese are reported from time to time. vaughan has succeeded in showing that this condition is due to the formation of a highly poisonous alkaloid which he has isolated, and which he calls _tyrotoxicon_.[220] this poisonous ptomaine has also been demonstrated in milk and other milk products, and is undoubtedly due to the development of various putrefactive bacteria that find their way into the milk. it seems quite probable that the development of these toxic organisms can also go on in the cheese after it is taken from the press. ~prevention or cheese defects.~ the defective conditions previously referred to can rarely be overcome in cheese so as to improve the affected product, for they only become manifest in most cases during the later stages of the curing process. the only remedy against future loss is to recognize the conditions that are apt to prevail during the occurrence of an outbreak and see that the cheese are handled in such a way as to prevent a recurrence of the difficulty. many abnormal and undesirable results are incident to the manufacture of the product, such as "sour" or "mealy" cheese, conditions due to the development of too much acid in the milk or too high a "cook." these are under the direct control of the maker and for them he alone is responsible. the development of taints due to the growth of unwelcome bacteria that have gained access to the milk while it is yet on the farm are generally beyond the control of the cheese maker, unless they are so pronounced as to appear during the handling of the curds. if this does occur he is sometimes able, through the intervention of a starter or by varying some detail in making, to handle the milk in such a way as to minimize the trouble, but rarely is he able to eliminate it entirely. one of the most strenuous duties which the maker must perform at all times is to point out to his patrons the absolute necessity of their handling the milk in such a way as to prevent the introduction of organisms of a baleful type. footnotes: [178] russell, 13 rept. wis. expt. stat., 1896, p. 112; campbell, trans. high. & agr. soc. scotland, 5 ser., 1898, 10:181. [179] winkler, milch zeit. (hildesheim), nov. 24, 1900. [180] campbell, no. brit., agric., may 12, 1897. [181] weigmann, milch zeit., no. 50, 1889. [182] klein, milch zeit. (hildesheim), no. 17, 1900. [183] adametz, landw. jahr., 18:256. [184] van slyke and hart, bull. 214, n. y. expt. stat., july 1902. [185] milch zeit., 1898, no. 49. [186] lafar, technical mycology, p. 216. [187] adametz, landw. jahr., 18:228. [188] freudenreich, landw. jahr. d. schweiz, 4:17; 5:16. [189] russell, 13 rept. wis. expt. stat., 1896, p. 95. [190] harrison and connell, rev. gen. du lait, nos. 4, 5, 6, 7 and 8, 1903-04. [191] lloyd, bath and west of eng. soc. rept., 1892, 2:180. [192] freudenreich, landw. jahr. d. schweiz, 1900; adametz, oest. molk. zeit., 1899, no. 7. [193] russell, 14 wis. expt. stat., 1897, p. 203. harrison and connell, rev. gen. du lait nos. 4, etc., 1903-04. [194] babcock and russell, 18 rept. wis. expt. stat., 1901. dean, harrison and harcourt, bull. 121, ont. agr'l. coll., june 1902. [195] schaffer, milch zeit., 1889, p. 146. [196] adametz, landw. jahr., 18:261. [197] duclaux, le lait, p. 213. [198] adametz, oest. molk. zeit., 1900, nos. 16-18. [199] freudenreich, landw. jahr. d. schweiz, 1897, p. 85. [200] weigmann, cent. f. bakt., ii abt., 1898, 4:593; also 1899, 5:630. [201] gorini, abs. in expt. stat. rec., 11:388. [202] babcock and russell, 14 rept. wis. expt. stat., 1897, p. 161. [203] jensen, cent. f. bakt., ii abt., 3:752. [204] freudenreich, cent. f. bakt., ii abt., 1900, 6:332. [205] jensen, ibid., 1900, 6:734. [206] 17 rept. wis. expt. stat., 1900, p. 102. [207] jensen, landw. jahr. d. schweiz, 1900. [208] babcock and russell, 18 rept. wis. expt. stat., 1901. [209] cent. f. bakt. 1899, p. 14. [210] bull. 128, wis. expt. stat., sept. 1905. [211] babcock and russell, 18 rept. wis. expt. stat., 1901. [212] harding, rogers and smith, bull. 183, n. y. (geneva) expt. stat., dec., 1900. [213] guillebeau, landw. jahr., 1890, p. 27. [214] freudenreich, füehl. landw. ztg., 43:361. [215] harrison, bull. 123 ont. agr'l. coll., may, 1902. [216] bull. 183, n. y. (geneva) expt. stat., dec. 1900. [217] connell, bull. canadian dept. of agr., 1897. [218] schmöger, milch zeit., 1883, p. 483. [219] de vries, milch zeit., 1888, pp. 861, 885. [220] zeit. f. physiol. chemie, 10:146. index. acid, effect of, on churning, 137; in butter-making, 138. acid test, 52. aeration of milk, 59. aerobic bacteria, 7. alcoholic fermentation in milk, 72. anaerobic bacteria, 7. animal, influence of, on milk infection, 34. animal odor, 56. anthrax, 94. antiseptics, 9, 88. aroma, of butter, 140. bacillus: definition of, 2. _acidi lactici_, 64; _cyaneo-fuscus_, 188; _cyanogenus_, 74; _foetidus lactis_, 157; _lactis aerogenes_, 65; _lactis erythrogenes_, 74; _lactis saponacei_, 67; _lactis viscosus_, 71; _nobilis_, 162, 174; _prodigiosus_, 74; _rudensis_, 188; _synxanthus_, 75; _tuberculosis_, 84. bacteria: on hairs, 35; kinds in milk, 63; in barn air, 42; in milk pails, 27; in butter, 154; classification of, 4; in cheese, 160; culture of, 17; in cream, 128; discovery of, 1; external conditions affecting, 8; form of, 2; in butter, 142; in butter-making, 127; in centrifuge slime, 39; in fore milk, 28; in rennet, 163; in separator slime, 39; manure, 37; number of, in milk, 50. distribution of: milk of american cities, 50; european cities, 50; in relation to cheese, 168. of disease: anthrax, 94; cholera, 98; diphtheria, 99; lockjaw, 94; toxic, 100; tuberculosis, 84; typhoid fever, 98. methods of study of: culture, 15; culture media, 13; isolation, 14. bitter butter, 158; cheese, 189; milk, 72. bloody milk, 74. blue cheese, 191; milk, 74. bovine tuberculosis, 84. brie cheese, 182. butter: bacteria in, 154; bitter, 158; "cowy," 157; fishy, 159; lardy, 157; moldy, 158; mottled, 156; oily, 158; putrid, 156; rancid, 155; tallowy, 157; turnip flavor in, 157. making: aroma, 140; flavor in, 140; pure culture, 143; in ripening of cream, 136. butyric acid fermentation, 69. by-products of factory, methods of preserving, 25. casease, 68. caseone, 68. centrifugal force, cleaning milk by, 38. cheese: bacterial flora of, 168; bitter, 189; blue, 187; brie, 182; edam, 72, 162; emmenthaler, 185; flavor of, 179; gassy fermentations in, 183; gorgonzola, 180; molds on, 191; mottled, 189; "nissler," 185; poisonous, 192; putrid, 190; ripening of moldy, 180; ripening of soft, 181; roquefort, 180; rusty spot in, 188; stilton, 180; swiss, 185. making and curing: chemical changes in curing, 166; influence of temperature on curing, 169; influence of rennet, 177; physical changes in curing, 165; prevention of defects, 193; starters in, 161; temperature in relation to bacterial influence, 169. theories of curing: digestive, 173; galactase, 175, 177; lactic acid, 174. chemical changes in cheese-ripening, 166. chemical disinfectants in milk: bleaching powder, 81; corrosive sublimate, 81; formalin, 80; sulfur, 80; whitewash, 81; vitriol, 81. chemical preservatives, 80. children, milk for, 45. cholera in milk, 98. classification by separator, 38. coccus, definition of, 2. cold, influence on bacteria, 8, 48. contamination of milk through disease germs, 95, 191. covered milk pails, 41. cream, bacterial changes in, 135; mechanical causes for bacteria in, 135; pasteurized, 113; restoration of consistency of pasteurized, 132. ripening of, 136; advantage of pure cultures in, 144; by natural starters, 142; characteristics of pure cultures in, 145; objections to pure cultures in, 146; principles of pure cultures in, 143; propagation of pure cultures, 151; purity of commercial starters, 150; home-made starters in, 146. creaming methods, 134. curd test, 76. dairy utensils a source of contamination, 21. diarrhoeal diseases, 100. digesting bacteria, 67. digestibility of heated milk, 111. diphtheria, 99. dirt in milk, 34. dirt, exclusion of, 36. disease germs in milk, 95; effect of heat on, 91; origin of, 83. disinfectants, 9: carbolic acid, 81; chloride of lime, 81; corrosive sublimate, 81; formalin, 80; sulfur, 80; vitriol salts, 81; whitewash, 79. disinfectants in milk: alkaline salts, 106; boracic acid, 106; formalin, 106; preservaline, 107; salicylic acid, 106. domestic pasteurizing apparatus, 119. drugs, taints in milk due to, 56. drying, effect of, 8. edam cheese, 72, 162. emmenthaler cheese, 185. endospores, 3. enzyms, 10. factory by-products, 22; treatment of, 25. farrington alkaline tablet, 52. fecal bacteria, effect of, on butter, 35. fermentation: in cheese: gassy, 183. in milk: alcoholic, 72; bitter, 72; blue, 74; butyric, 69; digesting, 67; gassy, 66; kephir, 72; koumiss, 72; lactic acid, 63; lange-wei, 72; red, 74; ropy, 69; slimy, 69; soapy, 73; souring, 63; sweet curdling, 67; treatment of, 75. tests, 76; gerber's, 76; walther's, 76; wisconsin curd, 76. filtration of milk, 38. fishy butter, 159. flavor: of butter, 140; of cheese, 179. foot and mouth disease, 93. fore milk, 28. formaldehyde, 80. formalin, 80. fruity flavor in cheese, 188. galactase in cheese, 175. gassy fermentations: in cheese, 183; in milk, 67; in swiss cheese, 167. gläsler, 185. gorgonzola cheese, 180. growth of bacteria, essential conditions for, 4; in milk, 46. hair, bacteria on, 35. heat, influence on bacterial growth, 8. heated milk: characteristics of, 109; action toward rennet, 112; body, 110; digestibility, 111; fermentative changes, 111; flavor, 110; hydrogen peroxid test in, 23; storch's test, 23. hygienic milk, bacteria in, 45. infection of milk: animal, 34; dairy utensils, 21; fore milk, 28; milker, 36. isolation of bacteria, methods of, 14. kephir, 72. koumiss, 72. lactic acid: fermentation in milk, 63; theory in cheese-curing, 174. lange-wei, 72. lardy butter, 157. light, action on bacteria, 9. manure, bacteria in, 33. methods: of isolation, 14; culture, 15. _micrococcus casei amari_, 189. microscope, use of, 17. milk: a bacterial food medium, 19; bacteria in, 48. disease organisms in: anthrax, 94; cholera, 98; diphtheria, 99; foot and mouth disease, 93; poisonous, 101; ptomaines, 101; scarlet fever, 99; tuberculosis, 84; typhoid fever, 98. contamination, 20: from air, 42; from animal odors, 55; dirt, 34; distinction between bacterial and non-bacterial, 57; fore milk, 28; infection in factory, 59; milker, 36; relative importance of various kinds, 43; utensils, 21. milk fermentations: alcoholic, 72; bitter, 72; bloody, 74; blue, 74; butyric acid, 69; gassy, 66, 167; kephir, 72; koumiss, 72; lactic acid, 63; red, 72; ropy, 69; slimy, 69; soapy, 74; souring, 63; sweet curdling, 67; tests for, 76; treatment of, 75; yellow, 75. milk, heated: action towards rennet, 112; digestibility, 111; flavor of, 110; fermentative changes in, 111; hydrogen peroxid test, 110. milking machines, influence of, on germ content, 37. milk preservation: chemical agents in, 106; condensation, 107; freezing, 108; heat, 108; pasteurization, 113; sterilization, 112. milk-sugar as bacterial food, 19. mold, in butter, 158; in cheese, 191. mottled cheese, 189. "nissler" cheese, 185. odors, direct absorption of, in milk, 55. _oidium lactis_, 159. oily butter, 158. pasteurization of milk; acid test in, 128; bacteriological study of, 124, 126, 149; for butter, 147; for cheese, 162; for direct use, 113; of skim milk, 25; details of, 128; temperature and time limit in, 118. pasteurizing apparatus: continuous flow, 122; coolers, 131; danish, 123; domestic, 119; farrington, 122; intermittant flow, 121; miller, 122; potts, 121; regenerator, 122; reid, 126; russell, 121; testing rate of flow, 124. _penicillium glaucum_, 159, 180, 190. pepsin, 10. physical changes in cheese-ripening, 165. poisonous bacteria: in cheese, 192; in milk, 100, 101. preservaline, 167. preservation of milk: by exclusion, 103; chemical agents, 106; condensing, 107; filtration, 38; freezing, 108; pasteurization, 112; physical agents, 107; sterilization, 112. ptomaine poisoning, 101. pure cultures, 15. pure culture starters: advantages of, 144; characteristics of, 145; home-made cultures compared with, 146; propagation of, 151. putrid cheese, 190; butter, 156. rancidity in butter, 155. red milk, 74. rennet: action in heated milk, 112; bacteria in, 163; influence of, on cheese-ripening, 177. restoration of consistency in pasteurized cream, 132. ripening of cheese: moldy cheese, 180; soft cheese, 181. of cream, 136; artificial starters, 143; natural starters, 142; principles of pure culture starters in, 143. ropy milk, 69. roquefort cheese, 180. rusty spot in cheese, 190. rusty cans: effect of, on acidity, 53. sanitary milk, 45, 104. sanitary pails, 41. scarlet fever in milk, 99. separator slime: bacteria in, 39; tubercle bacillus in, 93. scalded layer, resistance of bacteria in, 91. skim-milk, a distributor of disease, 96. slimy milk, 69. soapy milk, 74. soft cheese, ripening of, 186. sources of contamination in milk: barn air, 42; dairy utensils, 21; dirt from animals, 34; factory cans, 25; fore-milk, 28; milker, 36. souring of milk, 63. spirillum, definition of, 2. spores, 3. starters: in cheese-making, 161; in butter-making, 142; propagation of, 151; pure cultures in cream-ripening, 143. sterilization of milk, 112. _streptococcus hollandicus_, 72, 162. stilton cheese, 181. storch's test, 23. sulfur as a disinfectant, 81. sweet curdling milk, 68. sweet flavor in cheese, 188. swiss cheese, 177; gassy fermentations in, 24, 185. taints, absorption of, 55. taints, bacterial vs. physical, 58. taints in milk, absorption of, 55. taints, use of starters in overcoming, 79. taints in butter: putrid, 156; rancidity, 155; turnip flavor, 157. tallowy butter, 157. temperature: effect on bacterial development, 6, 48; effect of low, 108; effect of high, 108; and time limit in milk pasteurization, 113. tests for milk: fermentation, 76; storch's, 23; acid, 52. theories in cheese-curing: digestive, 171; galactase, 175, 177; lactic acid, 174. trypsin, 10. tubercle bacillus: in milk, 88; in separator slime, 93; thermal death limits, 117. tuberculin test, 86. tuberculosis, bovine, 84. turnip flavor in butter, 157. typhoid fever, 98. tyrogen, 162. tyrotoxicon, 101, 190. udder: artificial introduction of bacteria into, 32; milk germ-free in, 19; infection of, 28; washing, 89; tuberculosis in, 87. viscogen, 132. water: as a source of infection, 61. whey, pollution of vats, 23; method of preserving, 25; treatment of, in vats, 25. whitewash, 81. wisconsin curd test, 76. yeasts: alcoholic ferments in milk, 73; fruity flavor in cheese, 186; gassy due to yeasts, 186; in bitter cheese, 189; in canned butter, 159; kephir, 72. outlines of dairy bacteriology a concise manual for the use of students in dairying by h. l. russell dean of the college of agriculture university of wisconsin and e. g. hastings professor of agricultural bacteriology university of wisconsin _tenth edition_ madison, wisconsin h. l. russell 1914 copyright 1914 by h. l. russell and e. g. hastings preface to the tenth edition. this text was originally the outgrowth of a series of lectures on the subject of dairy bacteriology to practical students in the winter dairy course in the university of wisconsin. the importance of bacteriology in dairy processes has now come to be so widely recognized that no student of dairying regards his training as complete until he has had the fundamental principles of this subject. the aim of this volume is not to furnish an exhaustive treatise of the subject, but an outline and sufficient detail to enable the general student of dairying to obtain as comprehensive an idea of the bacteria and their effects on milk and other dairy products as may be possible without the aid of laboratory practice. when possible the dairy student is urged to secure a laboratory knowledge of these organisms, but lacking this, the student and general reader should secure a general survey of the field of bacteriology in relation to dairying. in this, the tenth edition, the effort has been made to include all of the recent developments of the subject. especially is this true in regard to the subject of market milk, a phase of dairying that has gained greatly in importance in the last few years. the changes in the methods of handling market milk have been marked. the results of these changes in influencing the quality of milk offered to the consumer are fully discussed. h. l. r. e. g. h. contents structure, growth and distribution of bacteria 7 methods of studying bacteria 20 contamination of milk 28 infection of milk with pathogenic bacteria 62 fermentations of milk 82 preservation of milk 113 bacteria and butter making 136 bacteria and cheese making 161 bacteria in market milk 189 chapter i. structure, growth and distribution. =relation of bacteriology to dairying.= the arts which have been developed by mankind have been the outgrowth of experience. man first learned by doing, _how_ to perform these various activities, and a scientific knowledge of the underlying principles which govern these processes was later developed. the art of dairying has been practiced from time immemorial, but a correct understanding of the fundamental principles on which the practice of dairying rests is of recent origin. in working out these principles, chemistry has been of great service, but in later years, bacteriology has also been most successfully applied to the problems of modern dairying. indeed, it may be said that the science of dairying, as related to the problems of dairy manufacture is, in large degree, dependent upon an understanding of bacteriological principles. it is therefore essential that the student of dairying, even though he is concerned in large measure with the practical aspects of the subject, should acquire as complete an understanding of these principles as possible. while bacteriology is concerned primarily with the activities of those microscopic forms of plant life known as the bacteria, yet the general principles governing the life of this particular class of organisms are sufficiently similar to those governing the molds and other types of microscopic life that affect milk and its products to make it possible to include all of these types in a general consideration of the subject. =nature of bacteria.= the vegetable kingdom to which the bacteria belong consists of plants of the most varying size and nature. those of most common acquaintance are the green plants varying in size from those not visible to the naked eye to the largest trees. another class of plants known as fungi or fungous plants do not contain chlorophyll, the green coloring matter, but are usually colorless and, as a rule, of small size; among them are included such forms as the mushrooms, smuts, rusts and mildews, as well as the molds and yeasts. the bacteria are closely allied to this latter class. when first discovered they were thought to be animals because of the ability of some forms to move about in liquids. the bacteria, like other kinds of living organisms, possess a definite form and shape. they are the simplest in structure of all the plants, the individual organism consisting of a single cell. the larger and more highly organized forms of life are made up of many microscopic cells, and the life of the individual consists of the work of all the cells. the bacteria are very comparable to the single cells of the higher plants and animals, but in the case of the bacteria the single cell is able to exist apart from all other cells and to carry out all of its life processes including reproduction. =forms of bacteria.= with the multicellular organisms much variation in form is possible, but with these single-celled organisms the possible variation in form is greatly limited. three well marked types occur among the bacteria: the round or coccus form (plural cocci); the rod-shaped or bacillus (plural bacilli); and the twisted or spirillum type (plural spirilla). most organisms of special significance in dairying belong to the coccus or bacillus group. =size of bacteria.= the bacteria, as a class, are among the smallest of living objects. none of them are individually visible to the naked eye, and they can be so seen only when clumps or masses are formed in the process of growth. [illustration: fig. 1.--forms of bacteria. a, coccus; b, bacillus; c, spirillum.] while there is considerable relative variation in size, yet in actual dimensions, this difference is so small as to make careful microscopic determinations necessary. an average diameter may be taken as about one thirty-thousandth of an inch, while the length varies naturally several fold, depending upon whether the type under observation is a coccus or a bacillus. it is very difficult to conceive of the minuteness of the bacteria; the following may give some idea of their size. in a drop of cream ready for churning may be found as many as 10,000,000 and in a piece of fresh cheese as large as a cherry there may be as many living bacteria as there are people on our earth. while the bacteria are very minute, the effect which they exert in milk and other dairy products is great on account of their enormous numbers. =manner of growth.= the cells of which all plants and animals consist increase in numbers by the division of each cell into two cells through the formation of a division wall across the cell. the new cells divide and the plant or animal continues to grow. the same cell division occurs in the bacteria but since the bacteria are single celled, division of the cells means an increase in numbers rather than growth as in the higher forms of life. [illustration: fig. 2.--division of bacteria. the bacteria increase in numbers by the division of each cell into two cells. (after novy.)] in the case of those bacteria that have a greater length than diameter, the new wall is formed at right angles to the long axis of the cell. as soon as the division is complete each cell is a complete individual, capable of carrying on all of its life processes. the cells may, however, cohere and thus form distinctive groupings that may serve to identify certain types. some of the cocci form long chains and the term _streptococcus_ is applied to such. other groupings may be similar to a bale of twine or they may be massed in clusters with no regularity distinguishable. =spores.= just as ordinary plants form resistant structures, known as seeds, capable of retaining vitality under conditions unfavorable for growth thereby perpetuating the species, so with certain of the bacteria, definite structures, known as _spores_, that are analogous in some respects to the seeds of the higher plants, are produced within the mother cell. the spores are exceedingly resistant to the influence of an unfavorable environment, such as heat, cold, drying, and even chemical agents. it is this property of the spores which makes it so difficult to destroy the bacterial life in the process of sterilizing milk. the property of spore-formation is fortunately confined to a comparatively small number of different species of bacilli. =movement.= many of the bacteria are provided with vibratory organs of locomotion, known as _cilia_ (singular cilium) which are variously distributed on the surface of the cell. by the movement of these relatively long, thread-like appendages the individual cell is able to move in liquids. it must be remembered, when these moving cells are observed under the microscope, that their apparent rate of movement is magnified relatively as much as their size. =conditions for growth.= all kinds of living things need certain conditions for growth such as food, moisture, air and a favorable temperature. the bacteria prefer as food such organic matter as milk, meat, and vegetable infusions. those living on dead organic matter are known as _saprophytes_, while those which are capable of thriving in the tissues of the living plant or animal are known as _parasites_. certain of the parasitic forms are capable of causing disease in plants and animals. in the first group are embraced most of the bacteria that are able to develop in milk or its products, such as those forms concerned in the spoiling of milk or its fermentation. it is true that milk may contain disease-producing bacteria coming either from a diseased animal or from a diseased human being. it is also true that some of such harmful forms are able to grow in milk, such as the organisms causing typhoid fever and diphtheria. =food.= the bacteria like all other plants must have their food in solution. where they apparently live on solids, such as meats, fruits, etc., they dissolve the food substances before utilizing the same. if the solutions are highly concentrated, as in the case of syrups, preserves and condensed milk, the bacteria cannot readily grow, although all of the necessary food ingredients are present. when such concentrated solutions are diluted, bacterial growth will take place and the solutions will spoil. [illustration: fig. 3.--photomicrograph of lactic acid bacteria. each cell is an individual organism, magnified 1250 diameters.] generally speaking the bacteria grow best in a neutral or slightly alkaline solution rather than in acid liquids. =temperature.= one of the most important conditions influencing the rate of growth of bacteria is the temperature. each form has a _minimum_ temperature below which growth can not take place; also a _maximum_ above which growth is again impossible. for the majority of species the minimum temperature ranges from 40 to 45° f. the maximum from 105 to 110° f. growth takes place most rapidly at the optimum temperature, which, for each species, lies close to the maximum temperature at which growth can occur. most of the bacteria of importance in the dairy grow well at from 70 to 100° f. there are forms that can grow below the freezing point of water when they are in solutions that do not freeze at this temperature. there are still other bacteria that can grow at 140° f. a temperature that is quickly fatal to most forms. these are of importance in the dairy since they limit the temperatures at which milk can be stored for long periods of time. =air supply.= living organisms, both plant and animal, require air or oxygen for the combustion of their food and for the production of energy. most bacteria use, as do the green plants and animals, the free oxygen of the air for their respiration. such organisms are called _aerobic_ or air-living. a much smaller group possess the power of taking oxygen from organic compounds such as sugar and the like and therefore are able to live under conditions where air is excluded. these are called _anaerobic_ bacteria. a large number of bacteria are able to live either in the presence or in the absence of free oxygen. most of the bacteria of importance in the dairy are of this nature. =rate of growth.= when there is an abundant supply of food and when the temperature conditions are favorable, the bacteria increase in numbers with astounding rapidity. it has been determined by actual experiment that the process of cell division under favorable conditions takes place in a few moments. barber has shown that one of the forms of bacteria constantly found in milk will divide in 17 minutes at 98° f. and that a single organism kept at this temperature for ten hours would increase to 1,240,000,000. if the temperature is reduced to 50° f., the time required for division is increased to several hours. the explanation for the rapid spoiling of milk that is not well cooled is thus apparent. the initial rapid rate of increase cannot be maintained for any length of time as the conditions become more and more unfavorable as growth continues, due to the accumulation of the by-products of the cell activity. thus, the growth of acid-forming organisms in milk becomes checked by the formation of acid from the fermentation of the sugar. =detrimental effect of external conditions.= environmental conditions of a detrimental character are constantly at work tending to repress the activity of bacteria or to destroy them. these act more readily on the vegetating cells than on the more resistant spores. it is of the utmost importance that those engaged in dairy work be familiar with these antagonistic forces since it is constantly necessary to repress or to kill outright the bacteria in milk and other dairy products. in many lines of dairy work it is likewise important to be familiar with the conditions favorable for bacterial growth. =effect of cold.= while it is true that chilling largely prevents fermentative action, and actual freezing stops all growth processes, still it does not follow that exposure to low temperatures will effectually destroy the vitality of bacteria, even in the growing condition. numerous non-spore-bearing species remain alive in ice for a prolonged period, and experiments with liquid air show that even a temperature of-310° f. maintained for hours does not kill all exposed cells. =effect of heat.= high temperatures, on the other hand, will destroy any form of life, whether in the vegetative or latent spore stage. the temperature at which the vitality of the cell is lost is known as the _thermal death point_. this limit is dependent not only upon the nature of the organism, but upon the time of exposure and the condition in which the heat is applied. in a moist atmosphere, the penetrating power of heat is great, consequently cell death occurs at a lower temperature than in a dry atmosphere. an increase in time of exposure lowers the temperature point at which death occurs. for growing organisms, the thermal death point of most species ranges from 130° to 140° f. for ten minutes. when spores are present, resistance is greatly increased, some forms being able to withstand steam at 212° f. from one to three hours. in the sterilization of milk, it is often necessary to heat for several hours, where a single exposure is made, to destroy the resistant spores, that seem to be more abundant under summer than winter conditions. steam under pressure is a much more effective agent, as the temperature is thus raised considerably beyond 212° f. an exposure of twenty minutes, at a temperature of 230° to 240° f. will kill all spores. where heat is used in a dry state, it is much less effective, a baking temperature of 260° to 300° f. for an hour being necessary to kill spores. this condition is of the utmost importance in the destruction of bacteria in the dairy and creamery. =effect of drying.= the spore-bearing bacteria withstand effects of desiccation without serious injury, and many of the non-spore-producing types retain their vitality for some months. the bacteria found in the air are practically all derived from the soil, and exist in the air in a dried condition, in which they are able to remain alive for considerable periods of time. in a dried condition, active cell growth is not possible, but when other conditions, such as moisture and food supply are present, resumption of growth quickly begins. this property is also of importance in the dairy as in the preparation of dry starters for creameries and cheese factories. =effect of light.= bright sunlight exerts a markedly injurious effect on bacterial life, both in a spore and in a growing condition. where the direct sunlight strikes, more or less complete disinfection results in the course of a few hours, the effect being produced by the chemical or violet rays, and not by the heat or red rays of the spectrum. this action, however, does not penetrate opaque objects, and is therefore confined to the surface. in diffused light, the effect is much lessened, although it is exerted to some extent. sunlight exerts a beneficial effect on the general health and well-being of animal life, and is a matter of importance to be taken into consideration in the erection of buildings for animals as well as for people. =effect of chemicals.= a great many chemical substances exert a more or less powerful toxic action on various kinds of life. many of these are of great service in destroying bacteria or holding them in check. those that are toxic and result in the death of the cell are known as _disinfectants_; those that merely inhibit, or retard growth are known as _antiseptics_. all disinfectants must of necessity be antiseptic in their action, but not all antiseptics are disinfectants, even when used in large amounts. disinfectants have no place in dairy work, except to destroy disease-producing bacteria, or to preserve milk for analytical purposes. the so-called chemical preservatives used to "keep" milk depend for their effect on the inhibition of bacterial growth. in this country, most states prohibit the use of these substances in milk. their only function in the dairy should be to check fermentative and putrefactive processes outside of milk and so keep the air free from taints. =products of growth.= all bacteria, as a result of their growth in food substances, form more or less characteristic compounds that are known as _by-products_. the changes brought about are those of decomposition and are collectively known as _fermentations_; they are characterized by the production of a large amount of by-products as the result of the development of a relatively small amount of cell life. the souring of milk, the rotting of eggs, the spoiling of meats, the making of vinegar from cider are examples of fermentations caused by different bacteria. if the substances decomposed contain but little sugar, as do animal tissues, the conditions are favorable for the growth of the putrefactive bacteria, and foul-smelling gases are formed. when sugars are present, as in milk, the environmental conditions are most favorable for the acid-forming bacteria that do not as a rule produce offensive odors. many of the bacteria form substances known as enzymes which are able to produce certain decomposition changes in the absence of the living cells, and it is by virtue of these enzymes that the organisms are able to break down such enormous quantities of organic matter. most of these enzymes react toward heat, cold, and chemical poisons in a manner quite similar to the living cells. in one respect, they are readily differentiated, and that is, that practically all of them are capable of producing their characteristic chemical transformations under conditions where the activity of the cell is wholly suspended as in a saturated ether or chloroform atmosphere. the production of enzymes is not confined to bacteria, but they are found throughout the animal and plant world, especially in those processes that are concerned in digestion. rennet, used in cheese making, is an example of an animal enzyme. =distribution of bacteria.= as bacteria possess greater powers of resistance than almost any other form of life, they are found very widely distributed over the surface of the earth. in soil they are abundant, because of the fact that all of the conditions necessary for growth are here best satisfied. they are, however, distributed with reference to the layers of the soil; the soil proper, i.e., that turned over by the plow, is extremely rich in them on account of the abundance of organic matter. but at the depth of a few feet they decrease rapidly in numbers, and in the deeper layers, from six to ten feet, or more, they are normally not present, because of the lack of proper food supply and oxygen. the fertility of the soil is closely associated with their presence. the bacteria are found in the air because of their development in the soil below. they are unable to grow even in a moist atmosphere, but are so readily dislodged by wind currents from the soil that over land areas the lower strata of the air always contain them. they are more numerous in summer than in winter; city air contains larger numbers than country air. wherever dried fecal matter is present, as in barns, the air contains many forms. water generally contains enough organic matter in solution, so that certain types of bacterial life find favorable growth conditions. water in contact with the soil surface takes up many impurities, and is of necessity rich in bacteria. as the rain water percolates into the soil, it loses its germ content, so that the normal ground water, like the deeper soil layers, contains practically no bacterial life. springs, therefore, are relatively deficient in germ life, except as they become contaminated with soil organisms, as the water issues from the ground. wells vary in their germ content, depending upon manner of construction, ease of contamination at surface, etc. wells are too frequently insufficiently protected from surface leachings, and consequently may contain all kinds of organisms found in the surface soil. typhoid fever is very frequently disseminated in this way, as is cholera and a number of animal maladies. while the inner tissues of healthy animals are free from bacteria, the natural passages, as the respiratory and digestive tracts, being in more direct contact with the exterior, become readily infected. this is particularly true with reference to the intestinal tract, and in the undigested residue of the food, bacterial activity is at a maximum. the result is that fecal matter of all kinds contains enormous numbers of organisms so that the pollution of any food medium, such as milk, with such material is sure to introduce elements that seriously affect its quality. chapter ii. methods of studying bacteria. =necessity of artificial cultivation.= the bacteria are so extremely small, that it is impossible to study individual germs separately without the aid of powerful microscopes. little advance was made in the knowledge of these lower forms of plant life until the introduction of culture methods, whereby a single organism could be cultivated, and the progeny of this cell increased to such an extent in a short course of time that the resulting mass of cells would be visible to the unaided eye. this is done by growing the bacteria on various kinds of nutrient media that are prepared for the purpose, but inasmuch as bacteria are so universally distributed, it becomes an impossibility to cultivate any special form alone, unless the medium in which they are grown is first freed from all pre-existing forms of germ life. =food materials.= many kinds of food substances are used for the cultivation of bacteria in the laboratory. in fact, bacteria will grow on almost any organic substance, whether it is solid or liquid, provided the other essential conditions of growth are furnished. the food substances that are used for culture purposes are divided into two classes,--solids and liquids. solid culture media may be either permanently solid, like potatoes and coagulated egg, or they may retain their solid properties only at certain temperatures, like gelatin or agar. the latter two, which were devised by robert koch, are of utmost importance in bacteriological research, for their use permits the separation of the different forms of bacteria that may happen to be in any mixture. gelatin is advantageously used, because the majority of bacteria present wider differences, due to growth upon this medium, than upon any other. it remains solid at ordinary temperatures, becoming liquid at about 80° f. agar, a gelatinous product derived from a japanese seaweed, has a much higher melting point, and is used especially with those organisms whose optimum temperature for growth is above the melting point of gelatin. besides these solid culture media, different liquid substances are extensively used, such as beef broth, milk and infusions of various vegetable and animal tissues. skim milk is of especial value in studying the milk bacteria, and may be used in its natural condition, or a few drops of litmus solution may be added, in order to detect any change in its chemical reaction due to the bacteria. =sterilization.= the various ingredients that are used in the preparation of culture media are not free from micro-organisms, hence the media would soon spoil if they were not destroyed, and the media subsequently protected from contamination from the air, etc. the process of rendering the media free from living micro-organisms is known as _sterilization_. it may be accomplished in a number of ways, but most often is done by the use of heat. for culture material, which is always organic in character, moist heat is employed. the various culture media, in appropriate containers, are subjected to a thorough steaming in a steam cooker. this destroys all of the vegetating cells but not the resistant spores that may be present. the media are then stored, for twenty-four hours, at temperatures favorable for the germination of the spores and are then again heated. three such applications on successive days are usually sufficient to free the media from all living germs, since between the heating periods the spores germinate and the resulting vegetative cells are more easily destroyed. the sterile media will keep for an indefinite period in a moist place. the media are usually placed in glass containers which may be sterilized before use by heating them in an oven, it being possible to thus secure a much higher temperature than with streaming steam. all glass or metal articles may be sterilized by the use of dry heat but for organic media, to avoid burning, moist heat must be used. all kinds of materials may be sterilized by treatment with steam under pressure. an exposure for a few moments at 250° f., a temperature attained with 15 pounds steam pressure, will destroy all kinds of bacteria and their spores. this method of sterilization is used in the canning of meats and vegetables and in the preparation of evaporated milk. to avoid contamination of the media after sterilization, the flasks and tubes are, after being filled, stoppered with plugs of cotton-wool, which effectually filter out all bacteria and mold spores from the air, and yet allow the air to pass freely in and out of the containers. =methods of determining the number of bacteria.= the method of determining the number and kinds of bacteria in any substance can be illustrated by the process as applied to milk. for this purpose the method of procedure is as follows: sterile gelatin in glass tubes is melted and then cooled until it is barely warm. to this melted gelatin a definite quantity of milk is added. the medium is gently shaken, so as to thoroughly mix the milk and gelatine, and the mixture then poured into a sterile, flat, glass dish, and quickly covered, where it is allowed to cool until the gelatin hardens. after the culture plate has been left for twenty-four to thirty-six hours at the proper temperature, tiny spots will begin to appear on the surface, or in the depth of the culture-medium. these spots are called _colonies_, and are composed of an almost infinite number of individual cells, the result of the continued growth of a single organism that was in the drop of milk and which was firmly held in place when the gelatin solidified. the number of these colonies represents approximately the number of living bacteria that were present in the amount of milk added to the tube of gelatin. if the plate is not too thickly sown with the bacteria, the colonies will continue to grow and increase in size, and as they do, minute differences will begin to appear. these differences may be in the color, the contour, and the texture of the colony, or the manner in which it acts toward gelatin. [illustration: fig. 4.--plate culture. each of the dots is a colony that has been formed by the growth of an organism embedded in the solid culture-medium. by counting the colonies, the number of living bacteria in the amount of milk added to the culture is determined.] in order to make sure that the number of colonies is not so numerous as to prevent counting and further study of their characteristics, a series of plate cultures is usually made in which varying amounts of milk are added to the tubes of gelatine. this is attained by adding a definite amount of the milk or other substance to be examined to a measured amount of sterile water, e.g., one cubic centimeter of milk to ninety-nine cubic centimeters of water. one cubic centimeter of this mixture may be used for the inoculation of the plate culture. this dilution may be carried on to any desired extent; in the examination of many dairy products, it is necessary to use very minute quantities of material, often only one one-millionth of a cubic centimeter. to study further the peculiarities of the different bacteria, small portions of the individual colonies are transferred to tubes of sterile culture-media. in order to do this the colony is touched with a piece of platinum wire; the minute amount of growth that adheres to the wire is sufficient to seed the tube of fresh culture-medium. the inoculating needle must always be sterilized before use by passing it through a gas flame. a culture thus obtained is called a _pure culture_ since it contains but a single kind of an organism, as the colony is the result of the growth of a single cell. these cultures then serve as a basis for continued study, and must be planted and grown upon the different kinds of media that are obtainable. in this way the slightest variations in the growth of different forms are detected, and the peculiar characteristics are determined, so that the student is able to recognize this form when he meets it again. [illustration: fig. 5.--different kinds of bacteria growing in gelatin. a, meager growth, no liquefaction or surface growth; b, profuse surface growth, radiating filaments from the growth below the surface; c, a rapid liquefying form; d, a gas producer that grows equally well in the presence or absence of air; e, form that grows only in the absence of air, an anaerob.] these culture methods are of essential importance in bacteriology, as it is the only way in which it is possible to secure a quantity of germs in a pure state. =the microscope in bacterial investigations.= in order to verify the purity of the cultures, the microscope is in constant demand throughout all the different stages of the isolating process. for this purpose it is essential that the instrument used shall be one of high magnifying powers (600 to 800 diameters), combined with sharp definition. the microscopical examination of any germ is quite as essential as the determination of culture characteristics, in fact, the two must go hand in hand. the examination reveals not only the form and size of the individual germs but the manner in which they are united with each other, as well as any peculiarities of movement that they may possess. in carrying out the microscopical part of the work, not only is the organism examined in a living condition, but colored preparations are made by using solutions of anilin dyes as staining agents. these are of great service in bringing out almost imperceptible differences. the art of staining has been carried to the highest degree of perfection in bacteriology, especially in the detection of germs that are found in diseased tissues in the animal or human body. in studying the peculiarities of any special organism, not only is it necessary that these cultural and microscopical characters should be closely observed, but special experiments must be made in different ways, in order to determine any special properties that the germ may possess. thus, the ability of any form to act as a fermentative organism can be tested by fermentation experiments; the property of causing disease, studied by the inoculation of pure cultures into experimental animals, like rabbits, guinea pigs and white mice. the methods of the bacteriologist in his laboratory are in their effect not dissimilar to those which the farmer employs in securing his crop of pure-bred grain. the laboratory farmer kills the weed seeds in his culture field by the application of heat. his field, which is embraced in his culture dish, has been fertilized and prepared by the addition of certain favorable ingredients. when he has garnered his crop, he maintains its purity by keeping his selected seed, the pure culture, free from all contamination. the dairyman, even though he may not expect to carry on the detailed operations of the laboratory, will understand the reason for the directions which he is often required to follow much better if he knows how the simple operations of the laboratory are carried out. for a fuller knowledge of these matters, the reader is referred to the special texts on bacteriology. chapter iii. contamination of milk. =spoiling of milk.= materials of animal origin are peculiarly prone to undergo changes, rendering them unfit for use, and of these, milk is exceedingly susceptible to such changes. this is due to the fact that the composition of milk is especially adapted to bacterial growth, and that the opportunity for entrance of such organisms is likewise such as to permit of abundant contamination. the consequence is that milk readily undergoes fermentative changes, due to the development of one or another type of micro-organism. =milk, a suitable bacterial food.= while milk is designed by nature for the nourishment of mammalian life, it is, curiously enough, equally well adapted to the growth of these lowest forms of vegetable life. the nutritive substances required by bacteria are here sufficiently dilute to make possible rapid growth. milk also contains all the necessary chemical substances to make a suitable bacterial food supply. of the nitrogenous compounds, albumen is in a readily assimilable form. casein, the principal nitrogenous constituent of milk, exists in an insoluble condition, and cannot be directly utilized, until it is acted upon by digesting enzymes. the fat in milk does not readily decompose, and while there are a few bacteria capable of splitting this substance, the majority of organisms are unable to utilize it. milk sugar, on the other hand, is an excellent food for most species. [illustration: fig. 6.--fat globules and bacteria. note the relative size of the fat globules of milk and the lactic acid bacteria.] =sources of contamination.= inasmuch as milk is especially exposed to the inroads of bacterial growth, and because of the fact that much of the contamination can easily be prevented, it is highly important that the milk producer and dealer should be thoroughly cognizant of the various sources of contamination. the different factors concerned in contamination may be grouped as follows: the interior of the udder; utensils, including all apparatus with which the milk is brought in contact subsequent to withdrawal from the animal; infection coming from the animal herself, from the milker, and the surrounding air. =condition of milk when secreted.= immediately after withdrawal from the udder, milk always contains bacteria, yet in the secreting cells of the udder of a healthy cow, germ life does not seem to be present. only when the gland is diseased are bacteria found in any abundance. in the passage of the milk from the secreting cells to the outside, it receives its first infection, so that when drawn from the animal it generally contains a considerable number of organisms. a study of the structure of the udder shows the manner in which such infection occurs. =structure of the udder.= the udder is composed of secreting tissue (_gland cells_) that is supported by fibrous connective tissue. the milk is elaborated in these cells and is discharged into microscopic cavities, from whence it flows through the numerous channels (_milk sinuses_) that ramify through the substance of the udder, until finally it is conveyed into the _milk cistern_, a common receptacle holding about one half pint that is located just above the teat. this cavity is connected with the outside by a direct opening (_milk duct_) through the teat. during the process of milking, the milk is elaborated rapidly in the gland cells, and their contents upon rupture of the milk cells, flow down into the cistern. the normal contraction of the muscles at the lower opening of the outer duct prevents the milk from passing out except when pressure is applied, as in milking. the inner walls of the milk duct and cistern are always more or less moist, and therefore afford a suitable place for bacteria to develop, if infection once occurs, and conditions are favorable for growth. =manner of invasion.= two possible sources of invasion of the udder by bacteria may exist. if bacteria are present in the circulating blood, there is the possibility of organisms passing directly through the tissues into the milk-secreting cells. the other alternative is the possible direct contamination from the outside by organisms passing up through the milk duct, and so spreading through the open channels in the udder. [illustration: fig. 7.--sectional view of udder. teat with milk duct connecting the exterior with the milk cistern. milk sinuses which conduct the milk from the secreting tissue to the milk cistern. (after moore & ward.)] =number of bacteria in fore-milk.= if a bacteriological examination is made of the milk drawn from each teat at different periods during the milking process, it will be found that the fore-milk, _i.e._, the first few streams, contains, as a rule, many more organisms per cubic centimeter than that removed later. not infrequently thousands of organisms per cubic centimeter may be found in the first streams while the middle milk, or strippings, will contain much smaller numbers. =distribution and nature of bacteria in udder.= if the udder itself is carefully examined as to its bacterial content, it appears that the majority of organisms found is confined to the lower portion of this organ, in the teat, milk-cistern and large milk-ducts; while bacteria occur in contact with the secreting tissue, they are relatively less abundant. this would seem to indicate that the more probable mode of infection is through the open teat. while there is no constant type of bacteria found in the fore-milk, yet it is noteworthy that nearly all observers agree that the organisms most commonly found are not usually the acid-producing, or gas-generating type, so abundant on the skin or hairy coat of the udder and which predominate in ordinary milks. coccus forms, belonging to both liquefying and non-liquefying types are most generally present. many of these produce acid slowly and in small quantities. the bacteria coming from the interior of the udder are of small practical significance since they do not grow rapidly at the temperatures at which milk is stored. if the milk is protected from contamination from other sources, the bacteria from the udder will ultimately cause it to spoil, but under ordinary conditions other forms are present in such greater numbers, and grow so much more rapidly in milk, that the udder forms have small opportunity to exert any effect. it is interesting to note that the bacteria found in the udder are similar to those that seem to be most abundant in such glandular tissues as the liver and spleen. this fact increases the probability that these comparatively inert coccus forms of the udder may originate directly from the blood stream. the organisms that normally are found in the udder exert no harmful effects on the gland. it might be thought that due to the presence of abundant food and a favorable temperature that growth would be abundant, but such is not the case. at times the udder may be invaded by forms that are not held in check by the natural factors and an inflammation of the udder is likely to result. =germicidal property of milk.= it has been claimed that freshly drawn milk, like other body fluids, possesses germicidal properties, _i.e._, the power of destroying bacteria with which it may be brought in contact. if milk is carefully examined bacteriologically, hour by hour, after it is withdrawn from the udder, it will generally be found that there is at first not only no increase in number of organisms during a longer or shorter period when it is kept at temperatures varying from 40° to 70° f., but that an actual reduction not infrequently takes place. when cultures of bacteria, such as _b. prodigiosus_, a red organism, lactic acid organisms, and even the yellow, liquefying coccus, so commonly found in the fore-milk, are artificially introduced into the udder, it has been found that no growth occurs and that in the course of a few days the introduced organisms actually disappear. whether this failure to colonize can be regarded as evidence of a germicidal property or not is questionable. in fact, this question is a matter of but little practical importance in the handling of milk since, under the best of conditions, the keeping quality of the milk is not materially enhanced. it may be of importance in inhibiting growth in the udder. =rejection of fore-milk.= the fact that the fore-milk contains per cubic centimeter so much more germ life than the remainder of the milk has led some to advocate its rejection when a sanitary milk supply is under consideration. while from a purely quantitative point of view, this custom may be considered advantageous, in practice, however, it is hardly worth while since it is not at all certain that the rejection will have any effect on the keeping quality or healthfulness of milk. this is especially true if the ends of the teats are thoroughly cleaned before milking. it is true that the fore-milk is relatively deficient in fat so that the loss of butter fat occasioned by the rejection of the first few streams is comparatively slight. =contamination from utensils.= one of the most important phases of contamination is that which comes from the utensils used to hold the milk from the time it is drawn until it is utilized. not only is this important because it is a leading factor in the infection of milk, but because much improvement can be secured with but little trouble, and it is especially necessary that the dairy student should be made familiar with the various conditions that obtain. pails and cans used to hold milk may be apparently clean to the eye, and yet contribute materially to the germ content of the milk placed in them. not only does much depend upon their condition, but it is equally important to take into consideration their manner of construction. dairy utensils should be simple in construction, rather than complex. they should be made so that they can be readily and easily cleaned, or otherwise the cleaning process is apt to be neglected. of first importance are those utensils that are used to collect the milk and in which it is handled while on the farm. the warm milk is first received in pails, and unless these are scrupulously cleaned, an important initial contamination then occurs. as ordinarily washed, the process falls far short of ridding the utensils of the bacterial life that is adherent to the inner surface of the pail. then, too, all angles or crevices afford an excellent hiding place for bacteria, and it is very important to see that all seams are well soldered. round corners and angles flushed with solder greatly facilitate thorough cleaning of utensils. tin utensils are recognized as most satisfactory. shipping cans are likely to serve as greater infecting agents than pails for they are subject to more wear and tear and are harder to clean. as long as the surface is bright and smooth, it may be easily cleaned, but large utensils, such as cans, are likely to become dented and rusty in spots on the inner side. the storage of milk in such utensils results in its rapid deterioration. the action of rennet has been found to be greatly retarded where milk comes in contact with a rusty iron surface. it is also probable that some of the abnormal flavors in butter are due to the action of acid cream on iron or copper surfaces from which the tin has been worn. it is equally important that attention be paid to the care of strainers, coolers, and the small utensils. cloth strainers are more or less of a hotbed for bacterial growth, for unless they are boiled, and then dried quickly and thoroughly, germ growth will continue apace in them, as long as they contain any moisture. =milking machines and farm separators.= the introduction of these special types of dairy machinery in the handling of milk on the farm has materially complicated the question of the care of milk. both of these types of apparatus are much more complicated than the usual milk utensil; consequently, the danger of imperfect cleaning is thereby increased. this is still further accentuated by the fact that cleansing of utensils on the farm can never be done so well as at the factory or milk depot where steam is available. the milking machine may be easily kept in a comparatively germ-free condition, but unless this is done, it contributes its quota of germ life to the milk. the farm separator is more widely used than the milking machine and in actual practice the grossest carelessness prevails in the matter of its care. frequently it is not taken apart and thoroughly cleansed, but is rinsed out by passing water through the machine. it is impossible by such a treatment to remove the slime that collects on the wall of the bowl; the machine remains moist and bacterial growth can go on. such a machine represents a most important source of contamination of milk and cream and it is probable that the widespread introduction of the hand separator has contributed more to lower the quality of cream delivered at the factory than any other single factor. =contamination from factory by-products.= the custom of returning factory by-products in the same set of cans that is used to bring fresh milk is a prominent cause of bad milk. whey and skim milk are rich in bacterial life, and not infrequently are so handled as to become a foul, fermenting mass. if the cans used to transport this material are not scrupulously cleaned on the farm, transfer of harmful bacteria to the milk is made possible. in this way the carelessness of a single patron may be the means of seeding the whole factory supply. this custom is not only liable to produce a poor quality of milk, but it is more or less of a menace to all the patrons of a factory, inasmuch as the opportunity always obtains that disease-producing organisms may thus be introduced into the supply. not infrequently is tuberculosis thus spread through the medium of factory by-products. [illustration: fig. 8.--whey disposal. whey barrels at a wisconsin swiss cheese factory. each patron's share is placed in a barrel which is so situated that it is impossible to empty it completely; thus it is not cleaned during the season.] the manufacture of swiss cheese presents a striking example of the disregard which factory operators show toward the employment of bacteriological principles. in these factories, the custom is widely practiced of apportioning the patrons' allotment of whey into individual barrels which are supposed to be emptied each day. as these barrels are, however, rarely ever cleaned from the beginning to the end of the season, they become very foul, and the whey placed in them from day to day highly polluted. it is this material which is taken back to the farms in the same set of cans that is used for the fresh milk. when one recalls that the very best type of milk is essential for the making of a prime quality of swiss cheese, and that to secure such, the maker insists that the patron bring the product to the factory twice daily, the before mentioned practice appears somewhat inconsistent. =treatment of factory by-products.= to overcome the danger of infecting milk from factory by-products with either undesirable fermentative organisms, or disease-producing bacteria, the most feasible process is to destroy these organisms by the application of heat. in denmark, some portions of germany, and in some of the states in this country, laws exist which require the heating of all skim milk before it is returned to the farm. this is done by the direct use of exhaust steam, or running the product through heaters. the treatment of whey in cheese factory practice is especially important since the warm whey must be stored for a number of hours before it is returned to the farms. even under the best of conditions the whey is certain to be in an advanced state of fermentation when placed in the milk cans, and it only needs the infection of the whey tank with harmful bacteria to cause great loss on account of the injury of the product by these bacteria. among canadian factories the custom of heating the whey as it passes from the cheese vat to whey tank has been introduced, and where ever adopted has been retained, because, it has resulted in such an improvement of the cheese that the gain was much greater than the cost, which is estimated at not over fifty cents per ton of cheese. the whey is heated not to exceed 155° f.; the hot whey serves to scald the whey tank and as the mass of whey is usually quite large, it does not cool to a point where bacterial growth can take place for a number of hours. the whey is thus quite sweet when returned to the farm and has greater feeding value. the heating also prevents the creaming of the whey in the tank and thus avoids the soiling of the cans with grease which is most difficult to remove. where compulsory legislation is in force it is generally required that these by-products be heated to a temperature of at least 176° f. this is done so as to destroy effectually the organisms of tuberculosis, and especially to permit of the utilization of the so-called storch test,[1] which enables a person to determine quickly whether milk or whey has been heated or not. [1] storch (40 rept. expt. stat., copenhagen, 1898) has devised a test whereby it can be determined whether this treatment has been carried out or not; milk contains a soluble enzyme known as peroxidase which has the property of decomposing hydrogen peroxid. if milk is heated to 176° f., (80° c.) or above, this enzyme is destroyed, so that the above reaction no longer takes place. if potassium iodide and starch are added to unheated milk and the same treated with hydrogen peroxid, the decomposition of the latter agent releases oxygen which acts on the potassium salt, which in turn gives off free iodine that turns the starch blue. =cleaning utensils.= various processes are applied to dairy utensils to cleanse them. in removing visible dirt and foreign matter, much of the bacterial life is mechanically eliminated, but most of the cleaning processes fail to destroy the germ life in these utensils. in rinsing, washing, or even scalding, the water is not applied at a sufficiently high temperature to destroy effectively the bacteria. these processes are primarily used for the removal of dirt and other matter. to facilitate such removal, washing powders of various kinds are frequently employed; some of these possess considerable disinfecting action. all utensils after cleansing should be thoroughly rinsed in clean, hot water. even where no further treatment is given, a careful cleaning may so reduce the germ content on the inner surface of utensil as to render contamination therefrom relatively unimportant. most of the contamination in a well cleaned utensil comes from the cracks and angles, which permit of the collection of the dirt. if these are properly attended to, thorough cleaning and rinsing alone will accomplish much. to exert an actual germ-destroying effect on the bacterial content of the utensil, resort must be had to boiling or steaming. to treat utensils so as to render them wholly germ-free would be impractical under ordinary commercial conditions, as it would consume too much time, although with proper apparatus, this process is not impossible, but it is well within the limits of practicability in factory treatment to apply steam for a short period of time. where cans, pails and such utensils, are steamed for a minute or so after being thoroughly cleaned, the germ content is greatly reduced. in a series of tests by harrison, the germ content of a set of cans cleaned in an ordinary way was 442,000 bacteria per cubic centimeter in 100 cubic centimeters of wash water; in a set washed in tepid water and then scalded--the best farm practice--it was 54,000 per cubic centimeter, while in cans carefully washed and then steamed for 5 minutes, it was reduced to 880 per cubic centimeter. it would not be worth while to institute measures that would accomplish the destruction of this small residual content. the use of steam, therefore, is of great service in eliminating bacterial life in all utensils. in apparatus of at all complicated design, it is absolutely necessary. of course, ordinarily, steam can be applied only at the factory, as the farm does not usually afford facilities for its easy generation. this fact has led in some cases to the adoption of the method of cleaning and sterilizing the cans at the factory rather than to await their arrival at the farm. this custom is most frequently followed in milk supply plants. it is also very important in cleaning dairy utensils to see that they are rapidly and thoroughly dried after being washed and steamed. as pointed out above, the short period of steaming that can be followed in practice does not kill all the bacteria. if moisture is retained, conditions permit of the growth of the undestroyed organisms. tests made on glass milk bottles showed that considerable growth occurred in the condensation water even after quite thorough sterilization. some of the devices used for the sterilization of such utensils as milk cans are so arranged that, after steam has been introduced, hot air is passed into the can until it is thoroughly dried. other utensils such as cloth strainers become sources of contamination unless the articles are thoroughly and quickly dried after cleaning. in a general way, it may be said that whenever a utensil is so constructed and in such a condition that every portion of its surface can be reached by a cloth or a brush, it can be kept in a sanitary condition. but whenever any portion cannot be thus reached, whether it is an angle or a seam in a pail or can, the interior of the separator bowl, or in the pipes used for conducting milk, contamination is certain to result from such places, unless extreme care is taken to destroy the bacteria therein by steaming. =contamination from the animal.= in the process of milking, the bacterial content of the milk is materially increased. in part this comes from the utensils into which the milk is drawn, but the animal herself, the milker, as well as the surrounding air, also contribute to a varying extent. of these factors, the one fraught by far with the most consequence, is the influence of the animal herself. it is a popular belief that the organisms found in milk are derived from the feed and water which the animal consumes, but under normal conditions, the bacteria consumed in food pass through the intestinal canal and do not appear in the circulation. it must not be assumed, however, that the character of feed and water supply is of no moment. stock should be given pure and wholesome water and no decomposed or spoiled food should be used. the infection traceable directly to the cow is modified materially by the conditions under which the animal is kept and the character of the feed consumed. the nature of the fecal matter is in part dependent upon the character of the food. the more nitrogenous the ration fed, the softer are the fecal discharges, producing a condition which is more likely to soil the coat of the animal unless care is taken. the same is true with animals kept on pasture in comparison with those fed dry fodder. stall-fed animals, however, are more likely to have their flanks fouled, unless special attention is paid to the removal of the manure. all dairy stalls should be provided with a manure drop which should be cleaned as frequently as circumstances will permit. [illustration: fig. 9.--bacteria on hairs. each colony on the hair represents one or more bacteria that were adherent to the hair when it was placed on the surface of the solid culture-medium.] the animal contributes materially to the quota of germ life finding its way into the milk through the dislodgment of dust and filth particles adhering to its hairy coat. the nature of this coat is such as to favor the retention of these particles. unless care is taken, the flanks and udder become polluted with fecal matter, which upon drying is displaced with every movement of the animal. every hair or dirt particle so dislodged and finding its way into the milk-pail adds its quota of organisms to the liquid. this can be readily demonstrated by placing cow's hairs on the moist surface of gelatin culture plates. almost invariably bacteria will be found in considerable numbers adhering to such hairs, as is indicated in fig. 9. dirt particles are even richer in germ life. not only is there the dislodgment of hairs, epithelial scales, and masses of dirt and filth, but during the milking process, as at all other times, every motion of the animal is accompanied by a shower of _invisible_ particles, more or less teeming with bacterial life. all of this material contains organisms that are more or less undesirable in milk. bacteria concerned in gassy fermentations and those capable of producing obnoxious taints are particularly common, so that this type of pollution is especially undesirable in milk. =amount of dirt in milk.= when one remembers that the larger part of fresh manure is of such a nature that it does not appear as sediment, the presence of evident filth in milk must bespeak careless methods of handling. the sediment or dirt test is used quite extensively to ascertain the amount of dirt milk may contain. by means of a cotton filter, the insoluble residue is removed and is made evident upon a layer of absorbent cotton. milk that would show with difficulty any evidence of dirt upon ordinary examination reveals such defects very readily in this test. =exclusion of dirt.= it is better to keep bacteria out of milk, so far as practicable, rather than to attempt to remove them after they have once gained entrance. as is usual, prevention of trouble is much more easily accomplished than removing the difficulty after it once occurs. [illustration: fig. 10.--dirt from milk. the dirt adherent to each of the filters was obtained from one pint of milk. the milks tested were produced on different farms.] much reduction as to the amount of dirt that finds its way into milk may be accomplished by improved stable environment. the fouling of the udder and flanks comes from wading in dirty water, muddy yards, and from improper type of stalls. barnyards are often a disgrace through the accumulation of manure and seepage. cows wading in such mire cannot but accumulate mud and filth to a material degree on the teats and udder. greater care as to drainage of the barnyard and the paving of same with gravel, cinders, etc., will permit of its being kept clean, and so prevent the fouling of animals. but more important than the yard is the stall which the animal occupies in the stable. the essential feature is to have a stall of such construction as to keep the animal out of her own manure when she lies down. to accomplish this, it is necessary to have a manure drop behind the stall proper so that the feces and urine are kept out of the bed of the stall as much as possible. [illustration: fig. 11.--the model stall. a stall of this type keeps the animals clean, and thus aids greatly in producing good milk.] most of the stalls widely advertised in the farm press seek to accomplish this in one way or another, usually by some arrangement by which the cow is forced back when standing and drawn forward on lying down. in fig. 11 a type of stall is illustrated that accomplishes this most successfully; the essential feature being a 2×3-inch wood strip nailed to the stall floor immediately in front of the hind feet of the animal when in a standing position. when the animal lies down, she crowds forward to avoid lying on this strip, and thus is out of contact with the manure, except such as is carried onto the bedding by the hind feet. by the use of this stall it is possible to keep the animals free from all accumulations of manure. effort should be made to prevent fouling of the animals rather than in cleaning them after once soiled. it is very evident that where the cattle come to the milker with muddy udders, they will not be so cleaned before milking as to prevent a large amount of such dirt from entering the milk. however, when all that can be done towards keeping the cows clean has been accomplished, a small amount of grooming will greatly reduce the contamination coming from them. the kind of bedding used in the stalls may have a marked influence on the contamination coming from the animal. if the straw is dusty, partially rotten and moldy, the bacteria and molds adhere to the coat of the animal and are thus introduced into the milk. in the case of cattle on pasture, no visible evidences of dirt are usually present but the hair is covered with the dust coming from the soil. there is very good reason to believe that the quality of milk is influenced by the type of pasture on which the cows graze, due to the difference in the types of bacteria in the surface soil. the milk from animals on low land is more likely to show undesirable fermentations than that from those grazing on higher lands. this is not due to the influence of the feed as is often supposed but rather to the dirt from the coat of the animal. =washing the udder.= if a surface is moist, dust and the adherent bacteria cannot be easily dislodged. the air over snow-covered mountains or over oceans is relatively free from bacteria. the udder and flanks of the animals can be carded to remove the loose hairs and the evident dirt; the fine dust can now be removed by wiping with a clean damp cloth just before the milking process. the actual washing and wiping of the udder and flanks still further reduces the contamination coming from the animal; experiments show a reduction of fully three-fourths of total contamination. clipping the udder and flanks also aids in keeping the animal clean. it is often asserted that the treatment of the animals in these ways reduces the yield of milk. it is certain that such an effect will persist for only a short time and there is reason to believe that grooming increases the yield. [illustration: fig. 12.--sanitary milk pails. the small opening is very efficient in keeping the dirt out of milk.] =sanitary milk pails.= the entrance of organisms into the milk can be greatly reduced by lessening the area of the milk pail exposed to the dust shower. to accomplish this purpose a number of so-called sanitary or hygienic milk pails have been devised. in some cases, these are the regular type of pail provided with a cover having a small opening through which the milk is received. in other cases, a strainer is interposed so as to remove more effectually the coarse particles. while pails of this type are successful in the removal of a large part of the dirt, and consequently reduce materially the bacterial content of the milk, yet they must be of simple construction, so that they can be kept in a clean condition in order to adapt them for general practical use. the use of such a utensil increases materially the keeping quality of the milk. [illustration: fig. 13.--sanitary milk pails. the stadtmueller pail and the truman pail, two of the most practical of the small-topped pails.] stocking has shown that under ordinary barn conditions, the use of small-topped pails reduced the number of bacteria 95 per cent; with dirty cows the reduction in bacteria amounted to 97 per cent. a six-inch opening presents only one-fourth as large an exposure as a twelve inch, so that the reduction in bacterial content is greater than the lessening in the size of the openings of the pails. the ordinary pail receives dust not only from the udder, but also from the flank which is usually a more important source of contamination than the udder itself, while the small-topped pail receives only that from the udder. [illustration: fig. 14.--use of sanitary milk pails. the open pail is fully exposed to the falling dust while the hooded pail excludes much of the dust and dirt coming from the animal.] =milking machines.= where the milk is removed from the udder by machine methods, instead of by hand, it is possible to eliminate nearly all external contamination from the animal and her surroundings. the only opportunity for infection is then through the leakage of air around the teat cups. care should be taken to see that the teats are in a clean condition before applying the suction cups. the main problem in the use of a milking machine is to keep the apparatus in an aseptic condition. immersion of the teat cups and the rubber connections in lime water, brine solution, or other mild antiseptics, prevents bacterial development. hastings has found that milk having a germ content of less than 10,000 bacteria per cubic centimeter may be produced by the use of a properly handled milking machine. =contamination from the milker.= while the milker is a small factor in comparison with the animal in the matter of contamination, yet he can not be neglected, as it is within his power to affect profoundly the quality of the milk. his personal habits as to cleanliness and his appreciation of the precautions necessary in the production of clean milk have much to do with the contamination of the milk. the milking should be done with dry hands, although a little vaseline may be used with effect. the hands should be washed before milking as milk is certain to come in contact with them to some extent. the milking should be done with the whole hand rather than stripping between the thumb and finger; the clothing should be covered with clean overalls and jumper, or at least a clean apron should be worn during the milking. if these are of white material, more frequent laundering is likely to result. =contamination from air.= it is difficult to disassociate the contamination arising from the condition of the air from that derived directly from the animal. barn operations of various kinds result in the production of dust, particularly where dry forage, such as hay or straw, is handled. where manure is given an opportunity to dry, dust is readily produced, and such material is particularly replete with bacterial life. some kinds of dust, such as that originating from ground grains, or shavings that may be used for bedding, contain a small amount of bacterial life in comparison with the dust from hay, or other dry fodder. in a dried condition, the slightest movement is apt to dislodge these fine particles, and they float in the air for considerable periods of time. if milk is drawn and exposed to the air of the barn during the feeding operations, it is subject to the dust shower that is present. where the storage can is allowed to stand in the stable during the milking, even though it is covered with a strainer, this accumulation of microscopic particles is added to the milk, as they readily pass the meshes of the finest strainer. [illustration: fig. 15.--contamination from the air. this culture plate, three inches in diameter, was exposed for 30 seconds in the barn during feeding of dry fodder. a 12-inch pail exposes over 18 times the surface of this plate.] =removal of dirt after introduction.= the more primitive method of improving the quality of milk, so far as its dirt content is concerned, is to attempt to remove the grosser particles of contamination after entrance. in the case of straining, the method is usually applied at the time of milking, but in the case of filtering and clarifying, it is carried out at the milk station, in an effort to improve the appearance of milk and overcome the influence of careless methods of the producer. by the use of strainers, either metallic or cloth, it is possible to remove particles of hair, undissolved dirt and manure, but it must be remembered that these grosser _visible_ particles of pollution are not really the cause of the troubles which may ensue in improperly handled milk. the bacteria which are adherent to these foreign particles are in large measure washed off in the process of straining, and pass through the meshes of the finest strainer. the main service, therefore, of straining is to improve the appearance of the milk, and it has no effect on the quality in any way. =production of clean milk.= the problem of clean milk is important, whatever may be the use to which milk may be put. it is important in the manufacture of butter, but owing to the fact that the fat is not readily acted upon by bacteria, it is not so sensitive to bacterial conditions, as when the milk is made into cheese. in this product, the bacterial condition of the milk is a matter of prime importance. in milk destined for direct consumption, the exclusion of the bacteria becomes yet more important. while it is impossible to exclude bacteria so completely that milk will not undergo fermentative changes, yet for domestic consumption it is preferable to have milk with as low bacterial content as can readily be secured. the highest type of market milk, that known as sanitary, or certified, is produced under such extreme conditions of care as to contain the minimum germ content. to accomplish these results requires such stringent control as to increase greatly the cost of the product. pure, clean milk can be produced at a very slight increase in cost over the regular expense of milk production, if the right kind of attention is given to certain details of a practical character. improvement in our milk supplies must largely come from this source, for any improvement to be permanent must be made to pay, and it requires considerable education to secure the co-operation of consumers and their willingness to pay for any material increase in the quality of the product. in the foregoing factors concerned in the contamination of milk, it is of course impossible to measure accurately the influence of the different sources of infection, as these are continually subject to variation in every case. as a rule, the most important factors are those pertaining to the utensils and the condition of the animal herself. if these two factors are brought under reasonable control, the major portion of contamination that ordinarily obtains is done away with. the application of the remedial or preventive measures heretofore mentioned will greatly reduce the germ content of the milk. =cooling of milk on farm.= bacterial growth is directly related to temperature conditions, and with summer temperatures, such development goes on apace, unless it is checked by early cooling. the larger portion of bacteria that find their way into milk, especially those that are previously in contact with the air, are in a dormant condition, and are therefore not stimulated into immediate growth, unless reasonably high temperatures prevail. in milk, which comes from the animal at blood heat, this growth is greatly stimulated. to counteract this effect, milk should be chilled as soon after milking as possible. if the temperature is immediately lowered to 50° f., or lower, actual cell development is greatly retarded, and the rate of souring, and other fermentative changes thereby diminished. in this country ice is liberally used in accomplishing this result. in europe, the use of ice is much less common. the employment of such artificial means of refrigeration makes possible the shipment of milk for long distances by rail. new york city now receives milk that is produced in canada and northeastern ohio. [illustration: fig. 16.--effect of cooling milk.] =aeration of milk=. the custom has been extensively recommended of subjecting milk to the influence of air in the belief that such exposure permits of the interchange of gases that would improve the quality. in practice, this process, known as aeration, is carried on in different ways. in some cases, air is forced into the milk; in others, the milk is allowed to distribute itself in a thin sheet over a broad surface, falling in drops or tiny streams through the air. whenever this process is carried on at a temperature lower than that of the milk, it results in more or less rapid cooling. in earlier times, aeration was generally recommended and practiced, especially in connection with the cheese industry, but carefully controlled experiments fail to show that the process exerts any material influence on the rate of germ development. if it is carried out in an atmosphere more or less charged with bacteria, as in the barn or stable, it is more than likely to add to the bacterial content of the milk. while to some extent odors may be eliminated by the process, the custom is not followed so generally now as it used to be some years ago. =absorption of taints.= a tainted condition in milk may result from the development of bacteria, acting upon various constituents of the milk, and transforming these in such a way, as to produce by-products that impair the flavor or appearance of the liquid; or it may be produced by the milk being brought in contact with any odoriferous or aromatic substance, under conditions that permit of the direct absorption of such odors. this latter class of taints is entirely independent of bacterial action, and is largely attributable to the physical property which milk possesses of absorbing volatile odors. this direct absorption may occur before the milk is withdrawn from the animal, or afterwards if exposed to strong odors. it is not uncommon for the milk of animals advanced in lactation to have a more or less strongly marked odor and taste; sometimes it is apt to be bitter, at other times salty to the taste. it is a defect that is peculiar to individual animals, and is liable to recur at approximately the same period in lactation. the peculiar "cowy" or "animal odor" of fresh milk is an inherent peculiarity that is due to the direct absorption of volatile elements from the animal herself. many kinds of feed consumed by the animal produce a more or less pronounced taint or flavor in the milk. with some plants, such as garlic, leeks, turnips, and cabbage, the odor is so pronounced as to render the milk quite unfit for use. in some states along the atlantic seaboard, wild plants of this character in woodland pastures may be so abundant as to make it impossible to pasture milch animals. the difficulty in such cases is due to absorption of the volatile principles into the circulation of the animal, and if such feed is consumed shortly before milking, the characteristic odors appear in the milk. if consumed immediately after the milk is withdrawn from the animal, sufficient time may elapse so that the peculiar odors are dissipated before the milk is again secreted. the same principle applies in a lesser degree to the use of certain green fodders that are more suitable for feed, such as rape, green rye, or even silage. silage produces a distinct, but not unpleasant odor in milk, but newly pastured rye often confers so strong an odor as to render the milk unusable. where certain drugs are employed in the treatment of animals, such as belladonna, castor oil, sulfur, or turpentine, the peculiar odors may reappear in the milk. such mineral poisons as arsenic have been known to persist for a period of three weeks before elimination. on account of the elimination of many drugs, unchanged, from the animal in the milk, the milk of any animal that is receiving medicine should not be used for human food. when such milk is mixed with that of a number of other animals and when it is used by adults, no harm is likely to result, but when the dilution is not great and the milk is used for young children it may affect them through its content of the drug. the feed may not only affect the quality of milk but its value as food. one of the most prominent of american dairymen, who has for many years produced milk especially for children's use, has said that he could feed his cows so as to make ill every child receiving the milk. =absorption of odors after milking.= if milk is brought in contact with strong odors after being drawn from the animal, it will absorb them readily, as in the barn, where frequently it is exposed to the odor of manure and other fermenting organic matter. it has long been a popular belief that milk evolves odors and cannot absorb them so long as it is warmer than the surrounding air, but from experiments of one of us (r), it has been definitely shown that the direct absorption of odors takes place much more rapidly when the milk is warm than when cold, although under either condition, it absorbs volatile substances quite rapidly. the custom of straining the milk in the barn has long been deprecated as inconsistent with proper dairy practice, and in the light of the above experiments, an additional reason is evident why this should not be done. even after milk is thoroughly cooled, it may absorb odors, as is noted where the same is stored in a refrigerator with certain fruits, meats, fish, etc. =distinguishing bacterial from other taints.= in perfectly fresh milk it is relatively easy to distinguish between taints caused by the growth of bacteria and those attributable to direct absorption. if the taint is evident at time of milking, it is in all probability due to character of feed consumed, or possibly to medicines. if, however, the intensity of the taint grows more pronounced as the milk becomes older, then it is probably due to living organisms which require a certain period of incubation before their by-products are most evident. moreover, if the difficulty is of bacterial origin, it can be frequently produced in another lot of milk (heated or sterilized is preferable) by inoculating the same with some of the original milk. not all abnormal fermentations are able, though, to compete with the lactic acid bacteria, and hence outbreaks of this sort soon die out by the re-establishment of more normal conditions. =factory contamination.= as the time element is of importance in the production of troubles due to bacteria, it follows that infection of milk on the farm is fraught with more consequence than factory contamination, as the organisms introduced would have a longer period of development. nevertheless, the conditions in the factory are by no means to be ignored, as they not infrequently permit the milk to become seeded with highly undesirable types. a much more rigid control can be exercised in the factory, where steam is at hand as an aid in the destruction of organisms. in the cleaning of pumps and pipes, steam is absolutely necessary to keep such apparatus in a sanitary condition. the water supply of the factory is a matter of prime importance, as water is used so extensively in all factory operations. when taken from a shallow well, especially if surface drainage from the factory is possible, the water may be contaminated to such an extent as to introduce undesirable bacteria in such numbers that the normal course of fermentation may be changed. the quality of the water, aside from flavor, can best be determined by making a curd test (p. 99) which is done by adding some of the water to boiled milk, and incubating the same. if "gassy" fermentations occur, it signifies an abnormal condition. in deep wells, pumped as thoroughly as is generally the case with factory wells, the germ content should be very low, ranging from a few score to a few hundred bacteria per cubic centimeter at most. the danger from ice is much less, for the reason that good daily practice does not sanction using ice directly in contact with milk or cream. then, too, water is largely purified in the process of freezing, although if secured from a polluted source, reliance should not be placed in this method of purification, for even freezing does not destroy all vegetating bacteria. the ordinary house fly is an important source of contamination in creameries, cheese factories and city milk plants. they are of importance not only in increasing the number of fermentative bacteria in milk but they may serve to contaminate it with disease-producing organisms. the windows of all places where milk is handled, whether on the farm or elsewhere should be screened. it should be kept in mind in the handling of milk and other dairy products that human food is being prepared and that cleanliness is desirable from every point of view, and that the methods of handling and production should compare with those used in the preparation of foods which like milk cannot be cleaned when once polluted. desirability, keeping quality, healthfulness and the value of every product made from milk depends upon the extent and amount of contamination. chapter iv. infection of milk with pathogenic bacteria. that the disease-producing, or pathogenic bacteria, are able to infect milk supplies is shown by the fact that numerous epidemics of contagious disease have been directly traced to milk infection. milk is generally consumed in a raw state, and as a considerable number of this class of organisms are able not only to live but actually grow in milk, which is such an ideal culture-medium for the development of most bacteria, it is not surprising that disease processes should be traced to this source. the organisms in milk capable of causing disease do not alter or change its physical properties sufficiently to enable their presence to be detected by a physical examination. =origin of pathogenic bacteria in milk.= disease-producing bacteria may be grouped, with reference to their relation toward milk, into two classes, depending upon the manner in which infection occurs: class i. disease-producing bacteria capable of being transmitted directly from a diseased animal to man through the medium of infected milk. class ii. bacteria pathogenic for man but not for cattle, which are capable of thriving in milk after it is drawn from the animal. in the first group, the disease produced by the specific organism must be common to both cattle and man. the organism must live a parasitic life in the animal, developing in the udder, and so infect the udder. it may, of course, happen that diseases toward which domestic animals alone are susceptible may be spread from one animal to another in this way without affecting human beings. in the second group the bacterial species live a saprophytic existence, growing in milk, as in any other nutrient medium, if it happens to find its way therein. in such cases, milk indirectly serves as an agent in the dissemination of disease, by giving conditions favorable to the growth of the disease germ. by far the most important of diseases that may be transmitted directly from animal to man through a milk supply is tuberculosis, but in addition to this, foot and mouth disease (aphthous fever in children), malta fever, and acute enteric troubles have also been traced to a similar source of infection. the most important specific diseases that are disseminated through subsequent infection of the milk are typhoid fever, diphtheria, scarlet fever, and cholera, but, of course, the possibility exists that any disease germ capable of living and thriving in milk may be spread in this way. in addition to these diseases that are caused by the introduction of specific organisms (the causal organism of scarlet fever has not yet been definitely determined), there are a large number of more or less illy defined troubles of an intestinal character that occur especially in infants and young children that are undoubtedly attributable to the activity of micro-organisms that gain access to milk during and subsequent to the milking, and which produce changes in milk before or after its ingestion that result in the formation of toxic products. =tuberculosis.= this disease is by far the most important bacterial malady that affects man and beast. in man, it assumes a wide variety of phases, ranging from consumption, tuberculosis of the lungs, which is by far the most common type, to scrofulous glands in the neck, cold abscesses, hip-joint, and bone diseases, as well as affection of the bowels. these various manifestations are all produced by the inroads of the specific organism, bacillus tuberculosis. the bovine, as well as swine, fowls, and other warm-blooded animals, are also affected with similar diseases. in man, the importance of the malady is recognized when it appears that fully one-seventh of the human race die of this scourge. in cattle, the disease is equally widespread, particularly in those countries where live stock has been intensively developed. in the northern countries of europe, such as denmark, germany, england, france, and the netherlands, as well as in canada, and this country, this disease has been most widely disseminated. this has been occasioned, in large measure, because of the exceedingly insidious nature of the disease in cattle, thereby permitting interchange of such diseased stock without the disease being recognized. tuberculosis is found more abundantly in this country in dairy than in beef stock. dairy cattle are, however, not more susceptible, but the closer environment in which milch cattle are kept, and the fact that there has been greater activity in the matter of introducing improved strains, accounts for the larger percentage of affected animals. it has been a disputed question for some years whether the organisms producing bovine and human tuberculosis are identical or from the practical standpoint, whether the bovine type of disease is transmitted under natural conditions to man. the bacteriologist can readily detect differences in appearance, in growth of cultures, and in disease-producing properties between the two strains. of the two, the bovine is much the more virulent when inoculated into experimental animals. in a considerable number of cases, record of accidental infection from cattle to man has been observed. these have occurred in persons making postmortem examination on tuberculous animals, and the tubercular nature of the wound proven by excision and inoculation. more recently, since the agitation by robert koch of germany, a number of scientific commissions have studied particularly the problem of transmission. it is now estimated that perhaps seven per cent of the tuberculosis in man is of bovine origin. this is almost wholly confined to children. the portions of the body that become diseased, when the infection has resulted from the use of milk, are the glands of the neck and of the abdomen. =manner of infection in man.= in the main, the source of the malady may be traced either to air infection or to the food, if one disregards the comparatively small number of cases of wound infection. air is frequently a medium by which the germ is transferred from one person to another. the sputum is exceedingly rich in tubercle bacilli and since this material is carelessly distributed by tubercular people, the air of the cities, villages and public buildings will frequently contain tubercle organisms. some of the organisms in the air find their way into the lungs, there to develop and produce consumption. the organisms in the air may be deposited in the nasal passages and throat, and ultimately find their way into the tissues of the body by penetrating the walls of the throat or of the intestine. it is probable that the tubercle bacilli thus introduced may find their way to the lungs and there develop without leaving any trace of their path. food may also possibly serve as a medium of infection. the contamination of solid food from flies and other sources is, of course, a possibility, but tuberculous meat from cattle and swine is much more likely to occur, although it must be said that the processes of preparing such food for use (roasting, frying, and boiling) are sufficient to destroy the vitality of the causal organism. the fact that most food products of this character are now inspected renders this possibility less likely to occur. unquestionably, the likelihood of ingesting tubercle organisms is much greater with milk than with any other food supply, as milk is consumed usually in an uncooked state, and as microscopic and physiologic tests indicate that not infrequently milk from tuberculous animals contains these organisms. =distribution of the disease in animals.= as practically any organ of the body may be affected with tuberculosis, it naturally follows that the lesions of this disease are widely distributed. the disease germ is introduced, in the main, through the lymph and not the blood system; consequently, in the initial stages the evidence of tuberculosis is often comparatively slight, and the lesion is restricted in its development. where such a condition obtains, it is known as "closed," in contradistinction to "open" tuberculosis, where the diseased tissue is more or less broken down and is discharging into the circulation, or elsewhere. manifestly, the danger of spreading not only in the affected animal itself, but to the outside, is much greater in the case of the open lesion. especially is this true where the disease is present in the lungs or organs that have an exterior opening so that the material containing the organisms is discharged from the body in the sputum, manure, urine or milk. the intestines themselves are rarely affected, but the lymph glands associated with the intestinal tract are not infrequently involved. =infection of milk with tubercle bacilli.= in a small percentage of cases, the udder itself becomes involved. where this condition obtains, one or more hard lumps are formed, which slowly increase in size, usually being restricted to one quarter of the udder. sometimes the affected quarter may develop to an enormous size, producing a hard, painless tumor. not often does the affected tissue break down into pus; consequently, no abnormal appearance is to be noted in the milk secretion until the disease has made very extended progress, in which case the percentage of fat generally diminishes. whenever the udder shows physical manifestation of this disease, the milk almost invariably is rich in tubercle bacilli. tubercle organisms may also appear in milk of animals in which no physical symptoms of the disease are to be found. this fact has been demonstrated by microscopic and animal experiments, but it is also abundantly confirmed by the frequent contraction of the disease by calves and hogs when fed on factory by-products. this latter class of animals is particularly dangerous, because there is no way in which the danger can be recognized. [illustration: fig. 17.--a tuberculous animal. the animal appears perfectly healthy although she has had the disease for five years.] it has also been proven that milk may become infected through the feces. in coughing up material from the lungs and associated glands, the matter is swallowed, instead of expectorated, as in man. the organisms retain their vitality in the intestine, and are voided in the feces. under ordinary conditions, the flanks and udder become more or less polluted with such filth, and the evidence is conclusive that infection of milk is not infrequently occasioned in this way. the fact that hogs following tuberculous steers in the feeding lots are very likely to acquire the disease is explained by the presence of tubercle organisms in the manure of such animals. [illustration: fig. 18.--a tuberculous animal. the last stages of generalized tuberculosis. note the emaciated condition.] it must be kept in mind that many animals may be infected with tubercle bacilli and therefore have tuberculosis in the incipient stages, without their being able to disseminate the disease to others. in the early stages, they are bacillus-carriers without being necessarily dangerous at that particular time, but the possibility always exists, as the disease develops in the system, that the trouble may assume a more formidable character, and that slowly developing chronic lesions may become acute, and "open," in which case, the affected animal becomes a positive menace to the herd. as the time when the lesions change from the "closed" to the "open" type and the animal becomes a source of danger cannot be determined, the only safe way to do is to exclude the milk of all tuberculous animals from the general supply, whether for direct consumption, or for manufacture into dairy products and to look upon every diseased animal as a menace to the herd. this is rendered all the more necessary when the milk is used for the feeding of children, who are relatively more susceptible to intestinal infection than the adult. the early stages of the disease in cattle are, however, so insidious that no reliance can be placed upon the detection of the malady by physical means. fortunately, in the tuberculin test, a method is at hand, which in a simple, but effective manner, enables the disease to be distinguished in even the early stages, long before recognition is possible in any other way. =tubercle bacilli in dairy products.= when infected milk is used for the preparation of butter and cheese, the organisms inevitably are incorporated in them. in the separation of milk a relatively large part of the tubercle organisms in the milk appear in the cream. in the making of cheese even more of the organisms are held in the curd. in butter and cheese, as in milk, no growth of the organism can take place; however, the vitality of the organism is retained for a considerable number of months. it is not believed that these products are of much importance in the spread of tuberculosis in the human family, since they are not consumed by children to any extent. cream is to be considered as a means of distribution since it is often used by children. =treatment of tuberculous milk.= it is easily possible to treat milk or factory by-products so as to render them positively safe. the process of pasteurization or sterilization is applicable to whole milk, and when effectively done destroys entirely the vitality of any tubercle bacilli. in making such exposure, care should be taken to prevent the formation of the "scalded layer," as the resistance of the organism toward heat is greatly increased under these conditions. in a closed receptacle, 140° f. for 15 to 20 minutes has been found thoroughly effective in destroying this organism. a momentary exposure at 176° f. is likewise sufficient. this is the method that is almost universally used in denmark in the manufacture of the finest butter. in the treatment of factory by-products, heat should also be employed. in denmark, compulsory pasteurization at not less than 176° f. is required. this treatment prevents not only the dissemination of tuberculosis among hogs and young cattle, but is equally efficacious in preventing the spread of foot and mouth disease. the per cent of tuberculous milch cows varies widely in different sections of the country, being greatest in the older dairy sections, and in those supplying milk to the cities, on account of the constant buying and selling of animals, thus giving more frequent opportunity of introducing the disease into the herds. throughout the country at large, probably less than ten per cent of the cows are tuberculous, and it is estimated that at least one per cent of the diseased animals have tuberculous udders. it has been suggested that the dilution of the milk of such animals with that of healthy cows would remove a great part of the danger from milk. in the case where the milk of a large number of herds is mixed, this may be of some importance, but in no case is it safe to assume that dilution of the milk of tuberculous cows is any guarantee of safety. it has been shown that milk, perfectly normal in appearance, coming from a tuberculous udder could be diluted a million times and still produce the disease on inoculation into experimental animals. in the case of swine, the susceptibility is so great that a single feeding of infected milk, even in a very dilute condition, causes with certainty the production of the disease. some observers maintain that the contamination of the milk with the manure of tuberculous animals is of greater hygienic importance, than that coming from diseased udders, since the number of animals having tuberculosis of the lungs and intestines is far greater than those with diseased udders. =economic aspects of bovine tuberculosis.= not only is this disease invested with much importance because of its inter-relation with the human, but from an economic point of view alone, it is undoubtedly the greatest scourge that affects the dairyman. its insidiousness makes it exceedingly difficult to recognize. the consequence is that many fine herds become seriously involved before its presence is recognized. in the main, the disease is introduced into a herd by purchase, often by buying in pure-bred stock to improve the quality of the herd. where the disease has been established in a region for some time, there is also danger that unheated factory by-products, as skim milk and whey, may function in its spread. where such conditions prevail, the spread of the disease in the creamery district is exceedingly rapid. when once introduced into a herd, the disease sooner or later spreads from the originally affected animal to others in the herd. close contact, and close confinement in ill ventilated stables facilitate the spread of the disease, and sooner or later, other animals acquire the trouble. this may all occur while all animals appear in a healthy condition. the symptoms of the disease in the earlier stages are quite indefinite. as the disease progresses, the nutritive functions appear to be disturbed, and sooner or later, the body weight begins to decline, and finally marked emaciation ensues. accompanying this condition, especially when the disease is in the lungs, is a cough, which is generally aggravated with active exercise. while the run-down condition permits frequently of the detection of the disease in the advanced stages, it is wholly impossible with any accuracy to diagnose the trouble in the incipient stages. it is at this stage that the tuberculin test comes to the aid of the stockman. _tuberculin test._ this test is made by the injecting beneath the skin of the animal a small quantity (about 2 c. c.) of tuberculin, and noting the temperature of the animal, before and after the injection. tuberculin, a product of the growth of the tubercle bacillus, when injected into the body causes a marked rise in temperature, in the case of an animal affected with the disease, and no such elevation in the case of a healthy animal. the process of preparing tuberculin makes it absolutely free from danger, so far as liability of producing the disease, or in any way injuring the animal, is concerned. fig. 19 shows the temperature range of both reacting and non-reacting animals. while the test is not absolutely infallible, it is so far superior to any and all other methods of diagnosis that it should take precedence over them. =miscellaneous diseases.= there are a number of diseases that affect both human beings and cattle, the causal organisms of which may be transmitted through the milk. foot and mouth disease is one wide spread in european countries but which has not yet gained a permanent foothold in this country. the ingestion of the milk, which always contains the causal organism, produces the disease in both humans and cattle. in the human the disease is very similar to that in cattle; it may end in death. vesicles are produced in the mouth, on the lips, nose and fingers. the causal organism, which has not yet been demonstrated, may occur in butter or cheese. it is easily destroyed by pasteurizing the milk. [illustration: fig. 19.--temperature curves. 1, the temperature curve of a healthy animal after injection with tuberculin; 2 and 3, the temperature curves of tuberculous animals after injection with tuberculin. (after moore.)] anthrax, actinomycosis (lumpy jaw), rabies, and malta fever are diseases the organisms of which have been found in the milk of affected animals. in case of the first three, while the possibility exists of the infection of human beings by milk, it is improbable that such infection does normally occur. malta fever is becoming an important disease in portions of southern europe. it is produced in man by the use of milk of goats suffering from the disease. the organism causing contagious abortion in cattle is known to be present in the milk of the infected animal at the time of its withdrawal from the udder. it is not probable that the organism is of any sanitary significance as far as man is concerned. it has been shown that the organism is able to produce a disease in guinea pigs on artificial inoculation that is very similar, so far as the lesions are concerned, to tuberculosis. it is also probable that the by-products of creameries and cheese factories may serve to spread the disease from one herd to another. inflammation of the udder (garget) is a frequent trouble in every herd. it is marked by the swelling of one or more quarters, by the appearance of fever and changes in the appearance and composition of the milk. the inflammation may be caused by cold or injury, or by the invasion of the udder with pus-forming bacteria. in the first case the trouble is not likely to persist for any length of time, and does not spread to other members of the herd. the milk may be more or less stringy, and may show a slimy flocculent sediment. it cannot be asserted that such milk is harmful to man but it should be rejected on general sanitary grounds, and because it cannot always be differentiated from that coming from an udder in which the inflammation is produced by bacteria. inflammation caused by the invasion of the udder with specific bacteria is usually of greater severity, the entire gland often becoming involved. the secretion of milk may cease and the function of the diseased quarters may never be restored. the milk in the less severe cases may not be abnormal in appearance, but with increasing severity, the nature of the milk changes, until it may be a watery liquid. the milk of any animal suffering from any form of garget should be rejected, as it may cause trouble, especially in children. there is some reason to believe that organisms coming from cases of garget have been responsible for the extensive outbreaks of septic sore throat that have occurred in some parts of the country. the milk of animals suffering from indigestion, diarrhea, abscesses on any part of the body, as from those which have retained the afterbirth should be likewise rejected. in short only the milk of healthy animals should be used for human food; that from any animal suffering from any disease or which is receiving medical treatment should not be so used. =typhoid fever=. the most important disease germ, distributed through the medium of milk, that is unable to produce a diseased condition in the cow is the organism of typhoid fever. this malady is an intestinal affliction of man, and the germ causing the same is found abundantly in the dejecta, both solid and liquid, as well as in the blood in certain stages of the disease. while the causal organism does not leave the body through the expired air, it is found abundantly in both the urine and feces. therefore, the dejecta, and any articles that may be soiled with the same become a positive menace. many different methods of transmitting the contagion exist, such as water, food infected in various ways, contact with infected persons, and through the medium of milk. milk is not so frequently the cause of dissemination as the other factors, but where milk supplies become contaminated, epidemics of considerable magnitude are wont to occur. the danger from milk is also aggravated by the fact that the typhoid bacillus is capable of withstanding considerable amounts of acid, and consequently finds, even in raw milk containing the normal lactic acid bacteria, conditions favorable for its growth. in a considerable percentage of cases, the disease is not sufficiently severe to cause the patient to take to his bed. these so-called "walking typhoid" cases are particularly dangerous, because they serve to spread the disease organism more widely. a very considerable proportion of the people that recover from typhoid fever still continue to harbor the typhoid bacillus in their urinary and gall bladders. this condition may obtain for years, and since such individuals are in perfect health and are ignorant of their own condition, and since they give off the organisms more or less constantly, they are often the cause of extensive milk borne epidemics. such persons are known as "typhoid carriers" and constitute one of the gravest problems the public official has to contend with in his struggle to prevent the spread of typhoid fever. where outbreaks are caused by milk, they can readily be traced by means of the milk route, as there are always a sufficient number of susceptible persons, so that outbreaks of epidemic proportions develop. in the stamford, conn., outbreak in 1895, 386 cases developed on one milk route. in this case it was shown that the carrying cans were thoroughly washed, but were later rinsed out with _cold_ water from a polluted shallow well. the mode of infection of milk varies, but in general, the original pollution is occasioned by the use of infected water in washing the utensils, or a case of "walking typhoid" or bacillus carrier, who directly infects the milk. in case of sickness in rural families, some member of the household may serve in the dual capacity of nurse and milkmaid, thus establishing the necessary connection. busey and kober report twenty-one outbreaks, in which dairy employees also acted in the capacity of nurses. the fact that the urine of a convalescent may retain the typhoid germ in large numbers for some weeks renders the danger from this source in reality greater than from feces, as, naturally, much less care is exercised in the disposition of the urine. the house fly is now regarded as one of the important means of spreading typhoid fever, indeed it is often called the "typhoid fly." the infectious material deposited in an open vault may serve as a source from which the fly carries the organisms to milk and other foods in the house or elsewhere. the protection of vaults and the screening of every place where human food is handled or prepared is the only protection. it should be emphasized that in the case of the tubercle organism, no growth ever occurs in milk, but with the typhoid bacillus growth is possible. it thus needs but the contamination of the milk with the smallest particle of material containing them to seed the milk. by the time it is consumed it may contain myriads of the disease-producing organisms. =diphtheria.= this is a highly infectious disease, affecting children primarily and is characterized by the formation of membranous exudates in the throat and air passages, which are teeming with the causal organism, the diphtheria bacillus. this organism is capable of forming highly toxic products, and it is to the effect of these poisons that its fatal result is generally due. the organism is thrown out from the body, in the main, through the mouth, the surroundings of the patient being infected directly from the air, and indirectly, by contact with polluted hands, lips, etc. thus, the germ deposited from the lips of a case of the disease, on the common drinking cup, slate, lead pencils, toys, and the like, may easily pass from child to child. not infrequently, the causal organism persists in the throat long after all evidence of membranous growth has subsided, and so the child itself may act as a "bacillus carrier." not so many epidemics of diphtheria as of typhoid have been traced to milk, but the evidence is sufficient to indict milk as a disseminator of contagion. in several cases, the diphtheria germ has actually been isolated from infected milk supplies. actual growth of the diphtheria germ is said to take place in raw milk more rapidly than in sterilized. =scarlet fever.= while the germ of scarlet fever has not yet been isolated, and therefore its life history in relation to milk cannot be depicted so accurately, yet milk-borne epidemics of this disease are sufficiently abundant to leave no doubt but that this food medium may sometimes serve as a means of disseminating such troubles. infection of the milk doubtless comes in the case of this disease from direct contact with a person suffering from the malady. =cholera.= while this disease is of no practical importance in america, owing to its relative infrequency, yet outbreaks of cholera have been traced to milk, in spite of the fact that the causal organism is more sensitive to the action of acids than most disease-producing bacteria. in several outbreaks in india, milk has been the medium through which the disease was spread. generally, infection of the milk has been traced to the use of polluted water. =children's diseases.= an exceedingly high mortality exists among infants and young children in the more congested centers, especially during the summer months. in the main, the cause of these troubles is due to intestinal disturbances, and unquestionably, the character of the food enters largely into the problem. as milk constitutes such a large proportion of the diet of the young, and is so susceptible to bacterial invasion, it would appear probable that much of the trouble of this character is due to the condition of this food supply. this is rendered more probable when it is remembered that bottle-fed infants suffer a much higher mortality than breast-fed children, due probably to the fact that the lengthened period between the time the milk is drawn and consumed permits of abundant bacterial growth. much carelessness also prevails among the poor in cities, relative to the care of utensils used in feeding children. nursing bottles often serve to infect the milk. where milk is pasteurized, or properly heated, it has been found that the mortality rate has been greatly reduced, thus indicating that the condition of the milk was directly responsible for the death rate. in fact, the mortality from these indefinite intestinal troubles probably exceeds that from all of the specific infectious diseases combined. improved care in handling this sensitive food supply will do much to better conditions in this direction. =ptomaine poisoning.= acute poisoning affecting adults as well as children, not infrequently occurs from the use of foods of various kinds. cases of poisoning arising from the use of shell fish, canned meats, ice cream, cheese, and other dairy products, are from time to time reported. these troubles are due to the production of toxic compounds, in the main, probably caused by bacterial decompositions. often such troubles may affect a number of persons, as at banquets and such gatherings, thereby giving the semblance of an epidemic. while such troubles are doubtless to be ascribed to bacterial activity, they are not transmissible from person to person. in the case of troubles arising from ice cream and such confections, the probable cause is due to the storage of milk or cream under refrigerator conditions, where germ growth can go on in the product, and yet the temperature be sufficiently low to prevent the usual acid fermentations. chapter v. fermentations of milk. milk, under normal conditions, is always contaminated with bacteria coming from the most varied sources. if it is produced under clean conditions, the number of bacteria will be small, but in any case, the number of kinds of bacteria that find their way into milk will be large. many of them find in milk at ordinary temperatures suitable conditions for growth; they use a portion of some of the constituents of the milk as food, producing certain other compounds that are known as "by-products." these by-products impart to milk a taste and odor that is not found in fresh milk. the effect of the action of bacteria may also be made evident by the change in the appearance of the milk. when these various changes become evident to the senses, either by taste, smell or sight, the milk usually is so modified as to be unfit for many ordinary purposes. the preservation of milk, a subject to be treated later, is a study of the ways of preventing or retarding the growth of bacteria in milk, and thus delaying the time when evidences of their action first become apparent. each class of bacteria produces more or less specific changes in the milk as a result of their growth. certain bacteria are of the greatest benefit to the butter and cheese maker, while others are distinctly harmful to the manufacturer of dairy products. the changes produced by the different bacteria are called "fermentations" of milk, each being most commonly named from the most important by-product formed. =acid fermentation of milk.= fresh milk has a sweet taste and little or no odor, but if it is allowed to stand at ordinary temperatures, it sours; the taste is no longer sweet because the sweetness of the sugar of the milk is masked by the acid produced from the decomposition of a portion of the sugar by the bacteria. the change in odor and taste of milk is apparent long before the appearance is altered and increases in intensity as the acid-fermentation progresses. the first alteration in appearance is most usually one of consistency; the liquid milk is transformed into a semi-solid mass. the terms "curdling" and "sour" are usually synonymous. milk is, however, often said to be sour as soon as the acid fermentation has progressed to a point where it is evident to taste or smell. this process of souring, or the acid fermentation is so common a change that raw milk which does not show this type of fermentation is looked upon with suspicion, and, usually, justly so. the process in the past was thought to be something inherent in the milk, a natural and inevitable change. it is now known that this is not so, but that it is due to certain kinds of bacteria, and that if these are prevented from getting into milk, it will not sour, but will undergo some other less desirable type of decomposition. the acid-forming bacteria comprise but a very small part of the total number of organisms that find their way into the milk during its production on the farm, yet in sour milk scarcely any other kinds of bacteria can be found. at ordinary air temperatures, the acid-forming bacteria grow more rapidly in milk than do any other forms, and the acid produced by them renders the milk an unfavorable medium for the growth of other bacteria. this is the reason why milk practically always undergoes the acid fermentation, although it is contaminated with a host of other kinds of bacteria. if a mixture of seeds is sown on low wet ground, certain kinds will grow best; if the same mixture is sown on drier land, other types will find most favorable conditions for growth, and the plants which appeared on the low land will not appear. the same condition is found in milk where the environment is most favorable for the acid-forming bacteria. =amount of acid formed in milk.= in this country the acidity of milk is expressed as so many per cent of lactic acid. a milk that shows an acidity of one per cent should, theoretically, contain one pound of lactic acid in each one hundred pounds of milk. the acid determined does not actually represent lactic acid, as there are other substances in milk which act as acids, with the reagents used in the present methods of determining the acidity of milk. for instance, perfectly fresh milk has an apparent acidity of 0.13 to 0.18 per cent, although no fermentation has occurred. other acids than lactic are formed in the acid fermentation, but the entire acid content is referred to as lactic when speaking of the acidity of milk. when the developing acidity of milk reaches 0.25 to 0.3 per cent, a sour taste becomes evident and the milk will curdle on heating. when the acidity increases to 0.6 to 0.7 per cent, the milk curdles at ordinary temperatures. the acidity continues, however, to increase until it reaches about 1 per cent, which is the maximum amount that will be produced in milk by the ordinary acid-forming bacteria. milk contains about 4 per cent of milk sugar, all of which is fermentable. if this were all decomposed by bacteria, the acidity of the milk would actually exceed 4 per cent. it is thus evident that the reason why more acid is not formed in milk is not because of any lack of sugar. the bacteria, like all other kinds of living things, are injured by their own by-products, unless these are constantly removed in some way; in milk the bacteria cannot escape the action of the acid which they themselves have formed, consequently growth ceases. the amount of acid formed is dependent on the kind of bacteria present and on the composition of the milk. certain bacteria will not produce enough acid to cause the curdling of the milk; still others will form 2 or even 3 per cent. these types, however, do not play any important part in the spontaneous souring of milk. in milk the acid first formed combines with the ash constituents and the casein to form salts which do not seriously affect the growth of the bacteria. ultimately, the limit of the ash and casein to take up acid is reached, and free lactic acid which is harmful to bacterial growth appears. if the content of casein and ash constituents is high, a higher degree of acidity will be reached than in a milk with a lower content. if a large part of the volume of the milk is made up of a compound that has no role whatever in the acid fermentation, such as the butter fat in cream, the amount of acid formed per unit volume of milk will be reduced, since in determining the acidity, a definite volume of milk is taken, and the acidity is expressed, as such a per cent of this amount. =types of acid-forming bacteria.= when substances undergo decomposition, it is a common belief that compounds offensive to the odor and taste are formed; but such is not necessarily the case. the products of the decomposition may be as agreeable and as harmless as the compounds decomposed. whether the decomposition products of any substance are offensive or not is dependent on the kinds of micro-organisms acting on it. there are forms of acid-producing bacteria that change milk in odor, taste, and appearance, yet the sour milk is not offensive in any sense of the word. other bacteria also sour the milk, but produce offensive odors and a disagreeable taste. thus, the acid-forming bacteria may be divided into two main groups, which may be designated as desirable and undesirable. this division is of importance to the butter and cheese maker and to the consumer of milk. =desirable acid-forming bacteria.= if milk is produced under clean conditions, it is not likely to have a disagreeable odor or taste at any time, even when it is sour; rather the taste is agreeable like that of good butter milk. the curd is perfectly homogeneous, showing no holes or rents, due to the development of gas, and there is but little tendency for the whey to be expressed from the curd. this type of fermentation is largely produced by the group of bacteria to which has been given the name, _bacillus lactis acidi_. the main by-product of this group of bacteria is lactic acid; small amounts of acetic acid and alcohol, with traces of other compounds, are also formed. the agreeable odor and to some extent the flavor of milk fermented by these bacteria is due to other by-products than lactic acid, for this has no odor and only a sour taste. the acid fermentation of milk is often called the lactic acid fermentation. in reality only the fermentation produced by the desirable group in which lactic acid is the most evident by-product should be thus called. [illustration: fig. 20.--different types of curds. on the left a solid, homogeneous curd produced by desirable bacteria; on the right, the curd produced by harmful bacteria. note the gas holes and free whey.] the bacteria of this group may enter the milk from the dust coming from the coat of the cow. they are also found in the barn dust and on cultivated plants. under ordinary farm conditions, the larger part of those found in milk come directly from the utensils. if the milk is drawn under extremely clean conditions and care is taken to sterilize the utensils, but few acid-forming bacteria of any kind will enter the milk; under such conditions most of the acid-forming bacteria will belong to the group in question. they find, however, such favorable conditions for growth in milk that they develop more rapidly than most other types with which milk becomes seeded; consequently under normal conditions, they gain the ascendency and so control the type of fermentation. the desirable type of acid-forming bacteria do not form spores; hence, are easily killed by heating the milk. they can grow in the presence or in the absence of free oxygen. in the bottom of a can of milk or in the middle of a cheese, there is no air, yet these bacteria grow as well under these conditions, as in milk exposed to the air. the range of temperature for growth varies from 50° to 100° f. but development is most rapid at 90° to 95° f. and about 1 per cent of acid is formed. another group of bacteria which may be classed among the desirable acid-forming organisms is constantly found in milk. they have little to do with the ordinary acid fermentation as they grow very slowly at ordinary temperatures. if a sample of raw milk is placed at the temperature of the animal body, the acidity will reach 1 per cent in a few hours. thereafter the acidity will increase slowly and may reach three per cent or above. the continued increase in acid is due to the growth of long rods of the _bacillus bulgaricus_ type, which apparently enter the milk with the fecal matter. the nature of the change produced by them in milk is very similar to that caused by _bact. lactis acidi_ in that lactic acid is the chief product; no gas is produced and hence the curd is uniform in appearance. temperatures from 100° to 110° f. favor their development. organisms belonging to this group are used in the preparation of the fermented milks now so widely sold in the cities. these desirable, acid-forming bacteria are of the greatest service in every branch of the dairy industry, whether in butter or in cheese making, or in the sale of milk in the city. the dairy industry is dependent upon fermentative activity, as much as the manufacture of beer or wine, and the main basis of this is the acid fermentation of the milk by these desirable types of bacteria. although milk contains a large amount of nitrogenous substances (casein and albumen), it does not undergo putrid decomposition, as do meat and eggs, not because it is not fitted for the growth of the bacteria causing that type of change, but because the acid formed in it stops the growth of the putrefactive bacteria. if a sample of milk is placed in a stoppered bottle, it will have much the same taste and odor at the end of several months as at the end of a few days. the acid acts as a preservative, like the vinegar in pickles, or the acid in silage and in sauerkraut. meat placed in a stoppered bottle which is then filled with milk will be preserved. the products formed in the decomposition of meat and eggs are not only offensive but may also be injurious to the health of the consumer. milk that has been fermented by the desirable kinds of acid-forming bacteria is not harmful. it is consumed in a variety of forms (buttermilk, cottage cheese) as a common article of food and its use is rapidly increasing. the preparation of the pure culture buttermilks or artificially soured milks that are now so frequently recommended for digestive troubles rests upon an acid fermentation of this type. =undesirable acid-forming bacteria.= other types of bacteria capable of forming substances that impart to milk an offensive odor and a disagreeable taste not infrequently appear instead of the desirable group. instead of producing from the sugar of milk large quantities of lactic acid, these types generate other acids, such as acetic and formic, which impart a sharp taste to the milk. besides the acids the bacteria of this group form gases from the sugar of the milk. some produce small amounts of gas; others so much that the curd will be spongy and will float on the surface of the whey. the fermentation caused by them is often called a "gassy fermentation" and is dreaded by butter and cheese makers since the gas is indicative of bad flavors that will appear in the product. gas may also be produced in other types of fermentations to be discussed later. this class of bacteria enters the milk with the dust, dirt, and manure, in which materials they are especially abundant. no spores are formed; hence they are easily killed by heating the milk. they grow both in the presence and in the absence of free oxygen. high temperatures favor their growth, most rapid development taking place at 100° to 103° f. =spontaneous fermentation of milk.= the normal souring of milk is due to a mixture of these two groups of bacteria. the relative proportions existing between the two in any sample of milk is dependent on a number of factors, most important of which is the degree of cleanliness exercised in the production of the milk. where careless conditions obtain under which dust and manure particles find their way into milk, it becomes more abundantly seeded with gas-generating bacteria, and consequently, the type of fermentation is undesirable. if, however, the milk is drawn into clean utensils and care is taken to exclude dirt, the pure lactic acid types are able to control the character of the changes produced, and a clean, pleasant tasting liquid results. it will be seen that things are well arranged by nature; one of the most important food products undergoes a type of decomposition that is not offensive and when produced under clean conditions, the sour milk is as healthful a food as is the fresh product. thus there is every reason for cleanliness in the production of milk, for cleanliness' sake and because clean milk means better products, and greater returns to everyone, producer and dealer. there are other kinds of acid-forming bacteria in milk but they are of small importance compared with those just discussed. some of the bacteria derived from the inside of the udder of the cow form acid, but these forms grow very slowly in milk at ordinary temperatures, and have no influence on the keeping quality. [illustration: fig. 21.--different types of curds. the flask on the left shows the soft curd produced by the bacteria that curdle the milk without the production of acid. the flask on the right shows the gassy curd formed by butyric acid bacteria in heated milk.] =sweet curdling fermentation of milk.= samples of milk are sometimes found that are curdled, but which do not taste sour, or have the normal odor of sour milk. the curd is usually soft and the taste bitter. it is evident that the curdling cannot be due to the same factors as in the normal souring of milk. such a change is similar to the action of rennet which is used to curdle the milk in cheese making. this ferment will curdle perfectly sweet milk, producing a curd that looks like that formed in the acid fermentation of milk. the cause of these sweet curdling milks, which appear from time to time, is due to the introduction of certain bacteria which have the power of secreting an enzyme resembling that found in rennet. in such cases the milks curdle prematurely especially when warmed. the curd may gradually disappear, for the bacteria also produce another enzyme that digests the curd, and thus renders it soluble. when this advanced phase becomes evident, it is often called the _digestive fermentation_ of milk. this change is produced largely by putrefactive bacteria of various kinds that find their way into milk with dust and dirt. many of them are spore formers; hence, are not killed when milk is heated, as in pasteurization, while the acid-formers are destroyed. pasteurized milk is thus likely to undergo the sweet-curdling fermentation, if it is kept for any length of time. raw milk rarely undergoes this type of decomposition, since the rennet-forming bacteria under ordinary conditions are unable to develop in competition with the acid-forming bacteria. =butyric acid fermentation of milk.= a fermentation that is much less frequently noted than the two previously discussed is known as the butyric fermentation, since butyric acid is the principal by-product. the causal bacteria cannot compete with the ordinary acid-forming bacteria in raw milk; hence it is most frequently noted in pasteurized milk, since the organisms produce spores and are not killed by the heating. pasteurized milk under the action of the butyric acid bacteria undergoes a gassy fermentation, developing a pronounced acidity and the disagreeable odor of butyric acid, which resembles that of rancid butter. the butyric acid bacteria are anaerobic, and thus can grow in butter and cheese away from the air. =slimy or ropy fermentation of milk.= a slimy or ropy condition of milk is frequently noted on the farm and in the dairy. several causes for this abnormal condition exist. sometimes the milk may be slimy when milked from the cow. this occurs most frequently in the case of inflammation of the udder which may or may not be due to bacteria. the direct cause of the abnormal condition in milk is the presence of fibrin and white corpuscles from the blood which form masses of slimy material; in such cases the trouble does not increase in intensity with age, nor can it be propogated by transference to another sample of fresh milk. [illustration: fig. 22.--slimy milk. it does not mix with water when poured into it.] another type of slimy milk is produced by the growth of certain types of bacteria which enter the milk after it is drawn from the udder. these may come from various sources. the bacteria concerned belong to two groups: (1) those that grow best in the air and do not form acid; (2) those that grow in the absence of air, throughout the entire mass of milk and which form acid. the slimy condition is noted in the milk only after the milk has been stored for some time; it usually increases with the age of the milk and can be produced in a second sample by transferring a little of the slimy milk to it. the fermentation produced by the aerobic bacteria is most often met in bottled milk and cream during the warmer times of the year. on account of their relation to oxygen, the growth is confined to the surface of the milk and only the upper layer becomes slimy; thus when the cream is removed, the abnormal condition is noted. the sliminess is due to the mass of bacterial growth rather than to the production of any specific substance in the milk. this trouble may be of considerable economic importance to the dealer, as such abnormal milk is objectionable for ordinary use, but as far as is known, it is incapable of affecting the health of the consumer. in numerous outbreaks of this trouble the source of contamination has been traced to infection from well water or a stream, as the organisms causing the trouble are found naturally in water. keeping the milk in a tank in the pump house sometimes permits of troubles of this sort, the water used for cooling giving opportunity for contamination. cattle wading in a stream sometimes pollute their udders and so indirectly infect the milk. such outbreaks rarely persist for any considerable length of time as the common acid organisms soon regain the ascendency. creameries and cheese factories are sometimes troubled with sliminess in starters. this seems to be due to some change which the ordinary lactic acid bacteria undergo on long propagation rather than to contamination of the starter. there are, however, types of acid-producing bacteria that are able to form specific substances in milk that are slimy in character. two of these forms of slimy milk are of economic importance. the slimy whey (lange wei) of holland is added to milk in the manufacture of edam cheese, apparently serving the same purpose as the addition of the pure culture starter in cheddar cheese making. in norway, a sour, slimy milk (taettemjolk) is used as food. it is produced by the addition of some previously fermented milk. this beverage is also used in some of the norwegian settlements of wisconsin, the original seed having been brought from norway, and the bacteria maintained by constant propagation from one sample of milk to another. the milk has the odor and taste of butter milk, but is not especially appetizing in appearance to any one not accustomed to it; it is, however, as harmless to health as is any other form of sour milk. it is not known that any of these forms of slimy milk are distinctly harmful to the quality of butter or cheese. =alcoholic fermentation of milk.= the bacteria as a class are incapable of producing alcohol in appreciable amounts. the alcoholic beverages, beer, wine, and cider, are produced by the growth of yeast, in such sugar containing liquids as fruit juices, extracts of grains, etc. the common types of yeasts are incapable of acting on milk sugar, but they can ferment glucose, maltose, and cane sugar, forming equal amounts of alcohol and carbonic acid gas, which causes the effervescence of fermented and carbonated drinks. there are, however, some types of yeasts found in milk and its products that are able to ferment milk sugar. all yeasts grow best in an acid medium, hence those fermenting milk sugar find suitable conditions for growth in sour milk or whey. they may at times become of economic importance in the cheese industry, because of the contamination of the milk with large numbers of them. the arrangement of the whey vat is often such that it cannot be completely emptied and cleaned; the sour whey thus presents favorable conditions for the growth of the lactose-fermenting yeasts. the return of the whey to the farm in the milk can that is often imperfectly cleaned may serve to contaminate the milk with the yeast. in the making of swiss cheese the whey is often so handled as to favor especially the growth of such yeasts, and since this type of cheese is prepared from sweet milk, the competition between the yeast and the acid-forming bacteria is not so sharp as in the making of cheddar cheese. the writers have found several instances where considerable loss was occasioned in the swiss cheese industry through the development of gassy cheese due to this type of fermentation. the yeasty or alcoholic fermentation may also be of importance in butter making. in many sections of the country the milk is separated on the farm and the cream is forwarded to the creamery at more or less infrequent intervals. it becomes sour and if it has become contaminated with yeasts, they will find favorable conditions for growth in the acid medium. a large amount of carbon dioxide gas is produced. cans of gathered cream often foam to such an extent as to run over, and in some cases actual explosions have occurred on account of the great pressure caused by the gas. =bitter fermentation of milk.= bitterness in milk may be due to bacteria that enter the milk after it is drawn from the cow, or it may be caused by the feed consumed by the animal. it has been previously shown that certain specific substances contained in the food may be absorbed and reappear in the milk. if the animal eats ragweed, lupines, or other plants containing bitter substances, the milk is likely to have a bitter taste, which will be noticeable at the time the milk is drawn. the milk of cows at certain advanced stages of lactation may show a bitter taste, due to a change in the ash constituents of the milk in which the lime salts are largely replaced by salts of sodium. there are many bacteria that will impart to milk a bitter taste. milk that has undergone the sweet-curdling fermentation is likely to be bitter, as is the ease with pasteurized milk. some of the acid-forming bacteria are able to develop a bitter principle, the milk retaining a pleasant odor and having the normal amount of acid, while the taste is intensely bitter. one of the authors (h) found in the case of a wisconsin brick cheese factory, that the usual acid organism was almost wholly replaced by a bitter type. storage of milk at very low temperatures is conducive to the appearance of a bitter taste in milk, the explanation in this case being that the acid-forming bacteria are unable to grow at a low temperature, while some of the putrefactive forms can multiply and develop these astringent or bitter by-products. =miscellaneous fermentations of milk.= there are a number of other abnormal fermentations in milk that occur so rarely as to be of but little economic importance. some, as the colored milks, are however, quite striking, and on this account have had much attention directed to them in the past. there are bacteria that are able to produce various colored substances, such as red, yellow, and blue. in case milk becomes seeded with large numbers of any of these kinds, it is very likely to be colored by the growth. red milk may be due to bacteria, but more frequently is caused by the actual presence of blood in the milk, due to a wound in the udder, or the effect of a severe case of inflammation of this gland. such a condition may be readily distinguished by allowing the milk to stand for a short time, in which case, if due to blood, the red corpuscles will soon settle to the bottom of the container, while bacterial troubles producing a red coloration are more evident on the surface. it is also claimed that certain bacteria may impart a soapy taste or turnip flavor to milk. =cycle of fermentations in milk.= if a sample of milk is allowed to stand, it will undergo a certain sequence of fermentations that well illustrates the principle that one type of organisms is dependent on some other type to furnish suitable conditions for its development. this cycle of changes that normally occurs in milk is as follows: (1) the bacteria that come from the interior of the udder are the first to develop, but usually the change they produce is not evident. (2) of the types that gain admission, subsequent to the milking, the acid-producing species are able to adjust themselves most perfectly to the conditions that obtain in milk. within a few hours they greatly predominate and soon the milk curdles under the production of acid. their growth, however, is soon stopped by the accumulation of their own by-products. (3) the semi-solid curdled milk, on account of its acid reaction then becomes a favorable medium for the growth of molds; a prevalent form, known as _oidium lactis_ usually develops as a white velvety layer. the molds in their growth form alkaline by-products, which tend to neutralize the acid reaction, so that in the course of two to three weeks, if the layer of the milk is not too deep (an inch or less), the chemical reaction of the milk becomes neutral or alkaline. (4) the putrefactive bacteria which found their way into milk when it was first drawn, and which have remained dormant in the sour milk, now find favorable conditions for growth. as a result of their activity, the milk soon undergoes a putrid decomposition, which is marked by offensive odors. if the milk is placed under such conditions as will exclude the growth of the mold, such as where the air is excluded from the surface, the sour milk will remain in that condition for an indefinite period, since the putrefactive bacteria are inhibited in their development by the acid, in a manner comparable to the preservation of pickles in vinegar, or the keeping of silage because of the acid that is produced as a result of the changes that the plant tissue undergoes when excluded from the air. the preservative effect of acids is of much importance in the case of certain dairy products (see chapter viii). =fermented drinks from milk.= within the last few years a great deal of attention has been directed toward the preparation of various kinds of drinks from milk. the use of such beverages has rapidly increased. butter milk is one which meets with the greatest approval. the true butter milk from cream that has been soured by the desirable acid-forming bacteria has a mild agreeable acid taste, wholly free from any sharpness that is often noted in butter milk made from cream in which considerable numbers of the undesirable acid-forming bacteria have grown. butter milk made from pasteurized cream soured with pure cultures will have good keeping qualities and is a most healthful drink for all classes of people, even for young children. butter milk is also prepared by allowing milk to sour and then breaking up the curd by stirring. if the type of fermentation is controlled as may be done (see chapter vii), such a form of fermented milk is a most desirable drink. it is probably as healthful and has all the therapeutic properties that are ascribed to other forms of fermented milks such as the bulgarian "yoghurt." this type of fermented milk is produced by an acid-forming organism that can form large amounts of acid, 2.0 or 3.0 per cent. the casein is dissolved to some extent and the remainder so changed, that it will remain in suspension for a long time in a finely divided form, after the curd has been broken up. such milk is sold under various names at home and abroad. one of the authors (h) has found such organisms in practically all milks examined. if raw milk is kept warm (98° to 100° f.) in a stoppered bottle which is filled full, the acidity will be found to increase slowly from day to day, reaching a maximum in ten to fourteen days. if the milk is then examined, it will be found to contain large numbers of an acid-forming organism very different in appearance from the bacteria causing the rapid souring of milk at ordinary temperatures. this organism is very similar if not identical with the one found in the bulgarian milk to which the name _b. bulgaricus_ has been given. the use of the milk fermented by this organism has spread rapidly because it is claimed by certain european bacteriologists that it has a favorable effect on the health of people, especially those suffering from intestinal troubles. it is not at all certain that ordinary sour milk or butter milk will not have the same effect; in fact in many of the fermented milks sold in europe, _b. bulgaricus_ has not been found, but only the ordinary lactic acid bacteria. several alcoholic drinks made from milk, such as kefir and koumiss, have been originated among the nomadic tribes of western asia. kefir is prepared from cow's milk by adding the kefir ferment in the form of grains which contain a number of kinds of bacteria and a yeast. the acid-forming bacteria impart a sour taste to the fermented milk, while the yeast forms carbon dioxide and about two per cent of alcohol. if the milk is allowed to ferment in stoppered bottles, the resulting product will be an acid effervescing drink, which is claimed to be more easily digested than sweet milk. this drink is used frequently in the treatment of invalids but it is improbable that it is more easily digested than ordinary soured milk or butter milk. the grains are removed from the fermented milk, and are then added to a quantity of fresh milk, or they may be dried and kept for future use. when needed again, they are soaked in water, then added to the milk. koumiss is made in russia from mare's milk and has much the same composition as kefir. in america and europe it is made from cow's milk, by adding cane sugar and compressed yeast. the yeast ferments the cane sugar while the acid-forming bacteria ferment the milk sugar. there is thus obtained a drink that is similar in composition to the real koumiss, in which both the acid and the alcohol come from the fermentation of the milk sugar. in koumiss and kefir the curd is very finely divided and will remain in suspension for a long time as with butter milk. =determination of the cause of taints in milk.= it is often of the greatest importance to be able to locate the cause of abnormal odors or tastes in milk, since methods for overcoming the trouble can be intelligently applied only when the actual cause is known. an abnormal condition may be caused either by the direct absorption of odors before or after the milk is drawn from the animal, or it may be due to bacteria. if the milk appears bad-flavored when first drawn, and if such taint becomes less pronounced as the milk becomes older, it is likely that the trouble is due to some characteristic of the feed. certain feeds, like green rye, rape, cabbage, and certain of the root crops, like turnips, impart a strong odor to milk, if the same are fed shortly before milking. if the tainted condition appears only some time after the milk is drawn, it may be due to the direct absorption of taints from the surroundings in which the milk is kept, or it may be caused by bacteria. these causes can often be differentiated, by noting whether the taint tends to increase in intensity with age. if such is the case, it is likely that the cause is of germ origin, but if the reverse is true, it cannot be ascribed with certainty to bacteria and recourse must be had to other methods, such as the transfer of a small quantity of the tainted milk to a sample of perfectly fresh milk, or preferably to some milk that has been heated to the boiling point and then cooled. in the case of an odor due to direct physical absorption, it will not appear in the inoculated sample, since the small amount transferred is not sufficient to be noted. if it is due to living organisms, the inoculation of the smallest quantity into a fresh sample is likely to reproduce the same change as originally noted. =tests for the bacteriological condition of milk.= within certain limits milk can be indirectly examined as to its bacterial content without any special equipment. milk when drawn from the cow has an apparent acidity ranging from 0.16 to 0.18 per cent. by the use of any of the methods of determining acidity in milk, much can be told concerning the number of bacteria in the milk, and hence concerning its keeping quality. milk that has an acidity of over 0.2 per cent is certain to contain many bacteria, and consequently will keep poorly. such milk is of low value for market milk, but may not be objectionable for butter or cheese making. if the acidity is below 0.2 per cent, but little can be told as to the numbers of bacteria, since any increase in acid is always preceded by an enormous increase in the numbers of acid-forming bacteria. a more important test than the acid test, from the standpoint of the butter and cheese maker, and even the milk dealer, is the fermentation test. in its simplest form, it consists in placing a sample of the milk to be tested in a warm place and noting the time required to curdle and the type of curd formed. in this country the fermentation test has been largely supplanted by the wisconsin curd test which possesses the advantage of detecting the presence of bacteria harmful in cheese making, especially the gas forming bacteria. the curd test is helpful in detecting the source of an abnormal condition in a milk supply coming from diverse sources. the milk furnished by each patron can be tested separately and the trouble located, perhaps in an individual herd; the offending herd determined, the test may then be used on the milk of individual cows. the cheese maker and the milk dealer should be able not only to detect which of the patrons furnish him poor milk, but he should be able to give the patron definite instructions how to avoid the sources of such trouble. this information can be given only when the source is positively known. [illustration: fig. 23.--curd test. a good curd obtained from milk containing no harmful bacteria but many desirable acid-forming organisms.] the wisconsin curd test is made as follows: samples of the milk to be tested are placed in sterile pint fruit jars. the milk is warmed to 90° f., ten drops of rennet are added to each sample, and as soon as the curd is solid, it is cut into small pieces with a case knife so as to facilitate the expulsion of the whey. as the curd settles to the bottom of the vessel, the whey is poured off at intervals so that a pat of firm curd is left. as the milk curdles the bacteria are enmeshed and are carried with the curd. the jars are kept at a temperature of 100° to 105° f., since this temperature is favorable to growth of the bacteria that are sought, the gas-forming organisms. at the end of ten to twelve hours, the jars are examined; if the curd is solid, the texture firm, not mushy or slimy on the surface, if the odor is agreeable, it indicates that the milk contains few or none of the undesirable forms of bacteria. if the curd is full of gas holes, it is apparent that undesirable bacteria are present and under such circumstances the curd will not have an agreeable odor. if the gas-forming bacteria are numerous, the curd may even be spongy from the abundance of gas holes, and the undesirable odor more pronounced. such curds are tough and rubbery. in some cases a bad flavor or odor is apparent even though the texture of the curd is not open and full of holes. the curd, the surface of which is slimy indicates undesirable organisms. a solid curd of agreeable odor is indicative of the presence of the desirable acid-forming bacteria. such a milk is excellent from the standpoint of the butter or cheese maker, but may not be so desirable from the standpoint of the milk dealer on account of its poor keeping qualities. on the other hand a milk suitable from the standpoint of the milk dealer, on account of its low germ content, and hence good keeping quality, may give a poor curd test. it is certain to contain some bacteria, especially those from the interior of the udder while it may contain none of the desirable acid-forming organisms without which a curd of good texture and flavor can not be obtained. the bacteria in the clean milk will grow rapidly at the high temperatures at which the curds are kept and the changes they will produce as to flavor and odor may be undesirable. the milk might be judged as poor when in reality it might be a most excellent sample, and if kept at the ordinary storage temperatures, it might keep for days. the test when used for market milk should be interpreted with this in mind. [illustration: fig. 24.--curd test. the curd obtained from milk containing many gas-forming bacteria. the irregular, angular holes are mechanical, due to the imperfect fusion of the pieces of curd.] if the results are to be of any value, the test must be made with care to avoid all sources of error; the tester must know that the bacteria causing the gas and bad flavors in the sample were originally present in the milk at the time the sample was taken, and that they have not come from the containers used or from other sources. to insure these conditions the jars must be thoroughly cleaned and then sterilized just before use by placing them in cold water and bringing them to the boiling point, or sterilized by a thorough steaming. the sample of milk of a patron must be taken so as to avoid contamination from the milk of the other patrons. this can best be done by filling the jars as the milk is poured from the patron's can into the weigh can. in cutting the curds, the knife used must be dipped in hot water between each test to cleanse the same. in short, the test should be carried out with great care so that the tester is certain of the results obtained. other tests for the bacteriological condition of milk will be described in chapter ix. =overcoming abnormal fermentations.= the lactic acid bacteria are often looked upon as normal to milk, and it is certain that they are to be classed as harmful, only as they injure the keeping qualities of milk. in milk designed for butter and cheese their presence is necessary. at times these desirable forms of bacteria may disappear, and be replaced by less desirable types. in one case it was observed that the usual lactic bacteria had been replaced in a cheese factory supply by an acid-forming organism that produced an intensely bitter taste in the milk, thus rendering the cheese of no value. when such harmful forms appear, they must be overcome, and the normal types of bacteria replaced. a thorough cleaning of the milk utensils, attention to the cattle and all places from which such bacteria may find their way into the milk is often sufficient to cause a disappearance of the trouble. if the acid-forming bacteria have disappeared, the inoculation of the milk with cultures in ways later to be discussed is often of advantage. at times more stringent measures must be employed in order to destroy the harmful bacteria, such as the use of strong disinfectants. =disinfection and disinfectants.= if any building or room becomes infected with disease-producing bacteria, or if organisms causing abnormal fermentations become established in a factory, the use of a disinfectant that will destroy with great rapidity the life of bacteria is necessary. the disinfection of all types of dairy apparatus and utensils can be accomplished by thorough cleansing, and by the use of steam or boiling water. the disinfection of rooms and stables cannot be so readily accomplished. consideration must always be given to the resistance of the organism it is desired to destroy. those that form spores are very resistant toward all chemical agents, while those that do not produce these resistant bodies are easily killed. in the dairy and factory, it is often necessary to destroy the organisms that develop in decomposing organic matter. here, as in all disinfection, a thorough cleaning should precede the application of any disinfectant. some chemicals act as deodorants, _i.e._, destroy the offensive odor, without removing the cause. it is impossible effectually to destroy bacteria embedded in a mass of organic matter, and through the removal of the material itself, the larger part of the bacteria will be removed. the disinfectant then comes in direct contact with the surface to be disinfected, consequently destroys the bacteria not removed in the cleaning. all places in which dairy work of any kind is done should be provided with an abundance of light and air. the direct rays of the sun have a powerful disinfecting action, and light makes evident accumulations of dirt that in a darker room would be unnoticed. ventilation keeps the rooms dry and thus prevents the growth of mold and the development of a musty odor. disinfectants are divided into two classes: (1) solid materials used in suspension, or in watery solutions; (2) gaseous substances. the latter are preferable for room disinfection when their use is permissible, for the gas penetrates to every part of the space, even into the cracks. gaseous disinfectants can only be used when the space is tightly closed, for the gas must be confined for several hours in the room, in order to make the process effective. such disinfectants can often be used to advantage in the treatment of refrigerators and cheese rooms to destroy mold spores. in less tightly closed spaces, reliance must be placed on the use of the solid or liquid disinfectants. =lime.= quick lime or stone lime has a considerable disinfecting action. on exposure to the air, quick lime becomes air slaked, and then has no disinfecting action whatever. water-slaked lime used in the form of white wash, lime water, or the powder is effective. air-slaked and water-slaked lime are similar in appearance, but a difference can be noted by placing a particle of each on the tongue; the air-slaked tastes like chalk while the water-slaked material causes the tongue to burn. white wash is one of the most effective agents that can be used in the disinfection of barns, milkrooms, etc. besides being a fairly strong disinfectant, it has a tendency to absorb odor, it encrusts the walls and lightens the interior of rooms. it can be applied with a brush or with a spray pump. =carbolic acid and cresol compounds.= these substances are among the cheapest and best disinfectants, but their use in the dairy is not advisable, on account of the penetrating and lasting odor. they can be used to advantage on the farm. some of the proprietary compounds, as zenoleum, kresol, etc., are easily applied, since they mix readily with water in all proportions, forming a milky-white emulsion that can be easily applied. they are less caustic and less poisonous than carbolic acid. =corrosive sublimate.= corrosive sublimate is the most efficient disinfectant under ordinary conditions. it is such an intense poison that it must be used with caution in places to which stock have access, or in the dairy. a solution of one part of the salt to a thousand parts of water (half ounce to 4 gallons of water) is the standard generally used. for gutters, drains, and waste pipes in factories, ferrous sulphate (green vitriol), and copper sulphate (blue vitriol), can be used to advantage. they are to be classed as deodorants rather than as true disinfectants. since they have no odor of their own, they can be used in any amount in the dairy. =sulphur= can be used to advantage in the destruction of mold spores in cheese rooms, but the effect of the vapors of burning sulphur on germ life is relatively slight, unless there is an abundant supply of moisture in the air of the enclosed space, in which case sulphurous acid is formed which has a much greater effect. to have the desired effect sulphur should be burned at the rate of three pounds to each one thousand cubic feet of space, and the room kept sealed for at least twelve hours. if the sulphur is placed in an iron kettle which is set in a vessel of water, danger from fire will be avoided, and the heat generated by the burning sulphur will evaporate sufficient water to increase the effect of the fumes. =formalin.= another disinfectant that may be used as a liquid or as a gas is formalin, which is a watery solution of the gas, formaldehyde. it is much more powerful in its action than sulphur, and has a great advantage over corrosive sublimate and other strong disinfectants in that it is not so poisonous to animals as it is to bacteria and fungi. it can be used as a solution (one to five per cent) for the washing of woodwork, or for the treatment of any object, since it has no corrosive action. it can also be employed as a gaseous disinfectant for the treatment of rooms. it is most conveniently applied by suspending large cloths in the room and spraying them with the solution, then closing the room for a number of hours. =bleaching powder.= chloride of lime, or bleaching powder as it is often called, is a good disinfectant, as well as a deodorant. it is used as a wash in the proportion of four to six ounces to a gallon of water. it must be used with care in factories since the free chlorine that is given off has a penetrating odor. chapter vi. preservation of milk. it has been shown in a previous chapter that milk becomes contaminated with a multitude of bacteria not only on the farm where it is produced, but during the various stages prior to its use. many of the bacteria which find their way into milk are readily able to develop, and by their growth, render the milk unfit, or even harmful for human food. with the most stringent precautions that can reasonably be taken, it is impossible to avoid all contamination; hence, all grades of milk will soon spoil, unless some means of preservation is employed. indeed, of all the foods classed as perishable, milk is the one that most rapidly deteriorates. produced under ordinary conditions, it is unfit for ordinary use in a few hours if kept at 70° f. there are three possible ways by which milk may be preserved: (1) the removal of bacteria that have gained entrance to it; (2) the prevention of growth of the contained bacteria; (3) the destruction of the contained organisms. in practice at least two and sometimes all of these methods are employed. the prevention of contamination, a subject discussed in chapter iii is in reality one of the most efficient means of preserving milk. in milk production, as elsewhere, prevention is preferable to cure. milk produced under such conditions that its germ content is but a few thousand per cubic centimeter will keep much longer than that handled in the ordinary manner. it might naturally be supposed that any method by which dirt is removed from milk would improve the keeping quality of milk, due to the reduction of bacteria, yet while the straining of the milk at the time of milking removes dirt of various kinds, it does not appreciably enhance the keeping quality, owing to the fact that the bacteria adherent to the dirt particles are washed off in straining, and pass through the pores of the strainer. =filtration of milk.= it is possible to remove all bacteria from water and other fluids and thus render them sterile by passing through filters of unglazed porcelain. this process can not be used with milk for the fat globules are larger than the bacteria (see fig. 6) and any process that would remove the latter would also remove the former. the term "filtration" is applied to a process used in some european cities for the removal of the insoluble dirt that has been introduced into the milk. suitable containers are filled with layers of coarse sand at the bottom and with finer sand at the top. the milk is introduced at the bottom and is forced upward through the sand. such a filtering process is a very efficient means of removing the dirt; but unless the filters are kept scrupulously clean, the bacteria are likely to grow in the filtering material, so that the number of organisms in the milk may actually be increased by the filtering process. it is necessary to remove the sand daily and thoroughly wash and sterilize the same. the extra care required in keeping these sand filters in sanitary condition has been the great objection to their employment in this country. filters of other material such as cellulose have been employed but with no marked success. =clarifying milk.= a much more efficient and less troublesome means of removing the insoluble foreign particles from milk is to pass it through a cream separator, allowing the cream and skim milk to mix in the same container. the slime that collects on the wall of the separator bowl is made up of dirt, casein, bacteria, and the cellular debris from the interior of the udder. the bacteria are heavier than the milk serum, and would, therefore, be deposited on the wall of the bowl were it not for other factors that in a measure prevent this. the movement of the fat toward the center of the bowl carries into the cream a considerable proportion of the bacteria in the milk. the slime will always contain many more bacteria than the milk, but the per cent of bacteria thus removed is relatively low, due to the small amount of slime obtained from the milk, so that the actual effect of clarification on the keeping quality of milk is insignificant. the complete removal of all insoluble and therefore visible dirt is, however, regarded of sufficient value to warrant the use. machines designed especially for the clarification of milk are now widely used. they differ from the cream separator in that the milk is introduced at the outside of the bowl and hence there is no separation of the fat from the serum. it is claimed that the removal of the dirt, cells from the interior of the udder and bacteria is as efficiently done as when the separator is used. the advantages claimed for the machine are that it has no effect on the subsequent gravity creaming of the milk and that less power is demanded than for the separator. from the standpoint of the consumer, all processes by which dirt is removed from milk are objectionable, since they make the milk appear cleaner and better than it really is, the harm having been done when the dirt with the adherent bacteria found its way into the milk. the removal of the foreign matter that has been introduced into the milk will have but little effect in reducing the number of bacteria, since a large part of the organisms will have been washed off the insoluble material. all of these processes improve the appearance of the milk but have little or no influence in increasing its keeping quality or its healthfulness. =preservation by cold.= the only legitimate way of preventing the growth of bacteria in milk is by holding it at temperatures at which the ordinary forms of bacteria cannot thrive. bacterial growth is greatly checked at temperatures approximating 50° f., or below, although certain types multiply at the freezing point or slightly above. if food products are actually congealed, no germ growth occurs, and they may be kept quite indefinitely, but this process cannot be successfully applied to milk, as the fat and casein are physically changed, so that a normal emulsion can not again be made when the frozen milk is melted. the fat separates in visible masses as though the milk had been partially churned. on account of this fact milk must be stored at temperatures above the freezing point. in denmark efforts have been made to preserve milk, that is to be shipped long distances, by freezing a portion of the milk, and placing a block of the frozen milk in each can after cooling the main mass of milk nearly to the freezing point. even this method has not proven practical, and at present reliance is placed on thorough chilling of the milk. at 32° f., the lactic bacteria cannot grow, but other types, such as certain of the putrefactive forms grow slowly; the milk may, therefore, have no objectionable odor or taste and yet be swarming with bacteria. in cities the practice is followed of placing cream in cold-storage during the cooler periods of summer in preparation for an increased demand, during hot weather or on holidays. it seems probable that poisoning from ice cream may, at times, be due to the use of such cream. =preservation by the use of antiseptics.= many chemical substances prevent the growth of bacteria when added to food supplies; such substances thus used are called _preservatives_. in the past some of these have been used in milk to a great extent, but at present, on account of stringent pure food laws, they are employed only to a slight extent. there is a great temptation for the small milk dealer in the city to employ them to preserve the excess of milk from day to day, as through the use of a few cents worth of some preparation, many dollars worth of milk may be kept from spoiling until it can be sold to the unsuspecting consumer. formalin has been most widely used in milk because it is a most efficient preservative; it is cheap and cannot be detected by the consumer, although it injures the digestibility of the casein. one ounce will keep one thousand pounds of milk sweet for twenty-four to forty-eight hours. borax, boric acid, and salicylic acid have also been used, but these substances must be employed in much larger quantities than formalin. bicarbonate of soda has sometimes been used although it is not a true preservative. its effect is based upon the neutralization of the acid produced by bacterial growth. the treated milk does not taste sour so quickly, and the curdling of the milk is also delayed. many proprietary compounds for milk preservation have been placed on the market in the past, but the use of all of these is illegal in most states. the federal law also prohibits their use in all dairy products that pass into interstate commerce. within recent years a method for the preservation of milk was introduced by a danish engineer, budde, which consists of adding to milk a very small amount of peroxid of hydrogen which is a very efficient antiseptic. the peroxid is decomposed by some substance in the milk; the products of decomposition being water and free oxygen. the peroxid together with the application of heat at a comparatively low temperature (122° f.) is sufficient to destroy the larger part of the bacteria in the milk. practical difficulties are encountered in the commercial application, so that it is probable the process will never be a commercial success. for the preservation of composite samples of milk for analytical purposes, such as the babcock test, strong disinfectants, as corrosive sublimate, are employed. this material is very poisonous, and leaves the milk unchanged in appearance. some coloring matter is therefore usually mixed with the sublimate in making the preservative tablets, so as to render their use more conspicuous. corrosive sublimate not only stops all bacterial growth, but quickly destroys the life of the cells. bichromate of potash is generally employed in the preservation of composite samples for the hart casein test. =destruction of bacteria in milk.= actual destruction of the life of bacterial cells by heat is one of the most important ways for preserving milk. heat easily destroys the vegetating, growing bacteria, while the spores, of which there are always a number in milk, are very resistant. if, however, the growing organisms are destroyed, the milk will keep much longer than if it had not been so treated. the process of pasteurization was first used by the french bacteriologist, pasteur, for the treatment of the wines of his native district which were likely to undergo undesirable types of fermentations due to bacteria. from the wine industry it was applied in the brewing industry, and was later found to be of the greatest service in the dairy industry. the process of pasteurization may be briefly defined, as the heating of milk to temperatures, varying from 140° f. and upward for a longer or shorter time, and subsequently cooling to a low temperature, so as to prevent the germination of the spores that are not destroyed by the heating. =effect of heat on milk.= when milk is heated it undergoes more or less profound changes, depending on the temperature and time of heating. some of these changes are of practical importance, since they are more or less evident, and objectionable to the consumer. in raw milk the fat globules are largely found in larger or smaller aggregates, rather than uniformly distributed throughout the serum. the surface of a mass of fat globules is smaller in proportion to the volume of the mass than is the case with single globules, hence globule clusters encounter less resistance in their passage through the serum, either as they rise to the surface in gravity creaming, or in the separator bowl. if these clusters are broken up, so that the globules are uniformly distributed, the milk will cream much less rapidly and completely. in the process known as "homogenization" of milk, the individual fat globules are broken into such small globules, that they cannot overcome the viscosity of the serum, and they remain distributed throughout the milk. in such cases, no cream rises, and even the cream separator is unable to remove the fat from such milk. in selling bottled milk, it is highly desirable that the cream line should show distinctly. in normal milk, this line forms in a few hours, but where milk is heated to a high temperature, and agitated at the same time, the clusters of fat globules are broken apart and the creaming power injured. this physical change is dependent not only on the temperature, but also on the time of exposure. a momentary exposure at 160° f., or for 20 minutes at 145° f., is about the maximum limit which can be applied to milk without material injury to the creaming property. [illustration: fig. 25.--fat globules in raw milk. in raw milk the fat globules are in masses of varying sizes. these rise to the surface quickly in gravity creaming.] the body or consistency of pasteurized cream may be restored by allowing the cream to stand for several days at low temperatures, or by the addition of a small amount of sucrate of lime. this substance, known to the dairy trade as "viscogen," is made by adding to a thick solution of cane sugar, some freshly slaked lime. the sugar solution permits of the dissolving of a much larger amount of the lime than is possible in water. when the liquid is allowed to settle, the clear solution is then decanted off and is used at the rate of about one part to 100 to 150 parts of cream. the fat globules are, by its action, brought into aggregates and the body of the cream thus restored. viscogen contains nothing that is at all harmful, but milk and cream to which it is added must be sold under some distinctive name as "visco-cream," since the laws of practically all states do not allow the addition of any substance whatever to milk or cream. [illustration: fig. 26.--fat globules in heated milk. when milk is heated the masses of globules are broken up and fat globules are uniformly distributed throughout the milk.] [illustration: fig. 27.--creaming of milk. the cylinder on the left contains raw milk; that in the center, milk heated to 140° f. for twenty minutes; on the right, milk heated to 160° f. for twenty minutes. the dark line indicates the depth of the cream after twenty-four hours. the breaking up of the fat globule clusters delays greatly the rising of the cream.] heated milk has a taste unlike that of raw milk; to one not accustomed to it the taste is objectionable. this change is due to some extent to the expulsion of the carbon dioxide from the milk. the insipid taste of boiled water is, in part, due to its freedom from carbon dioxide. the production of this cooked flavor is dependent upon the time and temperature of exposure. it has been claimed that heated milk is less digestible than raw, and a considerable amount of experimental work has been done, both on animals and children, in order to determine the relative digestibility of heated and raw milk. the results obtained have been contradictory. it is claimed that heated milk causes such diseases as rickets, scurvy and marasmus in children. it is probably true that milk heated to the boiling point is less fitted as food for the young child than raw milk, but, on the other hand, it has not been proven that properly pasteurized milk is an unsuitable food for children. the best evidence has been accumulated in recent years, in many of the large cities of this country and of europe, where pasteurized milk has been used with the greatest success in the feeding of children of all ages. the heated milk does not curdle readily when rennet is added due to the precipitation of the lime salts by heat. the curdling power can be restored by the addition of soluble lime salts or of acids. =purpose of pasteurization.= there are two reasons for the pasteurization of milk: (1) to improve the keeping quality; (2) to destroy any pathogenic bacteria it may contain. the first may be called the economic reason; the second, the hygienic reason fur pasteurization. in the selection of a proper pasteurizing temperature, two factors must be taken into account: first, the effect of heat on milk, and second, the temperature necessary to destroy those forms of bacteria that are of the greatest importance, as far as the keeping properties are concerned, and the pathogenic bacteria that might possibly be present in the milk. the lactic acid bacteria are non-spore-bearing and are not resistant to heat. most of them are destroyed when the milk is heated to 140° f. for fifteen minutes or to 160° f. for a moment. to insure proper keeping quality, somewhat higher temperatures must be employed, such as 145° to 150° f. for fifteen to twenty minutes. milk pasteurized at these temperatures will, as a rule, undergo an acid fermentation in much the same manner as will raw milk. the rate with which the acid develops is of course much slower than in the raw milk, due to the destruction of 95 to 99 per cent of the acid-forming bacteria. if the milk has been pasteurized at higher temperatures, the acid fermentation may not appear. the spores of the spore-bearing organisms will be left; these may germinate and cause their characteristic change in milk, which, as previously noted, is usually a sweet-curdling or a digesting fermentation. since the changes they produce in the milk are not evident at first, it might be used as food even though it was so far advanced in decomposition as to be undesirable or even harmful as food. indeed one of the objections urged against pasteurization is that it destroys the natural safe guard, the acid-forming bacteria. many people are so accustomed to use this as the indication of spoiled milk that they will use milk long after it should be used if it does not show an acid fermentation. the butyric acid organisms are spore forming and may at times produce their characteristic fermentation in pasteurized milk. the milk shows gas formation and develops an objectionable odor. the pathogenic bacteria most likely to be present in the milk are the typhoid and the tubercle organisms. the typhoid bacillus is no more resistant to heat than the ordinary acid-forming bacteria, and all milk that has been heated, so as to impart to it satisfactory keeping properties, will certainly be free from typhoid bacilli. it has sometimes been asserted that the tubercle bacillus is very resistant to heat; some claiming that it is necessary to heat milk to 200° f. in order to destroy it. other experimenters have asserted that lower temperatures would suffice, but the temperatures were still above those at which the milk is physically and chemically changed by the heating process. more recent work has shown that not all sources of error were avoided in the earlier attempts to determine the thermal death point of the tubercle bacillus, as, for example, it has been shown by the authors that the "scalded film" that forms on the surface of milk when heated in an open vessel will protect the bacteria imbedded in it. it has also been shown by the authors that a temperature of 140° f., for twenty minutes or 160° f. for one minute will destroy the tubercle bacilli in milk, in case the heating is done with sufficient thoroughness to insure all particles of the milk being heated to the same temperature for these periods of time. the pasteurization of milk can be done in such a manner as to impart to it good keeping qualities and to insure its freedom from pathogenic bacteria, and yet not impair its physical and chemical properties, but much of the so-called pasteurized milk placed on the market is not treated in accordance with proper hygienic methods. [illustration: fig. 28.--the pott's discontinuous pasteurizer. the milk is placed in the inner compartment. for heating and cooling, hot or cold water is passed between the jackets.] =methods of pasteurization.= in order to destroy the bacteria in milk, it is necessary that the milk be heated for a varying time dependent upon the temperature employed. a lower temperature for a considerable period may exert the same effect on the bacteria as a higher temperature for a shorter time. in practice, two types of pasteurizing machines are employed, depending on the temperature at which the milk is to be treated. the discontinuous machines or intermittently operated pasteurizers are those in which the milk is heated for any desired time at any temperature. such machines consist of jacketed containers the inner receptacle being filled with milk, while the outer space between the walls is filled with circulating hot water or steam. the milk is kept agitated by the rotation of the machine. after it is heated, it is cooled in the same container by replacing the hot water first with cold water, then ice water. the disadvantage of this process is that the capacity of the machine is limited which precludes its use in places where large quantities of milk or cream are handled; for the pasteurization of limited quantities, it is very successful, as every particle of milk or cream is under the direct control of the operator and may be thoroughly and efficiently treated. as pasteurization was introduced for the treatment of market milk, and for the preparation of cream for butter, machines have been devised which permit large quantities, as thousands of pounds, to be handled per hour. it is evident under these conditions that the milk must be heated for only a short time, and hence a higher temperature must be employed. these machines are called "continuous flow" pasteurizers since the milk passes through them in a constant stream. the period of exposure is very short, in some only a few seconds; hence, they are sometimes called "flash" pasteurizers. [illustration: fig. 29.--a continuous pasteurizer. the milk is exposed but a short time since it flows through the heater in a constant stream.] all machines of this type possess the obvious disadvantage that it is impossible to heat all of the milk for a uniform period. the milk in contact with the walls of the machine flows much more slowly than in the middle of the stream, just as the current near the bank is less rapid than in mid-stream. in none of the machines yet devised have the designers been able to overcome this disadvantage. in a test of one of the most widely used pasteurizers of this type, it was found that some of the milk passed through the machine in 15 seconds, while the larger part of it was held for about 30 seconds, and some as long as forty-five to sixty seconds. if the temperature employed had been such as to destroy the bacteria in that part of the milk heated for the minimum time, hygienic safety would be assured, but in order to avoid injuring the physical properties of the milk, the tendency is to use as low a temperature as possible, so that the milk heated for the minimum time may often contain organisms that have passed through the machine uninjured. many devices have been proposed for the heating and cooling of the milk. in many of the pasteurizers, the milk flows in a thin stream over a metal surface, on the opposite side of which is the heating agent, usually steam; while in others, the milk is allowed to flow through a vat in which revolve a series of discs into which steam is passed. the discs are of considerable size; thus, making a large heating surface; the milk is thus heated quickly, and is constantly stirred by the rotation of the heating discs. in other types the milk passes into the bottom of a chamber in which a dasher revolves at a rapid rate. this catches the milk, throwing it in a thin film onto the wall of the chamber, which is heated with steam on the opposite side. from such machines, of which the fjord, the jensen, and the reid machines are types, the milk may be forced to a considerable height. these are widely used in this country for the pasteurization of milk and cream for butter making. milk that has been heated must be cooled at once by the use of cold water and ice. in order to economize in the use of both steam and cooling agents, the so-called regenerative machines were devised. the essential feature of these machines lies in the fact that the cold milk inlet and the hot milk outlet are on opposite sides of a single partition; thus the inflowing cold milk is partially heated by means of the already treated hot milk which it is desired to cool. in order to avoid the disadvantages of the continuous machines, viz., lack of control, an apparatus has recently been devised which can handle large quantities of milk, heating the same to any temperature for any desired time. in such a machine the milk is first heated in a continuous heater, and is then passed into large tanks in which it is allowed to remain for the desired time, and from which it flows over the coolers. such an apparatus is called a "holding" machine, and is probably the most feasible type of pasteurizer now on the market, when all factors are considered. in some of the continuous machines, an attempt is made to accomplish the same result, by building the machine so that the milk requires fifteen to twenty minutes for passage through the machine, but in all such cases the same disadvantage of variation in rate of flow, as in other continuous flow type of machines obtains. =tests of pasteurizing machines.= it is possible for the operator to test the rate of flow in a machine, so as to determine whether all of the milk is heated for a uniform time. this is done most easily in the following manner: the machine is first filled with water, heating the same to the desired temperature, and regulating the rate of flow as it would be if milk was used. the flow of water is then turned off, and a stream of milk containing a known per cent of fat admitted to the machine. the time elapsing between the admission of milk to the machine, and that at which the first sign of turbidity is noted at the outlet, will be the minimum period necessary for any portion of the milk to flow through the machine. at frequent intervals thereafter, samples of the outflowing liquid may be collected, noting the time at which each sample is taken. the percentage of fat in the various samples is determined by the babcock test; at the moment when all of the water has been removed, the sample taken will show the same fat content as the milk used. the samples taken previous to this will show a lower fat test, dependent upon the relative amount of water and milk. in this manner, the minimum, the maximum, and the average period of exposure of milk in the machine tested, can be determined with exactness. the accompanying table gives results that were obtained in the testing of one of the continuous types of machines. the machine in question required about three hundred pounds of milk to fill it and was supposed to handle 1,000 pounds per hour. thus theoretically it should require twenty minutes for any portion of the milk to pass through the machine. as will be seen from the data, some of the milk passed through within seven minutes after the water was shut off and the milk turned on. the figures also show that not all of the water had been replaced by the milk in even 45 minutes. in actual practice like results will be obtained, and a portion of the milk will be heated to the temperature employed but a short time. in this, the vegetating bacteria will not be wholly destroyed. ========================================================= trial | |per cent of fat in milk coming from | | machine at following times |per cent|-------------------------------------- | of fat| minutes | in milk|-------------------------------------- | | 7 | 11 | 15 | 19 | 23 | 27 | 36 | 47 --------+--------+----+----+----+----+----+----+----+--- no. i | 4.0 | 0.2| 0.8| 1.6| 2.0| 2.4| 2.6| | no. ii | 3.8 | 0.2| 0.6| 1.5| 1.8| 2.2| 2.6| 3.0| 3.4 no. iii | 3.5 | 0.7| 1.9| 2.4| 2.8| 2.8| 3.0| 3.4| 3.4 ========================================================= =pasteurization of small quantities of milk.= it is often desirable to treat a small quantity of milk for home use, in which case the commercial types of pasteurizers are out of the question. this treatment can be done in a number of ways, consideration always being paid to the manner of heating which should be done under such conditions, as have been shown to be necessary for efficient pasteurization. milk may be heated in tall, narrow cans which are placed in hot water. in the household, milk may be treated by placing the filled bottle in a pail having a false bottom so the bottle shall not be broken when the pail is placed on the stove. the pail should be filled with water so that its level is about the same as that of the milk. the water is then heated to the desired temperature, maintained for the requisite period of time, and is then cooled as rapidly as possible. during the heating, the mouth of the bottle should be covered, either with an inverted glass tumbler, or the paper cap may be left in place, simply punching a small hole through it so as to permit of the insertion of a thermometer. [illustration: fig. 30.--a pasteurizer for use in the home. a milk bottle with a tumbler for a cover. the cover prevents the formation of the "scalded layer" on the milk during the heating and also protects the mouth of the bottle from dust.] =efficiency of pasteurizing.= it is easy to destroy over 99 per cent of the bacteria present by the use of any of the modern types of machines. the number remaining after treatment will be largely dependent, other things being equal, upon the number of bacteria before pasteurization. the pasteurizing process is not one by which poor milk can be changed into good milk, nor is it legitimate to use the process in place of cleanliness, as is sometimes done. there is a legitimate field for the process in the handling of market milk, as well as in the creamery; but it should be used to improve the keeping quality, and to insure the freedom of the milk from pathogenic bacteria, when other protective measures have been carried as far as possible under the prevailing conditions. =details of process.= if the process is to be successful, due attention must be given to certain details. in the treatment of market milk, care should be taken to use only that in which the acidity has not materially increased. a fair standard is about 0.2 per cent. high acid milk usually means old milk or dirty milk, either of which is very likely to contain many more spore-bearing bacteria than clean, fresh milk. the greater the number of spores, the more rapidly will the pasteurized milk spoil. if it is possible to exercise any selection of milk prior to pasteurization, the rapid test for determination of acidity will prove of great advantage. care should be taken to prevent fluctuations in the temperature to which the milk is heated. with varying steam pressure and variations in the rate of flow of milk, these fluctuations may be very considerable. regulators are now made that will control the temperature within narrow limits. in all pasteurized milk as it flows from the machine, there will remain some living bacteria. the spores will not be destroyed by any pasteurizing process, and under commercial conditions, vegetating bacteria are also present. if the milk is not quickly chilled after heating, these forms will grow, and their development is particularly hastened by the destruction of the lactic bacteria, the acid of which would otherwise hold them in check. the result is that, unless immediately chilled, pasteurized milk spoils almost as rapidly as though it had not been heated at all. efficient and rapid cooling are, therefore, as essential a portion of the process as the heating itself. care should also be taken to protect the milk from contamination after treatment. every utensil with which it comes in contact should be sterilized. the bottles should be thoroughly washed and sterilized and subsequently protected from dust until used. =sterilization of milk.= it is possible to render milk sterile by the use of temperatures above the boiling point of water, where it is heated in a closed vessel, in which steam under pressure is generated. such milk is often found in the european markets. in our own country, the only milk of this kind is the so-called "evaporated milk." in this process sweet fresh milk is evaporated in vacuum pans to about one-third of the original volume. this is then placed in tin cans, which are treated, as in the canning of such vegetables as peas and corn, by heating the milk to 230° or 240° f. for a few minutes. in this process, the bacteria (spores as well as vegetating forms) are completely killed, and the milk acquires a brownish tint, due to the caramelization of the sugar. the appearance of the product is very similar to cream, and previous to the passage of the pure food law, it was sold as evaporated cream. condensed milk is not wholly free from bacteria, but is sufficiently thick, by reason of its treatment so that the contained bacteria cannot grow. they remain dormant in the milk, but as soon as it is diluted to a normal consistency, growth takes place, and the milk rapidly spoils. condensed milk is prepared by adding cane sugar to fresh sweet milk, then evaporating the mixture to one-third the original volume, forming a semi-solid product. syrups owe their keeping qualities to the same factor, as condensed milk, _i.e._, the high consistency. milk is also preserved by wholly evaporating the water, thus leaving a dry powder, which on being mixed with water again will have much the same properties as the original milk. various methods have been devised for the preparation of these milk powders, all of which have been patented by the inventors. if the powder is to be kept for long periods, skim milk must be used, since the fat slowly undergoes changes which cause it to have a rancid odor. these dry preparations are largely used by bakers in place of fresh milk. chapter vii. bacteria and butter making. in the making of butter it is necessary to concentrate the milk fat into a small volume. this process, known as creaming, may be accomplished by gravity, if the milk is allowed to stand undisturbed, the fat globules rising slowly to the surface. much more rapid separation may be secured, by placing the milk in a rapidly revolving container in which it is subjected to centrifugal force, which causes the heavier parts of the milk to pass to the outside of the bowl, while the lighter part, the fat, collects at the center of the revolving bowl. there is an enormous number of fat globules in milk, over 5,000,000,000 in each cubic centimeter, and as these move through the milk serum, they carry with them many of the bacteria. the cream is thus much richer in bacteria than is the skim milk, or even the milk before separation. besides the mechanical separation in the manner described, the method of creaming is of importance, in determining not only the number but also the kind of bacteria in the cream. =methods of creaming.= in the shallow-pan method of creaming, the milk is kept at ordinary room temperatures. these temperatures favor especially the growth of the acid-forming bacteria. the milk is usually sour by the time the cream is removed from it; consequently, the bacterial content of the cream is high. moreover, the cream is exposed to air contamination, and is thus seeded with molds, and those forms of bacteria that are always found in the air. the cream obtained in this manner is likely to contain not only numerous bacteria, but a great variety of forms, some of which undoubtedly are the cause of the poor keeping qualities of butter made from such cream. in the more modern method of gravity creaming, in which the milk is placed in deep narrow cans kept in cold water, the conditions are not favorable for the growth of acid-forming bacteria. if the milk is produced under clean conditions, and is placed in cold water at once, the bacterial content of the cream will be low, and it will be less likely to contain undesirable forms than the cream which is obtained from the shallow pans. in separator cream the bacteria will be represented by the kinds present in the milk at time of separation. if this milk is quite old, the cream will contain large numbers of bacteria; if, however, early separation is made and the milk is clean, the bacterial content of the cream will be low. =types of butter.= butter may be divided into two types--acid or sour-cream, and sweet-cream, depending upon whether the cream is allowed to undergo the acid fermentation or not before it is churned. in southern europe, it is the custom to churn the cream as sweet as possible, and the resulting product possesses only the natural, or primary milk flavor. to one accustomed to butter made from sour or ripened cream, this taste is flat, and if the butter is free from salt, may remind one of grease. sweet-cream butter has a delicate flavor when it is made from good milk, and the taste for it is rapidly acquired. in some centers, as in paris, the market demands this type of butter quite exclusively. if the cream is allowed to undergo the acid fermentation before churning, the butter has a much higher degree of flavor and one that differs materially in kind. under primitive methods, it was difficult to keep the cream sweet until it could be churned. on the small farm with gravity creaming in shallow vessels and infrequent churning, the cream was certain to be sour when churned. undoubtedly, the making of butter from sour cream came into use because of its greater convenience; people became accustomed to sour-cream butter, and at the present time it is used in the greater part of the world, and is the type made in all of the great dairy countries. =ripening of cream.= in modern dairy practice the souring of the cream is called the _ripening_ process, and is, where the best methods are employed, largely under the control of the butter maker. the changes that go on in the ripening process are the same as have been discussed in the acid fermentation of milk. the increase in acid is accompanied by an enormous increase in the number of bacteria; the ripe cream will contain hundreds of millions of bacteria in each cubic centimeter. the effect of this germ life is to improve or injure the butter, depending upon the class of bacteria to which it belongs. the problem of the modern butter maker is to control the kinds of bacteria growing in the cream. the temperature at which cream is held during the ripening process is favorable to the growth of the acid-forming bacteria; hence, in ripe cream, they are practically the only kind of bacteria to be found. it must be remembered however, that there are different classes of acid-forming organisms, some of which produce desirable flavors, while others are distinctly harmful. the intensity of flavor of butter is, in a general way, directly related to the amount of acid that is formed in the cream. a low acidity at time of churning is usually associated with a mild flavor, while a higher degree of acidity, up to a certain point, imparts a more pronounced flavor to the product. if cream is over-ripened, the quality of the flavor is seriously impaired. in determining the acidity of cream, a definite volume is taken, and the acidity determined by titration, expressing the results as such a per cent of lactic acid. manifestly, the amount of fat in the cream influences the apparent per cent of acidity. the acidity will not usually exceed 0.5 to 0.7 per cent, but in reality the serum will contain more than this, as the acid is formed in the serum, the butter fat having no role whatever. in a very rich cream, 40 to 50 per cent fat, it is impossible to develop more than 0.4 to 0.5 per cent of acidity, and the flavor of the butter will be low, because of the relation between the amount of acid and fat, while in a thin cream having the same acidity, the ratio between the amounts of fat and acid will be very different. for example, in one hundred pounds of 50 per cent cream of 0.5 per cent acidity there will be one-half pound of acid and fifty pounds of fat; in the same quantity of cream containing 20 per cent of fat and having an acidity of 0.5 per cent there will be one-half pound of acid to twenty pounds of fat. the flavor of the butter from the rich cream will be quite different in intensity from that made from the thinner cream. the acidity of cream cannot be determined with any degree of accuracy by the taste or odor. every butter maker should have some method of determining the degree of acidity in his cream, so that he may better control the flavor of his product. several methods have been devised for this purpose and the necessary apparatus is sold by all dairy supply houses. the effect of the ripening of the cream is shown not only in the flavor of the product, but in a number of other ways. sour cream churns more easily, and more exhaustively than does sweet cream. it is supposed that the fat globules are surrounded by a film of albuminous material which prevents their coalescing readily. during the ripening process, the action of the acid apparently dissolves this enveloping substance, and the globules cohere more easily in the churning process. when raw cream is used the ripened-cream butter keeps better than that made from sweet cream. in sweet cream there are few lactic bacteria, the majority of the bacteria present being of various kinds, many of which may be injurious, so far as the keeping quality is concerned. in sour-cream butter the lactic bacteria make up over 99 per cent of the bacteria present, and their presence tends to prevent the development of undesirable non-acid forms. =source of butter flavor.= the flavor of ripened-cream butter has been shown to be directly connected with the acid-fermentation of the cream. the amount of lactic acid formed from the sugar fermented is dependent upon the kind of bacteria present. the acid-producing organisms that are desirable from the standpoint of the butter maker form comparatively small amounts of other by-products, but these undoubtedly affect the flavor of the butter. as fats have the power of absorbing odors, the butter fat absorbs some of the by-products of the acid fermentation, thus acquiring a certain aroma and flavor. it is not necessary that the cream be ripened, in order to have the fat acquire a flavor, for if sweet cream is churned with a considerable proportion of sour milk, the butter will have much the same flavor, both as to intensity and kind, as though the cream had been allowed to sour naturally. a process of butter making known as the leclair method is based on this principle. the flavor-producing substances can also be absorbed by the butter after it is churned, by working the butter in contact with sour milk. attempts have been made to add pure lactic acid to the cream, instead of allowing the acid to be formed by the bacteria, but while the physical effect on the cream is the same, the flavor and aroma of the butter are deficient, because the acid itself does not supply the necessary aromatic products. this emphasizes the importance of the by-products of the acid fermentation other than the lactic-acid. in the past numerous attempts have been made to find organisms that might be added to the cream, in order to produce the delicate flavor characteristic of the best type of butter. some bacteriologists have claimed that the source of the flavor-giving substance was to be found in the decomposition products of the nitrogenous constituents of the milk. none of these attempts have stood the test of practical use in creameries, and it has been demonstrated that the finest type of butter can be made by the use of lactic bacteria alone. formerly, when butter was made wholly from cream soured under natural conditions, a much higher degree of flavor was developed. under present market demands, a less pronounced flavor is desired, a condition more readily met by the use of modern methods. =importance of butter flavor.= the importance of flavor in determining the commercial value of butter is evidenced by the relatively high value placed upon this factor in scoring, viz., flavor, 45 points; body or texture, 25 points; color 15; salt 10; and package 5 points. the factors on which butter is judged, are with the exception of flavor, wholly under the control of the maker, but as the production of flavor is dependent on the kind of bacteria present in the cream, it is a far more difficult matter to control, and yet it is of the utmost importance in determining the value of the product. the flavor of the butter is dependent on the quality of the cream. if this is dirty and sour, the maker has little control over the type of fermentation, and hence, little control of the flavor of the butter. this has led in some cases to the grading of the cream, basing the division on the acidity, flavor, and fat content. such practice is entirely justifiable, as a better quality of butter can be made from fresh, sweet cream than from that already fermented. it is noteworthy that the quality of butter has not improved since the introduction of the centralizer system, in which cream is shipped for long distances. =control of the type of fermentation.= in the older methods of butter making, there was little or no control of the type of fermentation that took place in the cream. where milk is produced under clean conditions, and kept at ordinary temperatures, it will generally undergo fermentation changes, due to the desirable type of acid-forming organisms. in milk, which is less carefully handled, the undesirable bacteria are more abundant and the quality of the butter of lower grade. when butter was made on the farm, before the development of the factory system, it was not a question of vital importance whether the product was uniform from day to day, but with the advent of the modern creamery, turning out thousands of pounds of butter per day, and with the extension of the markets for the product, the question of uniformity came to be of much importance. a uniform product can be secured only by the control of the type of fermentation in the cream, or by the control of the kinds of bacteria that cause the souring of the cream. modern methods of butter making have been devised on the basis of an improvement in the ripening process. =starters.= from the earliest practice of allowing the cream to stand until sufficient quantity had accumulated for churning, it was only a step, but a most important one, to the addition of sour milk, sour cream, or butter milk, to hasten the ripening process. this was the beginning of the modern starter. experience demonstrated that the addition of these already fermented liquids exercised a desirable effect upon the production of butter flavor, even though, at that time, the phenomenon of milk fermentation was not satisfactorily understood, and the relation of bacterial by-products to the production of flavor in butter was not recognized. as a result of experience alone, improvements in the development of the "home made" starter took place. by careful selection of clean milk, and the natural fermentation of this under carefully controlled conditions, as well as the control of the temperature of the cream during the ripening, improvement in the technique of cream ripening gradually developed. more and more attention was given to the preparation of the starter, and its propagation from day to day, under conditions which would prevent its deterioration. this method of utilizing naturally fermented milk or cream was gradually extended, until it became almost universal in the larger butter-producing districts. in 1890 a more refined and scientific process was introduced by the danish bacteriologist, storch. recognizing the fact that butter flavor was attributable to the development of the bacteria present in the ripening cream, he conceived the idea of isolating the various types of organisms found in milk and testing them as to their effect on the quality of flavor. selection was then made of the most favorable flavor-producing types, and these were propagated in suitable culture media, such as skim milk, which was rendered more or less perfectly sterile by pasteurization or sterilization. under such conditions the addition of a selected ferment could be made to the fresh cream, and so control the type of fermentation which occurred therein. an essential requisite in any organism used for this purpose must be the ability to produce relatively large amounts of acid rapidly at ordinary ripening temperatures, and also to form sufficient quantities of the proper flavor-producing substances to impart a suitable flavor to the butter fat. such starters are known as pure culture or commercial starters, and are prepared in both liquid and dry form. at present they are used to a greater or less extent in all of the leading dairy districts. liquid starters consist of a mass of sterile nutrient medium, milk or beef broth, inoculated with the pure culture. the dry starters are made by adding liquid cultures, containing the growing bacteria, to some absorbing material, such as milk sugar, milk powder, or starch, the whole mass being dried at low temperatures, so as not to injure the bacteria. under such conditions the bacteria, exist in a dormant state, and are protected from their own by-products, to which they would be exposed if maintained in liquid cultures. the keeping quality, therefore, of dry cultures, is much better than that of liquid cultures. by the use of the pure-culture starters, the butter maker is able to add to his cream the same kind of bacteria from day to day, and the butter will be more uniform than when the less constant home-made starter is employed. in cream to which the starter is added, there are present a greater or less number of acid-forming bacteria, depending upon the age of the cream, and upon the condition under which it was produced. these will grow during the ripening process, and the flavor of the product will be the result of the mixture of the bacteria in the cream. the maker can not, therefore, be certain that the addition of a pure culture to raw cream will effectively control the type of fermentation. this can be secured only by first destroying the existing bacteria in the cream, before the selected culture is added. heating the cream accomplishes this; and in cream thus freed from the various kinds of bacteria, the butter maker can insure the dominance of the desirable types, contained in the pure-culture starter. if the cream can be obtained in a sweet condition, the maker through this process of pasteurization, and the use of pure cultures, secures almost perfect control over the type of fermentation that occurs in the cream, and thus exercises control over the degree and kind of flavor of the product. this most scientific type of butter making is now used by the most progressive butter makers in the leading butter-producing regions of the world. pasteurization of the cream also distinctly improves the keeping quality of butter, a condition doubtless due to the freedom of the same from organisms other than the lactic bacteria. this is a factor of as much importance as uniformity, because under modern business conditions, the surplus production must be kept in storage, and it is essential that the quality should not deteriorate materially during this time. =process of pasteurization for butter making.= in the pasteurization of market milk, it is necessary to take into account the effect of heating on the physical and chemical properties of the milk, and the degree of heat that can be employed is limited. in pasteurizing cream for butter, there is no such limitation, and the cream may be heated to any temperature desired. in denmark where the process of pasteurization has been used most extensively, temperatures ranging from 176° f. to 190° f. are used. the machines are of the "continuous flow" type, and the cream rather than the whole milk is treated. to prevent the spread of tuberculosis and other diseases, the danish government requires that all cream and milk be heated to 176° f., before the skim milk or butter milk is returned to the farms. the heating of the butter fat to high temperatures has an injurious effect on the texture of the butter, unless the cream is cooled to 50° f., for a period of at least two hours previous to churning. =propagation of starters.= as has been previously shown, the quality of butter depends on the kind of bacteria in the cream or in the starter added. the commercial starters contain lactic acid bacteria that have been selected with especial care; most of the starters now sold contain but a single kind of bacteria; hence, are often called pure-culture starters. the package purchased contains but a small quantity, and before the starter can be used in the ripening of cream, it must be increased in amount. it must also be propagated from day to day so that a fresh starter shall be available daily for addition to the cream. the propagation of the starter must be done with especial reference to keeping it in good condition and in as high a state of purity as possible. in the past the starter was propagated, by adding the contents of the bottle purchased to a small amount of milk that had been heated and cooled; this, if kept in a warm place, would be curdled in twenty-four hours, and could be used for the inoculation of a large mass of milk, that had been treated in a like manner, and which, when curdled, was added to the cream; a small amount was saved for the purpose of again inoculating a mass of milk that had been heated and cooled. following this method it was very difficult to keep the culture from becoming contaminated with other forms of bacteria. more recently the most successful butter makers have propagated the so-called "mother starters" in small vessels, and have used the larger mass of starter for the inoculation of the cream alone. glass vessels are preferable for the propagation of the mother starters since they are impervious and through the transparent wall the condition of the ripened starter can be more easily determined than in a metal or earthenware vessel. an ordinary milk bottle with an inverted tumbler for a cover, to protect the starter from contamination from the air, is a most convenient vessel. the starters may be propagated either in whole or skim milk; the former is preferable since, in most creameries, it can be more easily selected. the quality of the milk used has much to do with the quality of the starter; it should be as fresh and clean as it is possible to obtain. the clean bottle should be filled half to two-thirds full, covered and heated in some manner so that the milk shall be at a temperature close to the boiling point for fifteen to twenty minutes. the heating may be done by placing the bottles in water, which is heated on a stove or by steam, or the bottles may be subjected to streaming steam. the milk is cooled quickly and the contents of the package purchased added and well mixed with the milk. in the case of the dry starters, the mixing should be done with especial care. the bottle is kept in a warm place and in twenty-four to thirty-six hours, the milk should be curdled. a second bottle must be treated as before and inoculated from the first, and the process repeated daily since the bacteria must have fresh food, if they are to be maintained in good condition. in order to accomplish this, the maker must be able to maintain constant conditions from day to day, especially with reference to the amount of the ripened starter that is transferred to the fresh bottle of milk, and the temperature at which the bottles are kept. a spoon, arranged as shown in fig. 31, enables one to carry a definite amount of the ripened starter to the bottle of milk to be inoculated and a constant temperature box (fig. 32) permits of the maintenance of the same temperature from day to day. through careful supervision of these points, and by taking care at every step to avoid the introduction of contaminating organisms, the purity of the culture can be maintained, and the bacteria kept in a healthy condition. the starter is used because of the acid-forming bacteria it contains; it is said to be ripe and in the best condition for use at the time it contains the greatest number of living bacteria. it has been found by experiment that this is at the time the milk curdles at ordinary temperature, or when the acidity is about 0.6-0.7 per cent. if the acidity is allowed to increase to 0.8 or 0.9 per cent, the number of bacteria will be less and a larger amount of the starter must be used in order to ripen a definite amount of cream in the desired time. the use of an overripe starter may also have an injurious effect on the flavor. [illustration: fig. 31.--bottle for mother starters. a milk bottle with a tumbler for a cover and a spoon for inoculating the other bottles enables the butter maker to propagate the starters without contamination.] the ripened starter should be perfectly homogeneous, showing no bubbles of gas or free whey; the odor should be agreeable and the acid taste mild; on shaking, the curd should break up into a smooth, creamy liquid free from lumps. this is especially important in the starter that is to be added to the cream, since otherwise the starter cannot be uniformly mixed with it and white specks of curdled casein will be noted in the butter. [illustration: fig. 32.--an incubating chamber for starters. the inner compartment will hold a pail of water and the bottles for the mother starters. the temperature can be kept at any desired point by the use of warm or cold water. the four-inch space between the walls is filled with hay or mineral wool.] the firmness of the curd is not so dependent on the amount of acid formed as upon other factors. if the curd shrinks to any extent and the whey is expressed, it is certain to produce a starter that will contain lumps that cannot be broken up. with a pure culture of lactic bacteria, there is little difficulty in this regard, but as soon as gas-forming bacteria are introduced, trouble is likely to result. in the propagation of starters, it is always to be remembered that the bacteria, although invisible to the eye, are living things, and unless conditions are favorable in every particular, it is impossible to keep them in a healthy condition, so that growth in the cream is rapid, producing the acid demanded for churning, and imparting to the butter the desired flavor, both as to degree and kind. no part of the daily routine of the butter maker should be performed with more care than the preparation of the starters, both the mother starters, and the larger one for addition to the cream. the latter can best be made in one of the many forms of starter cans now on the market, since by their use, the maker can heat and cool the milk with little trouble, and can maintain the starter at any desired temperature. better starters cannot be made in them than by the use of simple and improvised apparatus, but better results can be obtained with the same expenditure of time and labor. in the handling of the large starter, care should be used not to overripen, since the larger quantity is more likely to "whey off" than is the smaller starter. skim milk rather than whole should be used for this. it should be selected with care and heated to 200° f. for thirty minutes. when it is impossible to secure fresh milk for starter making purposes, either condensed skim milk or milk powder may be used. the condensed milk is diluted with water until its volume is about the same as the milk before concentration; the mixture is then treated the same as fresh milk, being heated and cooled before inoculation. in the case of milk powder, one part of the powder is added to ten or twelve parts of water, allowed to dissolve as far as possible, and the mixture heated and cooled. either of these liquids will give satisfactory starters; the cost however is high, and in most places milk can be obtained more cheaply. the inoculation and the temperature should be so controlled, as to ripen the starter at the time it is to be needed. these conditions must be determined by the maker for himself. it should be remembered that the bacteria grow much more rapidly, as the temperature is increased; and hence, the amount of inoculation is dependent on the temperature at which the starter is to be kept. when the starter is propagated under practical conditions, it sooner or later deteriorates, either in acid production, or in flavor, and a new pure culture must be procured from the manufacturer. it is impossible to give a hard and fast rule as to the length of time a starter can be kept in good condition. it will depend on how well the maker satisfies the conditions necessary for maintaining its purity and strength. the use of imperfectly sterilized milk, or dirty utensils soon contaminates it; overripening is likely to injure the flavor. one of the most frequent troubles encountered is the appearance of a slimy or ropy condition in the starter, although the acidity developed may be normal and the flavor desirable. it has been found that this condition is not necessarily due to contamination, as was considered true in the past, but rather to some change in the lactic bacteria themselves. if the propagation is continued, the slimy condition will often disappear. =starters in "process" butter and oleomargarine.= the advance which has recently been made in the science and practice of cream ripening and butter production is utilized most effectively in the treatment of cream in the renovating process. old, soured, and stale cream is reduced in acidity by the addition of lime. the cream is then pasteurized and aerated to expel the odors as much as possible. a large amount of starter is then added and the cream immediately churned. under these conditions, the bad flavors are materially reduced in intensity, and desirable flavors absorbed by the fat from the selected starter used. it is thus possible to produce butter of good quality from cream that would at first be regarded as quite unsuitable for butter production. in the manufacture of oleomargarine the same principle is utilized. the butter aroma and flavor is imparted to the neutral oils and tasteless fats by mixing the same with a properly prepared starter. renovated or process butter is given a desirable flavor in the same way. =wash water.= it has been found that the purity of the water used in washing the granular butter has a marked influence on the keeping quality. if the water is from a shallow well into which surface water finds its way, it is certain to contain large numbers of those types of bacteria that are found in the soil, while if it comes from a deep well that is properly protected from surface contamination, the bacterial content of the water will be low and no injurious effect on the butter will be noted. when it is impossible to obtain pure water for washing purposes, a proper supply may be secured by sterilizing the water. the most convenient way of heating the water is by the direct injection of steam. it is necessary to use that coming directly from the boilers and not the exhaust from the engine, since the latter is likely to contain small amounts of oil that will impart to the butter an objectionable flavor. after cooling, the water is ready for use. it has been shown that the cost of treating an impure water is more than covered by the increased returns from the product. a pure and healthful water supply should be one of the essential things of every dairy, creamery, and cheese factory, not only for the sake of the quality of the product, but also to avoid contamination of products with disease-producing bacteria. =bacteria in butter.= the germ content of butter will depend on the type of cream. sweet-cream butter contains but few bacteria. in sour-cream butter the content in bacteria will be greatly increased, especially as to lactic organisms. often, it may amount to several millions of organisms per gram. the germ content of butter is said to be greater on the outside of a package than within the mass, due doubtless to the free access of air, thus favoring the growth of the aerobic forms. the composition of normal butter does not favor the growth of the majority of kinds of bacteria that are contained in it. the washing process removes much of the material suitable as food for the bacteria, such as sugar and albumen. if considerable butter milk is left in the butter, the growth of bacteria will be quite rapid, at first, but does not continue for any considerable length of time. the addition of salt also tends to restrain the growth of most kinds of bacteria. butter is at its best when it is perfectly fresh. deterioration begins within a short time and the rapidity with which the changes go on is dependent on the temperature at which the butter is stored. the temperature of the butter rooms in the large cold storage plants is kept below 0° f. the butter in such rooms will deteriorate very slowly, but on removal from the cold rooms and in storage at ordinary temperatures deterioration goes on more rapidly than would have been the case when the butter was fresh. at the temperature of an ordinary refrigerator the changes go on much more rapidly. this fact has often been looked on as indicating that the factors causing the changes are biological ones. the influence of temperature in accelerating the changes would be the same if no biological factor were active. that biological factors are of importance is indicated by the fact that the keeping quality of the product is profoundly affected by the quality of the cream. butter made from sweet, fresh cream, that has been thoroughly pasteurized, has the best keeping quality, while butter made from such cream, but not pasteurized, has the poorest keeping quality, especially when no salt is added. every process by which the desirable lactic bacteria are increased in proportion to other kinds has a marked effect in enhancing the keeping quality of the butter. thus, the use of pure cultures in raw cream, and pasteurization together with the pure cultures, have a marked beneficial effect. the addition of preservatives exerts an effect on keeping quality. borax is the chemical most frequently employed for this purpose. its use is allowed in australia and new zealand in butter that is shipped to england, but the use of all preservatives is forbidden in the united states. the size of the package also has an effect on the keeping quality; the smaller the package, the greater is the surface exposed to the air and the more rapidly the butter deteriorates. butter used in the united states navy is packed in hermetically sealed cans so as to exclude the air as far as possible. from the fact that any condition which restrains or inhibits the growth of micro-organisms has a tendency to improve the keeping quality of butter, it would appear that the detrimental changes in the quality of butter are due to biological causes. the most common defect known is that usually referred to as rancidity. there are, however, different types of changes that are probably included under this head and it is very probable that different causes are operative in their production. true rancidity is probably due to biological causes; the so-called tallowy change, in which the butter acquires the odor of tallow is probably due to the combined action of light and air on the fat. =bacterial defects in butter.= there are a number of defects in butter that are positively known to be due to the growth of bacteria in the milk or cream, or in the butter itself. the lack of flavor is looked upon as a defect in the case of ripened-cream butter. it may be due to insufficient ripening of the cream, or to the lack of acid-forming bacteria that produce the desirable flavor-forming compounds. not all acid-forming bacteria are able to produce favorable, flavor-giving compounds; hence, sour cream butter may sometimes be deficient in flavor by reason of this fact. =putrid butter.= this specific butter trouble has been observed in denmark, where it was first studied by jensen. butter affected by it rapidly acquires a peculiar putrid odor that ruins it for table use. sometimes this flavor may be developed in the cream previous to churning. it may be caused by a number of bacteria. =turnip flavored butter.= butter sometimes acquires a flavor resembling turnips. this trouble may be due to the feeding of such roots, the aromatic substances peculiar to them being absorbed directly by the milk and thus transferred to the butter. weigmann traced a similar flavor to certain bacteria that entered the milk from barn filth. =cowy odor in butter.= there is sometimes to be noted an odor in butter as in milk that resembles that of the cow stable. usually this defect has been ascribed to the absorption of these odors directly by the milk. organisms have also been described that impart to the butter a very similar odor. bitter butter may be due to the feed that is consumed by the cow, or it may be due to those forms of bacteria that produce a bitter fermentation of the milk. =other abnormal flavors.= among the numerous abnormal flavors that have been noted in butter is one of quite frequent appearance, the so called "fishy" flavor. it is now believed by many that this flavor is due to the presence of small amounts of iron or copper salts that have been introduced into the milk from utensils from which the protective coating of tin has been worn. if the milk or cream stored in such utensils develops any marked degree of acidity, the acid will dissolve a small amount of the iron or copper. the fishy flavor has not been found in sweet-cream butter as would be expected from the above explanation. in fresh butter a metallic taste is sometimes present. it is believed by some that on storage this flavor changes to the fishy flavor. all utensils used for the storage of milk and cream should be kept in good condition so as to prevent the acid milk or cream from coming in contact with iron or copper. [illustration: fig. 33.--moldy butter. the mold grows on the paper in which the butter is wrapped rather than on the butter. the print on the left was wrapped in the same paper as the print on the right except that the parchment cover had been steamed for a few moments.] =moldy butter.= a defect that causes a great amount of loss is the development of mold on the surface of the butter, either in tubs or in prints. this trouble is easily prevented. butter is not well suited to the growth of mold, but the paper used for lining the tubs, or wrapping the prints is an excellent medium for mold growth. the wood of the tub also furnishes ample food for this type of life, especially where the wood contains any sap. one other essential condition for mold growth is a supply of oxygen. the mold spores are widely disseminated, and are always to be found on the butter tubs and on the paper. the number is not likely to be sufficient to cause trouble unless the tubs and paper have been kept under such conditions, as to allow growth to take place on them before use. during damp, hot weather, the amount of moisture absorbed by these materials is often sufficient to allow molds to grow on them. this trouble can be prevented by the storage of tubs and paper in a clean dry place, or by a disinfecting treatment which will destroy the mold spores. the most successful method of treatment of tubs is to apply paraffin to the inner surface, which can be easily done by the use of some one of the various machines now on the market. the thin layer of paraffin excludes the moisture from the wood, and also prevents the mold from obtaining a supply of oxygen for its growth. the tubs may be steamed, treated with hot water, or filled with a dilute solution of formaldehyde, and allowed to stand overnight. soaking in brine as is usually done in the creameries is of some effect, but will not completely kill mold spores. [illustration: fig. 34.--moldy butter. the butter was placed in a paraffined tub, but the paper was not treated so as to destroy the mold spores thereon.] butter may mold where the tubs have been thoroughly treated, because of the mold spores on the paper used for the lining. one of the black molds is able to thrive on parchment paper whenever the air is damp. in the past but little attention has been paid to the paper as a source of trouble. it is certain that it is often at fault, and that as much attention should be paid to the paper as to the tub. a most efficient way of treating paper, either for tub liners or print wrappers is to place same in boiling water for a few minutes. chapter viii. bacteria and cheese making. butter, such as that of the sweet-cream type that is highly esteemed in many parts of the world, may be made without the aid of bacteria, but no important kind of cheese can be made under commercial conditions without them. =types of cheese.= cheese consists of the fat and the precipitated casein of milk, together with a large amount of water and the salts found in milk. the numerous types of cheese may be divided into two groups, depending on the manner in which the curdling of the milk is brought about. sour-milk cheese is made from curd, formed as a result of the acid fermentation of the milk. thus, at the very first stage in the making of this type, the importance of bacteria is apparent. the second type is that made from curd, which is precipitated by the addition of rennet to the milk. this type may also be divided into two groups, depending upon their texture; the hard cheese, and the soft cheese. the ordinary cheddar, the common american type, is the most important example of the hard cheese; limburger, of the soft cheese. cheese are designated as hard or soft, depending upon the amount of whey that is retained in them during the making process. the moisture content has an important influence on the type and amount of life that develops on and in the curd mass, and as will be seen, the ripening and flavor of the cheese are dependent upon these biological factors. the two groups of hard and soft cheese have no sharply defined limits, but merge into each other. the extreme types of the hard cheese are so dry and firm that they can be cut only with difficulty. such cheese are used primarily as condiments to impart a flavor to certain dishes, as macaroni, and for this purpose are grated. the extreme type of soft cheese is a soft, pasty mass and can be easily spread with a knife. hard cheese, because the ripening process goes on uniformly throughout the entire mass of cheese, may be made of any size which permits of commercial handling. they can also be kept for long periods and preserve their good qualities. soft cheese are made in small sizes, since on account of their consistency, they could not otherwise be handled, and also because of the manner of ripening. the ripening is due to the action of organisms developing on the surface, the by-products of which diffuse into the curd. if the cheese are too large, the outer layers become overripe, while the interior remains more or less unchanged, or insufficiently changed. soft cheese mature much more rapidly than hard cheese; consequently they are short lived. although made from the same substance, milk, it is noteworthy that there are over four hundred varieties of cheese produced. most of these find only a local market where made. less than a dozen varieties are to be regarded as general articles of commerce. =quality of milk.= in the making of butter there are a number of processes that the maker can use when he finds himself obliged to utilize poor milk. the milk can be pasteurized and the harmful bacteria thus destroyed; desirable kinds can then be added in the form of a pure-culture starter. pasteurization also drives off some of the volatile by-products of the first acid fermentation. by the use of these means, the maker can prepare a very good product from poor material. in the making of most kinds of cheese, especially those of the greatest commercial importance, the cheese maker can call to his help no such aids, but must use the milk as it is brought to him. it is possible to prepare certain kinds of soft cheese from pasteurized milk that differ in no essential point from the same cheese made from raw milk. hard cheese are also made from pasteurized milk, but in most cases such cheese differ, especially in the degree of flavor, from that made from unheated milk. it is quite probable that, as the factors concerned in the ripening of cheese become better known, methods will be evolved for the successful production of many kinds of cheese from pasteurized milk. it has been shown that the quality of milk is almost wholly dependent upon the number and kinds of bacteria it contains. these bacteria pass into the cheese, and there produce the same products as they would have done in the milk itself. in butter making, practically all processes are under the control of the maker, until the product is ready for the market; but cheese, on the other hand, passes through a complicated series of changes after it has left the maker's control. during the manipulation of the milk and the curd in the vat, he can exert some influence on the quality of the product, but he is much more dependent on the quality of the milk than is the case in butter making. every effort should therefore be made to furnish to the cheese maker the quality of milk from which he can prepare fine cheese. in other words, the milk should be produced under clean conditions and carefully cooled and handled until delivered to the maker. poor milk from a single farm may have such an effect upon the cheese made from the milk of twenty farms as to depreciate the selling value of the entire product several cents per pound. the tests that have been previously described (p. 105) have been devised especially for testing the quality of the milk for cheese making purposes, and are of the greatest service to the maker in tracing the source of poor milk. =cheddar cheese.= the first step in the making of cheddar cheese is the "ripening" of the milk, or the development of a small amount of acid. in this fermentation, the development of acid is preceded by an enormous increase in the number of acid-forming bacteria. milk for cheese making should show an acidity of about 0.2 per cent or slightly more than in fresh milk. in other words, the maker wishes the milk to be in such condition, bacteriologically, that if kept at a temperature favorable for the growth of the acid-forming bacteria, the acidity will increase rapidly. the curdling of the milk to precipitate the cheese solids is produced by the addition of rennet, which is obtained by extracting the fourth stomach of the young calf with a solution of common salt. in the past the maker prepared his own rennet solution from the dried stomachs ("rennets"), but at present, the extract is prepared commercially, in a much more uniform manner. the rapidity of the curdling is dependent upon the acidity of the milk. in order to secure proper rennet action, a slight increase of acid over that found in fresh milk is usually necessary; thus at the very beginning of the process of making cheddar cheese, the bacteria are of importance. as the milk curdles, the bacteria are enclosed in the curd as are the fat globules. the curd is cut into small fragments by means of a curd knife, and as the mass is warmed, the acid develops, causing the curd particles to shrink, thus expressing the whey. within a short time, the volume of the curd is not more than one-eighth that of the milk, but in the curd are held over 75 per cent of the bacteria of the milk. to secure rapid curdling in the vat, the milk is warmed to 85° to 90° f., a temperature that is most favorable for the growth of the lactic bacteria. since there is a large number of bacteria concentrated in a small volume, and the temperature, as well as all other conditions, is favorable to growth, multiplication of the bacteria goes on rapidly, and as a consequence, acid is formed in large amounts, as is shown by the following figures given by publow for the manufacture of the export type of cheddar cheese: acidity of milk before adding rennet .2 to .21 per cent acidity of whey before heating curd .14 to .145 " acidity of whey before removing from curd .16 to .18 " acidity of whey coming from the curd after removal of whey and curd is packed .24 to .30 " acidity of whey coming from curd before milling .65 to .75 " acidity of whey coming from curd before salting .90 to 1.10 " if the milk had been kept at the same temperature as the curd, the acidity would have increased much more slowly since the acid would have been distributed through a larger volume. in the cheese curd the same amount of acid is probably formed, as would have been produced in the total amount of milk during the same interval. the acid produced by this bacterial activity has a most marked effect on the curd. at first the curd masses are tough and firm, the particles showing no tendency to adhere to each other. as the acid increases in amount, the curd becomes plastic, the outer surface of the particles adhering or "matting," as the maker expresses it. the result is a solid coalescent mass of curd, which is cut into small pieces, _i.e._, "milled," before it is put to press. the acid allows the blending of the pieces under the influence of the pressure so that a cheese is one single mass. under certain abnormal conditions, the development of acid may be interfered with and the particles of curd fail to mat, in which case, the cheese will be crumbly when it is cut. the determination of the proper time for pressing is made by the application of what is known as the hot iron test. this is made by determining the length of the "strings" or "threads" which can be drawn from a mass of curd when it is brought in contact with a hot iron at a cherry red heat, the length of the curd threads being a measure of the amount of acid that has been formed in the curd. the rate of acid formation within the curd particles is also measured by determining the acidity of the whey as it comes from the curd at different stages in the making. this test, which is often used in place of the "hot iron" test is carried out in the same manner, as in determining the acidity of milk or cream. the quality of the cheese, both as to texture and flavor, is dependent to a great degree upon the amount of acid that is formed during the various stages in making; hence, the successful maker must follow closely by some means the acid formation in the curd until it is put to press. it is very necessary that the milk shall contain a sufficient number of acid-forming bacteria to produce the required amount of acid. if a sufficient number of bacteria are not present in the milk as it is received, as is the case with very sweet milk, they must be added by the maker in the form of a starter, or the process of making will be much prolonged. [illustration: fig. 35.--bacteria in cheese. a photomicrograph of curd just after curdling has taken place. note the few lactic acid bacteria embedded in the curd.] =starters in cheese making.= the starters used in cheese making, are identical with those employed in butter making and the same precautions should be observed in their propagation. it is important that the starters should not be such as to form a hard curd that cannot be mixed uniformly with the milk, since the curd particles would appear as white specks in the cheese. the starter should be added to the milk through a hair sieve, and well mixed with the milk, so as to distribute the bacteria uniformly. amounts varying from 0.5 to 2 per cent are used. in butter making, it is essential that the bacteria of the starter be able to form not only acid, but sufficient flavor-forming substances to impart to the butter a desirable flavor. in cheese making it is not probable that this latter characteristic is of any particular importance. [illustration: fig. 36.--bacteria in cheese. a photomicrograph of curd at the time the salt is added. the lactic acid bacteria have increased materially in numbers.] it is desirable that the process of cheese making shall conform as closely as possible to that which experience has shown to give the best results. the rate at which acid is developed in the curd and the rapidity with which the whey is expelled therefrom should bear a certain ratio to each other. if the milk has too high a degree of acidity, _i.e._, is overripe, the acidity developed in the curd will be too high before the curd is sufficiently firm; with a very sweet milk, the reverse may be true. it is desirable for the cheesemaker to obtain as good an idea as possible of the condition of the milk with reference to its bacterial content, since this will determine the rate at which acid will be formed in the curd. if the milk is too sweet, _i.e._, too low in acid-forming bacteria, a starter should be added. the only methods by which this information can be obtained by the maker is by determining the acidity by the usual method or better by the use of the rennet test by which is ascertained the time required for a given amount of rennet to curdle a definite quantity of milk at a standard temperature. the varying factor in the test will be the acidity of the milk. very slight differences influence profoundly the time of curdling. if, working under standard conditions, it is found that the time of curdling of one sample is 10 seconds and of another sample, 20 seconds, it is proof that the acidity of the first is higher than that of the second, that its bacterial content is greater and that acidity will develop in the curd more rapidly. the first may need a small amount of starter, the second a larger quantity. working with milk from the same source, the maker, from his experience, will know how much starter should be added to milk that has given a certain result with the rennet test in order that the acid shall be developed in the curd at a desired rate. =ripening of cheese.= the curd at the time it is put to press is tough and rubbery, and has none of the characteristic flavor of cheddar cheese; it is also quite insoluble and indigestible. before the cheese is fit to eat it must pass through a complex series of changes which are collectively known as _ripening_. in these changes there is not only a breaking down of the casein into soluble compounds, which process makes the cheese soft and plastic under pressure, but the characteristic flavor is developed in greater or less degree. a very considerable part of the cheese thus becomes soluble in water, and it is much more easily digested than in an unripened condition. the different factors that are operative in the ripening changes are not yet fully known, but in recent years as a result of scientific study, material progress in the study of the changes has been made. =rennet.= the commercial rennet extract when in condition for use contains very few bacteria. a preservative, boric acid, is added by the manufacturer to restrain the bacteria, otherwise the extract would soon be unfit for use. the bacteria in the commercial rennet extract are too few to be of any importance whatever in the ripening process. rennet extract contains an enzyme, rennin, that causes the milk to curdle; also another enzyme, pepsin, that exerts a digestive action on the curdled casein. pepsin is always found in the stomach juices of all animals, but no digestive action takes place, unless the reaction is distinctly acid, as is the ease under normal conditions, since hydrochloric acid is excreted by the walls of the stomach. outside of the stomach, the same conditions must obtain with reference to the presence of acid, if pepsin is to exert a digestive effect. in the cheese curd, the milk sugar is rapidly changed into lactic acid by the action of the bacteria. this gives the proper chemical reaction for peptic action, and the enzyme is then able to act on the paracasein, the nitrogenous part of the cheese. if milk contains no acid-forming bacteria, conditions will not permit of peptic action, and as a consequence, the ripening processes do not take place. if the sugar is fermented by some organism that does not form acid, as the lactose-fermenting yeasts, the cheese does not ripen. the lactic bacteria are therefore an essential factor in inaugurating the ripening changes in all types of rennet cheese. =preservative action of acid.= in a previous chapter it was shown that raw milk does not undergo putrefaction because of the restraining effect of the acid formed by the lactic bacteria on the putrefactive organisms. this same phenomenon is noted in cheese. milk always contains putrefactive bacteria which pass into the cheese, but they cannot grow therein because of the high acidity. in the absence of the acid-forming organisms in the cheese, the cheese may remain tough and rubbery, on account of the lack of suitable conditions for the action of the pepsin of the rennet extract, or when the milk contains large numbers of digesting organisms, the cheese may develop a putrefactive condition, as noted by the offensive odor and soft pasty texture. =other factors concerned in cheese ripening.= there are other factors that are also concerned in the complex series of ripening changes noted in cheddar cheese. all animal fluids and tissues, if kept under perfectly sterile conditions at ordinary temperatures, will undergo a certain amount of decomposition, due apparently to their content in enzymes that have a digestive action. meat kept in storage becomes more tender due to the softening of the connective tissue. milk, derived as it is from actively secreting cell tissue, gives certain reactions that are common to living material. if chloroform, which restrains the action of bacteria, but does not prevent the activity of enzymes, is added to it, it will curdle in the course of a few weeks and will become partially digested. this digesting ferment found in milk is known as _galactase_. compounds are formed in milk thus preserved that are similar to those found in a ripe cheddar cheese. many experiments have been made with pasteurized milk, but it has not been possible to produce typical, normal cheese from thoroughly pasteurized milk. such cheese are markedly deficient in the typical flavor of cheddar cheese. from this fact it is believed that the inherent enzymes of milk are a factor of some importance in the ripening of this type of cheese at least, if not of all types. in the past, other factors have been thought to be of importance. duclaux, a french bacteriologist, considered that the enzymes formed by the digesting bacteria are responsible for the ripening. it is now known that they can have but little if any part in the process, since they are not present in all cheese in sufficient numbers to have any marked effect, and since the acidity of the cheese mass will not permit of their development. other types of bacteria have been considered by bacteriologists to be of importance in the ripening process, but it is certain that the purely digestive change in the mass of the cheese can be accounted for through the action of the factors already noted. =flavor production.= the flavor of any type of cheese is the most important characteristic, just as it is in butter, for it is largely the flavor that determines the selling value of the product, and is the most difficult thing to control. it has been thought that the flavor-producing substances were derived from the paracasein of the curd and were produced by the factors that are concerned in the digestion of the paracasein. it has been shown that a cheese may be thoroughly ripened as far as its physical properties are concerned; that it may contain the end products of casein digestion, and yet be low in flavor. from recent researches it seems probable that the production of flavor is connected with the change that the sugar undergoes in the acid fermentation, as volatile acids, acetic, formic, etc., as well as alcohols and esters are formed in increasing amounts as the ripening progresses. these may have come from the decomposition of the milk sugar, or from a secondary change in the products of the lactic fermentation. there are organisms in both milk and cheese that do not grow on the ordinary culture media used by the bacteriologist, and it may well be that some of these are of importance in flavor production. their destruction in pasteurization is likely to be one of the reasons for the failure of cheese made from pasteurized milk to develop typical flavor. =effect of temperature on ripening.= the temperature at which the ripening cheese is kept has been found to be of the greatest importance in determining the quality of the product. if the cheese is kept at high temperatures, the ripening proceeds rapidly; the cheese is short lived, and has a sharp, strong flavor, and generally a more or less open texture. unless the cheese is made from the best quality of milk, it is likely to undergo undesirable fermentations when ripened at high temperatures. within recent years it has been found possible to ripen cheese at temperatures that were previously thought to be certain to spoil the product. much of the cheese is now ripened at temperatures below 50° f. the ripening goes on more slowly than at higher temperatures, but the flavor of the cheese is clean and entirely devoid of the sharp undesirable tang that is so frequently noted in old cheese, and the texture is solid and meaty. ripening at low temperatures, when the milk is not of the best quality, is certain to result in a much better product than when higher temperatures are employed. =abnormal fermentations in cheese.= as has been previously shown, it is necessary to have an abundant supply of acid-forming bacteria in the milk from which cheese is to be made. if these bacteria are supplanted by other kinds, the product will be more or less abnormal either in texture or in flavor, or possibly in both. many of these abnormal fermentations have been studied and the organisms concerned in the changes found. if the milk is handled carelessly, it will contain many bacteria able to form acid and gas. as noted previously, these organisms form products in milk that have an offensive odor and a disagreeable taste. in cheese the gases cause the formation of holes, more or less numerous, depending on the number of the gas-forming bacteria in the milk. where these bacteria are abundant, gas may appear while the curd is in the vat, causing it to float in the whey, when it is known as a "floater." again, the gas may not become evident until the cheese is in the press or on the curing shelf, when it becomes apparent by the swelling or bulging of the cheese. such cheese is termed "huffed" or "swelled." the internal pressure may be so great as to cause the cheese to crack and to force out some of the curd. the presence of gas holes is indicative of a poor cheese, because the formation of gas is always accompanied by the presence of other undesirable compounds. pure culture starters are often used to overcome gassy fermentations. in cheese a certain amount of acid can be produced by the acid-forming bacteria. when the pure lactic bacteria alone are present, the cheese is very likely to be of good quality. if the sugar is fermented by gas-forming organisms, the curd will be full of holes and the flavor poor, while if the sugar is fermented by a mixture of the desirable and undesirable bacteria, the quality of the product will depend on the relation of the two types. if through the addition of a pure-culture starter, the proportion of desirable bacteria is increased, the gas will be lessened in amount and the cheese improved. it was formerly supposed that the lactic bacteria had an injurious effect on the gas-forming organisms. there is no good reason to believe that this is the case, but that both grow in the milk and cheese, but since only a certain amount of acid can be produced, it is important to have as much of it formed by the lactic bacteria as possible, since the amount of injurious products in the cheese will thus be limited. [illustration: fig. 37.--gassy cheese. such a cheese is worthless on account of its poor flavor. the irregular holes are mechanical. the crack on the upper side is due to the pressure of the gas which has caused the cheese to bulge at this point.] the gas formed in the curd before the cheese is put to press can be gotten rid of by proper manipulation of the curd. while this treatment may improve the appearance of the cheese, it does not eliminate the substances that impart to the cheese undesirable qualities. gassy curds have also been treated by washing the curd with cold water. care must be taken in applying this method for the removal of too much of the sugar and acid from the curd by the washing will permit the growth of injurious forms of bacteria. the addition of salt or of saltpeter has also been made to the milk in order to overcome gassy conditions in the milk. in the handling of gassy milk, the usual practice has been to develop a larger amount of acid before drawing the whey than in the case of good milk. this was done with the idea that acid suppressed gas formation. it has been shown previously that this is not the case. it has also been shown by doane that the development of too much acid before drawing the whey is likely to result in undesirable flavors, producing what is known as "high-acid" or "sour" cheese. the gas-forming bacteria grow best at high temperatures; hence, cheese kept under these conditions are more likely to be affected by this trouble than are those kept at lower temperatures. the most successful method of preventing trouble with gassy milk in cheese making is to eliminate undesirable milk by frequent testing of the supply of the different patrons by means of the wisconsin curd test. not only gas-forming bacteria may be the cause of gassy cheese, but the lactose-fermenting yeasts may cause similar trouble. if these are abundant in the milk, a considerable part of the sugar may be fermented by them, in which case, carbon dioxide gas is abundantly formed. the cheese thus rendered gassy will present the same appearance to the eye as where the gas is formed by bacteria, but will have a different flavor. the odor of alcohol may be evident, and if most of the sugar has been fermented by the yeast, the acidity of the cheese may not be sufficient for the pepsin to exert its digestive action. milk containing many gas-forming bacteria occurs most frequently in summer. it is claimed by some that the milk of cattle pastured on low lands is more likely to contain the gas-forming organisms than that from cattle running on higher lands. if this is true, it must be due to the bacterial content of the soil; the udders of the animals become soiled as they lie on the ground, and during the milking, the dust finds its way into the pail. many cheese makers think that the milk from an animal suffering from a garget may be the cause of the huffing of cheese. this belief is undoubtedly well founded, as some of the bacteria known to be the cause of garget are gas-forming. =bitter cheese.= in a previous chapter the bitter fermentation of milk has been discussed. if milk containing large numbers of such organisms is made into cheese, the bitterness is very likely to be noted in it. cheese made from milk containing few or no lactic bacteria is likely to develop a bitter taste, due to the growth of the digestive bacteria that are able to grow through the lack of acid in the cheese. if the milk contains considerable numbers of yeasts, a sweet or fruity flavor is apt to develop, due to the products of the fermentation of the sugar by the yeast. this flavor resembles that of fermented fruit, or the bouquet of certain kinds of wine. =putrid cheese.= in the absence of acid-forming bacteria, the cheese may develop a putrid or rotten odor, due to the growth of some types of putrefactive or digesting bacteria. this trouble is very infrequent in cheddar cheese, since this is made from ripened milk, but occurs more frequently in those types in which no acid is developed. bacteria develop in the cheese in colonies or masses, just as they do in the plate cultures of the bacteriologist, made with transparent media, such as gelatin. cheese is opaque; therefore, the growing colonies cannot be readily discovered, but when pigment-forming bacteria grow in the cheese, their presence is likely to be noted, because of the colored spots that are formed. =rusty spot.= the "rusty spot" that has been encountered in new york and canada is due to one of the colored bacteria which produces an orange or yellowish-red pigment. various other pigment-forming organisms have been met in cheese, each producing its colored colony which differentiates itself from the mass of the cheese. if the pigment is produced in considerable quantities, and is soluble in any of the constituents of the cheese, the color will not appear in spots but will be more diffuse, or may impart a color to the entire mass. cases of acute poisoning arising from the ingestion of cheese are not infrequently reported; similar instances result from the use of ice cream. in both cases it is believed that poisonous products have been formed by bacteria, probably by some of the putrefactive forms. from what has been said with reference to the abnormal fermentations of cheese, it will be seen that they are always due to the lack of acid-forming bacteria, or to their partial replacement by other types. in order to prevent such troubles, it is necessary to insure that the milk has been produced under clean conditions, from healthy cows, and has been handled in such a manner as to reach the maker in as sweet and fresh condition as possible. the maker can, by the use of proper starters, control the kinds of bacteria essential for the ripening process. a well trained maker should be able to prepare from such milk a uniform product of the highest quality. the effort of cheese makers at the present time is to handle milk of more or less objectionable quality so as to secure from it as good cheese as is possible. but cheese is so sensitive as to character of milk used that greater effort should be spent in securing an improved supply. =moldy cheese.= in the case of the cheddar cheese and other types of hard cheese, it is essential that their surfaces be kept clean, and not discolored by the growth of molds, which find favorable conditions for growth on the surface of the cheese in the moist atmosphere of the curing room. the molding of cheddar cheese can be prevented by covering the cheese with a layer of paraffin which stops the development of the mold spores, by shutting off the necessary supply of oxygen. for this purpose the cheese are dipped in melted paraffin when a few days old. in the case of types of cheese which are salted by applying the salt to the surface, or with soft cheese which ripen from the outside, other methods of mold prevention are employed, such as rubbing and washing the cheese. the curing room itself may be freed from the mold spores by the use of such standard disinfectants as formalin or sulphur. =swiss cheese.= one of the most important kinds of hard cheese, is the swiss or emmenthaler, so named, from the country and valley in which the cheese was first made. in america, this type was introduced by swiss immigrants, and is being made in constantly increasing quantities in ohio and wisconsin. swiss cheese is a hard firm type, appearing in the markets in the form of the flat circular "drum" cheese, two to three feet in diameter, and six to eight inches thick, or in the smaller "block" form. in this country the cheese is prepared twice a day, since it is necessary to work up the milk while it is perfectly sweet. indeed, the milk is received at the factories while it is still warm, and within five or six hours after it is drawn from the cow the cheese is on the press. if the attempt is made to prepare swiss cheese from the kind of milk that is best suited for cheddar purposes, _i.e._, milk in which the acidity has increased to some extent, the flavor of the resulting product is likely to approximate a cheddar cheese rather than that of a swiss. in the salting process, the salt is not mixed with the curd before it is pressed, but is applied by immersing the cheese for a few days in a saturated brine, and then rubbing salt over the surface of the cheese. in this way the salt gradually diffuses quite uniformly through the cheese. the method of salting has apparently a marked influence on the ripening process, since if the salt is added in the same way, and in amounts used in the cheddar process, the flavor will not be that of a swiss cheese but will resemble a cheddar. in cheddar cheese, the whey is expelled from the curd by means of the acid which is developed in the curd, and by heating the curd to a temperature of 95° f. to 100° f. in swiss cheese the development of acid during the making process is prevented, because of the smaller number of acid-forming bacteria in the milk; other factors must therefore contribute to the expulsion of the whey to secure a firm curd. this is accomplished by cutting the curd into very small pieces and by briskly stirring it during the making, heating it during this process for a period of 20 to 30 minutes at 130° to 140° f. it might be thought that this high temperature, which is approximately that used in pasteurization would destroy the acid-forming bacteria, but these are apparently protected as they are within the curd. during the time the cheese is being pressed, the contained bacteria begin to grow and the whey coming from the cheese toward the end of the pressing shows a high acidity. if it does not show such a development of acid, the maker has reason to believe that the cheese may never ripen in a typical manner. it has been mentioned that the milk contains but few acid-forming bacteria. the maker, however, attempts to insure the presence of a sufficient number by the use of "home-made" rennet. this is prepared by placing a piece of dried rennet, _i.e._, the stomach of the calf, in whey, keeping the same in a warm place for twenty-four to thirty-six hours. as the rennet contains acid-forming organisms, these grow rapidly in the warm whey, so that by adding this sour whey to the milk, the maker is not only adding rennet, that is to curdle the milk, but also a small starter of lactic bacteria. if the rennet thus prepared contains no harmful bacteria and the milk is of good quality, the cheese is likely to ripen in a normal manner. the rennet should be prepared with due regard to bacteriological principles, a condition that is rarely met in swiss factories in this country. swiss cheese has two striking characteristics, the flavor and the presence of holes or "eyes." the flavor is sweetish rather than the sharp and pungent flavor of cheddar cheese. the bacteria concerned in its production are not known, but it is certain that specific organisms play some role, since if the flora of the cheese is changed by salting the curd or by the use of milk containing large numbers of lactic bacteria, the flavor will also be changed. this role of the acid-forming bacteria in swiss is the same as in cheddar, _i.e._, through the acid, conditions are established for peptic action, the curd being partially digested while at the same time the curd mass is protected from putrefactive processes. in swiss cheese during the ripening process, holes about the size of a large cherry develop which should be quite uniformly distributed throughout the cheese. the inner surface of the hole is glistening and, in a well-ripened cheese, a small quantity of clear brine, _i.e._, "tears" may be noted. these holes or "eyes" may be called the trade mark of the swiss cheese, since without them the product has a lessened commercial value, even if it possesses the typical flavor. the "eyes" are caused by bacteria that ferment the lactic acid produced by the lactic bacteria, forming from it propionic acid and carbon dioxide, the latter gas being the cause of the hole or "eye." [illustration: fig. 38.--swiss cheese. normal development of "eyes" in a swiss cheese. the eyes are generally as large as a cherry.] the "eye"-forming organisms cannot grow in the presence of any amount of salt, hence, if salt is added directly to the curd, the cheese is likely to be "blind" or free from holes. the eyes are formed not at the time gas holes are produced in a cheddar cheese, _i.e._, early in the ripening process, but after a lapse of three or four weeks. they are most abundant in the middle of the cheese since the manner of salting is such as to inhibit their formation near the surface. the eye-forming bacteria may have some effect on the flavor of the cheese. the swiss maker encounters the same troubles as does the cheddar maker. gassy cheese is more prevalent in the swiss than in the cheddar industry, since the maker cannot call to his aid the methods used by the cheddar maker, viz., the addition of a heavy starter, the washing of the curd, etc. it is especially important that the quality of the milk be first class in every respect, and yet customs prevail in the swiss industry that are directly inimical to the production of good milk. the grossest carelessness prevails at the factories in the matter of handling the whey. it is often kept in individual barrels for each patron. (see fig. 8.) these are not kept thoroughly clean and the result is that the whey taken back to the farm in the cans that are used to bring the fresh milk is often in an advanced stage of fermentation. there are many other kinds of hard cheese; but in each, so far as is known, the role of the acid-forming bacteria is identical with that noted in cheddar and swiss cheese, viz, in preparing conditions favorable for peptic action, and preventing the development of putrefactive bacteria present in the curd. =roquefort cheese.= among the more important foreign types of cheese that are characterized by the development of mold is roquefort, so named from the district in france in which it is made. this cheese is made from sheep's milk, in much the same manner as cheddar. the characteristic process in its preparation is the inoculation of the curd, at the time it is put to press, with the spores of a particular kind of mold, a type closely related to the ordinary green mold of bread and cheese. the mold for inoculation is grown on bread, the whole mass being dried so that it can be powdered; then the ground-up material is sprinkled on the curd as it is placed in the press hoops. the first stage in the ripening of roquefort is probably identical with that of the types of hard cheeses already considered, the breaking-down of the curd being due to the pepsin of the rennet used, which action is made possible by the acid formed by the bacteria. the second stage in ripening, and one in which the characteristic flavor of the cheese is developed, is due to the growth of the mold with which the cheese is seeded. molds can grow only in the presence of air, and in order to provide this condition, the cheese are run through a machine having a series of needle-like projections which fills the cheese with fine holes. this allows the air to penetrate the cheese and the mold to grow, the fruiting of which develops the characteristic flavor. the changes produced by the mold are not well understood, but the flavor is evidently connected with its development since in the absence of mold, it does not appear. the cheese must be cured under carefully controlled conditions, as to temperature and moisture; in france these are secured by curing the cheese in limestone caves that are highly saturated with moisture. attempts have been made to make roquefort cheese in other parts of the world, but they have never been successful, due undoubtedly to the fact that the proper environment and conditions for the development of the various types of organisms necessary in the ripening process have not been met. this cheese is sold for 50 to 75 cents per pound in the markets of the world. there are two other kinds of cheese that are closely related to roquefort, as to the manner of ripening, viz., the gorgonzola of italy and the stilton of england, both of which possess their characteristic flavors by reason of the development of molds. in stilton cheese the mold is not intentionally added, the maker relying on the contamination that comes from the factory for the usual seeding. if this does not develop, it is sometimes inoculated by exchanging plugs with a well-ripened stilton. this method is not so certain as in the inoculation of roquefort. =camembert cheese.= a typical example of soft cheese is one of the french types, known as camembert. this cheese is prepared from cow's milk which is curdled by rennet. the curd is not cut but is dipped into the forms, which condition, taken with the absence of pressure in forming the cheese, accounts for the large quantity of whey in it. the finished cheese are about one inch in thickness and three inches in diameter. in the ripening, the moisture and temperature of the curing room are very carefully regulated. the first stage in the ripening is due to the rennet and the lactic bacteria. later there appears on the surface of the moist cheese, a moldy growth. in this, there are at least two kinds of molds, the ordinary mold that appears on sour milk, _oidium lactis_, and another that is related to the bread mold but which has a white instead of a green fruiting stage. these molds are confined to the surface of the cheese but the enzymes which they produce diffuse into the substance, changing the color from a dull, opaque white to a translucent yellow. the acid that has been formed by the lactic bacteria is gradually used up by the growth of the mold, and conditions then become favorable for the growth of putrefactive bacteria which digest the curd. the cheese is ready for use when the action of the mold has penetrated to the center of the cheese, and before any pronounced putrefaction has taken place. the production of the typical flavor is dependent upon there being a definite relation between the growth of the molds and bacteria. this relation is dependent largely upon the moisture and temperature of the curing room. these cannot always be regulated with exactness; and hence, much of this type of cheese is not of first quality, and must be sold for a low price. while such fancy cheeses, as camembert, bring fifty cents and upward per pound, and the yield from the milk is much greater than with the hard type of cheese, yet the difficulties of successful manufacture are such as to make success less easily attained than with the other types. there are many other kinds of soft cheese that depend for their ripening upon factors similar to those concerned in the ripening of camembert; most of them are, however, of small importance from a commercial standpoint. =limburger cheese.= a very famous cheese is one originally made in germany to which the name limburger is given. it is classed as a soft cheese although it is much firmer than camembert. this cheese is made from cow's milk and is pressed very lightly or not at all, which condition accounts for its high per cent (50 per cent) of moisture. the surface is kept moist by repeated washing of the cheese, and by keeping the air of the curing room very moist. a yellowish, slimy, bacterial layer soon develops on the surface under these conditions. the enzymes produced by this external growth gradually diffuse to the center of the cheese, when it is regarded as ripe. the odor of the matured product is somewhat putrefactive, but is not so offensive as is usually supposed. definite knowledge concerning the types of organisms concerned in the surface layer is very limited. it is not certain whether the same kinds of organisms must always be present. limburger is much easier to make than camembert, due possibly to the fact that there are not needed definite forms of life and that the balance between them is not so delicate. a cheese known as brick is closely related to limburger in its method of making and of ripening but is less pronounced in flavor. in the manufacture of all of these types of cheese, troubles are likely to develop, due to an abnormal bacterial condition of the milk. it will be seen from what has been said that the bacteria are essential factors in cheese ripening, and that the cheese industry, like the butter industry, may be called a true fermentation industry. close co-operation must exist between the milk producer, and the maker so that the type of fermentation that goes on in the milk can be controlled. a recognition of the fundamental principles governing these fermentations, both normal as well as abnormal, is now regarded as an essential part of the training of the dairy manufacturer of today. chapter ix. bacteria in market milk. within the last decade attention has been especially directed toward the quality of milk that is furnished to the people in the cities. this has come about, in part, in connection with the demands made for better and purer food of every kind. these demands are reflected in the pure-food laws enacted by the federal government, and by the various states and municipalities. another factor that has focused attention on the milk supplies has been the belief that it plays an important role in the production and distribution of disease, especially among children. the rapid growth of cities in all of the great countries of the world, the higher standard of living, and the greater demand for milk and other dairy products, has, of necessity, widened the zone from which the milk supply of any particular city must be drawn. milk is now an article of export and of import; some of the great cities draw a portion of their supply from farms hundreds of miles away. this means that a longer time must elapse between the time of production and consumption, necessitating the exercise of greater care in production and handling in order to preserve the milk until it reaches the consumer. in the past in the cities, as in the smaller towns at present, the supply was largely furnished by the producer directly to the consumer. this direct contact afforded the consumer the opportunity of informing himself of the conditions under which his milk supply was produced if he desired. the advent of the middleman in the business, and the gathering of the milk from many hundreds of farms, and its redistribution to thousands of homes has made it impossible for the individual consumer to learn anything of the conditions surrounding production. when the individual cannot protect himself against fraud and unhealthful conditions, it is the duty of the government to protect him. this is the theory underlying the modern control of food supplies, water supplies, and of living conditions in general. acting on this basis the cities are seeking to control, to an increasing degree, the healthfulness and cleanliness of the milk supply. formerly such control as was given was largely with reference to the composition of the milk, the regulations providing that it should contain not less than a minimum amount of fat and other solids, and be free from preservatives. the more modern regulations are much more complex and touch every phase of production and handling that can, in any way, affect the value of the milk as human food. =municipal regulations.= the different cities vary widely in the methods employed to secure a satisfactory milk supply. rules and regulations are adopted to which the producer and dealer must conform. in order to ascertain whether the regulations are being obeyed, two types of examinations may be made: first the inspection of the farms and of the plants of the dealers; second the examination of the milk itself with reference to its chemical composition, bacterial content and temperature. the city of new york is doing more to safeguard and to improve its milk supply than any other large city in this country. a brief summary of its regulations and methods follow. a copy of the rules is furnished to each dairyman and is supposed to be posted in the stable. the cows. 1. the cows must be kept clean, and manure must not be permitted to collect upon the tail, sides, udder and belly of any milch cow. 2. the cows should be groomed daily, and all collections of manure, mud or other filth must not be allowed to remain upon their flanks, udders or bellies during milking. 3. the clipping of long hairs from the udder and flanks of the cows is of assistance in preventing the collection of filth which may drop into the milk. the hair on the tails should be cut, so that the brush will be well above the ground. 4. the udders and teats of the cow should be thoroughly cleaned before milking; this to be done by thorough brushing and the use of a cloth and warm water. 5. to prevent the cows from lying down and getting dirty between cleaning and milking, a throat latch of rope or chain should be fastened across the stanchions under the cow's neck. 6. only feed which is of good quality and only grain and coarse fodders which are free from dirt and mould should be used. distillery waste or any substance in a state of fermentation or putrefaction must not be fed. 7. cows which are not in good flesh and condition should be immediately removed and their milk kept separate until their health has been passed upon by a veterinarian. 8. an examination by a veterinary surgeon should be made at least once a year. the stable. 9. no stagnant water, hog-pen, privy or uncovered cesspool or manure pit should be maintained within 100 feet of the cow stable. 10. the cow stable should be provided with some adequate means of ventilation, either by the construction of sufficient air chutes extending from the room in which the cows are kept to the outside air, or by the installation of muslin stretched over the window openings. 11. windows should be installed in the cow barn to provide sufficient light (2 sq. feet of window light to each 600 cubic feet of air space the minimum) and the window panes be washed and kept clean. 12. there should be at least 600 cubic feet of air space for each cow. 13. milch cows should be kept in a place which is used for no other purpose. 14. stable floors should be made water-tight, be properly graded and well drained, and be of some non-absorbent material. cement or brick floors are the best, as they can be more easily kept clean than those of wood or earth. 15. the feeding troughs and platforms should be well lighted and kept clean at all times. 16. the ceiling should be thoroughly swept down and kept free from hanging straw, dirt and cobwebs. 17. the ceiling must be so constructed that dust and dirt therefrom shall not readily fall to the floor or into the milk. if the space over the cows is used for storage of hay, the ceiling should be made tight to prevent chaff and dust from falling through. 18. the walls and ledges should be thoroughly swept down and kept free from dust, dirt, manure or cobwebs, and the floors and premises be kept free from dirt, rubbish and decaying animal or vegetable matter at all times. 19. the cow beds should be so graded and kept that they will be clean and sanitary at all times. 20. stables should be whitewashed at least twice a year unless the walls are painted or are of smooth cement. 21. manure must be removed from the stalls and gutters at least twice daily. this must not be done during milking, nor within one hour prior thereto. 22. manure should be taken from the barn, preferably drawn to the field. when the weather is such that this cannot be done, it should be stored not nearer than 200 ft. from the stable and the manure pile should be so located that the cows cannot get at it. 23. the liquid matter should be absorbed and removed daily and at no time be allowed to overflow or saturate the ground under or around the cow barn. 24. manure gutters should be from six to eight inches deep, and constructed of concrete, stone or some non-absorbent material. 25. the use of land plaster or lime is recommended upon the floors and gutters. 26. only bedding which is clean, dry and absorbent should be used, preferably sawdust, shavings, dried leaves or straw. no horse manure should be used as bedding. 27. the flooring where the cows stand should be so constructed that all manure may drop into the gutter and not upon the floor itself. 28. the floor should be swept daily. this must not be done within one hour prior to milking time. 29. if individual drinking basins are used for the cows, they should be frequently drained and cleaned. 30. all live stock other than cows should be excluded from the room in which the milch cows are kept. (calf or bull pens may be allowed in the same room if kept in the same clean and sanitary manner as the cow beds.) 31. the barnyard should be well drained and dry, and should be as much sheltered as possible from the wind and cold. manure should not be allowed to collect therein. 32. a suitable place in some separate building should be provided for the use of the cows when sick, and separate quarters must be provided for the cows when calving. 33. there should be no direct opening from any silo or grain pit into the room in which the milch cows are kept. the milk house. 34. a milk house must be provided which is separated from the stable and dwelling. it should be located on elevated ground, with no hog-pen, privy or manure pile within 100 feet. 35. it must be kept clean and not used for any purpose except the handling of milk. 36. the milk house should be provided with sufficient light and ventilation, with floors properly graded and made water-tight. 37. it should be provided with adjustable sashes to furnish sufficient light and some proper method of ventilation should be installed. 38. the milk house should be provided with an ample supply of clean water for cooling the milk, and if it is not a running supply, the water should be changed twice daily. also a supply of clean ice should be provided to be used for cooling the milk to 50 degrees within two hours after milking. 39. suitable means should be provided within the milk house, to expose the milk pails, cans and utensils to the sun or to live steam. 40. facilities consisting of wash basins, soap and towel should be provided for the use of milkers before and during milking. during the summer months the milk house should be properly screened to exclude flies. the milkers and milking. 41. any person having any communicable or infectious disease, or one caring for persons having such disease, must not be allowed to handle the milk or milk utensils. 42. the hands of the milkers must be thoroughly washed with soap and water, and carefully dried on a clean towel before milking. 43. clean overalls and jumpers should be worn during the milking of the cows. they should be used for no other purpose, and when not in use should be kept in a clean place protected from dust. 44. the hands and teats should be kept dry during milking. the practice of moistening the hands with milk is to be condemned. 45. the milking stools should be at all times kept clean, and iron stools are recommended. 46. the first streams from each teat should be rejected, as this fore milk contains more bacteria than the rest of the milk. 47. all milk drawn from the cows 15 days before, or 5 days after parturition should be rejected. 48. the pails in which the milk is drawn should have as small an opening at the top as can be used in milking; top opening preferably not to exceed 8 inches in diameter. this lessens the contamination by dust and dirt during milking. 49. the milking should be done rapidly and quietly, and the cows should be treated kindly. 50. dry fodder should not be fed to the cows during or just before milking, as dust therefrom may fall into the milk. 51. all milk utensils, including pails, cans, strainers, and dippers, must be kept thoroughly clean and must be washed and scalded after each using, and all seams in these utensils should be cleaned, scraped and soldered flush. the milk. 52. milk from diseased cows must not be shipped. 53. the milk must not be in any way adulterated. 54. the milk as soon as drawn should be removed to the milk house and immediately strained and cooled to the proper temperature. 55. all milk must be cooled to a temperature below 50 degrees f., within two hours after being drawn, and kept thereafter below that until delivered to the creamery. 56. the milk should be strained into cans which are standing in ice water which reaches the neck of the can. the more rapidly the milk is cooled, the safer it is, and longer it will keep sweet. ice should be used in cooling milk, as very few springs are cold enough for the purpose. 57. if aerators are used, they should stand where the air is free from dust or odors, and on no account should they be used in the stable or out of doors. 58. milk strainers should be kept clean; scalded a second time just before using, and if cloth strainers are used, several of them should be provided, in order that they may be frequently changed during the straining of the milk. 59. the use of any preservative or coloring matter is adulteration, and its use by a producer or shipper will be a sufficient cause for the exclusion of his product from the city of new york. water supply. 60. the water supply used in the dairy and for washing utensils should be absolutely free from any contamination, sufficiently abundant for all purposes, and easy to access. 61. this supply should be protected against flood or surface drainage. 62. the privy should be located not nearer than 100 feet of the source of the water supply, or else be provided with a water-tight box that can be readily removed and cleaned, and so constructed that at no time will the contents overflow or saturate the surrounding ground. 63. the source of the water supply should be rendered safe against contamination by having no stable, barnyard, pile of manure or other source of contamination located within 200 feet of it. in order that the farm inspection shall be as effective as possible, and to make the work of the several inspectors as uniform as may be, the dairies are scored. a copy of the score card follows. department of health the city of new york =division of general= =sanitary inspection= =dairy report= inspection no. ... time...... a. p. m. date......191.. 1 =dairyman=.................. =owner= ..................... 2 =p. o. address=............. =p.o. address=.......state... 3 =county=..... state..... =party interviewed=............ 4 milk delivered to creamery at.......... formerly at......... 5 operated by.................. address....................... 6 distance of farm from creamery..... occupied farm since..... 7 no. cows....... no. milking...... no. qts. produced......... 8 all persons in the households of those engaged in producing or handling milk are............free from all infectious disease. weekly reports are..................being filed .......................................................... 9 date and nature of last case on farm........................ 10 =water supply= for utensils is from a............... located .......... feet deep and apparently is............ pure and wholesome............ state any possible contamination located within 200 feet of source of water supply or if water supply is not protected against surface drainage ........................................................... ........................................................... 11 water supply on this farm analyzed.... 191.. result........ 12 style of cow barn.... length.... ft. width.... ft. height of ceiling.... ft. 13 =dairy rules= of the department of health are........ posted .................. 14 =dairy herd= examined by............. on.............. 191.. report............ ================================================================= |perfect| allow | ---------------------------------------------------------------- equipment | | | | | | 15 =cow stable= is.......located on elevated | | | ground with no stagnant water, hog-pen, | | | privy, uncovered cesspool or manure pit | | | within 100 feet | 1 | ..... | | | | 16 =floors=, other than cow beds, are | | | of concrete or some non-absorbent material | 2 | ..... | | | | 17 floors are...properly graded and water-tight | 2 | ..... | | | | 18 =cow beds are=...of concrete or planks | | | laid on concrete | 2 | ..... | | | | 19 =drops= are.....constructed of concrete, | | | stone or some non-absorbent material | 2 | ..... | | | | 20 drops are......water-tight and space beneath | | | is clean and dry. | 2 | ..... | | | | 21 =ceiling= is constructed of.......and is | | | tight and dust proof | 2 | ..... | | | | 22 =windows= no.......total square feet | | | there is...........2 square feet of window | | | light for each 600 cu. ft. air space (1 | | | sq. ft. per each 600 cu. ft.--1) | 2 | ..... | | | | 23 =ventilation= consists of ......sq. ft. muslin| | | in ceiling or..........which is sufficient | | | 3, fair 2, poor 1, insufficient 0 | 3 | ..... | | | | 24 =air space= is......cu. ft. per cow (600 and | | | over--3) (500 to 600--2) (400 to 500--1) | | | (under 400--0) | 3 | ..... | | | | 25 =live stock=, other than cows, are....excluded| | | from rooms in which milch cows | | | are kept | 2 | ..... | | | | 26 there is..........direct opening from stable | | | into silo or grain pit | 1 | ..... | | | | 27 separate quarters are...........provided for | | | cows when calving or sick | 1 | ..... | | | | 28 =cow yard= is..............properly graded and| | | drained | 2 | ..... | | | | 29 =water supply= for cows is..........unpolluted| | | and plentiful | 1 | ..... | | | | 30 =milk house= has...........direct opening into| | | cow barn or other building | 1 | ..... | | | | 31 milk house has..........sufficient light and | | | ventilation | 1 | ..... | | | | 32 floor is.................properly graded and | | | water-tight | 1 | ..... | | | | 33 milk house is...........properly screened to | | | exclude flies | 1 | ..... | | | | 34 milk pails are............of smoothly tinned | | | metal in good repair | 1 | ..... | | | | 35 =milk pails= have...........all seams soldered| | | flush | 2 | ..... | | | | 36 milk pails are..........of the small mouthed | | | design, top opening not exceeding 8 inches | | | in diameter. diameter | 2 | ..... | | | | 37 =racks are=........provided to hold milk pails| | | and cans when not in use | 2 | ..... | | | | 38 =special milking suits= are......provided | 1 | ..... | |-------|-------| | 40 | | | | | =methods= | | | | | | 39 =stable interior= painted or whitewashed | | | on.......which is satisfactory 3, fair 2, | | | unsatisfactory 1, never 0 | 3 | ..... | | | | 40 =feeding troughs=, platforms or cribs are | | | ......well lighted and clean | 1 | ..... | | | | 41 =celling= is..........free from hanging straw,| | | dirt or cobwebs | 3 | ..... | | | | 42 =window panes= are.............washed and kept| | | clean | 1 | ..... | | | | 43 =walls and ledges= are...............free from| | | dirt, dust, manure or cobwebs | 2 | ..... | | | | 44 =floors and premises= are.......free from | | | from dirt, rubbish or decayed animal or | | | vegetable matter | 2 | ..... | | | | 45 =cow beds= are.........clean, dry and no horse| | | manure used thereon | 2 | ..... | | | | 46 =manure= is.......removed to field daily 4, | | | to at least 100 feet from barn 2, stored | | | less than 100 feet or where cows can get | | | at it 0 | 4 | ..... | | | | 47 =liquid matter= is....... allowed to saturate | | | ground under or around cow barn | 2 | ..... | | | | 48 =milking stools= are.......clean | 1 | ..... | | | | 49 =cow yard= is.......clean and free from | | | manure | 2 | ..... | | | | 50 =cows= have......been tuberculin tested and | | | all tuberculous cows removed | 7 | ..... | | | | 51 cows are.....all in good flesh and condition | | | at time of inspection | 2 | ..... | | | | 52 cows are.....all free from clinging | | | manure and dirt. (no. dirty.....) | 4 | ..... | | | | 53 =long hairs= are.....kept short on belly, | | | flanks, udder and tail | 1 | ..... | | | | 54 =udder and teats= of cows are...... | | | thoroughly brushed and wiped with a | | | clean damp cloth before milking | 3 | ..... | | | | 55 =all feed= is.....of good quality and | | | distillery waste or any substance in a state | | | of putrefaction is......fed | 2 | ..... | | | | 56 =milking= is.....done with dry hands | 2 | ..... | | | | 57 =fore milk= or first few streams from each | | | teat is.....discarded | 2 | ..... | | | | 58 =clothing= of milkers is.....clean | 1 | ..... | | | | 59 facilities for washing hands of milkers are | | | ......provided in cow barn or milk | | | house | 2 | ..... | | | | 60 =milk= is strained at.....and.....in | | | clean atmosphere | 1 | ..... | | | | 61 milk is.....cooled within two hours after | | | milking to 50 degrees f. 3, to 55 degrees | | | f. 2 to 60 degrees f. 1 | 3 | ..... | | | | 62 ice is.....used for cooling milk | 1 | ..... | | | | 63 =milk house= is.....free from dirt, rubbish | | | and all material not used in the | | | handling and storage of milk | 1 | ..... | | | | 64 =milk utensils= are.....rinsed with cold | | | water immediately after using and washed | | | clean with hot water and washing solution | 2 | ..... | | | | 65 utensils are.....sterilized by steam or | | | boiling water after each using | 2 | ..... | | | | 66 =privy= is.....in sanitary condition, with | | | vault and seats.....covered and protected | 1 | ..... | | | | |-------|-------| | 60 | | remarks equipment 40 per cent. score .... per cent methods 60 per cent. score .... per cent perfect dairy 100 per cent. score .... per cent a copy of the completed report is left with the dairyman. before the farm inspection is carried out the creameries to which the milk is delivered by the farmers are inspected at the time the milk is being delivered. the temperature of the milk and its cleanliness are noted. in the creamery the straining, cooling and handling of the milk are observed as well as the washing of the milk cans and other utensils, and the construction and condition of the creamery, the opportunity for the water supply to become contaminated, and the presence of infectious diseases among the employees. =grades of milk.= three grades of milk have been established. each dealer is required to state which grade or grades he expects to handle. the specifications for the different grades are as follows. _grade a. guaranteed milk._ guaranteed milk is that produced at farms holding permits therefor from the department of health and produced and handled in accordance with the following minimum requirements, rules and regulations: 1. only such cows shall be admitted to the herd as have not re-acted to a diagnostic injection of tuberculin. 2. all cows shall be annually tested with tuberculin, and all re-acting animals shall be excluded from the herd. 3. no milk from re-acting animals shall be shipped to the city of new york for any purpose whatever. 4. the milk shall not contain more than 30,000 bacteria per c. c. when delivered to the consumer, or at any time prior to such delivery. 5. the milk shall be delivered to the consumer only in sealed bottles, which have been sealed at the dairy. 6. the milk shall be delivered to the consumer within 30 hours of the time at which it was drawn. _grade a. certified milk._ certified milk is milk certified by a milk commission appointed by the medical society of the county of new york, or the medical society of the county of kings, as being produced under the supervision and in conformity with the requirements of that commission as laid down for certified milk, and sold under a permit therefor issued by the board of health. no milk shall be held, kept, offered for sale, or sold and delivered as certified milk in the city of new york which is produced under requirements less than those for guaranteed milk. _grade a. inspected milk--raw._ inspected milk (raw) is milk produced at farms holding permits therefor from the board of health, and produced and handled in accordance with the following minimum requirements, rules and regulations: 1. only such cows shall be admitted to the herd as have not re-acted to a diagnostic injection of tuberculin. 2. all cows shall be tested annually with tuberculin, and all re-acting animals shall be excluded from the herd. 3. no milk from re-acting animals shall be shipped to the city of new york for any purpose whatsoever. 4. the farms at which the milk is produced must obtain at least 75 points in an official score of the department of health. these 75 points shall be made up as follows: a minimum of 25 points for equipment, and 50 points for method. 5. the milk shall not contain more than an average of 60,000 bacteria per c. c. when delivered to the consumer, or at any time prior thereto. 6. unless otherwise specified in the permit, the milk shall be delivered to the consumer only in bottles. _grade a. selected milk--pasteurized._ selected milk (pasteurized) is milk produced at farms holding permits therefor from the board of health, and produced and handled in accordance with the following requirements, rules and regulations: 1. the farms at which the milk is produced must obtain at least 60 points in an official score of the department of health. of these 60 points, a minimum of 20 points shall be required for equipment and a minimum of 40 points for method. 2. all milk of this grade shall be pasteurized, and said pasteurization shall be carried on under a special permit issued therefor by the board of health, in addition to the permit for "selected milk (pasteurized.)" 3. the milk shall not contain more than an average of 50,000 bacteria per c. c. when delivered to the consumer, or at any time after pasteurization and prior to such delivery. 4. unless otherwise specified in the permit, the milk shall be delivered to the consumer only in bottles. 5. all containers in which pasteurized milk is delivered to the consumer shall be plainly labeled "pasteurized." labels must also bear the date and hour when pasteurization was completed, the place where pasteurization was performed, and the name of the person, firm or corporation performing the pasteurization. 6. the milk must be delivered to the consumers within 30 hours after the completion of the process of pasteurization. 7. no milk shall be pasteurized more than once. 8. no milk containing in excess of 200,000 bacteria per c. c. shall be pasteurized. _general regulations for grade a_-1. the caps of all bottles containing milk of grade a shall be white, and shall contain the words "grade a" in black letters, in large type. 2. if cans are used for the delivery of milk for grade a, the said cans shall have affixed to them white tags, with the words "grade a" printed thereon in black letters, in large type, together with the designation "inspected milk (raw)" or "selected milk (pasteurized)," as the quality of the contents may require. _grade b. selected milk--raw._ selected milk (raw) is milk produced at farms holding permits therefor from the board of health, and produced and handled in accordance with the following minimum requirements, rules and regulations: 1. only such cows shall be admitted to the herd as have been physically examined by a regularly qualified veterinarian and declared by him to be healthy, and free from tuberculosis in so far as a physical examination may determine that fact. 2. the farms at which the milk is produced must obtain at least 68 points in an official score of the department of health. these 68 points shall be made up as follows: a minimum of 25 points for equipment, and a minimum of 43 points for method. 3. the milk shall not contain an excessive number of bacteria when delivered to the consumer, or at any time prior thereto. _grade b. pasteurized milk._ pasteurized milk (grade b) is milk produced under a permit issued therefor by the board of health, and produced and handled in accordance with the following minimum requirements, rules and regulations and in further accordance with the special rules and regulations relating to the pasteurization of milk. 1. the milk after pasteurization must be at once cooled and placed in sterilized containers, and the containers immediately closed. 2. all containers in which pasteurized milk is delivered to the consumer shall be plainly labeled "pasteurized". labels must also bear the date and hour when the pasteurization was completed, the place where pasteurization was performed, and the name of the person, firm or corporation performing the pasteurization. 3. the milk must be delivered to the consumer within 36 hours after the completion of the process of pasteurization. 4. no milk shall be pasteurized more than once. 5. no milk containing an excessive number of bacteria shall be pasteurized. _general regulations for grade b_-1. caps of bottles containing milk of grade b shall be white and marked "grade b" in bright green letters of large type. 2. the necks and shoulders of cans containing grade b milk shall be painted bright green, and a metal tag shall be attached to each can with the words "grade b" in large type, and the words of the subdivision to which the quality of the milk in said can conforms. _grade c._ grade c is to be used for cooking and manufacturing purposes only. it includes all raw milk that does not conform to the requirements of any of the subdivisions of grade a or grade b. 1. the caps of all bottles containing milk of grade c shall be white and shall contain in red the words "grade c" in large type and "for cooking" in plainly visible type. 2. cans containing milk of grade c shall be painted red on necks and shoulders and shall have in red the words "grade c" in large type and the words "for cooking" in plainly visible type affixed to each can. all creameries handling milk of different grades will be required to demonstrate to the department of health that they are capable of keeping the grades separate, and must keep records satisfactory to the department of health concerning the amount of milk of each grade handled each day. it is to be noted that the grades of milk are based on the bacterial content of the milk and on the opportunity for the milk to become contaminated with pathogenic organisms. from the statements made in a previous chapter it is evident that the number of bacteria in any sample of milk is dependent upon (1) the original amount of contamination, (2) the age of the milk, and (3) the temperature at which it has been held. a high bacterial content is indicative of poor milk, while a low bacterial content can be obtained, in the case of raw milk, only where due attention is paid to cleanliness and cooling. this relation between the quality of milk and its bacterial content has led many cities to adopt numerical bacterial standards, even when grades of milk have not been established. boston requires that the milk shall not contain more than 500,000 bacteria per cubic centimeter. rochester, n. y., has a standard of 100,000 per cubic centimeter, while chicago requires that the milk on arrival in the city shall not contain more than 1,000,000 per cubic centimeter from may first to september thirtieth, and not over 500,000 between october first and april thirtieth. the sale of milk containing more than 3,000,000 bacteria per cubic centimeter is prohibited. it has been urged that bacterial standards are not of value since the healthfulness of milk depends on the kind of bacteria present rather than on the number. it is well recognized that milk containing millions of acid-forming organisms, butter milk, is a healthful food, while that containing many less bacteria may contain some disease-producing organisms. it has been urged that a qualitative standard should supplant the quantitative. the consumer desires milk that has been produced under clean conditions, and which has good keeping qualities. the harmless forms of bacteria exert the greatest influence on the keeping quality. experience has shown that the quantitative examination of the milk supply as it comes from the farm is the most feasible method of determining, in the laboratory, whether the farmer has obeyed the rules with reference to cleanliness and cooling of the milk. the bacteriological examination also gives an indication as to whether the large number of bacteria is due to gross contamination of the milk with mud and manure, or actual growth of bacteria as in old milk. in the latter case the ordinary acid-forming bacteria will usually predominate in the milk, while in the former, the number of kinds of bacteria and the proportion between the kinds will be changed. it is of course evident that the quantitative standards should be applied with judgment. it is also claimed that the delay in securing the results in the quantitative examination of milk is an objection to the bacterial standard, since the milk is consumed before the laboratory findings can be obtained. it is true that it does not protect the community as far as the particular sample is concerned, but it is also true that the examination is not made for the purpose of determining the condition of the particular sample, so much as it is to determine the methods that are employed on any particular farm, and these do not vary widely from day to day. thus, if a number of samples give high results, it is evident that conditions surrounding production need investigation. if the milk is well cooled on the farm, and kept cold while being shipped, the growth of bacteria will be slow, and the condition of the milk as far as keeping quality is concerned, much better than if less care is used. some cities have temperature standards; new york requires that the milk shall be cooled to 50° f. on the farm, and shall not be above 50° f. on arrival in the city. others require that it shall not be above 50° f. on delivery to the consumer. =certified milk.= in many cities the medical societies have appointed milk commissions, that adopt rules and regulations, concerning the production of milk that shall receive the certificate of the commission. producers, who desire to have their milk thus certified, must satisfy the commission that they are able to conform to the rules. the commission appoints a physician to examine the personnel of the farm, a veterinarian to make frequent examinations of the herd, a chemist to examine the milk as to its contents in fat and other solids, and a bacteriologist to determine the bacterial content of the milk. the rules are very stringent and cover every point that may influence, in any way, the value of the milk as human food. in order to conform to these requirements, a heavy expenditure must be incurred, and the business must pay for such expert service; hence, certified milk must be sold at high prices, twelve to twenty-five cents per quart. this price makes it a special product and its use is confined mainly to infant feeding. the bacterial standard for certified milk is usually 10,000 bacteria per cubic centimeter. it is only by the exercise of the greatest care at every point that the bacterial content can be kept below this maximum. the term "certified milk" has been registered by mr. francisco of new jersey, who was the first to engage in the production of such milk under the direction of the medical milk commission of essex county, new jersey. the use of the term is allowed when the milk is produced under the regulation of any medical milk commission. most certified milk is now produced on fancy dairy farms conducted by wealthy men. the barns and other equipment are the best that can be obtained, and the methods employed, as far as cleanliness is concerned, are extreme. in some of the dairies the bacterial content is reduced to a few hundred per cubic centimeter, or to that which is derived from the interior of the udder. such milk will, when well refrigerated, keep for long periods of time. it is a not uncommon thing for such milk to keep perfectly sweet for ten to fifteen days. =tests for the quality of milk.= at the milk depot and elsewhere, it is frequently desired to determine the bacterial condition of the milk in a less refined manner than by the plate cultures of the bacteriologist, which require a large amount of time for their preparation and do not yield any positive information for at least twenty-four hours. there are a number of such tests that may be applied. [illustration: fig. 39.--sediment testers. in the use of the apparatus on the right, increased air pressure is used to hasten the filtering process; the same is accomplished in the apparatus shown in the center by warming the milk by the injection of steam between the walls of the double jacket.] 1. _dirt or sediment test._ this is made by filtering a pint of the mixed milk through a small disc of absorbent cotton. the insoluble dirt is retained and imparts a color to the cotton, the shade of which is dependent on the amount of dirt (p. 45). since it is impossible to have dirt without bacteria, it is evident that milks containing a large amount of dirt will be high in bacteria. the reverse, however, is not necessarily true. [illustration: fig. 40.--good milk. a plate culture inoculated with 1/100 of a cubic centimeter of milk containing 67 colonies, which equals 6,700 bacteria per cubic centimeter of milk. such milk will keep well.] 2. _acidity test._ the acidity of the milk is also an indication of its bacterial content. if the acidity has increased, above the normal for fresh milk, the bacterial content is certain to be high, and the keeping quality poor. an acidity above 0.2 per cent in market milk is to be avoided, as an increase in acidity is always preceded by a great increase of bacteria. whether the acidity is above or below this point can be rapidly and easily determined at the receiving station by a modification of the farrington acid test. dissolve one alkaline tablet in an ounce of water. a unit volume of this solution added to a unit volume of milk is equal to 0.1 per cent of acidity. if two measures are provided,--one for the alkaline solution holding just twice as much as that used for the milk, the approximate acidity can be quickly determined by mixing a measure of each in a common white cup. if the acidity is above 0.2 per cent the color will remain white; if a pink color develops, it indicates an acidity less than this amount. this test is also useful in the selection of milk or cream that is to be used for special purposes, such as pasteurization. [illustration: fig. 41.--poor milk. a plate culture inoculated with 1/1000 of a cubic centimeter of market milk containing 1,680 colonies, which equals 1,680,000 bacteria per cubic centimeter. such milk has poor keeping qualities.] 3. _alcohol test._ a test giving similar information is made by adding two parts of 70 per cent alcohol to one part of milk, and noting whether curdling occurs. 4. _curd test._ the curd test described on p. 100 gives no indication of the number of bacteria present, only concerning the types present. it has been proposed to combine the fermentation test with the reduction test referred to below and thus gain some idea of, not only the number, but the kinds of bacteria present. 5. _reduction test._ the reduction test is made by adding to twenty cubic centimeters of milk, one-half cubic centimeter of a solution of methylene blue, a coal tar dye. a saturated solution of the dye is made in alcohol, and 2.5 per cent of this solution added to water. the time required for the reduction of the dye or the change of the color from blue to white when the samples are placed in tubes and kept at 98 to 100° f., is dependent upon the number of bacteria present. by allowing the tubes to stand until curdling occurs, and noting the nature of the curd, whether the solid curd of the desirable acid-forming bacteria or the gassy curd of the harmful types is produced, knowledge is gained of the kinds of bacteria present. according to barthel, milks that reduce the methylene blue within fifteen minutes contain hundreds of thousands of bacteria per cubic centimeter. those that require from fifteen minutes to one hour for the disappearance of the color are also high in bacteria, and are to be classed as a poor grade of market milk. if one to three hours is required, the milk is comparatively low in bacteria, and is to be classed as a good grade of market milk. when more than three hours elapse before the disappearance of the blue color, the bacterial content is low and the milk is to be placed in the highest grade. the time of reduction is only a rough index of the number of bacteria present, but it gives a good idea of the keeping quality of the milk, and of the conditions of production and handling. of the above tests the sediment and acid tests are more frequently used. =examination of milk sediments.= in the modern municipal laboratory, efforts are made to determine, as far as possible, the conditions of production on the farms, by an examination of the milk in the laboratory. the samples of milk are sedimented in a small centrifuge, and an examination of the sediment made with the microscope. the types of bacteria and the number of body cells found is an indication as to whether any of the animals of the herd are suffering from inflammation of the udder. the test also gives information similar to the dirt test since the insoluble dirt will be thrown down and will impart a color to the sediment. =pasteurization of market milk.=.the spread of the pasteurizing process as applied to market milk has been rapid. this has been due to the recognition of the fact that only by this process can a safe milk _i.e._, one free from pathogenic bacteria, be obtained. as previously mentioned a small proportion of all human beings that have suffered from typhoid fever become bacillus carriers. it is impossible to examine all persons who may be concerned in the handling of milk in order to ascertain whether they belong to this dangerous and unfortunate class of people. the larger cities have also recognized the impossibility of requiring the tuberculin test of all cattle furnishing milk. pasteurization remains the only safeguard, and it is probable that within a short time all the larger cities will require the pasteurization of all milk, except that produced under strict supervision. as previously mentioned heating causes certain changes in milk. in the treatment of market milk it is desirable to use as low temperatures as will suffice to destroy the disease-producing bacteria. it is fortunate that temperatures that will insure this result have little effect on the milk. the temperatures now recommended for pasteurization are as follows: 158 degrees f. for 3 minutes. 155 degrees f. for 5 minutes. 152 degrees f. for 10 minutes. 148 degrees f. for 15 minutes. 145 degrees f. for 18 minutes. 140 degrees f. for 20 minutes. in actual practice the milk is heated to 145 degrees for 25 to 30 minutes. the acid-forming bacteria are not completely destroyed and the pasteurized milk as a rule will undergo the same type of fermentation as raw milk. it is, however, deemed essential that all pasteurized milk be sold as such; that it be delivered to the consumer within twenty-four hours after pasteurization and that no milk be pasteurized a second time. the continuous pasteurizing machines have the disadvantage that a small portion of the milk passes through so quickly that all pathogenic bacteria therein might not be destroyed, (p. 131). this has led to the use of the "holding" process in which the milk is heated to the desired temperature and then placed in tanks where it remains at this temperature for any desired time. every portion is thus treated in a uniform manner. if the milk is bottled after pasteurization, there remains opportunity for reinfection, possibly with typhoid bacilli. pasteurization in the final container, the bottle, is being recommended. this is possible only when a special bottle is used with a metal cap lined with paper. =milk distribution.= until within recent years in the cities and at present in smaller towns, milk is largely retailed from cans which are carried on the wagons or are kept in stores. this exposes the milk to contamination from street dust and from the container furnished by the consumer. it is well recognized that every utensil with which milk is brought in contact adds more or less bacteria to it, and the less milk is handled, the better will be its condition when it reaches the consumer. milk is now largely retailed in glass bottles which are closed with pulp caps. in some cities the bottling is mainly done in the country at the bottling station to which the milk is brought by the farmers; or it may be shipped by the producer to a distributing company, and all subsequent treatment, as pasteurization and bottling done in the city. milk plants are now generally equipped for the rapid and economical handling of large quantities of milk in a most sanitary manner. the bottles as they are returned from the consumer are washed in a continuously-acting automatic washer which washes, rinses and sterilizes the bottles without their being removed from the cases in which they are carried on the wagons. these machines are effective, if not run at too rapid a rate, so that the bottles are not exposed for a sufficiently long period of time to sterilize them. the bottles are then filled and the paper caps inserted by machinery. the caps can now be obtained from the manufacturers in sealed tubes in which they have been sterilized so that the contamination from this source is avoided. the shipping cans are washed and sterilized with live steam, and in many plants are thoroughly dried, by passing hot air into them. under these conditions they then reach the farmer with none of the musty and disagreeable odor that frequently is present when the can contains a small quantity of water, condensed from steam. the top of the milk bottle over which the milk is poured is exposed to contamination from the hands of the deliveryman. trouble from this source can be avoided if the consumer cleans the lip of the bottle before removing the cap. the better grades of milk are dispensed in bottles, the top of which is protected by an additional cover of paper or tin foil which reaches to the neck of the bottle and is held in place by a crimped metal band. =milk supply of the small cities.= it is true that the quality of milk supplied to the large cities by the great milk companies is generally much superior to that sold in the smaller cities and villages. many of the smaller places are however, attempting in various ways to improve their supply. it is evident that methods will be successful here that can not be employed in the larger places. a detailed and careful farm inspection by a tactful, capable inspector, coupled with proper publicity will do much to improve conditions. the publication of the scores of the different farms, and the demonstration of the sediment test as applied to their product attracts favorable attention to the good dairies and unfavorable attention to the poor. this usually has an effect on the trade sufficient to cause the negligent producer and dealer to improve. it is also becoming recognized that high grade milk can be produced with very simple equipment. in fact the small farm is often more successful in producing high grade milk than is the large farm on which the work must be done by hired help for here the personality of the owner can not make itself felt as where the producer is doing a portion of the work about the barn and dairy himself. it is becoming more and more evident that the chief factor in the production of clean milk is the personality of the producer; he should be one who gets enjoyment out of his clean stables and cows and his high grade product. the man who is producing milk for the city market is but one of many and his individual efforts can not make themselves felt. the dairyman who is marketing his own product is in a position where his efforts to produce a fine product should prove of distinct advantage to him in enabling him to sell it for a higher price than that obtained for ordinary milk. it should be remembered that the production of clean, healthful milk is not a question of equipment, but of methods and of additional work. the cows must be fed, the stables must be cleaned, the cows milked, and the milk delivered to the consumer. if beyond this unavoidable labor a small additional amount is expended, the improvement in the product will be great. it is necessary that the additional work be placed where it will do the most good, in keeping the cows clean both summer and winter so that little need be done in cleaning them before milking, the pails and other utensils kept clean and sterilized, and the milk cooled as soon as possible and kept cold until delivered to the consumer. the delivery should be made within the shortest practicable time after the milk is drawn. in order that the healthfulness of the milk may be beyond question, the herd must be kept free from tuberculosis and some attention should be paid to the health of the men, especially with reference to whether they may be typhoid carriers or not. the necessary labor should not increase the cost of the milk over one cent per quart. it has been shown in many cases that such a product can be marketed at a price that will more than compensate for the additional cost. clean, fresh, rich milk is being sold in villages and small cities located in the great butter and cheese producing sections of the country for eight to ten cents per quart. =the duty of the consumer.= the educational campaign that has been carried on by the health departments with reference to farm conditions and methods of handling has been most effective in improving the milk supply. many cities are now extending this to the consumer, recognizing that as much harm may be done in the home as on the farm. the importance of keeping the milk cold, of not allowing it to stand exposed in open vessels, of thoroughly cleaning the vessel in which it is kept, or the milk bottle before returning it to the milkman are especially emphasized. moreover, it must be impressed upon the consumer that all of these improvements, not only on the farm where the milk is produced, but in the hands of the distributing companies in the cities, involve much expense, and cannot be carried out, unless the consumer is willing to pay their cost. more objection seems to be raised over an increase in the price of milk than any other food stuff. the consumer therefore needs education along the line of higher prices for milk. dairy products of all types have increased much in value in recent years, so that at present prices milk, sold directly as milk, is relatively cheaper than in any form, when prevailing prices are compared with those that obtained a decade ago. index. abnormal fermentations, overcoming of, 108. abortion, contagious, 75. acid, amount of formed in milk, 84. acidity test, 211. actinomycosis, 75. aeration of milk, 55. aerobic bacteria, 13. air, contamination of milk from, 51. alcohol test, 213. alcoholic fermentation, 96. anaerobic bacteria, 13. animal, contamination of milk from, 42. anthrax, 75. antiseptics, 16, 117. b. bacillus bulgaricus, 89, 101. bacillus lactis acidi, 86. bacteria, aerobic, 13; anaerobic, 13; culture media for, 20; desirable acid-forming, 86; determining number of, 22; distribution of, 18; effect of cold on, 14; effect of heat on, 15; food of, 12; forms of, 8; manner of growth of, 9; movement of, 11; nature of, 8; parasitic, 11; products of, 17; pure cultures of, 25; rate of growth of, 13; relation to air, 13; relation to chemicals, 16; relation to drying, 15; relation to light, 16; relation to temperature, 12; size of, 9; saprophytic, 11; spores of, 10; types of acid-forming, 86; undesirable acid-forming, 90. bedding, 47. bitter fermentation, 97. bleaching powder, 112. bloody milk, 99. butter, bacteria in, 154; bacterial defects in, 156; cowy odor in, 157; deterioration of, 155; fishy, 157; metallic, 157; molding of, 157; preservatives in, 155; putrid, 156; source of flavor, 140; turnip flavored, 156; types of, 137. butter-milk, 100. butyric fermentation, 93. boric acid, 117. borax, 117. c. carbolic acid, 111. cheese, abnormal fermentations of, 174; bitter, 177; camembert, 186; cheddar, 164; colored, 178; flavor production in, 172; gassy, 174; gorgonzola, 185; limburger, 187; moldy, 179; preservation of by acid, 171; putrid, 178; quality of milk for, 162; ripening of, 169; roquefort, 184; stilton, 185; swiss, 180; temperature of ripening, 173; types of, 161. children, diseases of, 80. chloride of lime, 112. cholera, 80. cleaning utensils, 39. clean milk, production of, 53. cold, effect of, on bacteria, 14. colored milk, 98. condensed milk, 135. contagious abortion, 75. contamination of milk, from milking machine, 50; in factory, 59. cooling of milk, 54. corrosive sublimate, 111. cream, control of fermentation of, 142; pasteurization of, 146; ripening of, 138; separators, 36. cresol, 111. curd test, 104. cycle of fermentations, 99. d. deodorants, 109. digestive fermentation, 93. diphtheria, 79. dirt, exclusion of, 44; removal of from milk, 53. disinfectants, 16, 109. disinfection, 109. distribution of bacteria, 18. dried milk, 135. drugs, excretion of in milk, 58. drying, effect of on bacteria, 15. e. emmenthaler cheese, 180. evaporated milk, 134. f. factory by-products, 36; treatment of, 38. feeds, effect of on milk, 57. fermentation test, 104. fermented milks, 100. fly, contamination of milk by, 60; means of spreading typhoid fever, 78. foot and mouth disease, 74. fore milk, 31; rejection of, 34. formalin, 112. g. galactase, 172. garget, 75. germicidal action of milk, 33. gorgonzola cheese, 185. h. hairs, bacteria on, 43. heat, effect on bacteria, 15. heated milk, detection of, 39. hydrogen peroxide, 118. k. kefir, 102. koumiss, 102. l. lange wei, 95. light, effect on bacteria, 16. limburger cheese, 187. lime, 110. lumpy jaw, 75. m. malta fever, 75. market milk, municipal regulations concerning, 190; pasteurization of, 214. milk, acid fermentation of, 83; aeration of, 55; affected by feed, 57; alcoholic fermentation of, 96; bacterial standards for, 206; bitter fermentation of, 93; certified, 202; butyric fermentation of, 93; certified, 202, 208; clarifying of, 115; condition of when secreted, 29; contamination of from animal, 42; from by-products, 36; from utensils, 34; contamination of with tubercle bacilli, 67; cooling of, 54; creaming of, 136; culture medium for bacteria, 28; cycle of fermentation in, 99; distribution of, 216; digestive fermentation of, 93; dirt in, 44; effect of heat on, 119; filtration of, 114; germicidal action of, 33; grades of, 201; guaranteed, 201; inspected, 202; miscellaneous fermentations of, 98; pasteurization of, 120; pasteurization of in home, 131; preservation of by antiseptics, 117; preservation of by cold, 116; relation to children's diseases, 80; ropy fermentation, 94; sediments, examination of, 214; selected, 203; slimy, 94; spontaneous fermentation of, 91; sterilization of, 134; straining of, 153; supply of small cities, 217; sweet curdling fermentation of, 92; tainted, 56, 58; temperature standards for, 208; tests for quality of, 209. milk pails, sanitary, 48; small topped, 48. milker, factor in contamination of milk, 51. milking-machines, 36, 50. mold on butter, 177; on cheese, 179. o. odors, absorption of, 56, 58. oidium lactis, 186. oleomargarine, 152. p. pasteurization, 120; efficiency of, 133; purpose of, 123; methods of, 125. pasteurized milk, fermentations in, 124. pasteurizing machines, tests of, 130. process butter, 152. ptomaine poisoning, 81. pure cultures, 25. r. rabies, 75. reduction test, 213. rennet, 170. ropy fermentation, 94. roquefort cheese, 184. rusty spot in cheese, 178. s. salicylic acid, 117. scarlet fever, 79. score card for dairies, 198. sediment test, 210. skim milk, heating of, 38. slimy fermentation, 94. spores of bacteria, 10. stalls, 46. starters, 143; for cheese, 167; propagation of, 146. sterilization, 21, 134. stilton cheese, 185. storch test, 39. straining of milk, 33. sulphur, 111. sweet curdling of milk, 92. swiss cheese, 180. t. taints, determination of cause of, 58, 103. temperature effect on growth, 12. tubercle bacilli, destruction of, 71; in butter, 70; in cheese, 70; in milk, 67. tuberculin test, 73. tuberculosis, 64; closed, 70; distribution of disease in animal, 66; economic aspects of, 72; open, 70. typhoid fever, 76. u. udder, inflammation, 75; invasion of by bacteria, 30; number and kind of bacteria from, 32; structure of, 30; washing of, 47; cleaning of, 39. utensils, contamination from, 34. w. water, effect on butter, 153; supply, 59; testing of, 60. whey, heating of, 38. wisconsin curd test, 104. y. yeast fermentation, 96. yoghurt, 101. note: project gutenberg also has an html version of this file which includes the original illustrations. see 27713-h.htm or 27713-h.zip: (http://www.gutenberg.net/dirs/2/7/7/1/27713/27713-h/27713-h.htm) or (http://www.gutenberg.net/dirs/2/7/7/1/27713/27713-h.zip) transcriber's note: text enclosed by tilde marks was in bold face in the original (~bold~). text enclosed by underscore marks is in italics (_italics_). the italic designation for single italized letters (such as variables in equations) and "foreign" abbreviations has been omitted for ease of reading. in numbers, equations, and chemical formulas, an underscore indicates that the following term enclosed within curly brackets is a subscript. examples: co_{2}, h_{2}so_{4}. a carat character indicates that the following term enclosed within curly brackets is a superscript. for example, 11.1^{3} is 11.1 to the third power. minor typographical errors have been corrected. the elements of bacteriological technique a laboratory guide for medical, dental, and technical students by j. w. h. eyre, m.d., m.s., f.r.s. (edin.) director of the bacteriological department of guy's hospital, london, and lecturer on bacteriology in the medical and dental schools; formerly lecturer on bacteriology at charing cross hospital medical school, and bacteriologist to charing cross hospital; sometime hunterian professor, royal college of surgeons, england second edition rewritten and enlarged philadelphia and london w. b. saunders company 1913 copyright, 1902, by w. b. saunders and company revised, entirely reset, reprinted, and recopyrighted july, 1913 copyright, 1913, by w. b. saunders company registered at stationers' hall, london, england printed in america press of w. b. saunders company philadelphia to the memory of john wichenford washbourn, c.m.g., m.d., f.r.c.p. physician to guy's hospital and lecturer on bacteriology in the medical school, and physician to the london fever hospital my teacher, friend, and co-worker preface to the second edition bacteriology is essentially a practical study, and even the elements of its technique can only be taught by personal instruction in the laboratory. this is a self-evident proposition that needs no emphasis, yet i venture to believe that the former collection of tried and proved methods has already been of some utility, not only to the student in the absence of his teacher, but also to isolated workers in laboratories far removed from centres of instruction, reminding them of forgotten details in methods already acquired. if this assumption is based on fact no further apology is needed for the present revised edition in which the changes are chiefly in the nature of additions--rendered necessary by the introduction of new methods during recent years. i take this opportunity of expressing my deep sense of obligation to my confrère in the physiological department of our medical school--mr. j. h. ryffel, b. c., b. sc.--who has revised those pages dealing with the analysis of the metabolic products of bacterial life; to successive colleagues in the bacteriological department of guy's hospital, for their ready co-operation in working out or in testing new methods; and finally to my chief laboratory assistant, mr. j. c. turner whose assistance and experience have been of the utmost value to me in the preparation of this volume. i have also to thank mrs. constant ponder for many of the new line drawings and for redrawing a number of the original cuts. john w. h. eyre. guy's hospital, s. e. _july, 1913._ preface to the first edition in the following pages i have endeavoured to arrange briefly and concisely the various methods at present in use for the study of bacteria, and the elucidation of such points in their life-histories as are debatable or still undetermined. of these methods, some are new, others are not; but all are reliable, only such having been included as are capable of giving satisfactory results even in the hands of beginners. in fact, the bulk of the matter is simply an elaboration of the typewritten notes distributed to some of my laboratory classes in practical and applied bacteriology; consequently an attempt has been made to present the elements of bacteriological technique in their logical sequence. i make no apology for the space devoted to illustrations, nearly all of which have been prepared especially for this volume; for a picture, if good, possesses a higher educational value and conveys a more accurate impression than a page of print; and even sketches of apparatus serve a distinct purpose in suggesting to the student those alterations and modifications which may be rendered necessary or advisable by the character of his laboratory equipment. the excellent and appropriate terminology introduced by chester in his recent work on "determinative bacteriology" i have adopted in its entirety, for i consider it only needs to be used to convince one of its extreme utility, whilst its inclusion in an elementary manual is calculated to induce in the student habits of accurate observation and concise description. with the exception of section xvii--"outlines for the study of pathogenic bacteria"--introduced with the idea of completing the volume from the point of view of the medical and dental student, the work has been arranged to allow of its use as a laboratory guide by the technical student generally, whether of brewing, dairying, or agriculture. so alive am i to its many inperfections that it appears almost superfluous to state that the book is in no sense intended as a rival to the many and excellent manuals of bacteriology at present in use, but aims only at supplementing the usually scanty details of technique, and at instructing the student how to fit up and adapt apparatus for his daily work, and how to carry out thoroughly and systematically the various bacterioscopical analyses that are daily demanded of the bacteriologist by the hygienist. finally, it is with much pleasure that i acknowledge the valuable assistance received from my late assistant, mr. j. b. gall, a. i. c., in the preparation of the section dealing with the chemical products of bacterial life, and which has been based upon the work of lehmann. john w. h. eyre. guy's hospital, s. e. contents page i. laboratory regulations 1 ii. glass apparatus in common use 3 the selection, preparation, and care of glassware, 8--cleaning of glass apparatus, 18--plugging test-tubes and flasks, 24. iii. methods of sterilisation 26 sterilising agents, 26--methods of application, 27--electric signal timing clock, 38. iv. the microscope 49 essentials, 49--accessories, 57--methods of micrometry, 61. v. microscopical examination of bacteria and other micro-fungi 69 apparatus and reagents used in ordinary microscopical examination, 69--methods of examination, 74. vi. staining methods 90 bacteria stains, 90--contrast stains, 93--tissue stains, 95--blood stains, 97--methods of demonstrating structure of bacteria, 99--differential methods of staining, 108. vii. methods of demonstrating bacteria in tissues 114 freezing method, 115--paraffin method, 117--special staining methods for sections, 121. viii. classification of fungi 126 morphology of the hyphomycetes, 126--morphology of the blastomycetes, 129. ix. schizomycetes 131 anatomy, 134--physiology, 136--biochemistry, 144. x. nutrient media 146 meat extract, 148--standardisation of media, 154--the filtration of media, 156--storing media in bulk, 159--tubing nutrient media, 160. xi. ordinary or stock culture media 163 xii. special media 182 xiii. incubators 216 xiv. methods of cultivation 221 aerobic, 222--anaerobic, 236. xv. methods of isolation 248 xvi. methods of identification and study 259 scheme of study, 259--macroscopical examination of cultivations, 261--microscopical methods, 272--biochemical methods, 276--physical methods, 295--inoculation methods, 315--immunisation, 321--active immunisation, 322--the preparation of hæmolytic serum, 327--the titration of hæmolytic serum, 328--storage of hæmolysin, 331. xvii. experimental inoculation of animals 332 selection and care of animals, 335--methods of inoculation, 352. xviii. the study of experimental infections during life 370 general observations, 371--blood examinations, 373--serological investigations, 378--agglutinin, 381--opsonin, 387--immune body, 393. xix. post-mortem examination of experimental animals 396 xx. the study of the pathogenic bacteria 408 xxi. bacteriological analyses 415 bacteriological examination of water, 416--examination of milk, 441--ice cream, 457--examination of cream and butter, 457--examination of unsound meats, 460--examination of oysters and other shellfish, 463--examination of sewage and sewage effluents, 466--examination of air, 468--examination of soil, 470--testing filters, 478--testing of disinfectants, 480. appendix 492 index 505 [illustration] bacteriological technique. i. laboratory regulations. the following regulations are laid down for observance in the bacteriological laboratories under the direction of the author. similar regulations should be enforced in all laboratories where pathogenic bacteria are studied. _guy's hospital._ ~bacteriological department.~ handling of infective materials. the following regulations have been drawn up in the interest of those working in the laboratory as well as the public at large, and will be strictly enforced. their object is to avoid the dangers of infection which may arise from neglect of necessary precautions or from carelessness. everyone must note that by neglecting the general rules laid down he not only runs grave risk himself, but is a danger to others. regulations. 1. each worker must wear a gown or overall, provided at his own expense, which must be kept in the laboratory. 2. the hands must be disinfected with lysol 2 per cent. solution, carbolic acid 5 per cent. solution, or corrosive sublimate 1 per mille solution, after dealing with infectious material, and ~before using towels~. 3. on no account must laboratory towels or dusters be used for wiping up infectious material, and if such towels or dusters do become soiled, they must be immediately sterilised by boiling. 4. special pails containing disinfectant are provided to receive any waste material, and nothing must be thrown on the floor. 5. all instruments must be flamed, boiled, or otherwise disinfected immediately after use. 6. labels must be moistened with water, and not by the mouth. 7. all disused cover-glasses, slides, and pipettes after use in handling infectious material, etc., must be placed in 2 per cent. lysol solution. a vessel is supplied on each bench for this purpose. 8. all plate and tube cultures of pathogenic organisms when done with, must be placed for immediate disinfection in the boxes provided for the purpose. 9. no fluids are to be discharged into sinks or drains unless previously disinfected. 10. animals are to be dissected only after being nailed out on the wooden boards, and their skin thoroughly washed with disinfectant solution. 11. immediately after the post-mortem examination is completed each cadaver must be placed in the zinc animal-box--_without removing the carcase from the post-mortem board_--and the cover of the box replaced, ready for carriage to the destructor. 12. dead animals, when done with, are cremated in the destructor, and the laboratory attendant must be notified when the bodies are ready for cremation. 13. none of the workers in the laboratory are allowed to enter the animal houses unless accompanied by the special attendant in charge, who must scrupulously observe the same directions regarding personal disinfection as the workers in the laboratories. 14. no cultures are to be taken out of the laboratory without the permission of the head of the department. 15. all accidents, such as spilling infected material, cutting or pricking the fingers, must be at once reported to the bacteriologist in charge. ii. glass apparatus in common use. the equipment of the bacteriological laboratory, so far as the glass apparatus is concerned, differs but little from that of a chemical laboratory, and the cleanliness of the apparatus is equally important. the glassware comprised in the following list, in addition to being clean, must be stored in a sterile or germ-free condition. ~test-tubes.~--it is convenient to keep several sizes of test-tubes in stock, to meet special requirements, viz.: 1. ~18 × 1.5~ cm., to contain media for ordinary tube cultivations. 2. ~18 × 1.3~ cm., to contain media used for pouring plate cultivations, and also for holding sterile "swabs." 3. ~18 × 2~ cm., to contain wedges of potato, beetroot, or other vegetable media. 4. ~13 × 1.5~ cm., to contain inspissated blood-serum. the tubes should be made from the best german potash glass, "blue-lined," stout and heavy, with the edge of the mouth of the tube _slightly_ turned over, but not to such an extent as to form a definite rim. (cost about $1.50, or 6 shillings per gross.) such tubes are expensive it is true, but they are sufficiently stout to resist rough handling, do not usually break if accidentally allowed to drop (a point of some moment when dealing with cultures of pathogenic bacteria), can be cleaned, sterilised, and used over and over again, and by their length of life fully justify their initial expense. a point be noted is that the manufacturers rarely turn out such tubes as these absolutely uniform in calibre, and a batch of 18 by 1.5 cm. tubes usually contains such extreme sizes as 18 by 2 cm. and 18 by 1.3 cm. consequently, if a set of standard tubes is kept for comparison or callipers are used each new supply of so-called 18 by 1.5 cm. tubes may be easily sorted out into these three sizes, and so simplify ordering. 5. ~5 × 0.7~ cm., for use in the inverted position inside the tubes containing carbohydrate media, as gas-collecting tubes. these tubes, "unrimmed," may be of common thin glass as less than two per cent. are fit for use a second time. [illustration: fig. 1.--bohemian flask.] [illustration: fig. 2.--pear-shaped flask.] [illustration: fig. 3.--erlenmeyer flask (narrow neck).] ~bohemian flasks~ (fig. 1).--these are the ordinary flasks of the chemical laboratory. a good variety, ranging in capacity from 250 to 3000 c.c., should be kept on hand. a modified form, known as the "pear-shaped" (fig. 2), is preferable for the smaller sizes--i. e., 250 and 500 c.c. ~erlenmeyer's flasks~ (fig. 3).--erlenmeyer's flasks of 75, 100, and 250 c.c. capacity are extremely useful. for use as culture flasks care should be taken to select only such as have a narrow neck of about 2 cm. in length. ~kolle's culture flasks~ (fig. 4).--these thin, flat flasks (to contain agar or gelatine, which is allowed to solidify in a layer on one side) are extremely useful on account of the large nutrient surface available for growth. a surface cultivation in one of these will yield as much growth as ten or twelve "oblique" tube cultures. the wide mouth, however, is a disadvantage, and for many purposes thin, flat culture bottles known as ~roux's bottles~ (fig. 5) are to be preferred. [illustration: fig. 4.--kolle's culture flask.] [illustration: fig. 5.--roux's culture bottle.] [illustration: fig. 6.--guy's culture bottle.] [illustration: fig. 7.--filter flask.] an even more convenient pattern is that used in the author's laboratory (fig. 6), as owing to the greater depth of medium which it is possible to obtain in these flasks an exceedingly luxuriant growth is possible; the narrow neck reduces the chance of accidental contamination to a minimum and the general shape permits the flasks to be stacked one upon the other. ~filter flasks or kitasato's serum flasks~ (fig. 7).--various sizes, from 250 to 2000 c.c. capacity. these must be of stout glass, to resist the pressure to which they are subjected, but at the same time must be thoroughly well annealed, in order to withstand the temperature necessary for sterilisation. all flasks should be either of jena glass or the almost equally well-known resistance or r glass, the extra initial expense being justified by the comparative immunity of the glass from breakage. ~petri's dishes or "plates"~ (fig. 8, a).--these have now completely replaced the rectangular sheets of glass introduced by koch for the plate method of cultivation. each "plate" consists of a pair of circular discs of glass with sharply upturned edges, thus forming shallow dishes, one of slightly greater diameter than the other, and so, when inverted, forming a cover or cap for the smaller. plates having an outside diameter of 10 cm. and a height of 1.5 cm. are the most generally useful. a batch of eighteen such plates is sterilised and stored in a cylindrical copper box (30 cm. high by 12 cm. diameter) provided with a "pull-off" lid. inside each box is a copper stirrup with a circular bottom, upon which the plates rest, and by means of which each can be raised in turn to the mouth of the box (fig. 9) for removal. ~capsules~ (fig. 8, b and c).--these are petri's dishes of smaller diameter but greater depth than those termed plates. two sizes will be found especially useful--viz., 4 cm. diameter by 2 cm. high, capacity about 14 c.c.; and 5 cm. diameter by 2 cm. high, capacity about 25 c.c. these are stored in copper cylinders of similar construction to those used for plates, but measuring 20 by 6 cm. and 20 by 7 cm., respectively. [illustration: fig. 8.--petri dish (a), and capsules (b, c).] [illustration: fig. 9.--plate box with stirrup.] ~graduated pipettes.~--several varieties of these are required, viz.: 1. pipettes of 1 c.c. capacity graduated in 0.1 c.c. 2. pipettes of 1 c.c. capacity graduated in 0.01 c.c. (fig. 10, a). 3. pipettes of 10 c.c. capacity graduated in 0.1 c.c. (fig. 10, b). these should be about 30 cm. in length (1 and 2 of fairly narrow bore), graduated to the extreme point, and having at least a 10 cm. length of clear space between the first graduation and the upper end; the open mouth should be plugged with cotton-wool. each variety should be sterilised and stored in a separate cylindrical copper case some 36 by 6 cm., with "pull-off" lid, upon which is stamped, in plain figures, the capacity of the contained pipettes. [illustration: fig. 10.--measuring pipettes, a and b.] the laboratory should also be provided with a complete set of "standard" graduated pipettes, each pipette in the set being stamped and authenticated by a certificate from one of the recognised physical measurement laboratories, such as charlottenburg. these instruments are expensive and should be reserved solely for standardising the pipettes in ordinary use, and for calibrating small pipettes manufactured in the laboratory. such a set should comprise, at least, pipettes delivering 10 c.c., 5 c.c., 2.5 c.c., 2 c.c., 1 c.c., 0.5 c.c., 0.25 c.c., 0.2 c.c., 0.1 c.c., 0.05 c.c., and 0.01 c.c., respectively. in the immediately following sections are described small pieces of glass apparatus which should be prepared in the laboratory from glass tubing of various sizes. in their preparation three articles are essential; first a three-square hard-steel file or preferably a glass-worker's knife of hard thuringian steel for cutting glass tubes etc.; next a blowpipe flame, for although much can be done with the ordinary bunsen burner, a blowpipe flame makes for rapid work; and lastly a bat's-wing burner. [illustration: fig. 11.--glass-cutting knife. a. handle. b. double edged blade. c. shaft. d. locking nut. e. spanner for nut.] 1. the glass-cutting knife. this article is sold in two forms, a bench knife (fig. 11) and a pocket knife. the former is provided with a blade some 8 cm. in length and having two cutting edges. the cutting edge when examined in a strong light is seen to be composed of small closely set teeth, similar to those in a saw. the knife should be kept sharp by frequent stroppings on a sandstone hone. the pocket form, about 6-cm. long over all, consists of a small spring blade with one cutting edge mounted in scales like an ordinary pocket knife. 2. for real convenience of work the blowpipe should be mounted on a special table connected up with cylindrical bellows operated by a pedal. that figured (fig. 12) is made by mounting a teak top 60 cm. square upon the uprights of an enclosed double-action concertina bellows (enfer's) and provided with a fletcher's universal gas blowpipe. 3. an ordinary bat's-wing gas-burner mounted at the far corner of the table top is invaluable in the preparation of tubular apparatus with sharp curves, and for coating newly-made glass apparatus with a layer of soot to prevent too rapid cooling, and its usually associated result--cracking. [illustration: fig. 12.--glass blower's table with enfer's foot bellows.] 6. ~sedimentation tubes 5×0.5~ cm., for sedimentation reactions, etc., and for containing small quantities of fluid to be centrifugalised in the hæmatocrit. these are made by taking 14-cm. lengths of stout glass tubing of the requisite diameter and heating the centre in the bunsen or blowpipe flame. when the central portion is quite soft draw the ends quickly apart and then round off the pointed ends of the two test-tubes thus formed. with the glass-cutting knife cut off whatever may be necessary from the open ends to make the tubes the required length. a rectangular block of "plasticine" (modelling clay) into which the conical ends can be thrust makes a very convenient stand for these small tubes. ~capillary pipettes or pasteur's pipettes~ (fig. 13 a).--these little instruments are invaluable, and a goodly supply should be kept on hand. they are prepared from soft-glass tubing of various-sized calibre (the most generally useful size being 8 mm. diameter) in the following manner: hold a 10 cm. length of glass tube by each end, and whilst rotating it heat the central portion in the bunsen flame or the blowpipe blast-flame until the glass is red hot and soft. now remove it from the flame and steadily pull the ends apart, so drawing the heated portion out into a roomy capillary tube; break the capillary portion at its centre, seal the broken ends in the flame, and round off the edges of the open end of each pipette. a loose plug of cotton-wool in the open mouth completes the capillary pipette. after a number have been prepared, they are sterilised and stored in batches, either in metal cases similar to those used for the graduated pipettes or in large-sized test-tubes--sealed ends downward and plugged ends toward the mouth of the case. [illustration: fig. 13.--capillary pipettes. a, b, c.] the filling and emptying of the capillary pipette is most satisfactorily accomplished by slipping a small rubber teat (similar to that on a baby's feeding bottle but _not perforated_) on the upper end, after cutting or snapping off the sealed point of the capillary portion. if pressure is now exerted upon the elastic bulb by a finger and thumb whilst the capillary end is below the surface of the fluid to be taken up, some of the contained air will be driven out, and subsequent relaxation of that pressure (resulting in the formation of a partial vacuum) will cause the fluid to ascend the capillary tube. subsequent compression of the bulb will naturally result in the complete expulsion of the fluid from the pipette (fig. 14). [illustration: fig. 14.--filling the capillary teat-pipette.] a modification of this pipette, in which a constriction or short length of capillary tube is introduced just below the plugged mouth (fig. 13, b), will also be found extremely useful in the collection and storage of morbid exudations. a third form, where the capillary portion is about 4 or 5 cm. long and only forms a small fraction of the entire length of the pipette (fig. 13, c), will also be found useful. ~"blood" pipettes~ (fig 15).--special pipettes for the collection of fairly large quantities of blood (as suggested by pakes) should also be prepared. these are made from _soft_ glass tubing of 1 cm. bore, in a similar manner to the pasteur pipettes, except that the point of the blowpipe flame must be used in order to obtain the sharp shoulder at either end of the central bulb. the terminal tubes must retain a diameter of at least 1 mm., in order to avoid capillary action during the collection of the fluid. [illustration: fig. 15.--blood pipettes and hair-lip pin in a test-tube.] [illustration: fig. 16.--blood-pipette in metal thermometer case.] for sterilisation and storage each pipette is placed inside a test-tube, resting on a wad of cotton-wool, and the tube plugged in the ordinary manner. as these tubes are used almost exclusively for blood work, it is usual to place a lance-headed hare-lip pin or a no. 9 flat hagedorn needle inside the tube so that the entire outfit may be sterilised at one time. for the collection of small quantities of blood for agglutination reactions and the like, many prefer a short straight piece of narrow glass tubing drawn out at either extremity to almost capillary dimensions. such pipettes, about 8 cm. in length over all, are most conveniently sterilized in ordinary metal thermometer cases (fig. 16). ~graduated capillary pipettes~ (fig. 17).--these should also be made in the laboratory--from manometer tubing--of simple, convenient shape, and graduated by the aid of "standard" pipettes (in hundredths) to contain such quantities as 10, 50, and 90 c. mm., and carefully marked with a writing diamond. these, previously sterilised in large test-tubes, will be found extremely useful in preparing accurate percentage solutions, when only minute quantities of fluid are available. [illustration: fig. 17.--capillary graduated pipettes.] ~automatic ("throttle") pipettes.~--these ingenious pipettes, introduced by wright, can easily be calibrated in the laboratory and are exceedingly useful for graduating small pipettes, for measuring small quantities of fluids, in preparing dilutions of serum for agglutination reactions, etc. they are usually made from the capillary pasteur pipettes (fig. 13, a). the following description of the manufacture of a 5 c. mm. pipette will serve to show how the small automatic pipettes are calibrated. 1. select a pipette the capillary portion of which is fairly roomy in bore and possesses regular even walls, and remove the cotton-wool plug from the open end. 2. heat the capillary portion near the free extremity in the by-pass flame of the bunsen burner and draw it out into a very fine hair-like tube and break this across. this hair-like extremity will permit the passage of air but is too fine for metallic mercury to pass. 3. from a standard graduated pipette deliver 5 c. mm. clean mercury into the upper wide portion of the pipette. 4. adjust a rubber teat to the pipette and by pressure on the bulb gradually drive the mercury in an unbroken column down the capillary tube until it is stopped by the filiform extremity. 5. cut off the capillary tube exactly at the upper level of the column of mercury, invert it and allow the mercury to run out. 6. snap off the remainder of the capillary tube from the broad upper portion of the pipette which is now destined to form the covering tube or air chamber, or what we may term the "barrel." this barrel now has the lower end in the form of a truncated cone, the upper end being cut square. remove the teat. 7. introduce the capillary tube into this barrel with the filiform extremity uppermost, and the square cut end projecting about 0.5 cm. beyond the tapering end of the barrel. [illustration: fig. 18.--throttle pipette--small capacity.] 8. drop a small pellet of sealing wax into the barrel by the side of the capillary tube and then warm the tube at the gas flame until the wax becomes softened and makes an air-tight joint between the capillary tube and the end of the barrel. 9. fit a rubber teat to the open end of the barrel, and so complete a pipette which can be depended upon to always aspirate and deliver exactly 5 cm. of fluid. slight modification of this procedure is necessary in making tubes to measure larger volumes than say 75 c. mm. thus to make a throttle pipette to measure 100 c. mm.: 1. take a short length of quill tubing and draw out one end into a roomy capillary stem, and again draw out the extremity into a fine hair point, thus forming a small pasteur pipette with a hair-like capillary extremity. 2. with a standard pipette fill 100 c. mm. into the neck of this pipette, and make a scratch with a writing diamond at the upper level (a) of the mercury meniscus (fig. 19, a). [illustration: fig. 19.--making throttle pipettes--large capacity] now force the mercury down into the capillary stem as far as it will go, so as to leave the upper part of the tube in the region of the diamond scratch empty (fig. 19, b). 3. heat the tube in the region of the diamond scratch in the blowpipe flame, and removing the tube from the flame draw it out so that the diamond scratch now occupies a position somewhere near the centre of this new capillary portion (fig. 19, c). 4. heat the tube in this position in the peep flame of the bunsen burner, and draw it out into a hair-like extremity. snap off the glass tube, leaving about 5 mm. of hair-like extremity attached to the upper capillary portion (fig. 19, d). allow the glass to cool. 5. lift up the bulb by the long capillary stem and allow the mercury to return to its original position--an operation which will be facilitated by snapping off the hair-like extremity from the long piece of capillary tubing. 6. mark on the capillary stem with a grease pencil the position of the end of the column of mercury (fig. 19, e.) 7. warm the capillary tubing at this spot in the peep flame of the bunsen burner, and draw it out very slightly so that when cut at this position a pointed extremity will be obtained. 8. with a glass-cutting knife cut the capillary tube through at the point "b," and allow the mercury to run out. 9. now apply a thick layer of sealing wax to the neck of the bulb. 10. take a piece of 5 mm. bore glass tubing and draw it out as if making an ordinary pasteur pipette. 11. break the capillary portion off so as to leave a covering tube similar to that already used for the smaller graduated pipettes. into this covering tube drop the graduated bulb and draw the capillary stem down through the conical extremity until further progress is stopped by the layer of sealing wax. 12. warm the pipette in the gas flame so as to melt the sealing wax and make an air-tight joint. 13. fit an india-rubber teat over the open end of the covering tube, and the automatic pipette is ready for use (fig. 19, f). ~sedimentation pipettes~ (fig. 20).--these are prepared from 10 cm. lengths of narrow glass tubing by sealing one extremity, blowing a small bulb at the centre, and plugging the open end with cotton-wool; after sterilisation the open end is provided with a short piece of rubber tubing and a glass mouthpiece. when it is necessary to observe sedimentation reactions in very small quantities of fluid, these tubes will be found much more convenient than the 5 by 0.5 cm. test-tubes previously mentioned. [illustration: fig. 20.--sedimentation pipette.] pasteur pipettes fitted with india-rubber teats will also be found useful for sedimentation tests when dealing with minute quantities of serum, etc. [illustration: fig. 21.--fermentation tubes.] ~fermentation tubes~ (fig. 21).--these are used for the collection and analysis of the gases liberated from the media during the growth of some varieties of bacteria and may be either plain (a) or graduated (b). a simple form (fig. 21, c) may be made from 14 cm. lengths of soft glass tubing of 1.5 cm. diameter. the bunsen flame is applied to a spot some 5 cm. from one end of such a piece of tubing and the tube slightly drawn out to form a constriction, the constricted part is bent in the bat's-wing flame, to an acute angle, and the open extremity of the long arm sealed off in the blowpipe flame. the open end of the short arm is rounded off and then plugged with cotton-wool, and the tube is ready for sterilisation. cleaning of glass apparatus. all glassware used in the bacteriological laboratory must be thoroughly cleaned before use, and this rule applies as forcibly to new as to old apparatus, although the methods employed may vary slightly. ~to clean new test-tubes.~-1. place the tubes in a bucket or other convenient receptacle, fill with water and add a handful of "sapon" or other soap powder. see that the tubes are full and submerged. 2. fix the bucket over a large bunsen flame and boil for thirty minutes--or boil in the autoclave for a similar period. 3. cleanse the interior of the tubes with the aid of test-tube brushes, and rinse thoroughly in cold water. 4. invert the tubes and allow them to drain completely. 5. dry the tubes and polish the glass inside and out with a soft cloth, such as selvyt. ~new flasks, plates, and capsules~ must be cleaned in a similar manner. ~to clean new graduated pipettes.~-1. place the pipettes in a convenient receptacle, filled with water to which soap powder has been added. 2. boil the water vigorously for twenty minutes over a bunsen flame. 3. rinse the pipettes in running water and drain. 4. run distilled water through the pipettes and drain. 5. run rectified spirits through the pipette and drain as completely as possible. 6. place the pipettes in the hot-air oven (_vide_ page 31), close the door, open the ventilating slide, and run the temperature slowly up to about 80° c. turn off the gas and allow the oven to cool. or 6a. attach each pipette in turn to the rubber tube of the foot bellows, or blowpipe air-blast, and blow air through the pipette until the interior is dry. glassware that has already been used is regarded as _infected_, and is treated in a slightly different manner. ~infected test-tubes.~-1. pack the tubes in the wire basket of the autoclave (having previously removed the cotton-wool plugs, caps, etc.), in the vertical position, and before replacing the basket see that there is a sufficiency of water in the bottom of the boiler. now attach a piece of rubber tubing to the nearest water tap, and by means of this fill each tube with water. 2. disinfect completely by exposing the tubes, etc., to a temperature of 120° c. for twenty minutes (_vide_ page 37). (if an autoclave is not available, the tubes must be placed in a digester, or even a large pan or pail with a tightly fitting cover, and boiled vigorously for some thirty to forty-five minutes to ensure disinfection.) 3. whilst still hot, empty each tube in turn and roughly clean its interior with a stiff test-tube brush. 4. place the tubes in a bucket or other convenient receptacle, fill with water and add a handful of sapon or other soap powder. see that the tubes are full and submerged. 5. fix the bucket over a large bunsen flame and boil for thirty minutes. 6. cleanse the interior of the tubes with the aid of test-tube brushes, and rinse thoroughly in cold water. 7. drain off the water and immerse tubes in a large jar containing water acidulated with 2 to 5 per cent. hydrochloric acid. allow them to remain there for about fifteen minutes. 8. remove from the acid jar, drain, rinse thoroughly in running water, then with distilled water. 9. invert the tubes and allow them to drain completely. dry the tubes and polish the glass inside and out with a soft cloth, such as selvyt. ~infected flasks, plates, and capsules~ must be treated in a similar manner. ~flasks~ which have been used only in the preparation of media must be cleaned immediately they are finished with. fill each flask with water to which some soap powder and a few crystals of potassium permanganate have been added, and let boil over the naked flame. the interior of the flask can then usually be perfectly cleaned with the aid of a flask brush, but in some cases water acidulated with 5 per cent. nitric acid, or a large wad of wet cotton-wool previously rolled in silver sand, must be shaken around the interior of the flask, after which rinse thoroughly with clean water, dry, and polish. ~infected pipettes.~-1. plunge infected pipettes immediately after use into tall glass cylinders containing a 2 per cent. solution of lysol, and allow them to remain therein for some days. 2. remove from the jar and drain. boil in water to which a little soap has been added, for thirty minutes. 3. rinse thoroughly in cold water. 4. immerse in 5 per cent. nitric acid for an hour or two. 5. rinse again in running water to remove all traces of acid. 6. complete the cleaning as described under "new pipettes." when dealing with graduated capillary pipettes employed for blood or serum work (whether new or infected), much time is consumed in the various steps from 5 onward, and the cleansing process can be materially hastened if the following device is adopted. fit up a large-sized kitasato's filter flask to a sprengel's suction pump or a geryk air pump (see page 43). to the side tubulure of the filter flask attach a 20 cm. length of rubber pressure tubing having a calibre sufficiently large to admit the ends of the pipettes. next fill a small beaker with distilled water. attach the first pipette to the free end of the rubber tubing, place the pipette point downward in the beaker of water and start the pump (fig. 22). [illustration: fig. 22.--cleaning blood pipettes.] when all the water has been aspirated through the pipette into the filter flask, fill the beaker with rectified spirit and when this is exhausted refill with ether. detach the pipette and dry in the hot-air oven. ~slides and cover-slips~ (fig. 23), when first purchased, have "greasy" surfaces, upon which water gathers in minute drops and effectually prevents the spreading of thin, even films. ~microscopical slides.~--the slides in general use are those known as "three by one" slips (measuring 3 inches by 1 inch, or 76 by 26 mm.), and should be of good white crown glass, with ground edges. ~new slides~ should be allowed to remain in alcohol acidulated with 5 per cent. hydrochloric acid for some hours, rinsed in running water, roughly drained on a towel, dried, and finally polished with a selvyt cloth. [illustration: fig. 23.--slides and cover-slips, actual size.] if only a few slides are required for immediate use a good plan is to rub the surface with jeweler's emery paper (hubert's 00). a piece of hard wood 76×26×26 mm. with a piece of this emery paper gummed tightly around it is an exceedingly useful article on the microscope bench. ~cover-slips.~--the most useful sizes are the 19 mm. squares for ordinary cover-glass film preparations, and 38 by 19 mm. rectangles for blood films and serial sections; both varieties must be of "no. 1" thickness, which varies between 0.15 and 0.22 mm., that they may be available for use with the high-power immersion lenses. cover-slips should be cleaned in the following manner: 1. drop the cover-slips one by one into an enamelled iron pot or tall glass beaker, containing a 10 per cent. solution of chromic acid. 2. heat over a bunsen flame and allow the acid to boil gently for twenty minutes. note.--a few pieces of pipe-clay or pumice may be placed in the beaker to prevent the "spurting" of the chromic acid. 3. turn the cover-slips out into a flat glass dish and wash in running water under the tap until all trace of yellow colour has disappeared. during the washing keep the cover-slips in motion by imparting a rotatory movement to the dish. 4. wash in distilled water in a similar manner. 5. wash in rectified spirit. 6. transfer the cover-slips, by means of a pair of clean forceps, previously heated in the bunsen flame to destroy any trace of grease, to a small beaker of absolute alcohol. drain off the alcohol and transfer the cover-slips, by means of the forceps, to a wide-mouthed glass pot, containing absolute alcohol, in which they are to be stored, and stopper tightly. note.--after once being placed in the chromic acid, the cover-slips must on no account be touched by the fingers. ~used slides and cover-slips.~--used slides with the mounted cover-slip preparations, and cover-slips used for hanging-drop mounts, should, when discarded, be thrown into a pot containing a 2 per cent. solution of lysol. after immersion therein for a week or so, even the cover-slips mounted with canada balsam can be readily detached from their slides. _slides._-1. wash the slides thoroughly in running water. 2. boil the slides in water to which "sapon" has been added, for half an hour. 3. rinse thoroughly in cold water. 4. dry and polish with a dry cloth. _cover-slips._-1. wash the cover-slips thoroughly in running water. 2. boil the cover-slips in 10 per cent. solution of chromic acid, as for new cover-slips. 3. wash thoroughly in running water. 4. pick out those cover-slips which show much adherent dirty matter, and rub them between thumb and forefinger under the water tap. the dirt usually rubs off easily, as it has become friable from contact with the chromic acid. 5. return all the cover-slips to the beaker, fill in _fresh_ chromic acid solution, and treat as new cover-slips. note.--_test-tubes, plates, capsules_, etc., which, from long use, have become scratched and hazy, or which cannot be cleaned in any other way, may be dealt with by immersing them in an enamelled iron bath, containing water acidulated to 1 per cent. with hydrofluoric acid, for ten minutes, rinsing thoroughly in water, drying, and polishing. plugging test-tubes and flasks. before sterilisation all test-tubes and flasks must be carefully plugged with cotton-wool, and for this purpose best absorbent cotton-wool (preferably that put up in cylindrical one-pound packets and interleaved with tissue paper--known as surgeons' wool) should be employed. 1. for a test-tube or a small flask, tear a strip of cotton-wool some 10 cm. long by 2 cm. wide from the roll. 2. turn in the ends neatly and roll the strip of wool lightly between the thumb and fingers of both hands to form a long cylinder. 3. double this at the centre and introduce the now rounded end into the open mouth of the tube or flask. 4. now, whilst supporting the wool between the thumb and fingers of the right hand, rotate the test-tube between those of the left, and gradually screw the plug of wool into its mouth for a distance of about 2.5 cm., leaving about the same length of wool projecting. [illustration: fig 24..--plugging test-tubes: a, cylinder of wool being rolled; b, cylinder of wool being doubled; c, cylinder of wool being inserted in tube.] the plug must be firm and fit the tube or flask fairly tightly, sufficiently tightly in fact to bear the weight of the glass plus the amount of medium the vessel is intended to contain, but not so tightly as to prevent it from being easily removed by a screwing motion when grasped between the fourth, or third and fourth, fingers, and the palm of the hand. for a large flask a similar but larger strip of wool must be taken; the method of making and inserting the plug is identical. iii. methods of sterilisation. sterilising agents. sterilisation--i. e., the removal or the destruction of germ life--may be effected by the use of various agents. as applied to the practical requirements of the bacteriological laboratory, many of these agents, such as electricity, sunlight, etc., are of little value, others are limited in their applications; others again are so well suited to particular purposes that their use is almost entirely restricted to such. the sterilising agents in common use are: ~chemical reagents.~--_disinfectants_ (for the disinfection of glass and metal apparatus and of morbid tissues). ~physical agents.~ heat.--(a) _dry heat:_ 1. naked flame (for the sterilisation of platinum needles, etc.). 2. muffle furnace (for the sterilisation of filter candles, and for the destruction of morbid tissues). 3. hot air (for the sterilisation of all glassware and of metal apparatus). (b) _moist heat:_ 1. water at 56° c. (for the sterilisation of certain albuminous fluids). 2. water at 100° c. (for the sterilisation of surgical instruments, rubber tubing, and stoppers, etc.). 3. streaming steam at 100° c. (for the sterilisation of media). 4. superheated steam at 115° c. or 120° c. (for the disinfection of contaminated articles and the destruction of old cultivations of bacteria). filtration.-1. cotton-wool filters (for the sterilisation of air and gases). 2. porcelain filters (for the sterilisation of various liquids). methods of application. ~chemical reagents~, such as belong to the class known as antiseptics (_i. e._, substances which inhibit the growth of, but do not destroy, bacterial life), are obviously useless. disinfectants or germicides (_i. e._, substances which destroy bacterial life), on the other hand, are of value in the disinfection of morbid material, and also of various pieces of apparatus, such as pipettes, pending their cleansing and complete sterilisation by other processes. to this class (in order of general utility) belong: lysol, 2 per cent. solution; perchloride of mercury, 0.1 per cent. solution; carbolic acid, 5 per cent. solution; absolute alcohol; ether; chloroform; camphor; thymol; toluol; volatile oils, such as oil of mustard, oil of garlic. formaldehyde is a powerful germicide, but its penetrating vapor restricts its use. these disinfectants are but little used in the final sterilisation of apparatus, chiefly on account of the difficulty of effecting their complete removal, for the presence of even traces of these chemicals is sufficient to so inhibit or alter the growth of bacteria as to vitiate subsequent experiments conducted by the aid of apparatus sterilised in this manner. note.--tubes, flasks, filter flasks, pipettes, glass tubing, etc., may be rapidly sterilised, in case of emergency, by washing, in turn, with distilled water, perchloride of mercury solution, alcohol, and ether, draining, and finally gently heating over a gas flame to completely drive off the ether vapor. chloroform or other volatile disinfectants may be added to various fluids in order to effect the destruction of contained bacteria, and when this has been done, may be completely driven off from the fluid by the application of gentle heat. ~dry heat.~--the _naked flame_ of the bunsen burner is invariably used for sterilising the platinum needles (which are heated to redness) and may be employed for sterilising the points of forceps, or other small instruments, cover-glasses, pipettes, etc., a very short exposure to this heat being sufficient. _ether flame._--in an emergency small instruments, needles, etc., may be sterilised by dipping them in ether and after removal lighting the adherent fluid and allowing it to burn off the surface of the instruments. repeat the process twice. it may then be safely assumed that the apparatus so treated is sterile. [illustration: fig. 25.--muffle furnace.] _muffle furnace_ (fig. 25).--although this form of heat is chiefly used for the destruction of the dead bodies of small infected animals, morbid tissues, etc., it is also employed for the sterilisation of porcelain filter candles (_vide_ p. 42). filter candles are disinfected immediately after use by boiling in a beaker of water for some fifteen or twenty minutes. this treatment, however, leaves the dead bodies of the bacteria upon the surface and blocking the interstices of the filter. to destroy the organic matter and prepare the filter candle for further use proceed as follows: 1. roll each bougie up in a piece of asbestos cloth, secure the ends of the cloth with a few turns of copper wire, and place inside the muffle (a small muffle 76×88×163 mm. will hold perhaps four small filter candles). 2. light the gas and raise the contents of the muffle to a white heat; maintain this temperature for five minutes. 3. extinguish the gas, and when the muffle has become quite cold remove the filter candles, and store them (without removing the asbestos wrappings) in sterile metal boxes. note.--the too rapid cooling of the candles, such as takes place if they are removed from the muffle before it has cooled down to the room temperature, may give rise to microscopic cracks and flaws which will effectually destroy their efficiency. _hot air._--hot air at 150° c. destroys all bacteria, spores, etc:, in about thirty minutes; a momentary exposure to a temperature of 175° to 180° c. will effect the same result and offers the more convenient method of sterilisation. this method is only applicable to glass and metallic substances, and the small bulk of cotton-wool comprised in the test-tube plugs, etc. large masses of fabric are not effectually sterilised by dry heat--short of charring--as its power of penetration is not great. sterilisation by hot air is effected in the hot-air oven (fig. 18). this is a rectangular, double-walled metal box, mounted on a stand and heated from below by a large bunsen burner. the interior of the oven is provided with loose shelves upon which the articles to be sterilised are arranged, either singly or packed in square wire baskets or crates, kept specially for this purpose. one of the sides is hinged to form a door. the central portion of the metal bottom, on which the bunsen flame would play, is cut away, and replaced by firebrick plates, which slide in metal grooves and are easily replaced when broken or worn out. the top of the oven is provided with a perforated ventilator slide and two tubulures, the one for the reception of a centigrade thermometer graduated to 200° or 250°c., the other for a thermo-regulator. an ordinary mercurial thermo-regulator may be used but it is preferable to employ a regulating capsule of the hearson type (see p. 219) with a spring arm adjusted to the lever so that when the boiling-point of the capsule (e. g., 175°c.) is reached the gas supply is absolutely cut off and the jet cannot again be lighted until the spring-arm has been readjusted by hand. the thermo-regulator is by no means a necessity, and may be replaced by a large bore thermometer with a sliding platinum point, connected with an electric bell, which can be easily adjusted to ring at any given temperature. even if the steriliser is provided with the capsule regulator above described the contact thermometer should also be fitted. [illustration: fig. 26.--hot-air oven.] to use the hot-air oven.-1. place the crates of test-tubes, metal cases containing plates and pipettes, loose apparatus, etc., inside the oven, taking particular care that none of the cotton-wool plugs are in contact with the walls, otherwise the heat transmitted by the metal will char or even flame them. to prepare a wire crate for the reception of test-tubes, etc., cover the bottom with a layer of thick asbestos cloth; or take some asbestos fibre, moisten it with a little water and knead it into a paste; plaster the paste over the bottom of the crate, working it into the meshes and smoothing the surface by means of a pestle. when several crates have been thus treated, place them inside the hot-air oven, close the door, open the ventilating slide, light the gas, and run the temperature of the interior up to about 160° c. after an interval of ten minutes extinguish the gas, open the oven door, and allow the contents to cool. the asbestos now forms a smooth, dry, spongy layer over the bottom, which will last many months before needing renewal, and will considerably diminish the loss of tubes from breakage. copper cylinders and large test-tubes intended for the reception of pipettes are prepared in a similar manner, in order to protect the points of these articles from injury. 2. close the oven door, and open the ventilating slide, in order that any moisture left in the tubes, etc., may escape; light the gas below; set the electric alarm to ring at 100°c. 3. when the temperature of the oven has reached 100°c., close the ventilating slide; reset the alarm to ring at 175°c. 4. run the temperature up to 175°c. 5. extinguish the gas at once, and allow the apparatus to cool. 6. when the temperature of the interior, as recorded by the thermometer, has fallen to 60°c.--_but not before_--the door may be opened and the sterile articles removed and stored away. note.--neglect of this precautionary cooling of the oven to 60° c. will result in numerous cracked and broken tubes. on removal from the oven, the cotton-wool plugs will probably be slightly brown in colour. metal instruments, such as knives, scissors, and forceps, may be sterilised in the hot-air oven as described above, but exposure to 175° c. is likely to seriously affect the temper of the steel and certainly blunts the cutting edges. if, however, it is desired to sterilise surgical instruments by hot air, they should be packed in a metal box, or boxes, and heated to 130° c. and retained at that temperature for about thirty minutes. ~moist heat.~--_water at 56° c._--this temperature, if maintained for thirty minutes, is sufficient to destroy the vegetative forms of bacteria, but has practically no effect on spores. its use is limited to the sterilisation of such albuminous "fluid" media as would coagulate at a higher temperature. method.-1. fit up a water-bath, heated by a bunsen flame which is controlled by a thermo-regulator, so that the temperature of the water remains at 56° c. 2. immerse the tubes or flasks containing the albuminous fluid in the water-bath so that the upper level of such fluid is at least 2 cm. below the level of the water. (the temperature of the bath will now fall somewhat, but after a few minutes will again rise to 56° c). 3. after thirty minutes' exposure to 56° c, extinguish the gas, remove the tubes or flasks from the bath, and subject them to the action of running water so that their contents are rapidly cooled. 4. the vegetative forms of bacteria present in the liquid being killed, stand it for twenty-four hours in a cool, dark place; at the end of that time some at least of such spores as may be present will have germinated and assumed the vegetative form. 5. destroy these new vegetative forms by a similar exposure to 56° c. on the second day, whilst others, of slower germination, may be caught on the third day, and so on. 6. in order to ensure thorough sterilisation, repeat the process on each of six successive days. this method of exposing liquids to a temperature of 56° c. in a water-bath for half an hour on each of six successive days is termed _fractional sterilisation_. _water at 100°c._ destroys the vegetative forms of bacteria almost instantaneously, and spores in from five to fifteen minutes. this method of sterilisation is applicable to the metal instruments, such as knives, forceps, etc., used in animal experiments; syringes, rubber corks, rubber and glass tubing, and other small apparatus, and is effected in what is usually spoken of as the "water steriliser" (fig. 27). [illustration: fig. 27.--water sterilizer.] this is a rectangular copper box, 26 cm. long, 18 cm. wide, and 12 cm. deep, mounted on legs, heated from below by a bunsen or radial gas burner, and containing a movable copper wire tray, 2 cm. smaller in every dimension than the steriliser itself, and provided with handles. the top of the steriliser is hinged to form a lid. method.-1. place the instruments, etc., to be sterilised inside the copper basket, and replace the basket in the steriliser. 2. pour a sufficient quantity of water into the steriliser, shut down the lid, and light the gas below. [illustration: fig. 28.--koch's steriliser.] [illustration: fig. 29.--arnold's steriliser.] 3. after the water has boiled and steam has been issuing from beneath the lid for at least ten minutes, extinguish the gas, open the lid, and lift out the wire basket by its handles and rest it diagonally on the walls of the steriliser; the contained instruments, etc., are now sterile and ready for use. 4. after use, or when accidentally contaminated, replace the instruments in the basket and return that to the steriliser; completely disinfect by a further boiling for fifteen minutes. 5. after disinfection, and whilst still hot, take out the instruments, dry carefully and at once, and return them to their store cases. _streaming steam_--i. e., steam at 100°c.--destroys the vegetative forms of bacteria in from fifteen to twenty minutes, and the sporing forms in from one to two hours. this method is chiefly used for the sterilisation of the various nutrient media intended for the cultivation of bacteria, and is carried out in a steam kettle of special construction, known as koch's steam steriliser (fig. 28) or in one of its many modifications, the most efficient of which is arnold's (fig. 29). the steam steriliser in its simplest form consists of a tall tinned-iron or copper cylindrical vessel, divided into two unequal parts by a movable perforated metal diaphragm, the lower, smaller portion serving for a water reservoir, and the upper part for the reception of wire baskets containing the articles to be sterilised. the vessel is closed by a loose conical lid, provided with handles, and perforated at its apex by a tubulure; it is mounted on a tripod stand and heated from below by a bunsen burner. the more elaborate steriliser is cased with felt or asbestos board, and provided with a water gauge, also a tap for emptying the water compartment. to use the steam steriliser.-1. fill the water compartment to the level of the perforated diaphragm, place the lid in position, and light the bunsen burner. 2. after the water has boiled, allow sufficient time to elapse for steam to replace the air in the sterilising compartment, as shown by the steam issuing in a steady, continuous stream from the tubulure in the lid. 3. remove the lid, quickly lower the wire basket containing media tubes, etc., into the sterilising compartment until it rests on the diaphragm, and replace the lid. 4. after an interval of twenty minutes in the case of fluid media, or thirty minutes in the case of solid media, take off the lid and remove the basket with its contents. 5. now, but not before, extinguish the gas. note.--after removing tubes, flasks, etc., from the steam steriliser, they should be at once separated freely in order to prevent moisture condensing upon the cotton-wool plugs and soaking through into the interior of the tubes. this treatment will destroy any vegetative forms of bacteria; during the hours of cooling any spores present will germinate, and the young organisms will be destroyed by repeating the process twenty-four hours later; a third sterilisation after a similar interval makes assurance doubly sure. the method of sterilising by exposure to streaming steam at 100° c. for twenty minutes on each of three consecutive days is termed _discontinuous_ or _intermittent sterilisation_. exposure to steam at 100° c. for a period of one or two hours, or _continuous sterilisation_, cannot always be depended upon and is therefore not to be recommended. _superheated steam_--i. e., steam under pressure (see pressure-temperature table, appendix, page 500) in sealed vessels at a temperature of 115° c.--will destroy both the vegetative and the sporing forms of bacteria within fifteen minutes; if the pressure is increased, and the temperature raised to 120° c., the same end is attained in ten minutes. this method was formerly employed for the sterilisation of media (and indeed is so used in some laboratories still), but most workers now realise that media subjected to this high temperature undergo hydrolytic changes which render them unsuitable for the cultivation of the more delicate micro-organisms. the use of superheated steam should be restricted almost entirely to the disinfection of such contaminated articles, old cultivations, etc., as cannot be dealt with by dry heat or the actual furnace. sterilisation by means of superheated steam is carried out in a special boiler--chamberland's autoclave (fig. 30). the autoclave consists of a stout copper cylinder, provided with a copper or gun-metal lid, which is secured in place by means of bolts and thumbscrews, the joint between the cylinder and its lid being hermetically sealed by the interposition of a rubber washer. the cover is perforated for a branched tube carrying a vent cock, a manometer, and a safety valve. the copper boiler is mounted in the upper half of a cylindrical sheet-iron case--two concentric circular rows of bunsen burners, each circle having an independent gas-supply, occupying the lower half. in the interior of the boiler is a large movable wire basket, mounted on legs, for the reception of the articles to be sterilised. to use the autoclave.-1. pack the articles to be sterilised in the wire basket. 2. run water into the boiler to the level of the bottom of the basket; also fill the contained flasks and tubes with water. 3. see that the rubber washer is in position, then replace the cover and fasten it tightly on to the autoclave by means of the thumbscrews. 4. open the vent cock and light both rings of burners. 5. when steam is issuing in a steady, continuous stream from the vent tube, shut off the vent cock and extinguish the outer ring of gas burners. 6. wait until the index of the manometer records a temperature of 120° c., then regulate the gas and the spring safety valve in such a manner that this temperature is just maintained, and leave it thus for twenty minutes. in the more expensive patterns of autoclave this regulation of the safety valve is carried out automatically, the manometer being fitted with an adjustable pointer which can be set to any required pressure-temperature and so arranged that when the index of the manometer coincides with the adjustable hand the safety valve is opened. 7. extinguish the gas and allow the manometer index to fall to zero. [illustration: fig. 30.--chamberland's autoclave.] 8. now open the vent cock slowly, and allow the internal pressure to adjust itself to that of the atmosphere. 9. remove the cover and take out the sterilised contents. ~sterilisation periods.~--an exceedingly useful device for the timing of sterilisation periods (and indeed for many other operations in the laboratory) is the electric signal timing clock. this is a clock of american type in which the face is surrounded by a metal plate having a series of 60 holes at equal distances apart, corresponding to the minutes on the dial. this plate is connected with one of the poles of a dry battery, the other pole of which is connected to the metal case of the clock for the purpose of actuating an ordinary magnet alarm bell. in the centre of each of the holes in the plate a metal rod is fixed, which then passes through an insulating ring and projects inside the clock face, where it makes contact with the hour hand. the clock is mounted on a heavy base, with a key-board containing 20 numbered plugs. if one of the plugs is inserted in a hole in the plate it makes contact with the rod, and when the hour hand of the clock touches the other end the circuit is completed and the bell starts ringing. the period of this friction contact is approximately 20 seconds. the clock can therefore be used for electrically noting the periods of time from one minute by multiples of one minute up to one hour. [illustration: fig. 31.--electric signal timing clock.] ~filtration.~--(a) _cotton-wool filter._--practically the only method in use in the laboratory for the sterilisation of air or of a gas is by filtration through dry cotton-wool or glass-wool, the fibres of which entangle the micro-organisms and prevent their passage. perhaps the best example of such a filter is the cotton-wool plug which closes the mouth of a culture tube. not only does ordinary diffusion take place through it, but if a tube plugged in the usual manner with cotton-wool is removed from the hot incubator, the temperature of the contained air rapidly falls to that of the laboratory, and a partial vacuum is formed; air passes into the tube, through the cotton-wool plug, to restore the equilibrium, and, so long as the plug remains dry, in a germ-free condition. if, however, the plug becomes moist, either by absorption from the atmosphere, or from liquids coming into contact with it, micro-organisms (especially the mould fungi) commence to multiply, and the long thread forms rapidly penetrate the substance of the plug, and gain access to and contaminate the interior of the tube. [illustration: fig. 32.--cotton-wool air filter.] method.-if it is desired to sterilise gases before admission to a vessel containing a pure cultivation of a micro-organism, as, for instance, when forcing a current of oxygen over or through a broth cultivation of the diphtheria bacillus, this can be readily effected as follows: 1. take a length of glass tubing of, say, 1.5 cm. diameter, in the centre of which a bulb has been blown, fill the bulb with dry cotton-wool (fig. 32), wrap a layer of cotton-wool around each end of the tube, and secure in position with a turn of thin copper wire or string; then sterilise the piece of apparatus in the hot-air oven. 2. prepare the cultivation in a ruffer or woodhead flask (fig. 33) the inlet tube of which has its free extremity enveloped in a layer of cotton-wool, secured by thread or wire, whilst the exit tube is plugged in the usual manner. [illustration: fig. 33.--ruffer's flask.] 3. sterilise a short length of rubber tubing by boiling. transfer it from the boiling water to a beaker of absolute alcohol. 4. when all is ready remove the rubber tube from the alcohol by means of a pair of forceps, drain it thoroughly, and pass through the flame of a bunsen burner to burn off the last traces of alcohol. 5. remove the cotton-wool wraps from the entry tube of the flask and from one end of the filter tube and rapidly couple them up by means of the sterile rubber tubing. 6. connect the other end of the bulb tube with the delivery tube from the gas reservoir. the gas in its passage through the dry sterile cotton-wool in the bulb of the filter tube will be freed from any contained micro-organisms and will enter the flask in a sterile condition. (b) _porcelain filter._--the sterilisation of liquids by filtration is effected by passing them through a cylindrical vessel, closed at one end like a test-tube, and made either of porous "biscuit" porcelain, hard-burnt and unglazed (chamberland system), or of kieselguhr, a fine diatomaceous earth (berkefeld system), and termed a "bougie" or "candle" (fig. 34). note.--in selecting candles for use in the laboratory avoid those with metal fittings, since during sterilisation cracks develop at the junction of the metal and the siliceous material owing to the unequal expansion. in this method the bacteria are retained in the pores of the filter while the liquid passes through in a germ-free condition. it is obvious that to be effective the pores of the filter must be extremely minute, and therefore the rate of filtration will usually be slow. chamberland filter candles possess finer channels than berkefeld candles and consequently filter much more slowly. to overcome this disadvantage, either aspiration or pressure, or a combination of these two forces, may be employed to hasten the process. doultons white porcelain filters it may be noted are as efficient as the chamberland candles and filter rather more rapidly. _apparatus required._-1. separatory funnel containing the unfiltered fluid. 2. sterile filter candle (fig. 34), the open end fitted with a rubber stopper (fig. 34, a) perforated to receive the delivery tube of the separatory funnel, and its neck passed through a large rubber washer (fig. 34, b) which fits the mouth of the filter flask. 3. sterile filter flask of suitable size, for the reception of the filtered fluid, its mouth closed by a cotton-wool plug. 4. water injector sprengel (see fig. 38, c) pump, or geryk's pump (an air pump on the hydraulic principle, sealed by means of low vapor-tension oil, fig. 35). if this latter is employed, a wulff's bottle, fitted as a wash-bottle and containing sulphuric acid, must be interposed between the filter flask and the pump, in order to prevent moist air reaching the oil in the pump. 5. air filter (_vide_ page 40) sterilised. 6. pressure tubing. 7. screw clamps (fig. 36). method.-1. couple the exhaust pipe of the suction pump with the lateral tube of the filter flask (first removing the cotton-wool plug from this latter), by means of pressure tubing, interposing, if necessary, the wash-bottle of sulphuric acid. [illustration: fig. 34.--porcelain filter candle.] [illustration: fig. 35.--geryk air pump.] 2. remove the cotton-wool plug from the neck of the filter flask and adjust the porcelain candle in its place. [illustration: fig. 36.--screw clamps.] 3. attach the nozzle of the separatory funnel to the filter candle by means of the perforated rubber stopper (fig. 37). [illustration: fig. 37.--apparatus arranged for filtering--aspiration.] 4. open the tap of the funnel, and exhaust the air from the filter flask and wash-bottle; maintain the vacuum until the filtration is complete. 5. when the filtration is completed close the tap of the funnel; adjust a screw clamp to the pressure tubing attached to the lateral branch of the filter flask; screw it up tightly, and disconnect the acid wash-bottle. 6. attach the air filter to the open end of the pressure tubing; open the screw clamp gradually, and allow filtered air to enter the flask, to abolish the negative pressure. 7. detach the rubber tubing from the lateral branch of the flask, flame the end of the branch in the bunsen, and plug its orifice with sterile cotton-wool. 8. remove the filter candle from the mouth of the flask, flame the mouth, and plug the neck with sterile cotton-wool. 9. disinfect the filter candle and separatory funnel by boiling. if it is found necessary to employ pressure in addition to or in place of suction, insert a perforated rubber stopper into the mouth of the separatory funnel and secure in position with copper wire; next fit a piece of glass tubing through the stopper, and connect the external orifice with an air-pressure pump of some kind (an ordinary foot pump such as is employed for inflating bicycle tyres is one of the most generally useful, for this purpose) or with a cylinder of compressed air or other gas. in order to filter a large bulk of fluid very rapidly it is necessary to use a higher pressure than glass would stand, and in these cases the metal receptacle designed by pakes (fig. 38, a), to hold the filter candle itself as well as the fluid to be filtered, should be employed. (a vacuum must also be maintained in the filter flask, by means of an exhaust pump, during the entire process.) this piece of apparatus consists of a brass cylinder, capacity 2500 c.c., with two shoulders; and an opening in the neck at each end, provided with screw threads. a nut carrying a pressure gauge fits into the top screw; and into the bottom is fitted a brass cylinder carrying the filter candle and prolonged downwards into a delivery tube. leakage is prevented by means of rubber washers. into the top shoulder a tube is inserted, bent at right angles and provided with a tap. all the brass-work is tinned inside (fig. 38, a). in use the reservoir is generally mounted on a tripod stand. ~to sterilise.~-1. insert the filter candle into its cylinder and screw this loosely on. [illustration: fig. 38.--pakes' filtering reservoir--pressure and aspiration.] 2. wrap a layer of cotton-wool around the delivery tube and fasten in position. 3. remove the nut carrying the pressure gauge and plug the neck with cotton-wool. 4. heat the whole apparatus in the autoclave at 120° c. for twenty minutes. method.-1. remove the apparatus from the autoclave, and allow it to cool. 2. screw home the box carrying the bougie. 3. set the apparatus up in position, with its delivery tube (from which the cotton-wool wrapping has been removed) passing through a perforated rubber stopper in the neck of a filter flask. [illustration: fig. 39.--closed candle arranged for filtering.] 4. fill the fluid to be filtered into the cylinder and screw on the nut carrying the pressure gauge. (this nut should be immersed in boiling water for a few minutes previous to screwing on, in order to sterilise it.) 5. connect the horizontal arm of the entry tube with a cylinder of compressed oxygen (or carbon dioxide, fig. 38, b), by means of pressure tubing. 6. connect the lateral arm of the filter flask with the exhaust pump (fig. 38, c) and start the latter working. 7. open the tap of the gas cylinder; then open the tap on the entry tube of the filter cylinder and raise the pressure in its interior until the desired point is recorded on the manometer. maintain this pressure, usually one or one and a half atmospheres, until filtration is completed, by regulating the tap on the entry tube. some forms of filter candle are made with the open end contracted into a delivery nozzle, which is glazed. in this case the apparatus is fitted up in a slightly different manner; the fluid to be filtered is contained in an open cylinder into which the candle is plunged, while its delivery nozzle is connected with the filter flask by means of a piece of flexible pressure tubing (previously sterilised by boiling), as in figure 39. iv. the microscope. the essentials of a microscope for bacteriological work may be briefly summed up as follows: [illustration: fig. 40.--microscope stand.] the instrument, of the monocular type, must be of good workmanship and well finished, rigid, firm, and free from vibration, not only when upright, but also when inclined to an angle or in the horizontal position. the various joints and movements must work smoothly and precisely, equally free from the defects of "loss of time" and "slipping." all screws, etc., should conform to the royal microscopical society's standard. it must also be provided with good lenses and a sufficiently large stage. the details of its component parts, to which attention must be specially directed, are as follows: [illustration: fig. 41.--foot, three types.] ~1. the base or foot~ (fig. 40, a).--two elementary forms--the tripod (fig. 41, a) and the vertical column set into a plate known as the "horse-shoe" (fig. 41, b)--serve as the patterns for countless modifications in shape and size of this portion of the stand. the chief desiderata--stability and ease of manipulation--are attained in the first by means of the "spread" of the three feet, which are usually shod with cork; in the second, by the dead weight of the foot-plate. the tripod is mechanically the more correct form, and for practical use is much to be preferred. its chief rival, the jackson foot (fig. 41, c), is based upon the same principle, and on the score of appearance has much to recommend it. ~2.~ the ~body tube~ (fig. 40, b) may be either that known as the "long" or "english" (length 250 mm.), or the "short" or "continental" (length 160 mm.). neither length appears to possess any material advantage over the other, but it is absolutely necessary to secure objectives which have been manufactured for the particular tube length chosen. in the high-class microscope of the present day the body tube is usually shorter than the continental, but is provided with a draw tube which, when fully extended, gives a tube length greater than the english, thus permitting the use of either form of objective. [illustration: fig. 42.--coarse adjustment.] [illustration: fig. 43.--fine adjustment.] for practical purposes the tube length = distance from the end of the nosepiece to the eyeglass of the ocular. this is the measurement referred to in speaking of "long" or "short" tube. ~3.~ the ~coarse adjustment~ (fig. 40, c) should be a rack-and-pinion movement, steadiness and smoothness of action being secured by means of accurately fitting dovetailed bearings and perfect correspondence between the teeth of the rack and the leaves of the pinion (fig. 42). also provision should be made for taking up the "slack" (as by the screws _aa_, fig. 42). ~4.~ the ~fine adjustment~ (fig. 40, d) should on no account depend upon the direct action of springs, but should be of the lever pattern, preferably the nelson (fig. 43). in this form the unequal length of the arms of the lever secures very delicate movement, and, moreover, only a small portion of the weight of the body tube is transmitted to the thread of the vertical screw actuating the movement. [illustration: fig. 44.--spindle head to fine adjustment.] a spindle milled head (fig. 44) will be found a very useful device to have fitted in place of the ordinary milled head controlling the fine adjustment. in this contrivance the axis of the milled head is prolonged upward in a short column, the diameter of which is one-sixth of that of the head. the spindle can be rapidly rotated between the fingers for medium power adjustments while the larger milled head can be slowly moved when focussing high powers. ~5.~ the ~stage~ (fig. 40, e) should be square in shape and large in area--at least 12 cm.--flat and rigid, in order to afford a safe support for the petri dish used for plate cultivations; and should be supplied with spring clips (removable at will) to secure the 3 by 1 glass slides. a mechanical stage must be classed as a necessity rather than a luxury so far as the bacteriologist is concerned, as when working with high powers, and especially when examining hanging-drop specimens, it is almost impossible to execute sufficiently delicate movements with the fingers. in selecting a mechanical stage, preference should be given to one which forms an integral part of the instrument (fig. 45) rather than one which needs to be clamped on to an ordinary plain stage every time it is required, and its traversing movements should be controlled by stationary milled heads (fig. 45, _aa'_). the shape of the aperture is a not unimportant point; it should be square to allow of free movement over the substage condenser. the mechanical stage should be tapped for three (removable) screw studs to be used in place of the sliding bar, so that if desired the vernier finder (fig. 45, _bb'_), such as is usually fitted to this class of stage, or a maltwood finder, may be employed. [illustration: fig. 45.--mechanical stage.] [illustration: fig. 46.--iris diaphragm.] ~6. diaphragm.~--separate single diaphragms must be avoided; a revolving plate pierced with different sized apertures and secured below the stage is preferable, but undoubtedly the best form is the "iris" diaphragm (fig. 46) which enters into the construction of the substage condenser. ~7.~ the ~substage condenser~ is a necessary part of the optical outfit. its purpose is to collect the beam of parallel rays of light reflected by the plane mirror, by virtue of a short focus system of lenses, into a cone of large aperture (reducible at will by means of an iris diaphragm mounted as a part of the condenser), which can be accurately focussed on the plane of the object. this focussing must be performed anew for each object, on account of the variation in the thickness of the slides. the form in most general use is that known as the abbé (fig. 47) and consists of a plano-convex lens mounted above a biconvex lens. this combination is carried in a screw-centering holder known as the substage below the stage of the microscope (fig. 40 f), and must be accurately adjusted so that its optical axis coincides with that of the objective. vertical movement of the entire substage apparatus effected by means of a rack and pinion is a decided advantage, and some means should be provided for temporarily removing the condenser from the optical axis of the microscope. [illustration: fig. 47--optical part of abbé illuminator.] with the oil immersion objective, however, an ~achromatic condenser~, giving an illuminating cone of about 0.9, should be used if the full value of the lens is to be obtained. it is generally assumed that a good objective requires an illuminating cone equivalent to two-thirds of its numerical aperture. the best abbé condenser transmits a cone of about .45 whilst the aperture of the 1/12 inch immersion lenses of different makers varies from 1.0 to 1.4, hence, the efficiency of these lenses is much curtailed if the condenser is merely the abbé. these improved condensers must be absolutely centered to the objective and capable of very accurate focussing otherwise much of their value is lost. ~8. mirrors.~--below the substage condenser is attached a gymbal carrying a reversible circular frame with a plane mirror on one side and a concave mirror on the other (fig. 40, g). the plane mirror is that usually employed, but occasionally, as for example when using low powers and with the condenser racked down and thrown out of the optical axis, the concave mirror is used. ~9. oculars, or eyepieces.~--those known as the huyghenian oculars (fig. 48) will be sufficient for all ordinary work without resorting to the more expensive "compensation" oculars. two or three, magnifying the "real" image (formed by the objective) four, six, or eight times respectively, form a useful equipment. as an accessory ~ehrlich's eyepiece~ is a very useful piece of apparatus when the enumeration of cells or bacteria has to be carried out. this is an ordinary eyepiece fitted with an adjustable square diaphragm operated by a lever projecting from the side of the mount. three notches are made in one of the sides of the square and by moving the lever square aperture can be reduced to three-quarters, one-half or one-quarter of the original size. ~10. objectives.~--three objectives are necessary: one for low-power work--e. g., 1 inch, 2/3 inch, or 1/2 inch; one for high-power work--e. g., 1/12 inch oil immersion lens; and an intermediate "medium-power" lens--e. g., 1/6 inch or 1/8 inch (dry). these lenses must be carefully selected, especial attention being paid to the following points: (a) _correction of spherical aberration._--spherical aberration gives rise to an ill-defined image, due to the central and peripheral rays focussing at different points. (b) _correction of chromatic aberration._--chromatic aberration gives rise to a coloured fringe around the edges of objects due to the fact that the different-coloured rays of the spectrum possess varying refrangibilities and that a simple lens acts toward them as a prism. (c) _flatness of field._--the ideal visual field would be large and, above all, _flat_; in other words, objects at the periphery of the field would be as distinctly "in focus" as those in the centre. unfortunately, however, this is an optical impossibility and the field is always spherical in shape. some makers succeed in giving a larger central area that is in focus at one time than others, and although this may theoretically cause an infinitesimal sacrifice of other qualities, it should always be sought for. successive zones and the entire peripheral ring should come into focus with the alteration of the fine adjustment. this simultaneous sharpness of the entire circle is an indication of the perfect centering of the whole of the lenses in the objective. [illustration: fig. 48.--huyghenian eyepiece.] (d) _good definition._--actual magnification is, within limits, of course, of less value than clear definition and high resolving power, for it is upon these properties we depend for our knowledge of the detailed structure of the objects examined. (e) _numerical aperture_ (_n. a._).--the numerical aperture may be defined, in general terms, as the ratio of the _effective_ diameter of the back lens of the objective to its equivalent focal length. the determination of this point is a process requiring considerable technical skill and mathematical ability, and is completely beyond the powers of the average microscopist.[1] although with the increase in power it is correspondingly difficult to combine all these corrections in one objective, they are brought to a high pitch of excellence in the present-day "achromatic" objectives, and so remove the necessity for the use of the higher priced and less durable apochromatic lenses. in selecting objectives the best "test" objects to employ are: 1. a thin (one cell layer), even } { 1", 2/3", 1/2": "blood film," stained with jenner's } for { 1/6", 1/8" or romanowsky's stain. } { 1/12" oil 2. a thin cover-slip preparation } of a young cultivation of } { 1/8" dry _b. diphtheriæ_ (showing } for { segmentation) stained with } { 1/12" oil methylene-blue. } ~accessories.~--_eye shade_ (fig. 49).--this piece of apparatus consists of a pear-shaped piece of blackened metal or ebonite, hinged to a collar which rotates on the upper part of the body tube of the microscope. it can be used to shut out the image of surrounding objects from the unoccupied eye, and when carrying out prolonged observations will be found of real service. _nosepiece._--perhaps the most useful accessory is a nosepiece to carry two of the objectives (fig. 50), or, better still, all three (fig. 51). this nosepiece, preferably constructed of aluminium, must be of the covered-in type, consisting of a curved plate attached to the lower end of the body tube--a circular aperture being cut to correspond to the lumen of that tube. to the under surface of this plate is pivoted a similarly curved plate, fitted with three tubulures, each of which carries an objective. by rotating the lower plate each of the objectives can be brought successively in to the optical axis of the microscope. [illustration: fig. 49.--eye shade.] for critical work and particularly for photo-micrography, however, the interchangeable nosepiece is by no means perfect as it is next to impossible to secure accurate centreing of each lens in the optical axis. for special purposes, therefore, it is necessary to employ a special nosepiece such as that made by zeiss or leitz into which each objective slides on its own carrier and upon which it is accurately centred. [illustration: fig. 50.--double nosepiece.] [illustration: fig. 51.--triple nosepiece.] _warm stage_ (fig. 52).--this is a flat metal case containing a system of tubes through the interior of which water of any required temperature can be circulated. it is made to clamp on to the stage of the microscope by the screws _a a'_, and is perforated with a large hole coinciding with the optical axis of the microscope; a short tube b, projecting from one end of the warm stage permits water of the desired temperature to be conducted from a reservoir through a length of rubber tubing to the interior of the stage and a similar tube at the other end _b'_ of the stage allows exit to the waste water. by raising the temperature of hanging-drop preparations, etc., placed upon it, above that of the surrounding atmosphere, the warm stage renders possible exact observations on spore germination, hanging-drop cultivations, etc. [illustration: fig. 52.--warm stage.] a better form is the electrical hot stage designed by lorrain smith;[2] it requires the addition of a lamp resistance and sliding rheostat, also a delicate ammeter reading to .01 of an ampère. it consists of a wooden frame supporting a flat glass bulb with a long neck bent upward at an obtuse angle (fig. 53). the bulb is filled with liquid paraffin, which rises in the open neck when expanded by heat. the neck also accommodates the thermometer. two coils of manganin wire run in the paraffin at opposite sides of the bulb (outside the field of vision), coupled to brass terminals on the wooden frame by platinum wire fused into the glass. the resistance of the two coils in series is about 10 ohms. a current of 2-1/2 ampères is needed, and is conducted to the coils in the stage through the rheostat. with the help of the ammeter any desired temperature can be obtained and maintained, up to about 200° c. if immersion oil contact is made between the top lens of the condenser and the lower surface of the bulb, this stage works very well indeed with the 1/12-inch oil immersion lens. [illustration: fig. 53.--lorrain smith's warm stage.] _dark ground or paraboloid condenser._--this is an immersion substage condenser of high aperture by means of which unstained objects such as bacteria can be shown as bright white particles upon a dense black background. the central rays of light are blocked out by means of an opaque stop while the peripheral rays are reflected from the paraboloidal sides of the condenser and refracted by the object viewed. to obtain the best results with this type of condenser a powerful illuminant--such as a small arc lamp or an incandescent gas lamp--is needed, together with picked slides of a certain thickness (specified for the particular make of condenser but generally 1 mm.) and specially thin cover-glasses (not more than 0.17 mm.) the objective must not have a higher na than 1.0, consequently immersion lenses must be fitted with an internal stop to cut down the aperture. _micrometer._--some form of micrometer for the purpose of measuring bacteria and other objects is also essential. details of those in general use will be found in the following pages. [illustration: fig. 54--diamond object marker.] _object marker_ (fig. 54).--this is an exceedingly useful piece of apparatus. made in the form of an objective, the lenses are replaced by a diamond point, set slightly out of the centre, which can be rotated by means of a milled plate. screwed on to the nosepiece in place of the objective, rotation of the diamond point will rule a small circle on the object slide to permanently record the position of an interesting portion of the specimen. the diamond is mounted on a spring which regulates the pressure, and the size of the circle can be adjusted by means of a lateral screw. methods of micrometry. the unit of length as applied to the measurement of microscopical objects is the one-thousandth part of a millimetre (0.001 mm.), denominated a _micron_ (sometimes, and erroneously, referred to as a micro-millimetre), and indicated in writing by the greek letter µ. of the many methods in use for the measurement of bacteria, three only will be here described, viz.: (a) by means of the camera lucida. (b) by means of the ocular or eyepiece micrometer. (c) by means of the filar micrometer (ramsden's micrometer eyepiece). for each of these methods a ~stage micrometer~ is necessary. this is a 3 by 1 inch glass slip having engraved on it a scale divided to hundredths of a millimetre (0.01 mm.), every tenth line being made longer than the intervening ones, to facilitate counting; and from these engraved lines the measurement in every case is evaluated. a cover-glass is cemented over the scale to protect it from injury. [illustration: fig. 55.--camera lucida, abbé pattern.] (a) by means of the camera lucida. 1. attach a camera lucida (of the wollaston, beale, or abbé pattern) (fig. 55) to the eyepiece of the microscope. 2. adjust the micrometer on the stage of the microscope and accurately focus the divisions. 3. project the scale of the stage micrometer on to a piece of paper and with pen or pencil sketch in the magnified image, each division of which corresponds to 10µ. mark on the paper the optical combination (ocular objective and tube length) employed to produce this particular magnification. 4. repeat this procedure for each of the possible combinations of oculars and objectives fitted to the microscope supplied, and carefully preserve the scales thus obtained. to measure an object by this method simply project the image on to the scale corresponding to the particular optical combination in use at the moment. read off the number of divisions it occupies and express them as _micra_. in place of preserving a scale for each optical combination, the object to be measured and the micrometer scale may be projected and sketched, in turn, on the same piece of paper, taking particular care that the centre of the eyepiece is 25 cm. from the paper on which the divisions are drawn. [illustration: fig. 56.--eyepiece micrometer, ordinary.] [illustration: fig. 57.--eyepiece micrometer, net.] (b) by means of the eyepiece micrometer. the ~eyepiece micrometer~ is a circular glass disc having engraved on it a scale divided to tenths of a millimetre (0.1 mm.) (fig. 56), or the entire surface ruled in 0.1 mm. squares (the net micrometer) (fig. 57). it can be fitted inside the mount of any ocular just above the aperture of the diaphragm and must be adjusted exactly in the focus of the eye lens. some makers mount the glass disc together with a circular cover-glass in such a way that when placed in position in any huyghenian eyepiece of their own manufacture, the scale is exactly in focus for normal vision. special eyepieces are also obtainable having a sledging adjustment to the eye lens for focussing the micrometer. the value of one division of the micrometer scale must first be ascertained for each optical combination by the aid of the stage micrometer, thus: 1. insert the eyepiece micrometer inside the ocular and adjust the stage micrometer on the stage of the microscope. 2. focus the scale of the stage micrometer accurately; the lines will appear to be immediately below those of the eyepiece micrometer. make the lines on the two micrometers parallel by rotating the ocular. 3. make two of the lines on the ocular micrometer coincide with those bounding one division of the stage micrometer; this is effected by increasing or diminishing the tube length; and note the number of included divisions. 4. calculate the value of each division of the eyepiece micrometer in terms of µ, by means of the following formula: x = 10 y. where x = the number of included divisions of the eyepiece micrometer. y = the number of included divisions of the stage micrometer. 5. note the optical combination employed in this experiment and record it with the calculated micrometer value. repeat this process for each of the other combinations. carefully record the results. to measure an object by this method read off the number of divisions of the eyepiece micrometer it occupies and express the result in _micra_ by a reference to the standard value for the particular optical combination employed. zeiss prepares a compensating eyepiece micrometer for use with his apochromatic objectives, the divisions of which are so computed that (with a tube length of 160 mm.) the value of each is equivalent to as many _micra_ as there are millimetres in the focal length of the objective employed. _wright's eikonometer_ is really a modification of the eyepiece micrometer for rapidly measuring microscopical objects by direct inspection, having previously determined the magnifying power of the particular optical combination employed. it is a small piece of apparatus resembling an eyepiece, with a sliding eye lens, which can be accurately focussed on a micrometer scale fixed within the instrument. when placed over the microscope ocular the divisions of this scale measure the actual size of the virtual image in millimetres. in order to use this instrument for direct measurement, it is first necessary to determine the magnifying power of each combination of ocular, tube length and objective. place a stage micrometer divided into hundredths of a millimetre on the microscope stage and focus accurately. rest the eikonometer on the eyepiece. observation through the eikonometer shows its micrometer scale superposed on the image of the stage micrometer. rotate the eikonometer until the lines on the two scales are parallel, and make the various adjustments to ensure that two lines on the eikonometer scale coincide with two lines on the stage micrometer. for the sake of illustration it may be assumed that five of the divisions on the stage micrometer accurately fill one of the divisions of the eikonometer scale; this indicates a magnifying power of 500 as the constant for that particular optical combination, and a record should be made of the fact. the magnification constants of the various other optical combinations should be similarly made and recorded. to measure any object subsequently it should be first focussed carefully in the ordinary way. the eikonometer should then be applied to the eyepiece and the size of the object read off on the eikonometer scale as millimetres, and the actual size calculated by dividing the observed size by the magnification constant for the particular optical combination employed in the observation. (c) by means of the filar micrometer. [illustration: fig. 58.--ramsden's filar micrometer.] [illustration: fig. 59.--ramsden's micrometer field, a, fixed wire; b, reference wire (fixed); c, travelling wire.] the ~filar~ or cobweb micrometer (ramsden's micrometer) eyepiece (fig. 58) consists of an ocular having a fine "fixed" wire stretching horizontally across the field (fig. 59), a vertical reference wire--fixed--adjusted at right angles to the first; and a fine wire, parallel to the reference wire, which can be moved across the field by the action of a micrometer screw; the drum head is divided into one hundred parts, which successively pass a fixed index as the head is turned. in the lower part of the field is a comb with the intervals between its teeth corresponding to one complete revolution of this screw-head. as in the previous method, the value of each division of the micrometer scale (i. e., the comb) must first be determined for each optical combination. this is effected as follows: 1. place the filar micrometer and the stage micrometer in their respective positions. 2. rotate the screw of the filar micrometer until the movable wire coincides with the fixed one, and the index marks zero on the drum head. (if when the drum head is at zero the two wires do not exactly coincide they must be adjusted by loosening the drum screw and resetting the drum.) 3. focus the scale of each micrometer accurately, and make the lines on them parallel. 4. rotate the head of the micrometer screw until the movable line has transversed one division of the stage micrometer. note the number of complete revolutions (by means of the recording comb) and the fractions of a revolution (by means of scale on the head of the micrometer screw), which are required to measure the 0.01 mm. 5. make several such estimations and average the results. 6. note the optical combination employed in this experiment and record it carefully, together with the micrometer value in terms of µ. 7. repeat this process for each of the different optical combinations and record the results. to measure an object by this method, simply note the number of revolutions and fractions of a revolution of the screw-head required to traverse such object from edge to edge, and express the result as _micra_ by reference to the recorded values for that particular optical combination. _microscope illuminant._--in tropical and subtropical regions diffuse daylight is the best illuminant. in temperate climes however daylight of the desirable quantity is not always available, and recourse must be had to oil lamps, gas lamps--preferably those with incandescent mantles--and electricity; and of these the last is undoubtedly the best. a handy lamp holder which can be manufactured in the laboratory is shown in fig. 60. it consists of a base board weighted with lead to which is attached the ordinary domestic lamp holder, and behind this is fastened a curved sheet-iron reflector. an obscured metal filament lamp of about 16 candle power gives the most suitable light, and if monochromatic light is needed, the blue grease pencil is streaked over the side of the lamp nearest the microscope; the current is switched on and when the glass bulb is warm, rubbing with a wad of cotton-wool will readily distribute the blue greasy material in an even film over the ground glass. [illustration: fig. 60.--electric microscope lamp.] footnotes: [1] its importance will be realised, however, when it is stated in the words of the late professor abbé: "the numerical aperture of a lens determines all its essential qualities; the brightness of the image increases with a given magnification and other things being equal, as the square of the aperture; the resolving and defining powers are directly related to it, the focal depth of differentiation of depths varies inversely as the aperture, and so forth." [2] made by mr. otto baumbach, 10, lime grove, manchester. v. microscopical examination of bacteria and other micro-fungi. apparatus and reagents used in ordinary microscopical examination. the following comprises the essential apparatus and reagents for routine work with which each student should be provided. 1. india-rubber "change-mat" upon which cover-glasses may be rested during the process of staining. 2. squares of blotting paper about 10 cm., for drying cover-slips and slides. (the filter paper known as "german lined"--a highly absorbent, closely woven paper, having an even surface and no loose "fluff" to adhere to the specimens--is the most useful for this purpose.) [illustration: fig. 61.--disinfectant jar.] 3. glass jar filled with 2 per cent. lysol solution for the reception of infected cover-glasses and infected pipettes, etc. 4. a square glazed earthenware box with a loose lining containing 2 per cent. lysol solution for the reception of infected material and used slides. the bottom of the lining is perforated so that when full the lining and its contents can be lifted bodily out of the box, when the disinfectant solution drains away and the slides, etc., can easily be emptied out. the empty lining is then returned to the box with its disinfectant solution (fig. 61). 5. bunsen burner provided with "peep-flame" by-pass. 6. porcelain trough holding five or six hanging-drop slides (fig. 62). [illustration: fig. 62.--hanging-drop slides: a, double cell seen from above; b, single cell seen from the side.] the best form of hanging-drop slide is a modification of boettcher's glass ring slide, and is prepared by cementing a circular cell of tin, 13 to 15 mm. diameter, and 1 to 2 mm. in height, to the centre of a 3 by 1 slip by means of canada balsam. it is often extremely convenient to have two of these cells cemented close together on one slide (fig. 62, a). another form of hanging-drop slide is made in which a circular or oval concavity or "cell" is ground out of the centre of a 3 by 1 slip. these are more expensive, less convenient to work with, and are more easily contaminated by drops of material under examination, and should be carefully avoided. 7. three aluminium rods (fig. 63), each about 25 cm. long and carrying a piece of 0.015 gauge platino-iridium wire 7.5 cm. in length. the end of one of the wires is bent round to form an oval loop, of about 1 mm. in its short diameter, and is termed a loop or an oese; the terminal 3 or 4 mm. of another wire is flattened out by hammering it on a smooth iron surface to form a "spatula"; the third is left untouched or is pointed by the aid of a file. these instruments are used for inoculating culture tubes and preparing specimens for microscopical examination. [illustration: fig. 63.--ends of platinum rods. a, loop; b, spatula; c, needle.] the method of mounting these wires may be described as follows: take a piece of aluminium wire 25 cm. long and about 0.25 cm. in diameter, and drill a fine hole completely through the wire about a centimetre from one end. sink a straight narrow channel along one side of the wire, in its long axis, from the hole to the nearest end, shallow at first, but gradually becoming deeper. on the opposite side of the wire make a short cut, 2 mm. in length, leading from the hole in the same direction. [the use of a fine dental drill and small circular saw, worked by a dental motor facilitates the manufacture of these aluminium handled instruments.] now pass one end of the platinum wire through the hole, turn up about 2 mm. at right angles and press the short piece into the short cut. turn the long end of the wire sharply, also at right angles, and sink it into the long channel so that it emerges from about the centre of the cut end of the aluminium wire (fig. 63). a few sharp taps with a watch maker's hammer will now close in the sides of the two channels over the wire and hold it securely. [illustration: fig. 64.--platinum rod in aluminium handle--method of mounting. the platinum wire may be fused into the end of a piece of glass rod, but such a handle is vastly inferior to aluminium and is not to be recommended.] 8. two pairs of sharp-pointed spring forceps (10 cm. long), one of which must be kept perfectly clean and reserved for handling clean cover-slips, the other being for use during staining operations. 9. a box of clean 3 by 1 glass slips. 10. a glass capsule with tightly fitting (ground on) glass lid, containing clean cover-slips in absolute alcohol. 11. one of faber's "grease pencils" (yellow, red, or blue) for writing on glass. 12. a wooden rack (fig. 65) with twelve drop-bottles (fig. 66) each 60 c.c. capacity, containing aniline water. gentian violet, saturated alcoholic solution. lugol's (gram's) iodine. absolute alcohol. methylene-blue, } fuchsin, basic, } saturated alcoholic solution. neutral red, 1 per cent. aqueous solution. leishman's modified romanowsky stain. carbolic acid, 5 per cent. aqueous solution. acetic acid, 1 per cent. solution. sulphuric acid, 25 per cent. solution. xylol. [illustration: fig. 65.--staining rack, rubber change mat and lysol pot.] [illustration: fig. 66.--drop bottle.] [illustration: fig. 67.--canada balsam pot.] and two pots with air-tight glass caps (fig. 67), each provided with a piece of glass rod and filled respectively with canada balsam dissolved in xylol, and sterile vaseline. methods of examination. bacteria, etc., are examined microscopically. 1. in the living state, unstained, or stained. 2. in the "fixed" condition (i. e., fixed, killed, and stained by suitable methods). the preparation of a specimen from a tube cultivation for examination by these methods may be described as follows: ~1. living, unstained.~--(a) _"fresh" preparation._-1. clean and dry a 3 by 1 glass slip and place it on one of the squares of filter paper. deposit a drop of water (preferably distilled) or a drop of 1 per cent. solution of caustic potash, on the centre of the slip, by means of the platinum loop. [illustration: fig. 68.--holding tubes for removing bacterial growth, as seen from the front.] technique of opening and closing a culture tube. 2. remove the tube cultivation from its rack or jar with the left hand and ignite the cotton-wool plug by holding it to the flame of the bunsen burner. extinguish the flame by blowing on the plug, whilst rotating the tube on its long axis, its mouth directed vertically upward, between the thumb and fingers. (this operation is termed "flaming the plug," and is intended to destroy any micro-organisms that may have become entangled in the loose fibres of the cotton-wool, and which, if not thus destroyed, might fall into the tube when the plug is removed and so accidentally contaminate the cultivation.) 3. hold the tube at or near its centre between the ends of the thumb and first two fingers of the left hand, and allow the sealed end to rest upon the back of the hand between the thumb and forefinger, the plug pointing to the right. keep the tube as nearly in the horizontal position as is consistent with safety, to diminish the risk of the accidental entry of organisms (fig. 68). 4. take the handle of the loop between the thumb and forefinger of the right hand, holding the instrument in a position similar to that occupied by a pen or a paint-brush, and sterilise the platinum portion by holding it in the flame of a bunsen burner until it is red hot. sterilise the adjacent portion of the aluminium handle by passing it rapidly twice or thrice through the flame. after sterilising it, the loop must not be allowed to leave the hand or to touch against anything but the material it is intended to examine, until it is finished with and has been again sterilised. 5. grasp the cotton-wool plug of the test-tube between the little finger and the palm of the right hand (whilst still holding the loop as directed in step 4), and remove it from the mouth of the tube by a "screwing" motion of the right hand. 6. introduce the platinum loop into the tube and hold it in this position until satisfied that it is quite cool. (the cooling may be hastened by touching the loop on one of the drops of moisture which are usually to be found condensed on the interior of the glass tube, or by dipping it into the condensation water at the bottom; at the same time care must be taken in the case of cultures on solid media to avoid touching either the medium or the growth.) 7. remove a small portion of the growth by taking up a drop of liquid, in the case of a fluid culture, in the loop; or by touching the loop on the surface of the growth when the culture is on solid medium; and withdraw the loop from the tube without again touching the medium or the glass sides of the tube. 8. replace the cotton-wool plug in the mouth of the tube. 9. replace the tube cultivation in its rack or jar. 10. mix the contents of the loop thoroughly with the drop of water on the 3 by 1 slide. 11. again sterilise the loop as directed in step 4, and replace it in its stand. 12. remove a cover-slip from the glass capsule by means of the cover-slip forceps, rest it for a moment on its edge, on a piece of filter paper to remove the excess of alcohol, then pass it through the flame of the bunsen burner. this burns off the remainder of the alcohol, and the cover-slip so "flamed" is now clean, dry, and sterile. 13. lower the cover-slip, still held in the forceps, on to the surface of the drop of fluid on the 3 by 1 slip, carefully and gently, to avoid the inclusion of air bubbles. 14. examine microscopically (_vide infra_). during the microscopical examination, stains and other reagents may be run in under a cover-slip by the simple method of placing a drop of the reagent in contact with one edge of the cover-glass and applying the torn edge of a piece of blotting paper to the opposite side. the reagent may then be observed to flow across the field and come into contact with such of the micro-organisms as lie in its path. the non-toxic basic dyes most generally employed for the intra-vitam staining of bacteria are neutral red, } quinoleine blue } methylene green } in 0.5 per cent. aqueous solutions. vesuvin, } _negative stain_ (burri).--by this method of demonstration the appearances presented by dark ground illumination (by means of a paraboloid condenser) are closely simulated, since minute particles, bacteria, blood or pus cells etc. stand out as brilliantly white or colourless bodies on a dark grey-brown background. _reagent required:_ any one of the liquid waterproof black drawing inks (chin-chin, pelican, etc.). this is prepared for use as follows: measure out and mix: liquid black ink, 25 c.c. tincture of iodine 1 c.c. allow the mixture to stand 24 hours, centrifugalise thoroughly, pipette off the supernatant liquid to a clean bottle and then add a crystal of thymol or one drop of formalin as a preservative. method.-1. with the sterilised loop deposit one drop of the liquid ink close to one end of a 3 by 1 slide. 2. with the sterilised loop deposit a drop of the fluid culture (or of an emulsion from a solid culture) by the side of the drop of ink (fig. 69, a); mix the two drops thoroughly by the aid of the loop. 3. sterilise the loop. 4. hold the slide firmly on the bench with the thumb and forefinger of the left hand applied to the end nearest the drop of fluid. 5. take another clean 3 by 1 slide in the right hand and lower its short end obliquely (at an angle of about 60°) transversely on to the mixed ink and culture on the first slide, and allow the fluid to spread across the slide and fill the angle of incidence. 6. maintaining the original angle, draw the second slide firmly and evenly along the first toward the end farthest from the left hand (fig. 69, b). 7. throw the second slide into a pot of disinfectant; allow the first slide to dry in the air. [illustration: fig. 69.--spreading negative film.] 8. place a drop of immersion oil on the centre of the film, lower the 1/12-inch objective into the oil and examine microscopically without the intervention of a cover-slip. (the film of ink may be covered with a long cover-glass and xylol balsam as a permanent preparation.) (<b) _hanging-drop preparation._-1. smear a layer of sterile vaseline on the upper surface of the ring cell of a hanging-drop slide by means of the glass rod provided with the vaseline bottle, and place the slide on a piece of filter paper. 2. "flame" a cover-slip and place it on the filter paper by the side of the hanging-drop slide. 3. place a drop of water on the centre of the cover-slip by means of the platinum loop. 4. obtain a small quantity of the material it is desired to examine, in the manner detailed above (pages 74-76, steps 2 to 11 must be followed in their entirety and with the strictest exactitude whenever tube contents are being handled), and mix it with the drop of water on the cover-slip. 5. raise the cover-slip in the points of the forceps and rapidly invert it on to the ring cell of the hanging-drop slide, so that the drop of fluid occupies the centre of the ring. (carefully avoid contact between the drop of fluid and either the ring cell or the layer of vaseline. should this happen, the now _infected_ hanging-drop slide and its cover-slip must be dropped into the pot of lysol and a new preparation made.) 6. press the cover-slip firmly down into the vaseline on to the top of the ring cell. (this spreads out the vaseline into a thin layer, and besides ensuring the adhesion of the cover-slip, seals the cells and so retards evaporation.) 7. examine microscopically. the examination of a "fresh" specimen or a "hanging-drop" preparation is directed to the determination of the following data: 1. the nature of the bacteria present--e. g., cocci, bacilli, etc. 2. the purity of the cultivation; this can only be determined when gross morphological differences exist between the organisms present. 3. the presence or absence of spores; when present, spores show their typical refrangibility exceedingly well by this method. 4. the presence or absence of mobility. in a hanging-drop specimen some form of movement can practically always be observed, and its character must be carefully determined by noting the relative positions of adjacent micro-organisms. (a) brownian or molecular movement. minute particles of solid matter (including bacteria), when suspended in a fluid, will always show a vibratory movement affecting the entire field, but never altering the relative positions of the bacteria. (cocci exhibit this movement, but with the exception of the micrococcus agilis, the cocci are non-motile.) (b) streaming movement. this is due to currents set up in the hanging drop as a result of jarring of the specimen or of evaporation, or to the fact that the cover-slip is not perfectly level, and although the relative positions of the bacteria may vary, still the flowing movement of large numbers of organisms in some one direction will usually be sufficient to demonstrate the nature of this motion. (c) locomotive movement, or ~true motility~, is determined by observing some one particular bacillus changing its position in the field independently of, and in a direction contrary to, other organisms present. when the examination is completed and the specimen finished with, the "fresh specimen"--i. e., the slide with the cover-slip attached--must be dropped into the lysol pot. in the hanging-drop specimen, however, the cover-slip only is infected, and this may be raised from the ring cell by means of forceps and dropped into the disinfectant. _permanent staining of the hanging-drop specimen._--occasionally it is necessary to fix and stain a hanging-drop preparation. this may be done as follows: 1. remove the cover-slip from the cell by the aid of the forceps. 2. if the drop is small, fix it by dropping it face downward, whilst still wet, on to the surface of some gulland's solution or corrosive sublimate solution (_vide_ page 82) in a watch-glass. if the drop is large, place it face upward on the rubber mat, cover it with an inverted watch-glass, and allow it to dry. then fix it in the alcohol and ether solution (_vide_, page 82). 3. dip the cover-glass into a beaker containing hot water in order to remove some of the vaseline adhering to it. 4. wash successively in alcohol, xylol, ether, and alcohol, to remove the last traces of grease. 5. wash in water. 6. stain, wash, dry, and mount as for an ordinary cover-slip film preparation (_vide_ pages 83-85). ~2. killed, stained.~--in this method three distinct processes are necessary: "preparing" and "fixing" the film. staining. mounting. _preparing the film._-1. flame a cover-slip and place it on a piece of filter paper. 2. place a drop of water on the centre of the cover-slip by means of platinum loop. 3. obtain a small quantity of the material to be examined upon a sterilised platinum loop (see pages 74-76, steps 2 to 11) and mix it with the drops of water on the cover-slip. 4. spread the drop of emulsion evenly over the cover-slip in the form of a square film to within 1 mm. of each edge of the cover-slip. 5. allow it to dry completely in the air. _fixing._--fix by passing the cover-slip, held in the fingers, three or four times through the flame of a bunsen burner. in some instances (e. g., when the films after staining are intended for micrometric observations) it is almost essential to fix by exposure to a uniform temperature of 115° c., for twenty minutes. this is best done in a carefully regulated hot-air oven. fixation may also be effected by immersing in some fixative fluid, such as one of the following: 1. absolute alcohol, for five to fifteen minutes. { equal parts, for five to thirty 2. absolute alcohol, { minutes (e. g., for blood or ether, { milk). 3. osmic acid, 1 per cent. aqueous solution, for thirty seconds. 4. corrosive sublimate, saturated aqueous solution, for five minutes. 5. corrosive sublimate (lang), for five minutes. this solution is prepared by dissolving: sodium chloride 0.75 gramme hydrarg. perchloride 12.00 grammes acetic acid 5.00 grammes in distilled water 100.00 c.c. filter. 6. gulland's solution, for five minutes. this solution is prepared by mixing: absolute alcohol 25.0 c.c. ether 25.0 c.c. corrosive sublimate, 20 per cent. alcoholic solution 0.4 c.c. 7. formalin 10 per cent. aqueous solution (= 4 per cent. aqueous solution of formaldehyde since formalin is a 40 per cent. solution of the gas in water). either of these methods of fixation coagulates the albuminous material and ensures perfect adhesion of the film to the cover-slip. _clearing._--wash the cover-slip thoroughly in running water and proceed with the staining. if the film has been prepared from broth, liquefied gelatine, or pus or other morbid exudations, saturate the film after fixation with acetic acid 2 per cent. and allow it to act for two minutes. wash with alcohol, then let the alcohol remain on the cover-slip for two minutes. (this will "clear" the groundwork and give a much sharper and cleaner film than would otherwise be obtained.) if the film has been prepared from blood or bloodstained fluid, treat with acetic acid 2 per cent. for two minutes after fixation. wash with water, dry, and proceed with the staining. (this will remove the hæmoglobin and facilitate examination.) _staining._-1. rest the cover-slip, film side uppermost, on the rubber mat. 2. by means of a drop-bottle, cover the film side of the cover-slip with the selected stain, allow it to act for a few minutes, then wash off the excess in running water. the penetrating power of stains is increased by (a) physical means--e. g., heating the stain; (b) chemical means--e. g., by the addition of carbolic acid, 5 per cent. aqueous solution; caustic alkalies, 2 per cent. aqueous solutions; water saturated with aniline oil; borax, 0.5 per cent. aqueous solution. the most commonly used dyes for cover-slip film preparations are the aniline dyes. (a) basic: (a) methylene-blue. (b) gentian violet. (c) fuchsin. these dyes are kept in saturated alcoholic (90 per cent.) solutions so that decomposition may be retarded. two or three drops of alcoholic solution of these dyes to, say, 4 c.c. water, usually makes a sufficiently strong staining fluid for cover-slip film preparations. carbolic methylene-blue (c.m.b.) and carbol fuchsin (c.f.) are prepared by covering the cover-slip with 5 per cent. solution of carbolic acid and adding a few drops of the saturated alcoholic solution of methylene-blue or fuchsin respectively to it. for aniline gentian violet (a.g.v.) the stain is added to a saturated solution of aniline oil in water. (d) thionine blue. (e) bismarck brown. (f) neutral red. (b) acid: (a) eosin, aqueous yellowish. (b) safranine. these dyes are kept in 1 per cent. aqueous solution to which is added 5 per cent. of alcohol, as a preservative. they are generally used in this form. a few nuclear stains (carmine, hæmatoxylin) are occasionally used more especially in "section" work. _decolourisation._--after overstaining, films may be decolourised by washing for a longer or shorter time in one of the following reagents arranged in ascending order of power 1. water. 2. chloroform. 3. acetic acid, 1 per cent. 4. alcohol. 5. alcohol absolute, } equal parts. acetic acid, 1 per cent., } {hydrochloric, 1 per cent. aqueous solution. {hydrochloric, 1 per cent. alcoholic { (90 per cent.) solution. 6. mineral acids: {sulphuric, 25 per cent. aqueous solution. {nitric, 33 per cent. aqueous solution. _counterstaining._--use colours which will contrast with the first stain; e. g., vesuvin, } neutral red, }for films stained by methylene-blue or eosin, }gram's method. fuchsin, } methylene-blue, }for films stained by fuchsin. gentian violet, } 8. _mounting._-1. wash the film carefully in running water. 2. blot off the superfluous water with the filter paper, or dry more completely between two folds of blotting paper. 3. complete the drying in the air, or by holding the cover-slip in the fingers at a safe distance above the flame of the bunsen burner. 4. place a drop of xylol balsam on the centre of a clean 3 by 1 glass slide and invert the cover-slip over the balsam, and lower it carefully to avoid the inclusion of air bubbles. note.--xylol is used in preference to chloroform to dissolve canada balsam, as it does not decolourise the specimen. ~impression films~ (_klatschpraeparat_) are prepared from isolated colonies of bacteria in order that their characteristic formation may be examined by higher powers than can be brought to bear on the living cultivation. they are prepared from plate cultivations (_vide_ page 230) in the following manner. 1. remove a clean cover-slip from the alcohol pot with sterile forceps and burn off the spirit. 2. open the plate and rest one edge of the cover-slip on the surface of the medium a little to one side of the selected colony. lower it cautiously over the colony until horizontal. avoid any lateral movement or the inclusion of bubbles of air. 3. make gentle vertical pressure on the centre of the cover-slip with the points of the forceps to ensure perfect contact with the colony. 4. steady one edge of the cover-slip with the forceps and pass the point of a mounted needle just under the opposite edge and raise the cover-slip carefully; the colony will be adherent to it. when nearly vertical, grasp the cover-slip with the forceps and remove it from the plate. re-cover the plate. 5. place the cover-slip, film uppermost, on the rubber mat, and cover it with an inverted watch-glass until dry. 6. fix by immersing in one of the fixing fluids previously mentioned (_vide_ page 82). 7. clear with acetic acid and alcohol. 8. stain and mount as an ordinary cover-slip film preparation, being careful to perform all washing operations with extreme gentleness. ~microscopical examination of the unstained specimens.~-1. place the body tube of the microscope in the vertical position. 2. arrange the hanging-drop slide on the microscope stage so that the drop of fluid is in the optical axis of the instrument, and secure it in that position by means of the spring clips. 3. use the 1/6-inch objective, rack down the body tube until the front lens of the objective is almost in contact with the cover-slip--that is, well within its focal distance. this is best done whilst bending down the head to one side of the microscope, so that the eyes are on a level with the stage. 4. apply the eye to the ocular and adjust the plane mirror to the position which secures the best illumination. 5. rack the condenser down slightly and cut down the aperture of the iris diaphragm so that the light, although even, is dim. 6. rack up the body tube by means of the coarse adjustment until the bacteria come into view; then focus exactly by means of the fine adjustment. some difficulty is often experienced at first in finding the hanging drop, and if the first attempt is unsuccessful, the student must not on any account, whilst still applying his eye to the ocular, rack the body tube down (for by so doing there is every likelihood of the front lens of the objective being forced through the cover-glass, and not only spoiling the specimen, but also contaminating the objective); but, on the contrary, withdraw his eye, rack the tube up, and commence again from step 2. ~dark ground illumination.~-1. set up the microscope stand in the vertical position and insert the highest eyepiece available. 2. remove the nosepiece from the microscope tube and fit the 2/3 inch objective in place. 3. remove the substage condenser and replace it by the dark ground condenser. 4. fit up the source of illumination some 30-50 cm. distant from the microscope. (this should be the liliput arc lamp (leitz), nernst lamp or incandescent gas lamp; if either of the two latter are employed, a bull's eye condenser to produce parallel rays must be interposed between light and microscope); and adjust illuminant and microscope so that the substage plane mirror is completely filled with light. 5. focus the two concentric rings engraved upon the upper surface of the condenser and centre them accurately by means of the centring screws. 6. prepare a "fresh" specimen (see pages 74-76) of the material it is desired to observe, using selected, new, 3 by 1 glass slips of less than 1 mm. thickness, and no. 1 cover-glasses (0.17 mm. thick), which should be cleaned with a piece of soft washleather and not with the emery paper, as scratches on the glass produce haziness in the preparation. 7. deposit a large drop of immersion oil (or pure water) on the upper surface of the condenser and rack it down a few millimetres. 8. adjust the fresh preparation on the microscope stage and fasten it in position with the stage clips. 9. rack up the condenser until the immersion fluid makes contact with the under surface of the slide; avoid the formation of air bubbles. 10. adjust the substage mirror so that the light is reflected upward. a bright spot will be seen on the fresh preparation near the centre of the field. 11. replace the 2/3-inch objective by the 1/12-inch oil immersion lens which has been fitted with the special stop to reduce its n. a.; place a drop of immersion oil upon the centre of the cover-glasses of the fresh preparation and lower the microscope tube until the front lens of the objective has entered the oil drop. 12. focus the bright spot referred to in step 10. if it no longer occupies the centre of the field, alter the angle of the substage mirror until it does. 13. now focus the lens accurately on the film, cautiously vary the height of the dark ground condenser until the best position is found. the intensely illuminated bacteria will stand out in vivid contrast to the dark background. [illustration: fig. 70.--immersion oil bottle.] ~microscopical examination of the stained specimen.~--(the body tube of the microscope may be vertical or inclined to an angle.) 1. secure the slide on the stage of the microscope by means of the spring clips. 2. place a drop of cedarwood oil on the centre of the cover-slip. the immersion oil is pure cedarwood oil, and is kept in a small bottle of stout glass (fig. 70), the cavity of which is shaped like an inverted cone, and is provided with a safety funnel (so that the oil does not escape if the bottle is accidentally overturned) and a dust cap of boxwood fitted with a wooden rod with which the drop of oil is applied to the cover-glass or lens. 3. use the 1/12-inch oil immersion lens of the microscope. rack down the body tube till the front lens of the objective is in contact with the oil and nearly touching the cover-slip. 4. rack up the condenser until it is in contact with the under surface of the slide. 5. apply the eye to the ocular and arrange the plane mirror so as to obtain the greatest possible amount of light. 6. rack up the body tube until the stained film comes into view. 7. focus the condenser accurately on the film. 8. focus the film accurately by means of the fine adjustment. vi. staining methods. in the following pages are collected the various "stock" stains in everyday use in the bacteriological laboratory, together with a selection of the most convenient and generally useful staining methods for demonstrating particular structures or differentiating groups of bacteria. the stains employed should either be those prepared by gruebler, of leipzig, or merck, of darmstadt. the methods printed in ordinary type are those which a long experience has shown to be the most reliable, and to give the best results--those relegated to small type comprise such as are not so generally useful, but give excellent results in the hands of the experienced worker. bacteria stains. ~methylene-blue.~-1. _saturated aqueous solution._ weigh out methylene-blue 1.5 grammes place in a stoppered bottle having a capacity of from 150 to 200 c.c. and add distilled water 100.0 c.c. allow the water to remain in contact with the dye for two weeks, shaking the contents of the bottle vigourously for a few moments every day. filter. 2. _saturated alcoholic solution._ weigh out methylene-blue 1.5 grammes place in a stoppered bottle of 150 c.c. capacity and add alcohol, 90 per cent 100.0 c.c. allow the alcohol to remain in contact with the dye for two hours, shaking vigourously every few minutes. filter. 3. _carbolic methylene-blue_ (kuehne). weigh out methylene-blue 1.5 grammes carbolic acid 5.0 grammes and dissolve in distilled water 100.0 c.c. and add absolute alcohol 10.0 c.c. filter. 4. _alkaline methylene-blue_ (loeffler). measure out and mix methylene-blue, saturated alcoholic solution 30.0 c.c. caustic potash, 0.1 per cent. aqueous solution 100.0 c.c. filter. ~gentian violet.~-5. _saturated aqueous solution._ weigh out gentian violet 2.25 grammes and proceed as in preparing the corresponding solution of methylene-blue. 6. _saturated alcoholic solution._ weigh out gentian violet 5.0 grammes and proceed as in preparing the corresponding solution of methylene-blue. 7. _carbolic gentian violet_ (nicollé). measure out and mix gentian violet, saturated alcoholic solution 10.0 c.c. carbolic acid, 1 per cent. aqueous solution 100.0 c.c. filter. 8. _anilin water solution_ (koch-ehrlich). measure out distilled water 100 c.c. add anilin oil drop by drop (shaking well after the addition of each drop) until the solution is opaque. filter until clear. and add absolute alcohol 10 c.c. saturated alcoholic solution gentian violet 11 c.c. filter. note.--this solution will not keep longer than 14 days. ~thionine blue (or lauth's violet).~-9. _carbolic thionine blue_ (nicollé). weigh out thionine blue 1.0 gramme carbolic acid 2.5 grammes and dissolve in distilled water 100.0 c.c. filter. before use dilute with equal quantity of distilled water and again filter. ~fuchsin (basic).~-10. _saturated aqueous solution._ weigh out basic fuchsin 1.5 grammes and proceed as in preparing the corresponding solution of methylene-blue (_q. v._). 11. _saturated alcoholic solution._ weigh out basic fuchsin 3.5 grammes and proceed as in preparing the corresponding solution of methylene-blue. 12. _carbolic fuchsin_ (ziehl). weigh out basic fuchsin 1.0 gramme carbolic acid 5.0 grammes dissolve in distilled water 100.0 c.c. and add absolute alcohol 10.0 c.c. filter. contrast stains. ~eosin.~--there are several commercial varieties of eosin, which, from the bacteriological point of view, possess very different values. gruebler lists four varieties, of which two only are useful for bacteriological work: eosin, aqueous yellowish. eosin, aqueous bluish. 13. _eosin aqueous solution_ (yellowish or bluish shade), 1 per cent. weigh out eosin, aqueous 1.0 gramme dissolve in distilled water 100.0 c.c. and add absolute alcohol 5.0 c.c. filter. 14. _eosin alcoholic solution_, 0.5 per cent. weigh out eosin, alcoholic 0.5 gramme and dissolve in alcohol (70 per cent.) 100.0 c.c. filter. ~safranine.~-15. _aqueous solution._ weigh out. safranine 0.5 gramme and dissolve in distilled water 100.0 c.c. filter. ~neutral red.~-16. _aqueous solution._ weigh out neutral red 1.0 gramme and dissolve in distilled water 100.0 c.c. filter. ~vesuvin (or bismarck brown).~-17. _saturated aqueous solution._ weigh out vesuvin 0.5 gramme and dissolve in distilled water 100.0 c.c. filter. tissue stains. ~aniline gentian violet~ (for weigert's fibrin stain).-weigh out gentian violet 1.0 gramme and dissolve in absolute alcohol 15.0 c.c. distilled water 80.0 c.c. then add aniline oil 3.0 c.c. shake well and filter before use. ~hæmatoxylin~ (ehrlich).-1. weigh out hæmatoxylin 2.0 grammes and dissolve in absolute alcohol 100.0 c.c. 2. weigh out ammonium alum 2.0 grammes and dissolve in distilled water 100.0 c.c. 3. mix 1 and 2, allow the mixture to stand forty-eight hours, then filter. 4. add glycerine 85.0 c.c. acetic acid, glacial 10.0 c.c. 5. allow the stain to stand for one month exposed to light; then filter again ready for use. ~hæmatin~ (mayer's).-a. weigh out hæmatin 1.0 gramme and dissolve in alcohol 90 per cent. (warmed to 37°c.) 50 c.c. b. weigh out potash alum 50 grammes and dissolve in distilled water 100 c.c. prepare these two solutions in separate flasks. take a clean flask of 250 c.c. capacity and insert a large funnel in its neck. pour the solutions a and b simultaneously and slowly into the funnel to mix thoroughly. store for future use. note.--if acid hæmatin is required, introduce glacial acetic acid (3 c.c.) into the mixing flask before adding the solutions a and b. ~alum carmine~ (mayer).-weigh out alum 2.5 grammes carmine 1.0 gramme and place in a glass beaker. measure out in a measuring cylinder, distilled water 100.0 c.c. place the beaker on a sand-bath, add the water in successive small quantities, and keep the mixture boiling for twenty minutes. measure the solution and make up to 100 c.c. by the addition of distilled water. filter. ~lithium carmine~ (orth).-weigh out carmine 2.5 grammes and dissolve in lithium carbonate, cold saturated solution 100.0 c.c. filter. ~picrocarmine.~-weigh out picrocarmine 2.0 grammes and dissolve in distilled water 100.0 c.c. blood stains when watery solutions of medicinal methylene blue and water soluble eosins are mixed a precipitate is formed which is soluble only in alcohol, and solutions of this precipitate impart a peculiar reddish-purple colour to chromatin. this compound was first used by romanowsky to demonstrate malarial parasites, but various modifications are now employed for staining blood films generally, and also for bacteria and protozoa. the best modifications of the original romanowsky are those of jenner and leishman--jenner being most suitable for the histological study of the blood, and leishman for the demonstration of protozoa. ~jenner's stain.~-a. weigh out: eosin aqueous yellow 6.0 grammes dissolve in distilled water (non-alkaline) 250 c.c. this will make a thick solution. b. weigh out: methylene blue (medicinally pure) hoechst 5.0 grammes dissolve in distilled water (non-alkaline) 250 c.c. 1. add b to a very slowly, stirring all the time. a viscous precipitate forms which frequently loses its viscosity when heat is applied. (this explains the necessity of mixing slowly). 2. evaporate slowly in a porcelain basin, stirring occasionally, on a water bath at 55° c. when a paste begins to form scrape and break up occasionally. (on no account must the paste be allowed to fuse.) 3. grind the resulting mass into an amorphous powder. 4. weigh out: amorphous powder 0.5 grammes dissolve in methylic alcohol (merck's puriss, for analysis) 100 c.c. allow time for true solution. (about three days is sufficient.) method.-1. prepare film, dry, but _do not fix_. 2. flood the unfixed film with the stain, allow it to act for 3 minutes (the methylic alcohol of the stain fixes the film). 3. pour off the stain and wash in distilled water until the film presents a pink colour. 4. dry and mount. ~leishman's stain.~-_a._ weigh out: methylene blue (medicinal) 1 gramme dissolve in sodium carbonate, 0.5 per cent. aqueous solution 100 c.c. keep at 65° c. for 12 hours in either a hot incubator or a water-bath; then stand in dark place at room temperature (20°c.) for ten days. _b._ weigh out: eosin, extra b. a. 0.1 gramme dissolve in distilled water 100 c.c. 1. mix the two solutions a and b in equal volumes, and allow the mixture to stand for 12 hours with occasional stirring. 2. filter, and collect precipitate on filter paper. 3. wash precipitate thoroughly with distilled water, and dry. 4. weigh out 0.15 gramme of the dried precipitate; rub up in a mortar with 5 c.c. of methylic alcohol (merck's puriss, for analysis). allow undissolved powder to settle, then decant the supernatant fluid to a clean 100 c.c. measuring cylinder. 5. add further 5 c.c. alcohol to sediment in mortar and repeat the process, and so on until all the sediment has been dissolved. 6. now make up the fluid in the measuring cylinder to 100 c.c. by the addition of more methylic alcohol. method.-1. prepare film, dry, but _do not fix_. 2. flood the unfixed film with stain, allow it to act 30 seconds. 3. add double the volume of distilled water to the stain on the film, and mix with glass rod or platinum loop. 4. allow this diluted stain to act five minutes. 5. wash off with distilled water. 6. leave some water on film for thirty seconds to intensify the colour contrasts. 7. dry and mount. methods of demonstrating structure of bacteria, etc. ~to demonstrate capsules.~ ~1. macconkey.~-_stain._-weigh out dahlia 0.5 gramme methyl green (00 crystals) 1.5 grammes rub up in a mortar with distilled water 100.0 c.c. add fuchsin, saturated alcoholic solution 10.0 c.c. and make up to 200 c.c. by the addition of distilled water 90.0 c.c. filter. allow the stain to stand for two weeks before use; keep in a dark place or in an amber glass bottle. owing to the unstable character of the methyl green, this stain deteriorates after about six months. method.-1. prepare and fix film in the usual manner. 2. flood the cover-slip with the stain and allow it to act for five to ten minutes. 3. wash very thoroughly in water; if necessary, direct a powerful stream of water on the film from a wash-bottle. 4. dry and mount. ~2. muir's method.~- 1. prepare, dry and fix film in the ordinary manner. 2. flood the film with carbolic fuchsin, warm until steam begins to rise. allow the stain to act for thirty seconds. 3. wash quickly with methylated spirit. 4. wash thoroughly with water. 5. subject the film to the action of the following mordant for five seconds: corrosive sublimate, saturated aqueous solution 2 c.c. tannic acid, 20 per cent. aqueous solution 2 c.c. potash alum saturated aqueous solution 5 c.c. 6. wash thoroughly in water. 7. treat with methylated spirit for about sixty seconds. (the preparation should now be pale red.) 8. wash thoroughly in water. 9. counterstain in methylene blue, aqueous solution thirty seconds. 10. wash in water. 11. dehydrate in alcohol. 12. clear in xylol and mount in xylol balsam. ~3. welch's method.~- 1. prepare and fix film in the usual manner. 2. flood the slide with acetic acid 2 per cent.; allow the acid to remain in contact with the film for two minutes. this swells up and fixes the capsule and enables it to take the stain. 3. blow off the acetic acid by the aid of a pipette. 4. immerse in aniline gentian violet, five to thirty seconds. 5. wash in water. 6. dry and mount. ~4. ribbert's method.~- _stain._- measure out and mix: acetic acid, glacial 12.5 c.c. alcohol, absolute 50.0 c.c. distilled water 100.0 c.c. warm to 36° c. (e. g., in the "hot" incubator) and saturate with dahlia. filter. method.- 1. prepare and fix films in the usual manner. 2. cover the film with the stain and allow it to act for one or two seconds only. 3. wash thoroughly in water. 4. dry and mount. ~to demonstrate flagella.~ ~1. muir's modified pitfield.~--this is the best method and gives the most reliable results, for not only is the percentage of successful preparations higher than with any other, but the bacilli and flagella retain their relative proportions. (a) ~mordant.~- tannic acid, 10 per cent. aqueous solution 10 c.c. corrosive sublimate, saturated aqueous solution 5 c.c. alum, saturated aqueous solution 5 c.c. carbolic fuchsin (ziehl) 5 c.c. mix thoroughly. a precipitate forms which must be allowed to settle for a few hours. decant off the clear fluid into tubes and centrifugalise thoroughly. this solution is at its best some four or five days after manufacture; it keeps for about a couple of weeks, but must be re-centrifugalised each time, before use. (b) _stain._- alum, saturated aqueous solution 25 c.c. gentian violet, saturated alcoholic solution 5 c.c. filter. this stain must be freshly prepared. method.--the cultivations employed should be smear agar cultures, twelve to eighteen hours old if incubated at 37°c, twenty-four to thirty hours if incubated at 22°c. 1. remove a very small quantity of the growth by means of the platinum spatula. 2. emulsify it with a few cubic centimetres of distilled water in a watch-glass, by gently moving the spatula to and fro in the water. do not rub up the growth on the side of the watch-glass. some workers prefer to use tap water, others employ normal saline solution, but distilled water gives the best emulsion. 3. spread a thin film of the emulsion on a newly flamed cover-slip, using no force, but rather _leading_ the drop over the cover-slip with the platinum loop. 4. allow the film to dry in the air, properly protected from falling dust. 5. fix by passing thrice through the bunsen flame, holding the cover-slip whilst doing so by one corner between the finger and thumb. 6. pour on the film as much of the mordant as the cover-glass will hold. grasp the cover-slip with the forceps and hold it, high above the flame, until steam rises. allow the steaming mordant to remain in contact with the film two minutes. 7. wash well in water and dry carefully. 8. pour on the film as much of the stain as the cover-glass will hold. steam over the flame as before for two minutes. 9. wash well in water. 10. dry and mount. ~2. "pitfield" original method.~- (a) _mordant._- tannic acid 1 gramme water 10 c.c. (b) _stain._- saturated aqueous solution of alum 10 c.c. saturated alcoholic solution of gentian violet 1 c.c. distilled water 5 c.c. mix equal parts of a and b before using. 1. prepare and fix the film in the manner described above. 2. boil the mixture and immerse the cover-slip in it, whilst still hot, for one minute. 3. wash in water. 4. examine in water; if satisfactory, dry and mount in canada balsam. ~3. maccrorrie's method.~- _mordant-stain._- measure out and mix. night blue, saturated alcoholic solution 10 c.c. potash alum, saturated aqueous solution 10 c.c. tannin, 10 per cent. aqueous solution 10 c.c. note.--the addition of gallic acid, 0.1 to 0.2 gramme, may improve the solution, but is not necessary. method.- 1. prepare and fix the films as above. 2. pour some of the mordant-stain on the film and warm gently, high above the flame, for two minutes (or place in the "hot" incubator for a like period). 3. wash thoroughly in water. 4. dry and mount. ~4. loeffler's method.~- (a) _mordant._- tannic acid, 20 per cent. aqueous solution 10 c.c. ferrous sulphate, saturated aqueous solution 5 c.c. hæmatoxylin solution 3 c.c. carbolic acid, 1 per cent. aqueous solution 4 c.c. this solution must be freshly prepared. _hæmatoxylin solution_ is prepared by boiling 1 gramme logwood with 8 c.c. distilled water, filtering and replacing the loss from evaporation. _alternative mordant_ (bunge's mordant).- tannic acid, 20 per cent. aqueous solution 10 c.c. ferrous sulphate, saturated aqueous solution 5 c.c. fuchsin, saturated alcoholic solution 1 c.c. (b) _stain._- weigh out methylene-blue } or methylene-violet } 4 grammes or fuchsin } and dissolve in aniline water, freshly saturated and filtered 100 c.c. method.-1. prepare and fix films as above. 2. pour the mordant on to the film and warm cautiously over the flame till steam rises; keep the mordant gently steaming for one minute. 3. wash well in distilled water till no more colour is discharged; if necessary, wash carefully with absolute alcohol. 4. filter a few drops of the stain on to the film, warm as before, and allow the steaming stain to act for one minute. 5. wash well in distilled water. 6. dry and mount. note.--the flagella of some organisms can be demonstrated better by means of an alkaline stain or an acid stain--a point to be determined for each. speaking generally, those bacilli which give rise to an acid reaction in the culture medium require an alkali; those which form alkali in cultivation require an acid. according to requirements, therefore, loeffler recommends the addition of sodium hydrate, 1 per cent. aqueous solution, 1 c.c.; or an equal quantity of an exactly comparable solution of sulphuric acid. ~5. van ermengem's method.~--this method, being merely a precipitation of a silver salt on the micro-organisms and not a true stain, creates a false impression as to the relative proportions of bacteria and flagella. (a) _fixing fluid._- osmic acid, 2 per cent. aqueous solution 10 c.c. tannic acid, 20 per cent. aqueous solution 20 c.c. acetic acid, glacial 1 c.c. the fixing fluid should be prepared some days before use and filtered as required. in colour it should be distinctly violet. (b) _sensitising solution._- silver nitrate, 0.5 per cent. aqueous solution. this solution must be kept in a dark blue glass bottle or in a dark cupboard. filter immediately before use. (c) _reducing solution._- weigh out gallic acid 5 grammes tannic acid 3 grammes potassium acetate, fused 10 grammes and dissolve in distilled water 350 c.c. filter. this solution will keep active for several days, but fresh solution must be used for each preparation. method.- 1. prepare emulsion, make and fix films as above in the preceding method, steps 1 to 4. 2. pour on the film as much of the fixing solution as the cover-glass will hold, heat carefully over the flame till steam rises, and allow the steaming fixing fluid to act for five minutes. 3. wash well in water. 4. wash in absolute alcohol. 5. wash in distilled water. 6. pour some of the sensitising solution on the film and allow it to act for from thirty seconds to one minute; blot off the excess of fluid with filter paper. 7. without washing, transfer the film to a watch-glass containing the reducing solution and allow it to remain therein for from thirty seconds to one minute; blot off the excess of fluid with filter paper. 8. without washing, again treat the film with the sensitising solution, this time until the film commences to turn black. 9. wash in distilled water. 10. dry and mount. ~to stain nuclei of yeast cells.~ 1. prepare and fix film in the usual manner. 2. soak in ferric ammonia sulphate 3 per cent. aqueous solution for two hours. 3. wash thoroughly in water. 4. stain in hæmatoxylin solution (see page 95) for thirty minutes. 5. wash in water. 6. differentiate in ferric ammonia sulphate solution for 1-1/2-2 minutes, examining wet under microscope during the process. ~to stain spores.~ ~1. single stain.~-1. prepare cover-slip film in the usual way. 2. in fixing, pass the cover-slip film fifteen or thirty times through the flame instead of only three. this destroys the resisting power of the spore membrane and allows the stain to reach the interior. 3. stain in the usual way with methylene-blue or fuchsin. 4. wash in water. 5. dry and mount. ~2. double stain.~-1. prepare and fix film in the usual way--i. e., pass three times through flame to fix. 2. cover the film with hot carbol-fuchsin and hold in the forceps above a small flame until the fluid begins to steam. set the cover-slip down and allow it to cool. repeat the process when the stain ceases to steam and continue to repeat until the stain has been in contact with the film for twenty minutes. (this stains both spores and bacteria.) 3. wash in water. 4. decolourise in alcohol, 2 parts; acetic acid, 1 per cent., 1 part. (this removes the stain from everything but the spores.) 5. wash in water. 6. mount the cover-slip in water and examine microscopically with the 1/6-inch objective. (spores should be red, and the rest of the film colourless or a very light pink.) if satisfactory, pass on to section 7; if unsatisfactory, repeat steps 2 to 5. 7. counterstain in weak methylene-blue. (now spores red, bacilli blue.) 8. wash in water. 9. dry and mount. the spores of different bacilli differ greatly in their resistance to decolourising reagents; even the spores of the same species of organisms vary according to their age. young spores are more easily decolourised than those more mature. sulphuric acid, 1 per cent. aqueous solution, and hydrochloric acid, 0.5 per cent. alcoholic (90 per cent.) solution, are useful decolourising reagents. ~3. moeller's method.~- 1. prepare and fix films in the usual manner. 2. immerse in absolute alcohol for two minutes, then in chloroform for two minutes; wash in water. this dissolves out any fat or crystals that might otherwise retain the "spore" stain. 3. immerse in chromic acid, 5 per cent. aqueous solution, for one minute; wash in water. 4. pour ziehl's carbolic fuchsin on the film, warm as in previous methods, and allow it to act for ten minutes. 5. wash in water. 6. decolourise in sulphuric acid, 5 per cent. aqueous solution, for five seconds. 7. wash in water. 8. counterstain with kuehne's carbolic methylene-blue for one or two minutes. 9. wash in water. 10. dry and mount. (spores red, bacilli blue.) ~4. abbott's method.~- 1. prepare and fix films in the usual manner. 2. pour loeffler's alkaline methylene-blue on the film; warm cautiously over the flame till steam rises and allow the hot steam to act for one to five minutes. 3. wash thoroughly in water. 4. decolourise in nitric acid, 2 per cent. alcoholic (alcohol 80 per cent.) solution. 5. wash thoroughly in water. 6. counterstain in eosin, 1 per cent. aqueous solution. 7. wash. 8. dry and mount. (spores blue, bacilli red.) differential methods of staining. ~gram's method.~--this method depends upon the fact that the protoplasm of some bacteria permits aniline gentian violet and lugol's iodine solution, when applied consecutively, to enter into a chemical combination which results in the formation of a new blue-black pigment, only very sparingly soluble in absolute alcohol. such organisms are said to "stain by gram," or to be "gram positive." 1. prepare a cover-slip film and fix in the usual way. 2. stain in aniline gentian violet three to five minutes. filter as much aniline water on to the cover-slip as it will hold; then add the smallest quantity of alcoholic solution of gentian violet which suffices to saturate the aniline water and form a "bronze scum" upon its surface--if too much of the alcoholic gentian violet is added the alcohol present redissolves this scum. to prepare aniline water, pour 4 or 5 c.c. aniline oil into a stoppered bottle and add distilled water, 100 c.c. shake vigourously and filter immediately before use. the excess of oil sinks to the bottom of the bottle and may be used again. 3. wash in water. 4. treat with lugol's iodine solution until the film is black or dark brown. to do this treat with iodine solution for a few seconds, wash in water, and examine the film over a piece of white filter paper. note the colour. repeat this process until the film ceases to darken with the fresh application of iodine solution. lugol's solution is prepared by dissolving iodine 1 gramme iodide of potassium 3 grammes in distilled water 300 c.c. 5. wash in water. 6. wash with alcohol until no more colour is discharged and the alcohol runs away clear and colourless. the following mixture may be substituted for absolute alcohol as a decolouriser acetone 10 c.c. absolute alcohol 100 c.c. 7. wash in water. 8. counterstain very lightly with aqueous solution of neutral red. other counterstains may be used such as dilute eosin, dilute fuchsin, or vesuvin. note.--this section may be omitted when dealing with films prepared from pure cultivations. 9. wash in water. 10. dry and mount. ~gram-claudius method.~-1. prepare a cover-slip film and fix in the usual way. 2. stain in methyl violet, 1 per cent. aqueous solution for three to five minutes. 3. treat with two lots picric acid, saturated aqueous solution. 4. wash in water and dry. 5. decolourise with clove oil. 6. wash off clove oil with xylol. 7. mount in xylol balsam. ~gram-weigert method.~-1-5. proceed as for the corresponding sections of gram's method (_quod vide_). 6. dry in the air. 7. wash in aniline oil, 1 part, xylol, 2 parts, until no more colour is discharged. 8. wash in xylol. 9. mount in xylol balsam. ~modified gram-weigert method.~--(to demonstrate trichophyta in hair.) 1. soak the hairs in ether for ten minutes to remove the fat. 2. stain thirty minutes in a tar-like solution of aniline gentian violet (prepared by adding 15 drops of the alcoholic solution of gentian violet to 3 drops of aniline water). 3. dry the hairs between pieces of blotting paper. 4. treat with perfectly fresh iodine solution. 5. again dry between blotting paper. 6. treat with aniline oil to remove excess of stain. (if necessary, add a drop or two of nitric acid to the oil.) 7. again treat with aniline oil. 8. treat with aniline oil and xylol, equal parts. 9. clear with xylol. 10. mount in xylol balsam. to obtain the best differentiation the preparation should be repeatedly examined microscopically (with a 1/6-inch objective) between steps 5 and 9, as the actual time involved varies with different specimens. ~ziehl-neelsen's method.~--(to demonstrate tubercle and other acid-fast bacilli.) 1. smear a thin, even film of the specimen on the cover-slip by means of the platinum loop. (in the case of sputum, if it is a very watery specimen, allow the film to dry, then spread a second and even a third layer over the first.) 2. fix by passing three times through the flame. 3. stain in hot carbol-fuchsin (as in staining for spores) for five to ten minutes. (this stains everything on the film.) avoid over-heating. 4. decolourise by dipping in sulphuric acid, 25 per cent. (this removes stain from everything but acid-fast bacilli; e. g., tubercle, leprosy, and smegma bacilli and the film turns yellow.) 5. wash in water. (a pale red colour returns to the film). 6. wash in alcohol till no more colour is discharged. (this often, but not invariably, removes the stain from acid-fast bacilli other than tubercle; e. g., smegma bacillus.) 7. wash in water. 8. counterstain in weak methylene-blue. (stains non-acid-fast bacilli, leucocytes, epithelial cells, etc.) 9. wash in water, dry, and mount. ~pappenheim's method.~-this method is supposed to differentiate between b. tuberculosis and other acid-fast micro-organisms. 1. prepare and fix film in the usual way. 2. stain in carbol-fuchsin _without heat_ for three minutes. 3. without previously washing in water treat the film with three or four successive applications of corallin (rosolic acid) solution. corallin 1 gramme methylene-blue (saturated alcoholic solution) 100 c.c. glycerine 20 c.c. 4. wash in water. 5. dry and mount. ~neisser's method--modified.~--(to demonstrate diphtheroid bacilli.) _stain i._-measure out and mix methylene-blue, saturated alcoholic solution 4.0 c.c. acetic acid, 5 per cent. aqueous solution 96.0 c.c. filter. _stain ii._-weigh out neutral red 2.5 grammes and dissolve in distilled water 1000 c.c. filter. method.-1. prepare and fix films in the usual way. 2. pour stain i on the film and allow it to act for two minutes. 3. wash thoroughly in water. 4. treat with lugol's iodine for ten seconds. 5. wash thoroughly in water. 6. pour stain ii on to the film and allow it to act for thirty seconds. 7. wash thoroughly in water. 8. dry and mount. note.--the cultivation from which the films are prepared must be upon blood-serum which has been incubated at 37°c. for from nine to eighteen hours. the bacilli are stained a light red by the neutral red, which contrasts well with the two or three black spots, situated at the poles and occasionally one in the centre representing protoplasmic aggregations (? metachromatic granules) stained by the acid methylene-blue. ~wheal and chown (oxford) method.~--(to demonstrate actinomyces.) 1. stain briefly with ehrlich's hæmatoxylin (until nuclei are faint blue after washing with tap water). 2. wash in tap water. 3. stain in hot carbol-fuchsin (as for tubercle bacilli) for five to ten minutes. 4. wash in tap water. 5. decolourise with spengler's picric acid alcohol. this is prepared by mixing: alcohol, absolute 20 c.c. picric acid, saturated aqueous solution 10 c.c. distilled water 10 c.c. during the progress of steps 1-5 the preparation must be repeatedly examined microscopically with the 1/6-inch objective. when properly differentiated the clubs appear brilliant red on greenish ground. 6. dehydrate in alcohol. 7. clear in xylol. 8. mount in xylol balsam. this method serves equally well for films and for sections. vii. methods of demonstrating bacteria in tissues. for bacteriological purposes, sections of tissue are most conveniently prepared by either the ~freezing method~ or the ~paraffin method~. the latter is decidedly preferable, but as it is of greater importance to demonstrate the bacteria, if such are present, than to preserve the tissue elements unaltered, the "frozen" sections are often of value. whichever method is selected, it is necessary to take small pieces of the tissue for sectioning,--2 to 5 mm. cubes when possible, but in any case not exceeding half a centimetre in thickness. post-mortem material should be secured as soon after the death of the animal as possible. the tissue is prepared for cutting by-(a) fixation; that is, by causing the death of the cellular elements in such a manner that they retain their characteristic shape and form. the fixing fluids in general use are: absolute alcohol; corrosive sublimate, saturated aqueous solution; corrosive sublimate, lang's solution (_vide_ page 82); formaldehyde, 4 per cent. aqueous solution. (of these, lang's corrosive sublimate solution is decidedly the best all-round "fixative.") (b) hardening; that is, by rendering the tissue of sufficient consistency to admit of thin slices or "sections" being cut from it. this is effected by passing the tissue successively through alcohols of gradually increasing strength: 30 per cent. alcohol, 50 per cent. alcohol, 75 per cent. alcohol, 90 per cent. alcohol, absolute alcohol. in both these processes a large excess of fluid should always be used. freezing method. 1. ~fixation.~ place the pieces of tissue in a wide-mouthed glass bottle and fill with absolute alcohol. allow the tissues to remain therein for twenty-four hours. 2. ~hardening.~ remove the alcohol (no longer absolute, as it has taken up water from the tissues) from the bottle and replace it with fresh absolute alcohol. allow the tissues to remain therein for twenty-four hours. [illustration: fig. 71.--washing tissues.] note.--if not needed for cutting immediately, the hardened tissues can be stored in 75 per cent. alcohol. 3. remove the alcohol from the tissues by soaking in water from one to two hours. remove the stopper from the bottle; rest a glass funnel in the open mouth and place under a tap of running water. the water of course, overflows, but the tissues remain in the bottle (fig. 71). 4. impregnate the tissues with mucilage for twelve to twenty-four hours, according to size. transfer the pieces of tissue to a bottle containing sterilised gum mixture. ~formula.~- gum arabic 5 grammes saccharose 1 gramme boric acid 1 gramme water 100 c.c. 5. place the tissue on the plate of a freezing microtome (cathcart's is perhaps the best form), cover and surround with fresh gum mixture; freeze with ether, or for preference, carbon dioxide, and cut sections. 6. float the sections off the knife into a glass dish containing tepid water and allow them to remain therein for about an hour to dissolve out the gum. (if not required at once, store in 90 per cent. alcohol.) 7. transfer to a glass capsule containing the selected staining fluid, by means of a section lifter. 8. transfer the sections in turn to a capsule containing absolute alcohol (to dehydrate) and to one containing xylol or oil of cloves (to clear). 9. mount in xylol balsam. _alternative rapid method._- 1. cut very small blocks of the tissue. 2. fix in formalin 10 per cent. aqueous solution (fixation fluid no. 7, page 82) for 24 hours. 3. transfer block to plate of freezing microtome and freeze with carbon dioxide vapour. 4. float the sections off the knife into a glass dish of tepid water. 5. stain the sections in glass capsules containing selected stains. 6. place the stained section in a dish of clean water and introduce a glass slide obliquely beneath the section; with a mounted needle draw the section on to the slide and hold it there; gently remove the slide from the water, taking care that any folds in the section are floated out before the slide is finally removed from the water. 7. drain away as much water as possible from the section. drop absolute alcohol on to the section from a drop bottle, to dehydrate it. 8. double a piece of blotting paper and gently press it on the section to dry it. 9. drop on xylol to clear the section. 10. place a large drop of xylol balsam on the section and carefully lower a cover-glass on to the balsam. paraffin method. 1. ~fixation.~ place the pieces of tissue, resting on cotton-wool, in a wide-mouthed glass bottle. pour on a sufficient quantity of the corrosive sublimate fixing fluid; allow the tissue to remain therein for twelve to twenty-four hours according to size. 2. pour off the fixing fluid and wash thoroughly in running water for twenty minutes to half an hour to remove the excess of corrosive sublimate. [illustration: fig. 72.--~l~-shaped brass moulds.] [illustration: fig. 73.--paraffin kettle.] 3. ~hardening.~ place the tissues in each of the following strengths of alcohol in turn for from twelve to twenty-four hours: 50 per cent., 75 per cent., 90 per cent., absolute. 4. ~dehydration~ is effected by transferring the tissues to fresh absolute alcohol. 5. ~clearing.~ half fill a wide-mouthed bottle with chloroform. on the surface of the chloroform float a layer of absolute alcohol about five to ten millimetres in depth. place the pieces of tissue in the layer of alcohol and when they have sunk through this layer, transfer them to pure chloroform for from six to twenty-four hours according to the size of the pieces. when "cleared," the tissue becomes more or less transparent. 6. ~infiltration.~ place the cleared tissues in fresh chloroform with several pieces of paraffin wax and stand in a warm place, such as on the top of the warm incubator. the warmth gradually melts the paraffin and the tissues should remain in the mixture about twenty-four hours. 7. transfer the tissues to a vessel containing pure melted paraffin. place this vessel in a paraffin water-bath regulated for 2° c. above the melting-point of the paraffin used, and allow the tissues to soak for some four to six hours to ensure complete impregnation. the paraffin used should have a melting-point of not more than 58° c. for all ordinary purposes 54°c. will be found quite high enough. 8. imbed in fresh paraffin in a metal (or paper) mould. (a) arrange a pair of ~l~-shaped pieces of metal on a plate of glass to form a rectangular trough (fig. 72). (b) pour fresh melted paraffin into the mould from a special vessel (fig. 73). (c) lift the piece of tissue from the paraffin bath and arrange it in the mould. (d) blow gently on the surface of the paraffin in the mould, and as soon as a film of solid paraffin has formed, carefully lift the glass plate on which the mould is set and lower plate and mould together into a basin of cold water. (e) when the block is cold, break off the metal ~l~'s; trim off the excess of paraffin from around the tissue with a knife, taking care to retain the rectangular shape, and store the block in a pill-box. when several pieces of tissue have to be imbedded at one time, shapes of stout copper, 10 cm., 5 cm., and 2.5 cm. square respectively, and 0.75 cm. deep (fig. 74) will be found extremely useful. these placed upon plates of glass replace the pair of l's in the above process. when the paraffin has set firmly the screw a should be loosened to allow the two halves of the flange b to separate slightly--this facilitates removal of the paraffin block. [illustration: fig. 74.--paraffin mould.] 8. cement the block on the carrier of a "paraffin" microtome (the minot, the jung, or the cambridge rocker) with a little melted paraffin. greater security is obtained if the paraffin around the base of the block is melted by means of a hot metal or glass rod. 9. cut sections--thin, and if possible in ribbands. ~mounting paraffin sections.~-1. place a large drop of 30 per cent. alcohol on the centre of a slide (or cover-slip) and float the section on to the surface of the drop, from a section lifter. 2. hold the slide in the fingers of one hand and warm cautiously over the flame of a bunsen burner, touching the under surface of the glass from time to time on the back of the other hand. as soon as the slide feels distinctly warm to the skin, the paraffin section will flatten out and all wrinkles disappear. (the slide with the section floating on it may be rested on the top of the paraffin bath for two or three minutes, instead of warming over the flame as here described.) 3. cautiously tilt up the slide and blot off the excess of spirit with blotting paper, leaving the section attached to the centre of the slide. 4. place the slide in a wire rack (fig. 75), section downward, in the "hot" incubator for twelve to twenty-four hours. at the end of this time the section is firmly adherent to the glass, and is treated during the subsequent steps as a "fixed" cover-glass film preparation. note.--if large, thick sections have to be manipulated, or if time is of importance or acids are used during the staining process, it is often advisable to add a trace of mayer's albumin to the alcohol before floating out the section. if this substance is employed, a sojourn of twenty minutes to half an hour in the "hot" incubator will be found ample to ensure firm adhesion of the section to the slide. the albuminous fluid is prepared as follows: [illustration: fig. 75.--section rack.] ~mayer's albumin.~- weigh out salicylate of soda 1 gramme and dissolve in glycerine 50 c.c. add white of egg 50 c.c. mix thoroughly by means of an egg whisk. filter into a clean bottle. as an alternative method paint a thin layer of schallibaum's solution on the slide with a camel's hair pencil; lay the section carefully on this film and heat gently to fix the section. _schallibaum's solution_: clove oil 30 c.c. collodion 10 c.c. keep in a dark blue bottle in a cool place. ~staining paraffin sections.~-1. warm paraffin section over the bunsen flame to soften (_but not to melt_) the paraffin, then dissolve out the wax with xylol poured on from a drop bottle. 2. remove xylol by flushing the section with alcohol. 3. if the tissue was originally "fixed" in a corrosive sublimate solution, the section must now be treated with lugol's iodine solution for two minutes and subsequently immersed in 90 per cent. alcohol to remove all traces of yellow staining. 4. wash in water. 5. stain deeply, if using a single stain, as the subsequent processes decolourise. 6. wash in water, decolourise if necessary. 7. flood with several changes of absolute alcohol to dehydrate the section. 8. clear in xylol. (oil of cloves is not usually employed, as it decolourises the section.) 9. mount in xylol balsam. special staining methods for sections. ~double-staining carmine and gram-weigert.~-1. prepare the section for staining as above, sections 1 to 3. 2. stain in lithium carmine (orth's) or picrocarmine for ten to thirty minutes, in a porcelain staining pot (fig. 76). 3. wash in picric acid solution until yellow. at this stage cell nuclei are red, protoplasm is yellow, and bacteria are colourless. picric acid solution is prepared by mixing picric acid, saturated aqueous solution 40 c.c. hydrochloric acid 1 c.c. alcohol (90 per cent.) 160 c.c. 4. wash in water. 5. wash in alcohol. 6. stain in aniline gentian violet. 7. wash in iodine solution till dark brown or black. 8. wash in water. 9. dip in absolute alcohol for a second. 10. decolourise with aniline oil till no more colour is discharged. [illustration: fig. 76.--staining pot.] 11. wash with aniline oil, 2 parts, xylol, 1 part. 12. clear with xylol. 13. mount in xylol balsam. ~alternative gram-weigert method for sections.~-1. fix paraffin section on slide and prepare for staining in the usual manner. 2. stain in alum carmine for about fifteen minutes. 3. wash thoroughly in water. 4. filter aniline gentian violet solution on to the section on the slide and allow to stain about twenty-five minutes. 5. wash thoroughly in water. 6. treat with lugol's iodine until section ceases to become any blacker. 7. wash thoroughly in water. 8. treat with a mixture of equal parts of aniline oil and xylol until no more colour comes away. 9. wash thoroughly with xylol. 10. decolourise and dehydrate rapidly with absolute alcohol until there remains only a very faint bluish tint. 11. clear with xylol. 12. mount in xylol balsam. (then fibrin and hyaline tissue are stained deep blue, whilst bacteria which "stain gram" appear of a deep blue-violet colour.) ~unna-pappenheim method.~-stain.-weigh out and mix methylene green 0.15 gramme pyronin 0.25 gramme and dissolve in carbolic acid 0.5 per cent. aqueous solution 78 c.c. measure out alcohol 2.5 c.c. } glycerine 20.0 c.c. } and add to the stain. ~method.~-1. place tissue in the above stain for ten minutes. 2. differentiate and dehydrate with absolute alcohol. 3. clear in xylol. 4. mount in xylol balsam. ~to demonstrate capsules.~-1. _macconkey's method._--stain precisely as for cover-slip films (_vide_ page 100). 2. _friedländer's method._-stain.- gentian violet, saturated alcoholic solution 50 c.c. acetic acid, glacial 10 c.c. distilled water 100 c.c. method.- 1. prepare the sections for staining, _secundum artem_. 2. stain sections in the warm (e. g., in the hot incubator) for twenty-four hours. 3. wash with water. 4. decolourise lightly with acetic acid, 1 per cent. 5. dehydrate rapidly with absolute alcohol. 6. clear with xylol. 7. mount in xylol balsam. ~to demonstrate acid-fast bacilli.~-1. prepare the sections for staining in the usual way. 2. stain with hæmatin solution ten to twenty seconds, to obtain a pure nuclear stain; then wash in water. 3. stain with carbolic fuchsin twenty to thirty minutes at 47°c.; then wash in water. 4. treat with aniline hydrochlorate, 2 per cent. aqueous solution, for two to five seconds. 5. decolourise in 75 per cent. alcohol till section appears free from stain--fifteen to thirty minutes. 6. dehydrate with absolute alcohol. 7. clear very rapidly with xylol. 8. mount in xylol balsam. ~to demonstrate spirochætes in tissues.~ ~piridin method (levaditi).~-1. cut slices of tissue 1 mm. thick. 2. fix in 10 per cent. formalin solution for twenty-four hours. 3. wash in water for one hour. 4. place in 96 per cent. alcohol for twenty-four hours. 5. measure into a dark green or amber bottle 100 c.c. silver nitrate solution 1 per cent., and 10 grammes pyridin puriss. transfer slices of tissue to this. stopper and keep at room temperature three hours, then in thermostat at 50° c. for four to six hours. 6. wash quickly in 10 per cent. pyridin solution. 7. reduce silver by transferring slices of tissue to following solution for forty-eight hours. pyrogallic acid 4 grammes acetone 10 c.c. pyridin puriss 15 grammes distilled water 100 c.c. 8. wash well in water. take through alcohols of increasing strength up to absolute, keeping in each strength for twenty-four hours. 9. clear, embed, cut very thin sections, mount, remove paraffin, again clear and mount in xylol balsam. the spirochætes if present are black and show up against the pale yellow color of the background. weak carbol fuchsin, neutral red or toluidin blue can also be used to stain the background if desired, after the removal of the paraffin in step 9. ~to demonstrate protozoa in sections (leishman).~-reagents required: leishman's polychrome stain. acetic acid 1 in 1500 aqueous solution. caustic soda 1 in 7000 aqueous solution. distilled water. 1. mount section, remove paraffin and take into distilled water as usual (_vide_ page 121). 2. drain off the excess of water. 3. cover the section with diluted leishman (1 part stain, 2 parts distilled water) and allow to act for five to ten minutes (until tissue appears a deep blue). 4. decolourise with acetic acid solution until only the nuclei appear blue (examine the section wet, with low power objective). 5. if the eosin colour is too well marked treat with the caustic soda solution until the desired tint is obtained (as seen with the 1/6-inch objective). 6. wash with distilled water. 7. rapidly dehydrate with alcohol. 8. clear with xylol. 9. mount in xylol balsam. ~viii. classification of fungi.~ for practical purposes fungi may be divided into: ~1. hymenomycetes~ (including the mushrooms, etc.). ~2. hyphomycetes~ (moulds). ~3. blastomycetes~ (yeasts and torulæ). ~4. schizomycetes~ (bacteria). note.--formerly myxomycetes were included in the fungi; they are now recognized as belonging to the animal kingdom, and are termed "mycetozoa." ~morphology of the hyphomycetes.~ at the commencement of his studies, the attention of the student is directed to the various non-pathogenic moulds and yeasts, not only that he may gain the necessary technique whilst handling cultivations of harmless organisms, but also because these very species are amongst the commonest of those that may accidentally contaminate his future preparations. the hyphomycetes are composed of a mycelium of short jointed rods or "hyphæ" springing from an axis or germinal tube which develops from the spore. hyphæ are-(a) nutritive or submerged. (b) reproductive or aerial. the protoplasm of these cells contains granules, pigment, oil globules, and sometimes crystals of calcium oxalate. ~reproduction.~--apical spore formation--asexual; zoospores--sexual. ~mucorinæ.~--_mucor_ (fig. 77).--note the branching filaments--"mycelium" (a), "hyphæ" (b). note the asexual reproduction. 1. a filament grows upward. at its apex a septum forms, then a globular swelling appears--"sporagium" (d). this possesses a definite membrane. 2. from the septum grows a club-shaped mass of protoplasm--"columella" (c). [illustration: fig. 77.--mucor mucedo.] [illustration: fig. 78.--aspergillus] 3. the rest of the contained protoplasm breaks up into "swarm spores" (e). finally the membrane ruptures and spores escape. ~perisporaceæ.~--_aspergillus_ (fig. 78).--note the branching filaments--"mycelium" (a). [illustration: fig. 79.--penicillium.] note the asexual reproduction. 1. a filament (b) grows upward, its termination becomes clubbed; on the clubbed extremity flask-shaped cells appear--"sterigmata" (c). 2. at free end of each sterigma is formed an oval body--a spore or "gonidium" (d), which, when ripe, is thrown off from the sterigma. two or more gonidia may be supported upon each sterigma. _penicillium_ (fig. 79).--note the branching filaments--"mycelium" (a) (frequently containing globules). note the asexual reproduction. 1. a filament grows upward--"goniodophore" (b)--and its apex divides up into several branches--"basidia" (c). 2. at the apex of each basidium a flask-shaped cell, "sterigma" (d), appears. 3. at the apex of each sterigma appears a row of oval cells--"spores" or "conidia" (e). these, when ripe, are cast off from the sterigmata. [illustration: fig. 80.--oïdium.] ~ascomycetæ.~--_oïdium_ (fig. 80).--(this family is perhaps as nearly related to the blastomycetes as it is to the hyphomycetes.) note the branching filaments--"pseudomycelium" (a). here and there filaments are broken up at their ends into oval or rod-shaped segments, "oïdia," and behave as spores. note the asexual reproduction. from the pseudomycelium arise true hyphæ (b), each of which in turn ends in a chain of spores (c). ~morphology of the blastomycetes.~ the blastomycetes are composed of spherical or oval cells (8 to 9.5µ in diameter), which, when rapidly multiplying by budding, may form a spurious mycelium. a thin cell-wall encloses the granular protoplasm, in which vacuoles and sometimes a nucleus may be noted. this latter is best seen when stained with hæmatoxylin (see page 105). during their growth and multiplication the blastomycetes split up solutions containing sugar into alcohol and co_{2}. ~saccharomyces~ (fig. 81).--note the round or oval cells of granular protoplasm (a) containing solid particles and vacuoles (c), and surrounded by a definite envelope. ~reproduction.~--budding; ascospores--asexual. note the asexual _reproduction_. 1. "gemmation"--that is, the budding out of daughter cells (b) from various parts of the gradually enlarging mother cell. these are eventually cast off and in turn become mother cells and form fresh groups of buds. [illustration: fig. 81.--saccharomyces with ascospores.] [illustration: fig. 82.--torula.] 2. spore formation--"ascospores" (e). these are formed at definite temperatures and within well-defined periods; e. g., saccharomyces cerevisiæ, thirty hours at 25° to 37°c., or ten days at 12°c. ~torulæ~ (fig. 82).--torulæ, whilst resembling yeasts in almost every other respect, never form endo-spores. note the elongated, sausage-shaped cells (a) the larger oval cells (b) and the globular cells (c) the former two often interlacing and growing as a film. note the absence of ascospore formation. ix. schizomycetes. ~classification and morphology.~--bacteria are often classified, in general terms, according to their life functions, into- _saprogenic_, or putrefactive bacteria; _zymogenic_, or fermentative bacteria; _pathogenic_, or disease-producing bacteria; or according to their food requirements into- _prototrophic_, requiring no organic food (e. g., nitrifying bacteria); _metatrophic_, requiring organic food (e. g., saprophytes and facultative parasites); _paratrophic_, requiring living food (obligate parasites); or according to their metabolic products into- _chromogenic_, or pigment-producing bacteria; _photogenic_, or light-producing bacteria; _aerogenic_, or gas-producing bacteria; and so on. such broad groupings as these have, however, but little practical value when applied to the systematic study of the fission fungi. on the other hand, no really scientific classification of the schizomycetes has yet been drawn up, and the varying morphological appearances of the members of the family are still utilised as a basis for classification, as under-~1. cocci.~ (fig. 83).--rounded or oval cells, subdivided according to the arrangement of the individuals after fission, into-_diplococci_ and _streptococci_, where division takes place in one plane only, and the individuals remain attached (a) in pairs or (b) in chains. _tetrads_, _merismopedia_, or _pediococci_, where division takes place alternately in two planes at right angles to each other, and the individuals remain attached in flat tablets of four, or its multiples. [illustration: fig. 83.--types of bacteria--cocci: 1, diagram of sphere indicating planes of fission; 2, diplococci; 3, streptococci; 4, tetrads; 5, sarcinæ; 6, staphylococci.] _sarcinæ_, where division takes place in three planes successively, and the individuals remain attached in cubical packets of eight and its multiples. [illustration: fig. 84.--types of bacteria--bacilli, etc.: 1, bacilli; 2, diplobacilli; 3 streptobacilli; 4, spirilla; 5, vibrios; 6, spirochætæ.] _micrococci_ or _staphylococci_, where division takes place in three planes, but with no definite sequence; consequently the individuals remain attached in pairs, short chains, plates of four, cubical packets of eight, and irregular masses containing numerous cocci. ~2. bacilli~ (fig. 84, 1 to 3).--rod-shaped cells. a bacillus, however short, can usually be distinguished from a coccus in that two sides are parallel. some bacilli after fission retain a characteristic arrangement and may be spoken of as _diplobacilli_ or _streptobacilli_. leptothrix is a term that in the past has been loosely used to signify a long thread, but is now restricted to such forms as belong to the leptothriciæ (_vide infra_). ~3. spirilla~ (fig. 84, 4 to 6).--curved and twisted filaments. classified, according to shape, into- spirillum. vibrio (comma). spirochæta. many spirochætes appear to belong to the animal kingdom and are grouped under protozoa; other organisms to which this name has been given are undoubtedly bacteria. higher forms of bacteria are also met with, which possess the following characteristics: they are attached, unbranched, filamentous forms, showing-(a) differentiation between base and apex; (b) growth apparently apical; (c) exaggerated pleomorphism; (d) "pseudo-branching" from apposition of cells; and are classified into- 1. beggiotoa. } free swimming forms, which 2. thiothrix. } contain sulphur granules. 3. crenothrix. } 4. cladothrix. } these forms do not contain 5. leptothrix. } sulphur granules. 6. streptothrix. a group which exhibits true but not dichotomous branching, and contains some pathogenic species. the morphology of the same bacterium may vary greatly under different conditions. for example, under one set of conditions the examination of a pure cultivation of a bacillus may show a short oval rod as the predominant form, whilst another culture of the same bacillus, but grown under different conditions, may consist almost entirely of long filaments or threads. this variation in morphology is known as "pleomorphism." some of the factors influencing pleomorphism are: 1. the composition, reaction, etc., of the _nutrient medium_ in which the organism is growing. 2. _the atmosphere_ in which it is cultivated. 3. _the temperature_ at which it is incubated. 4. exposure to or protection from _light_. the various points in the anatomy morphology and physiology of bacteria upon which stress is laid in the following pages should be studied as closely as is possible in preparations of the micro-organisms named in connection with each. ~anatomy.~ 1. _capsule_ (fig. 85, b).--a gelatinous envelope (probably akin to mucin in composition) surrounding each individual organism, and preventing absolute contact between any two. in some species the capsule (e. g., b. pneumoniæ) is well marked, but it cannot be demonstrated in all. in very well marked cases of gelatinisation of the cell wall, the individual cells are cemented together in a coherent mass, to which the term "zoogloea" is applied (e. g., streptococcus mesenteroides). in some species colouring matter or ferric oxide is stored in the capsule. 2. _cell wall_ (fig. 85, c).--a protective differentiation of the outer layer of the cell protoplasm; difficult to demonstrate, but treatment with iodine or salt solution sometimes causes shrinkage of the cell contents--"plasmolysis"--and so renders the cell wall apparent (_e. g._, b. megatherium) in the manner shown in figure 85. stained bacilli, when examined with the polarising microscope, often show a doubly refractile cell wall (e. g., b. tuberculosis and b. anthracis). in some of the higher bacteria the cell wall exhibits this differentiation to a marked degree and forms a hard sheath within which the cell protoplasm is freely movable; and during the process of reproduction the cell protoplasm may be extruded, leaving the empty tube unaltered in shape. [illustration: fig. 85.--dragrammatic sketch of composite bacterium to illustrate details of anatomical structure.] [illustration: fig. 86.--plasmolysis.] 3. _cell contents._--protoplasm (mycoprotein) contains a high percentage of nitrogen, but is said to differ from proteid in that it is not precipitated by c_{2}h_{6}o. it is usually homogeneous in appearance--sometimes granular--and may contain oil globules or sap vacuoles (fig. 85, d), chromatin granules, and even sulphur granules. sap vacuoles must be distinguished from spores, on the one hand, and the vacuolated appearance due to plasmolysis, on the other. the cell contents may sometimes be differentiated into a parietal layer, and a central body (e. g., beggiotoa) when stained by hæmatoxylin. 4. _nucleus._--this structure has not been conclusively proved to exist, but in some bacteria chromatin particles have been observed near the centre of the bacterial cell and denser masses of protoplasm situated at the poles which exhibit a more marked affinity than the rest of the cell protoplasm for aniline dyes. these latter are termed polar granules or _polkoerner_ (fig. 85, e). occasionally these aggregations of protoplasm alter the colour of the dye they take up. they are then known as metachromatic bodies or _ernstschen koerner_ (e. g., b. diphtheriæ). 5. _flagella_ (organs of locomotion, fig. 85, a).--these are gelatinous elongations of the cell protoplasm (or more probably of the capsule), occurring either at one pole, at both poles, or scattered around the entire periphery. flagella are not pseudopodia. the possession of flagella was at one time suggested as a basis for a system of classification, when the following types of ciliation were differentiated (fig. 87): [illustration: fig. 87.--types of ciliation.] 1. polar: (a) _monotrichous_ (a single flagellum situated at one pole; e. g., b. pyocyaneus). (b) _amphitrichous_ (a single flagellum at each pole; e. g., spirillum volutans). (c) _lophotrichous_ (a tuft or bunch of flagella situated at each pole; e. g., b. cyanogenus). 2. diffuse: _peritrichous_ (flagella scattered around the entire periphery e. g., b. typhosus). ~physiology.~ ~reproduction.~--_active stage._--vegetative, i. e., by the division of cells, or "fission." 1. the cell becomes elongated and the protoplasm aggregated at opposite poles. 2. a circular constriction of the organism takes place midway between these aggregations, and a septum is formed in the interior of the cell at right angles to its length. 3. the division deepens, the septum divides into two lamellæ, and finally two cells are formed. [illustration: fig. 88.--fission of cocci.] [illustration: fig. 89.--fission of bacteria.] 4. the daughter cells may remain united by the gelatinous envelope for a variable time. eventually they separate and themselves subdivide. cultures on artificial media, after growing in the same medium for some time--i. e., when the pabulum is exhausted--show "involution forms" (fig. 90), well exemplified in cultures of b. pestis on agar two days old, b. diphtheriæ on potato four to six days old. [illustration: fig. 90.--involution forms.] they are of two classes, viz.: (a) involution forms characterised by alterations of shape (fig. 90). (not necessarily dead.) (b) involution forms characterised by loss of staining power. (always dead.) _resting stage._--spore formation.--conditions influencing spore formation: in an old culture nothing may be left but spores. it used to be supposed that spores were _always_ formed, so that the species might not become extinct, when (a) the supply of nutrient was exhausted. (b) the medium became toxic from the accumulation of metabolic products. (c) the environment became unfavourable; e. g., change of temperature. this is not altogether correct; e. g., the temperature at which spores are best formed is constant for each bacterium, but varies with different species; again, aerobes require oxygen for sporulation, but anaerobes will not spore in its presence. (a) arthrogenous: noted only in the micrococci. one complete element resulting from ordinary fission becomes differentiated for the purpose, enlarges, and develops a dense cell wall. one or more of the cells in a series may undergo this alteration. this process is probably not real spore formation, but merely relative increase of resistance. these so-called arthrospores have never been observed to "germinate," nor is their resistance very marked, as they fail to initiate new cultures, after having been exposed to a temperature of 80° c. for ten minutes. (b) endogenous: the cell protoplasm becomes differentiated and condensed into a spherical or oval mass (very rarely cylindrical). after further contraction the outer layers of the mass become still more highly differentiated and form a distinct spore membrane, and the spore itself is now highly refractile. it has been suggested, and apparently on good grounds, that the spore membrane consists of two layers, the exosporium and the endosporium. each cell forms one spore only, usually in the middle, occasionally at one end (some exceptions, however, are recorded; e. g., b. inflatus). the shape of the parent cell may be unaltered, as in the anthrax bacillus, or altered, as in the tetanus bacillus, and these points serve as the basis for a classification of spore-bearing bacilli, as follows: (a) cell body of the parent bacillus unaltered in shape (fig. 91, a). (b) cell of the parent bacillus altered in shape. 1. _clostridium_ (fig. 91, b): rod swollen at the centre and attenuated at the poles; spindle shape; e. g., b. butyricus. 2. _cuneate_ (fig. 91, c): rods swollen slightly at one pole and more or less pointed at the other; wedge-shaped. [illustration: fig. 91--types of spore-bearing bacilli.] 3. _clavate_ (fig. 91, d): rods swollen at one pole and cylindrical (unaltered) at the other; keyhole-shaped; e. g., b. chauvei. 4. _capitate_ (fig. 91, e): rods with a spherical enlargement at one pole; drumstick-shaped; e. g., b. tetani. the endo-spores remain within the parent cell for a variable time (in one case it is stated that germination of the spore occurs within the interior of the parent cell--"endo-germination"), but are eventually set free, as a result of the swelling up and solution of the cell membrane of the parent bacillus in the surrounding liquid, or of the rupture of that membrane. they then present the following characteristics: 1. well-formed, dense cell membranes, which renders them extremely difficult to stain, but when once stained equally difficult to decolourise. 2. high refractility, which distinguished them from vacuoles. 3. higher resistance than the parent organism to such lethal agents as heat, desiccation, starvation, time, etc., this resistance being due to (a) low water contents of plasma of the spore. (b) low heat-conducting power } of the spore (c) low permeability } membrane. this resistance varies somewhat with the particular species--e. g., some spores may resist boiling for a few minutes--but practically all are killed if the boiling is continued for ten minutes. ~germination.~--when transplanted to suitable media and placed under favourable conditions, the spores germinate, usually within twenty-four to thirty-six hours, and successively undergo the following changes which may be followed in hanging-drop cultures on a warm stage: 1. swell up slowly and enlarge, through the absorption of water. 2. lose their refrangibility. 3. at this stage one of three processes (but the particular process is always constant for the same species) may be observed: (a) the spore grows out into the new bacillus without discarding the spore membrane (which in this case now becomes the cell membrane); _e. g._, b. leptosporus. (b) it loses its spore membrane by solution; e. g., b. anthracis. (c) it loses its spore membrane by rupture. in this process the rupture may be either polar (at one pole only _e. g._, b. butyricus), or bipolar (e. g., b. sessile), or equatorial; (e. g., b. subtilis). in those cases where the spore membrane is discarded the cell membrane of the new bacillus may either be formed from-(a) the inner layer of the spore membrane, which has undergone a preliminary splitting into parietal and visceral layers; e. g., b. butyricus. (b) the outer layers of the cell protoplasm, which become differentiated for that purpose; e. g., b. megatherium. the new bacillus now increases in size, elongates, and takes on a vegetative growth--i. e., undergoes fission--the bacilli resulting from which may in their turn give rise to spores. [illustration: fig. 92. simple.] [illustration: fig. 93. solution.] [illustration: fig. 94. polar.] [illustration: fig. 95. bipolar.] [illustration: fig. 96. equatorial.] ~food stuffs.~--1. _organic foods._-(a) the pure parasites (e. g., b. lepræ) will not live outside the living body. (b) both saprophytic and facultative parasitic bacteria agree in requiring non-concentrated food. (c) the facultative parasites need highly organised foods; e. g., proteids or other sources of nitrogen and carbon, and salts. (d) the saprophytic bacteria are more easily cultivated; e. g., 1. some bacteria will grow in almost pure distilled water. 2. some bacteria will grow in pure solutions of the carbohydrates. 3. _water_ is absolutely essential to the _growth_ of bacteria. food of a definite reaction is needed for the growth of bacteria. as a general rule growth is most active in media which react slightly acid to phenolphthalein--that is, neutral or faintly alkaline to litmus. mould growth, on the other hand, is most vigourous in media that are strongly acid to phenolphthalein. ~environment.~--the influence of physical agents upon bacterial life and growth is strongly marked. 1. _atmosphere._--the presence of _oxygen_ is necessary for the growth of some bacteria, and death follows when the supply is cut off. such organisms are termed _obligate aerobes_. some bacteria appear to thrive equally well whether supplied with or deprived of oxygen. these are termed _facultative anaerobes_. a third class will only live and multiply when the access of free oxygen is completely excluded. these are termed _obligate anaerobes_. 2. _temperature._--practically no bacterial growth occurs below 5°c, and very little above 40° c. 30°c. to 37° c is the most favorable for the large majority of micro-organisms. the maximum and minimum temperatures at which growth takes place, as well as the optimum, are fairly constant for each bacterium. bacteria have been classified, according to their optimum temperature, into- min. opt. max. 1. psychrophilic bacteria (chiefly water organisms) 0° c. 15° c. 30°c. 2. mesophilic bacteria (includes pathogenic bacteria) 15° c. 37° c. 45°c. 3. thermophilic bacteria 45° c. 55° c. 70°c. the thermal death-point of an organism is another biological constant; and is that temperature which causes the death of the vegetative forms when the exposure is continued for a period of ten minutes (see pages 298-301). 3. _light._--many organisms are indifferent to the presence of light. on the other hand, light frequently impedes growth, and alters to a greater or lesser extent the biochemical characters of the organisms--e. g., chromogenicity or power of liquefaction. pathogenic bacteria undergo a progressive loss of virulence when cultivated in the presence of light. 4. _movements._--movements, if slight and simply of a flowing character, do not appear to injuriously affect the growth of bacteria; but violent agitation, such as shaking, absolutely kills them. a condition of perfect rest would seem to be that most conducive to bacterial growth. ~the metabolic products of bacteria.~--_pigment production._--many micro-organisms produce one or more vivid pigments--yellow, orange, red, violet, fluorescent, etc.--during the course of their life and growth. the colouring matter usually exists as an intercellular excrementitious substance. occasionally, however, it appears to be stored actually within the bodies of the bacteria. the chromogenic bacteria are therefore classified, in accordance with the final destination of the colouring matter they elaborate, into-_chromoparous_ bacteria: in which the pigment is diffused out upon and into the surrounding medium. _chromophorous_ bacteria: in which the pigment is stored in the cell protoplasm of the organism. _parachromophorous_ bacteria: in which the pigment is stored in the cell wall of the organism. different species of chromogenic bacteria differ in their requirements as to environment, for the production of their characteristic pigments; e. g., some need oxygen, light, or high temperature; others again favor the converse of these conditions. _light production._--some bacteria, and usually those originally derived from water, whether fresh or salt, exhibit marked phosphorescence when cultivated under suitable conditions. these are classed as "photogenic." _enzyme production._--many bacteria produce soluble ferments or enzymes during the course of their growth, as evidenced by the liquefaction of gelatine, the clotting of milk, etc. these ferments may belong to either of the following well-recognised classes: proteolytic, diastatic, invertin, rennet. _toxin production._--a large number, especially of the pathogenic bacteria, elaborate or secrete poisonous substances concerning which but little exact knowledge is available, although many would appear to be enzymic in their action. these toxins are usually differentiated into-_extracellular_ (or soluble) toxins: those which are diffused into, and held in solution by, the surrounding medium. _intracellular_ (or inseparate) toxins: those which are so closely bound up with the cell protoplasm of the bacteria elaborating them that up to the present time no means has been devised for their separation or extraction. _end-products of metabolism._--under this heading are included-organic acids (e. g., lactic, butyric, etc.). alkalies (e. g., ammonia). aromatic compounds (e. g., indol, phenol). reducing substances (e. g., those reducing nitrates to nitrites). gases (e. g., sulphuretted hydrogen, carbon dioxide, etc.). and while the discussion of their formation, etc., is beyond the scope of a laboratory handbook, the methods in use for their detection and separation come into the ordinary routine work and will therefore be described (_vide_ page 276 _et seq._). x. nutrient media. in order that the life and growth of bacteria may be accurately observed in the laboratory, it is necessary-1. to _isolate_ individual members of the different varieties of micro-organisms. 2. to _cultivate_ organisms, thus isolated, apart from other associated or contaminating bacteria--i. e., in _pure culture_. for the successful achievement of these objects it is necessary to provide nutriment in a form suited to the needs of the particular bacterium or bacteria under observation, and in a general way it may be said that the nutrient materials should approximate as closely as possible, in composition and character, to the natural pabulum of the organism. the general requirements of bacteria as to their food-supply have already been indicated (page 142) and many combinations of proteid and of carbohydrate have been devised, from time to time, on those lines. these, together with various vegetable tissues, physiological or pathological fluid secretions, etc., are collectively spoken of as _nutrient media_ or _culture media_. the greater number of these media are primarily _fluid_, but, on account of the rapidity with which bacterial growth diffuses itself through a liquid, it is impossible to study therein the characteristics of individual organisms. many such media are, therefore, subsequently rendered solid by the addition of substances like gelatine or agar, in varying proportions, the proportions of such added material being generally mentioned when referring to the media; e. g., 10 per cent. gelatine, 2 per cent. agar. gelatine is employed for the solidification of those media it is intended to use in the cultivation of bacteria at the room temperature or in the "cold" incubator. in the percentages usually employed, gelatine media become fluid at 25°c.; higher percentages remain solid at somewhat higher temperatures, but the difficulty of filtering strong solutions of gelatine militates against their general use. media, on the other hand which have been solidified by the addition of agar, only become liquid when exposed to 90° c. for about ten minutes, and again solidify when the temperature falls to 40°c. when it becomes necessary to render these media fluid, heat is applied, upon the withdrawal of which they again assume their solid condition. such media should be referred to as _liquefiable media_; in point of fact, however, they are usually grouped together with the solid media. note.--it must here be stated that the designation 10 per cent. gelatine or 2 per cent. agar refers only to the quantity of those substances actually added in the process of manufacture, and _not_ to the percentage of gelatine or agar, as the case may be, present in the finished medium; the explanation being that the commercial products employed contain a large proportion of insoluble material which is separated off by filtration during the preparation of the liquefiable media. other media, again--e. g., potato, coagulated blood-serum, etc.--cannot be again liquefied by physical means, and these are spoken of as _solid_ media. the following pages detail the method of preparing the various nutrient media, in ordinary use (see also chapter xi), those which are only occasionally required for more highly specialised work are grouped together in chapter xii. it must be premised that scrupulous cleanliness is to be observed with regard to all apparatus, vessels, funnels, etc., employed in the preparation of media; although in the preliminary stages of the preparation of most media absolute sterility of the apparatus used is not essential. meat extract. a watery solution of the extractives, etc., of lean meat (usually beef) forms the basis of several nutrient media. this solution is termed "meat extract" and it has been determined empirically that its preparation shall be carried out by extracting half a kilo of moist meat with one litre of water. for many purposes, however, it is more convenient to have a more concentrated extract; one kilo of meat should therefore be extracted with one litre of water, to form "double strength" meat extract. it was customary at one time, and is even now in some laboratories to use either "shin of beef" or "beef-steak"--both contain muscle sugar which often needs to be removed before the nutrient medium can be completed. heart muscle (bullock's heart or sheep's heart) is much to be preferred and from the point of economy, ease and cleanliness of manipulation, and extractive value, the imported frozen bullock's hearts provide the best extract. meat extract (fleischwasser) is prepared as follows: 1. measure 1000 c.c. of distilled water into a large flask (or glass beaker, or enamelled iron pot) and add 1000 grammes (roughly, 2-1/2 pounds) of fresh lean meat--e. g., bullock's heart--finely minced in a mincing machine. 2. heat the mixture gently in a water-bath, taking care that the temperature of the contents of the flask does not exceed 40° c. for the first twenty minutes. (this dissolves out the soluble proteids, extractives, salts, etc.) 3. now raise the temperature of the mixture to the boiling-point, and maintain at this temperature for ten minutes. (this precipitates some of the albumins, the hæmoglobin, etc., from the solution.) 4. strain the mixture through sterile butter muslin or a perforated porcelain funnel, then filter the liquid through swedish filter paper into a sterile "normal" litre flask, and when cold make up to 1000 c.c. by the addition of distilled water--to replace the loss from evaporation. 5. if not needed at once, sterilise the meat extract in bulk in the steam steriliser for twenty minutes on each of three consecutive days. calf, sheep, or chicken flesh is occasionally substituted for the beef; or the meat extract may be prepared from animal viscera, such as brain, spleen, liver, or kidneys. note.--as an alternative method, 5 c.c. of brand's meat juice or 3 grammes of wyeth's beef juice, or 10 grammes liebig's extract of meat (lemco) may be dissolved in 1000 c.c. distilled water, and heated and filtered as above to form ordinary or single strength meat extract. media, prepared from such meat extracts are, however, eminently unsatisfactory when used for the cultivation of the more highly parasitic bacteria; although when working in tropical and subtropical regions their use is well-nigh compulsory. ~reaction of meat extract.~--meat extract thus prepared is acid in its reaction, owing to the presence of acid phosphates of potassium and sodium, weak acids of the glycolic series, and organic compounds in which the acid character predominates. owing to the nature of the substances from which it derives its reaction, the total acidity of meat extract can only be estimated accurately when the solution is at the boiling-point. moreover, it has been observed that prolonged boiling (such as is involved in the preparation of nutrient media) causes it to undergo hydrolytic changes which increase its acidity, and ~the meat extract only becomes stable in this respect after it has been maintained at the boiling-point for forty-five minutes~. although meat extract always reacts acid to phenolphthalein, it occasionally reacts neutral or even alkaline to litmus; and again, meat extract that has been rendered exactly neutral to litmus still reacts acid to phenolphthalein. this peculiar behaviour depends upon two factors: 1. litmus is insensitive to many weak organic acids the presence of which is readily indicated by phenolphthalein. 2. dibasic sodium phosphate which is formed during the process of neutralisation is a salt which reacts alkaline to litmus, but neutral to phenolphthalein. in order, therefore, to obtain an accurate estimation of the reaction of any given sample of meat extract, it is essential that-1. the meat extract be previously exposed to a temperature of 100° c. for forty-five minutes. 2. the estimation be performed at the boiling-point. 3. phenolphthalein be used as the indicator. the estimation is carried out by means of titration experiments against standard solutions of caustic soda, in the following manner: _method of estimating the reaction._-_apparatus required_: _solutions required_: 1. 25 c.c. burette graduated 1. 10n naoh, accurately in tenths of a centimetre. standardised. 2. 1 c.c. pipette graduated in 2. n/1 naoh, accurately hundredths, and provided standardised with rubber tube, pinch-cock, and delivery nozzle. 3. 25 c.c. measure (cylinder or 3. n/10 naoh, accurately pipette, calibrated for standardised. 98°c.--_not_ 15°c). 4. several 60 c.c. conical 4. 0.5 per cent. solution of beakers or erlenmeyer phenolphthalein in 50 per flasks. cent. alcohol. 5. white porcelain evaporating basin, filled with boiling water and arranged over a gas flame as a water-bath. 6. bohemian glass flask, fitted as a wash-bottle, and filled with distilled water, which is kept boiling on a tripod stand. method.--arrange the apparatus as indicated in figure 97. (a) 1. fill the burette with n/10 naoh. 2. fill the pipette with n/1 naoh. [illustration: fig. 97.--arrangement of apparatus for titrating media.] 3. measure 25 c.c. of the meat extract (previously heated in the steamer at 100° c. for forty-five minutes) into one of the beakers by means of the measure; rinse out the measure with a very small quantity of boiling distilled water from the wash-bottle, and then add this rinse water to the meat extract already in the beaker. 4. run in about 0.5 c.c. of the phenolphthalein solution and immerse the beaker in the water-bath, and raise to the boil. 5. to the medium in the beaker run in n/10 naoh cautiously from the burette until the end-point is reached, as indicated by the development of a pinkish tinge, shown in figure 98 (b). note the amount of decinormal soda solution used in the process. note.--just before the end-point is reached, a very slight opalescence may be noted in the fluid, due to the precipitation of dibasic phosphates. after the true end-point is reached, the further addition of about 0.5 c.c. of the decinormal soda solution will produce a deep magenta colour (fig. 98, c), which is the so-called "end-point" of the american committee of bacteriologists. [illustration: fig. 98.--a, sample of filtered meat extract or nutrient gelatine to which phenolphthalein has been added. the medium is acid, as evidenced by the unaltered colour of the sample. b, the same neutralised by the addition of n/10 naoh. the production of this faint rose-pink colour indicates that the "end-point," or neutral point to phenolphthalein, has been reached. if such a sample is cooled down to say 30° or 20° c., the colour will be found to become more distinct and decidedly deeper and brighter, resembling that shown in c. c, also if, after the end-point is reached, a further 0.5 c.c. or 1.0 c.c. n/10 naoh be added to the sample, the marked alkalinity is evidenced by the deep colour here shown.] (b) perform a "control" titration (occasionally two controls may be necessary), as follows: 1. measure 25 c.c. of the meat extract into one of the beakers, wash out the measure with boiling water, and add the phenolphthalein as in the first estimation. 2. run in n/1 naoh from the pipette, just short of the equivalent of the amount of _deci_-normal soda solution required to neutralise the 25 c.c. of medium. (for example, if in the first estimation 5 c.c. of n/10 naoh were required to render 25 c.c. of medium neutral to phenolphthalein, only add 0.48 c.c. of n/1 naoh.) immerse the beaker in the water-bath. 3. complete the titration by the aid of the n/10 naoh. 4. note the amount of n/10 naoh solution required to complete the titration, and add it to the equivalent of the n/1 naoh solution previously run in. take the total as the correct estimation. _method of expressing the reaction._-the reaction or _titre_ of meat extract, medium, or any solution estimated in the foregoing manner, is most conveniently expressed by indicating the number of cubic centimetres of normal alkali (or normal acid) that would be required to render _one litre_ of the solution exactly neutral to phenolphthalein. [illustration: fig. 99.--stock bottle for dekanormal soda solution.] the sign + (plus) is prefixed to this number if the original solution reacts acid, and the sign (minus) if it reacts alkaline. for example, "meat extract + 10," indicates a sample of meat extract which reacts acid to phenolphthalein, and would require the addition of 10 c.c. of _normal_ naoh per litre, to neutralise it. note.--such a solution would probably react alkaline to litmus. conversely, if as the result of our titration experiments we find that 25 c.c. of meat extract require the addition of 5 c.c. n/10 naoh to neutralise, then 1000 c.c. of meat extract will require the addition of 200 c.c. n/10 naoh = 20 c.c. n/1 naoh. and this last figure, 20, preceded by the sign + (i. e., +20), to signify that it is acid, indicates the reaction of the meat extract. note.--the standard soda solutions should be prepared by accurate measuring operations, controlled by titrations, from a stock solution of 10n naoh, which should be very carefully standardised. if a large supply is made or the consumption is small this stock solution must be kept in an aspirator bottle to which air can only gain access after it has been dried and rendered free from co_{2}. this may be done by first leading it over h_{2}so_{4} and soda lime, or soda lime alone, by some such arrangement as is shown in figure 99, which also shows a constant burette arrangement for the delivery of small measured quantities of the dekanormal soda solution. standardisation of media. differences in the reaction of the medium in which it is grown will provoke not only differences in the rate of growth of any given bacterium, but also well-marked differences in its cultural and morphological characters; and nearly every organism will be found to affect a definite "optimum reaction"--a point to be carefully determined for each. for most bacteria, however, the "optimum" usually approximates fairly closely to +10; and as experiment has shown that this reaction is the most generally useful for routine laboratory work, it is the one which may be adopted as the standard for all nutrient media derived from meat extract. briefly, the method of standardising a litre of media to +10 consists in subtracting 10 from the initial _titre_ of the medium mass; the remainder indicates the number of cubic centimetres of normal soda solution that must be added to the medium, per litre, to render the reaction +10. ~standardising nutrient bouillon.~--for example, 1000 c.c. bouillon are prepared; at the first titration it is found 1. 25 c.c. require the addition of 5.50 c.c. n/10 naoh to neutralise. two controls give the following results: 2. 25 c.c. require the addition of 5.70 c.c. n/10 naoh to neutralise. 3. 25 c.c. require the addition of 5.60 c.c. n/10 naoh to neutralise. averaging these two controls, 25 c.c. require the addition of 5.65 c.c. n/10 naoh to neutralise, and therefore 1000 c.c. require the addition of 226 c.c. n/10 naoh, or 22.60 c.c. n/1 naoh, or 2.26 c.c. n/10 naoh. initial _titre_ of the bouillon = +22.6, and as such requires the addition of (22.6 c.c. 10 c.c.) = 12.6 c.c. of n/1 naoh per litre to leave its finished reaction +10. but the three titrations, each on 25 c.c. of medium, have reduced the original bulk of bouillon to (1000 75 c.c.) = 925 c.c. the amount of n/1 naoh required to render the reaction of this quantity of medium +10 may be deduced thus: 1000 c.c.:925 c.c.::12.6 c.c.:x. then x = 11.65 c.c. n/1 naoh. whenever possible, however, the required reaction is produced by the addition of dekanormal soda solution, on account of the minute increase it causes in the bulk, and the consequent insignificant disturbance of the percentage composition of the medium. by means of a pipette graduated to 0.01 c.c. it is possible to deliver very small quantities; but if the calculated amount runs into thousandth parts of a cubic centimetre, these are replaced by corresponding quantities of normal or even decinormal soda. in the above example it is necessary to add 11.65 c.c. normal naoh or its equivalent, 1.165 c.c. dekanormal naoh. the first being too bulky a quantity, and the second inconveniently small for exact measurement, the total weight of soda is obtained by substituting 1.16 c.c. dekanormal soda solution, and either 0.05 c.c. of normal soda solution or 0.5 c.c. of decinormal soda solution. ~standardising nutrient agar and gelatine.~--the method of standardising agar and gelatine is precisely similar to that described under bouillon. the filtration of media. ~fluid media~ are usually filtered through stout swedish filter paper (occasionally through a porcelain filter candle), and in order to accelerate the rate of filtration the filter paper should be folded in that form which is known as the "physiological filter," not in the ordinary "quadrant" shape, as by this means a large surface is available for filtration and a smaller area in contact with the glass funnel supporting it. to fold the filter proceed thus: 1. take a circular piece of filter paper and fold it exactly through its centre to form a semicircle (fig. 100, a). 2. fold the semicircle exactly in half to form a quadrant; make the crease 2, distinct by running the thumbnail along it, then open the filter out to a semicircle again. 3. fold each end of the semicircle in to the centre and so form another quadrant; smooth down the two new creases 3 and 3a, thus formed and again open out to a semicircle. 4. the semicircle now appears as in figure 100, a, the dark lines indicating the creases already formed. 5. fold the point 1 over to the point 3, and 1a to 3a, to form the creases 4 and 4a, indicated in the diagram by the light lines. fold point 1 over to 3a, and 1a to 3, to form the creases 5 and 5a. [illustration: fig. 100.--filter folding: a, filter folded in half, showing creases; b, appearance of filter on completion of folding; c, filter opened out ready for use.] 6. thus far the creases have all been made on the same side of the paper. now subdivide each of the eight sectors by a crease through its centre on the opposite side of the paper, indicated by the faint broken lines in the diagram. fold up the filter gradually as each crease is made, and when finished the filter has assumed the shape of a wedge, as in figure 100, b. when opened out the filter assumes the shape represented in figure 100, c. the folded filter is next placed inside a glass funnel supported on a retort stand, and moistened with hot distilled water before the filtration of the medium is commenced. ~liquefiable solid media~ are filtered through a specially made filter paper--"papier chardin"--which is sold in boxes of twenty-five ready-folded filters. [illustration: fig. 101.--hot-water filter funnel and ring burner.] gelatine, when properly made, filters through this paper as quickly as bouillon does through the swedish filter paper, and does _not_ require the use of the hot-water funnel. agar, likewise, if properly made, filters readily, although not at so rapid a rate as gelatine. if badly "egged," and also during the winter months, it is necessary to surround the glass funnel, in which the filtration of the agar is carried on, by a hot-water jacket. this is done by placing the glass funnel inside a double-walled copper funnel--the space between the walls being filled with water at about 90° c.--and supporting the latter on a ring gas burner fixed to a retort stand (fig. 101). the gas is lighted and the water jacket maintained at a high temperature until filtration is completed. if the steam steriliser of the laboratory is sufficiently large, it is sometimes more convenient to place the flask and filtering funnel bodily inside, close the steriliser and allow filtration to proceed in an atmosphere of live steam, than to use the gas ring and hot-water funnel. storing media in bulk. after filtration fill the medium into sterile litre flasks with cotton-wool plugs and sterilise in the steamer for twenty minutes on each of three consecutive days. after the third sterilisation, and when the flasks and contents are cool, cut off the top of the cotton-wool plug square with the mouth of the flask; push the plug a short distance down into the neck of the flask and fill in with melted paraffin wax to the level of the mouth. when the wax has set the flasks are stored in a cool dark cupboard for future use. [illustration: fig. 102.--rubber cap closing store bottle. a, before, and b, after sterilizing.] this plan is not absolutely satisfactory, although very generally employed on occasion, and it is preferable to fill the medium into long-necked flint glass bottles (the quart size, holding nearly 1000 c.c., such as those in which pasteurised milk is retailed) and to close the neck of the bottle by a special rubber cap.[3] this cap is made of soft rubber, the lower part, dome-shaped with thin walls, being slipped over the neck of the bottle (fig. 102, a). the upper part is solid, but with a sharp clean-cut (made with a cataract or tenotomy knife) running completely through its axis from the centre of the disc to the top of the dome. during sterilisation the air in the neck of the bottle, expanded by the heat, is driven out through the valvular aperture in the solid portion of the stopper. on removing the bottle from the steam chamber, the liquid contracts as it cools, and the pressure of the external air drives the solid piece of rubber down into the neck of the bottle, and forces together the lips of the slit (fig. 102, b). thus sealed, the bottle will preserve its contents sterile for an indefinite period without loss from evaporation. tubing nutrient media. after the final filtration, the nutrient medium is usually "tubed"--_i. e._, filled into sterile tubes in definite measured quantities, usually 10 c.c. this process is sometimes carried out by means of a large separator funnel fitted with a "three-way" tap which communicates with a small graduated tube (capacity 20 c.c. and graduated in cubic centimetres) attached to the side. the shape of this piece of apparatus, known as treskow's funnel, renders it particularly liable to damage. it is better, therefore, to arrange a less expensive piece of apparatus which will serve the purpose equally well (fig. 103). a geissler's three-way stop-cock has the tube on one side of the tap ground obliquely at its extremity, and the tube on the opposite side cut off within 3 cm. of the tap. the short tube is connected by means of a perforated rubber cork with a 10 cm. length of stout glass tubing (1.5 cm. bore). the third channel of the three-way tap is connected, by means of rubber tubing, with the nozzle of an ordinary separator funnel. finally, the receiving cylinder above the three-way tap is graduated in cubic centimetres up to 20, by pouring into it measured quantities of water and marking the various levels on the outside with a writing diamond. fluid media containing carbohydrates are filled into fermentation tubes (_vide_ fig. 21); or into ordinary media tubes which already have smaller tubes, inverted, inside them (fig. 104), to collect the products of growth of gas-forming bacteria. when first filled, the small tubes float on the surface of the medium after the first sterilisation nearly all the air is replaced by the medium, and after the final sterilisation the gas tubes will be submerged and completely filled with the medium. [illustration: fig. 103.--separatory funnel and three-way tap arranged for tubing media.] [illustration: fig. 104.--gas tube (durham).] ~storing "tubed" media.~--media after being tubed are best stored by packing, in the vertical position, in oblong boxes having an internal measurement of 37 cm. long by 12 cm. wide by 10 cm. deep. each box (fig. 105) has a movable partition formed by the vertical face of a weighted triangular block of wood, sliding free on the bottom (fig. 105, a); or by a flat piece of wood sliding in a metal groove in the bottom of the box, which can be fixed at any spot by tightening the thumbscrew of a brass guide rod which transfixes the partition (fig. 105, b). the front of the box is provided with a handle and a celluloid label for the name of the contained medium. these boxes are arranged upon shelves in a dark cupboard--or preferably an iron safe--which should be rendered as nearly air-tight as possible, and should have the words "media stores" painted on its doors. [illustration: fig. 105.--medium box, showing alternative partitions a and b.] footnotes: [3] this rubber cap has been made for me by the holborn surgical instrument co., thavies inn, london, w. c. xi. culture media. ordinary or stock media. ~nutrient bouillon.~-1. measure out double strength meat extract, 500 c.c., into a litre flask and add 300 c.c. distilled water. 2. weigh out witté's peptone, 10 grammes (= 1 per cent.), salt, 5 grammes (= 0.5 per cent.), and mix into a smooth paste with 200 c.c. of distilled water previously heated to 60° c. (be careful to leave no unbroken globular masses of peptone.) 3. add the peptone emulsion to the meat extract in the flask and heat in the steamer for forty-five minutes (to completely dissolve the peptone, and to render the acidity of the meat extract stable). 4. estimate the reaction of the medium; control the result; render the reaction of the finished medium +10 (_vide_ page 155). 5. heat for half an hour in the steamer at 100°c. (to complete the precipitation of the phosphates, etc.). 6. filter through swedish filter paper into a sterile flask. 7. fill into sterile tubes (10 c.c. in each tube). 8. sterilise in the steamer for twenty minutes on each of three consecutive days--i. e., by the discontinuous method (_vide_ page 35). note.--as an alternative method when neither fresh nor frozen meat is available nutrient bouillon may be prepared from a commercial meat extract, as follows: ~lemco broth.~- 1. measure out 250 c.c. distilled water into a litre flask. 2. weigh out 10 grammes liebig's lemco meat extract on a piece of clean filter paper and add to the water in the flask. shake the flask well to make an even emulsion of the meat extract. 3. weigh out witté's peptone (10 grammes), salt (5 grammes). mix into smooth paste with 100 c.c. distilled water previously heated to 60°c. 4. add the peptone salt emulsion to the meat extract emulsion in the flask and add 650 c.c. distilled water. heat in the steamer for forty-five minutes. 5. standardise the medium and complete as for nutrient bouillon. ~nutrient gelatine.~-1. weigh a 2-litre flask on a trip balance (fig. 106) and note the weight, or counterpoise carefully. [illustration: fig. 106.--trip balance.] an extremely useful counterpoise is a small sheet-brass cylinder about 38 mm. high and 38 mm. in diameter, with a funnel-shaped top and provided with a side tube by which its contents, fine "dust" shot, may be emptied out (fig. 107). [illustration: fig. 107.--counterpoise; weight when empty, 35 grammes; when full of dust shot, 200 grammes.] 2. measure out double strength meat extract, 500 c.c., into the "tared" flask. 3. weigh out and mix 10 grammes of peptone, 5 grammes of salt, and make into a thick paste with 150 c.c. distilled water; then add the emulsion to the meat extract in the flask; also add 100 grammes sheet gelatine cut into small pieces; place the flask in the water-bath and raise to the boil. [illustration: fig. 108.--arrangement of steam can and water-bath for the preparation of media.] 4. arrange a 5-litre tin can (with copper bottom, such as is used in the preparation of distilled water) by the side of the water bath, fill the can with boiling water and place a lighted bunsen burner under it. fit a long safety tube to the neck of the can and also a delivery tube, bent twice at right angles; adjust the tube to reach to the bottom of the interior of the flask containing the gelatine, etc. (fig. 108). 5. keep the water in the steam can vigourously boiling, and so steam at 100°c, bubbling through the medium mass, for ten minutes, by which time complete solution of the gelatine is effected. a certain amount of steam will condense as water in the medium flask during this process--hence the necessity for the use of double strength meat extract--but if the water bath is kept boiling this condensation will not exceed 100 c.c. 6. weigh the flask and its contents; then (1115[4] grammes + weight of the flask) minus (weight of the flask and its contents) equals the weight of water required to make up the bulk to 1 litre. the addition of the requisite quantity of water is carried out as follows: in one pan of the trip balance place the counterpoise of the tared flask (or its equivalent in weights) together with the weights making up the _calculated medium weight_. in the opposite pan place the flask containing the medium mass. now add boiling distilled water from a wash bottle until the two pans are exactly balanced. 7. titrate and estimate the reaction of the medium mass; control the result. calculate the amount of soda solution required to make the reaction of the medium mass +10 (i. e., calculate for 1000 c.c., less the quantity used for the titrations). 8. add the necessary amount of soda solution and heat in the steamer at 100° c. for twenty minutes, to precipitate the phosphates, etc. 9. allow the medium mass to cool to 60° c. well whip the whites of two eggs, add to the contents of the flask and replace in the steamer at 100° c. for about half an hour (until the egg-albumen has coagulated and formed large, firm masses floating on and in clear gelatine). 10. filter through papier chardin into a sterile flask. 11. tube in quantities of 10 c.c. 12. sterilise in the steamer at 100° c. for twenty minutes on each of three consecutive days--i. e., by the discontinuous method. ~nutrient agar-agar.~-1. weigh a 2-litre flask and note the weight--or counterpoise exactly. 2. measure out double strength meat extract, 500 c.c., into the "tared" flask. 3. weigh out and mix 10 grammes of peptone, 5 grammes of salt, and 20 grammes of powdered agar, and make into a thick paste with 150 c.c. distilled water, and add to the meat extract in the flask; place the flask in a water-bath. 4. arrange the steam can and water-bath as already directed (for the preparation of gelatine) and figured. 5. bubble live steam (at 100° c.) through the medium mass, for twenty-five minutes, by which time complete solution of the agar is effected. 6. now weigh the flask and its contents; then (1035[5] grammes + weight of flask) minus (weight of flask and its contents) equals the weight of water required to make up the bulk of the medium to 1 litre. add the requisite amount (see preparation of gelatine, page 166, step 6). 7. titrate, and estimate the reaction of the medium mass; control the result. calculate the amount of soda solution required to make the reaction of the medium mass + 10 (i. e., calculated for 1000 c.c., less the quantity used for the titrations). 8. add the necessary amount of soda solution and replace in the steamer for twenty minutes (to complete the precipitation of the phosphates, etc.). 9. allow the medium mass to cool to 60° c. well whip the whites of two eggs, add to the contents of the flask, and replace in the steamer at 100° c. for about _one hour_ (until the egg-albumen has coagulated and formed large, firm masses floating on and in clear agar.) 10. filter through papier chardin, by the aid of a hot-water funnel, if necessary (fig. 101), into a sterile flask. 11. tube in quantities of 10 c.c. or 15 c.c. 12. sterilise in the steamer at 100° c. for thirty minutes on each of three consecutive days--i. e., by the discontinuous method. ~blood-serum (inspissated).~-1. sterilise cylindrical glass jar (fig. 109) and its cover by dry heat, or by washing first with ether and then with alcohol and drying. 2. collect blood at the slaughter house from ox or sheep in the sterile cylinder. 3. allow the vessel to stand for fifteen minutes for the blood to coagulate. (this must be done before leaving the slaughterhouse, otherwise the serum will be stained with hæmoglobin.) 4. separate the clot from the sides of the vessel by means of a sterile glass rod (the yield of serum is much smaller when this is not done), and place the cylinder in the ice-chest for twenty-four hours. 5. remove the serum with sterile pipettes, or syphon it off, and fill into sterile tubes (5 c.c. in each) or flasks. 6. heat tubes containing serum to 56° c. in a water-bath for half an hour on each of two successive days. 7. on the third day, heat the tubes, in a sloping position, in a serum inspissator to about 72° c. (a coagulum is formed at this temperature which is fairly transparent; above 72° c., a thick turbid coagulum is formed.) [illustration: fig. 109.--blood-serum jar with wicker basket for transport.] the serum inspissator (fig. 110) in its simplest form is a double-walled rectangular copper box, closed in by a loose glass lid, and cased in felt or asbestos--the space between the walls is filled with water. the inspissator is supported on adjustable legs so that the serum may be solidified at any desired "slant," and is heated from below by a bunsen burner controlled by a thermo-regulator. the more elaborate forms resemble the hot-air oven (fig. 26) in shape and are provided with adjustable shelves so that any desired obliquity of the serum slope can be obtained. 8. place the tubes in the incubator at 37° c. for forty-eight hours in order to eliminate those that have been contaminated. store the remainder in a cool place for future use. _alternative method._ _steps 1-5 as above._ 6. sterilise the serum by the fractional method--that is, by exposure in a water-bath to a temperature of 56° c. for half an hour on each of six consecutive days; store in the fluid condition. 7. coagulate in the inspissator when needed. [illustration: fig. 110.--serum inspissator.] ~serum water.~- this forms the basis of many useful media, and is prepared as follows: 1. collect blood in the slaughterhouse (see page 168) and when firmly clotted collect all the expressed serum and measure in a graduated cylinder. 2. for every 100 c.c. of serum add 300 c.c. distilled water and mix in a flask. 3. heat the mixture in the steamer at 100° c. for thirty minutes. (this destroys any diastatic ferment present in the serum and partially sterilises the fluid.) 4. filter if turbid. 5. if not needed at once complete the sterilisation of the serum water by two subsequent steamings at 100° c. for twenty minutes at twenty-four hour intervals. ~citrated blood agar. guy's.~-1. kill a small rabbit with chloroform vapour, and nail it out on a board (as for a necropsy); moisten the hair thoroughly with 2 per cent. solution of lysol. 2. sterilise several pairs of forceps, scissors, etc. by boiling. 3. reflect the skin over the thorax with sterile instruments. 4. open the thoracic cavity by the aid of a fresh set of sterile instruments. 5. open the pericardium with another set of sterile instruments. 6. sear the surface of the left ventricle with a red-hot iron. 7. take a sterile capillary pipette (fig. 13, c); break off the sealed extremity with a pair of sterile forceps. 8. steady the heart in a pair of forceps and thrust the point of the pipette through the wall of the ventricle and through the seared area, apply suction to the plugged end of the pipette and fill it with blood. 9. transfer the entire quantity of blood collected from the rabbit's heart to a small erlenmeyer flask containing a number of sterile glass beads and 5 c.c. concentrated sod. citrate solution. (see page 378.) 10. agitate thoroughly and set aside for a couple of hours. 11. melt up several tubes of nutrient agar (see page 167) and cool to 42° c. 12. with a sterile 10 c.c. graduated pipette transfer 1 c.c. citrated blood from the erlenmeyer flask to each tube of liquefied agar. rotate the tube between the hands in order to diffuse the citrated blood evenly throughout the agar. 13. place the tubes in a sloping position and allow the medium to set. 14. place tubes of blood agar for forty-eight hours in the incubator at 37° c. and at the end of that time eliminate any contaminated tubes. 15. store such tubes as remain sterile for future use. ~milk.~-1. pour 1 litre of fresh cow's or goat's milk into a large separating funnel, and heat in the steamer at 100° c. for one hour. 2. remove from the steamer and estimate the reaction of the milk (normal cows' milk averages +17). if of higher acidity than +20, or lower than +10, reject this sample of milk and proceed with another supply of milk from a different source. reject milk to which antiseptics have been added as preservatives. 3. allow the milk to cool, when the fat or cream will rise to the surface and form a thick layer. 4. draw off the subnatant fat-free milk into sterile tubes (10 c.c. in each). 5. sterilise in the steamer at 100° c. for twenty minutes on each of five successive days. 6. incubate at 37° c. for forty-eight hours and eliminate any contaminated tubes. store the remainder for future use. ~litmus milk.~-1. prepare milk as described above, sections 1 to 3. 2. draw off the subnatant fat-free milk into a flask. 3. add sterile litmus solution, sufficient to colour the milk a deep lavender. 4. tube, sterilise, etc., as for milk. ~nutrose agar (eyre).~-(this is a modification of the well known drigalski-conradi medium originally introduced for the isolation of b. typhosus). 1. collect 250 c.c. perfectly fresh ox serum (_vide_ blood serum, page 168, steps 1 to 5) and add to it 450 c.c. sterile distilled water. 2. weigh out agar powder, 20 grammes, and emulsify it with 250 c.c. of the cold serum water. 3. weigh out witté's peptone 10 grammes sodium chloride 5 grammes nutrose 10 grammes and dissolve in 200 c.c. of serum water heated to 80° c. 4. mix the agar emulsion and the peptone-nutrose solution in a "tared" flask of 2-litre capacity and add a further 100 c.c. serum water. 5. complete the solution of the various ingredients by bubbling live steam through the flask as in making nutrient agar. 6. add further 250 c.c. serum water. 7. weigh the flask and its contents: then (1045 grammes + weight of flask) minus (weight of flask and its present contents) = weight of fluid required to make up the bulk of the medium to 1 litre. add the requisite amount of sterile distilled water. 8. titrate and estimate the reaction of the medium mass. then standardise to reaction of +2.5. 9. clarify with egg, and filter as for nutrient agar. (in clarifying, after the addition of the egg white the mixture should be in the steamer for full two hours.) 10. after filtration is complete measure the filtrate, and to every 150 c.c. of the medium add: litmus solution (kahlbaum) 20 c.c. krystal violet aqueous solution (1:1000) (b. hoechst) 1.5 c.c. lactose 1.5 grammes 11. tube in quantities of 15 c.c. 12. sterilise in the steamer at 100° c. for thirty minutes on each of three successive days--i. e., by the discontinuous method for three days. ~egg medium (dorset).~-1. prepare 1000 c.c. of a 0.85 per cent. solution of sodium chloride in a stout 2-litre flask. 2. sterilise in the autoclave at 120° c. for twenty minutes. cool to 20° c. 3. take 12 fresh eggs; wash the shells first with water then with undiluted formalin: allow the shells to dry. 4. break the eggs into a sterile graduated cylinder and measure the total volume of the mixed whites and yolks. add one part sterile saline solution to three parts mixed eggs. 5. transfer this mixture to a large wide-mouthed stoppered bottle previously sterilised. add sterile glass beads and shake thoroughly in a mechanical shaker for about thirty minutes, or whip with an egg-whisk. 6. filter through coarse butter muslin into a sterile flask. note.--a few drops of alcoholic solution of basic fuchsin (sufficient to give a definite pink colour), or a few drops of waterproof chinese ink added to the medium at this stage facilitates the subsequent "fishing" of colonies. 7. tube in quantities of 10 c.c. 8. solidify in the sloping position in the inspissator at 75° c. for one hour. 9. place the tubes for forty-eight hours in the incubator at 37° c., and eliminate any contaminated tubes. to prevent drying, 0.5 c.c. glycerine bouillon (see page 209) may be added to each tube between steps 8 and 9. 10. cap those tubes of media which remain sterile with india-rubber caps and store for future use. ~potato.~-1. choose fairly large potatoes, wash them well, and scrub the peel with a stiff nail-brush. 2. peel and take out the eyes. 3. remove cylinders from the longest diameter of each potato by means of an apple-corer or a large cork-borer (i. e., one of about 1.4 cm. diameter). the reaction of the fresh potato is strongly acid to phenolphthalein. if, therefore, the potatoes are required to approximate +10, as for the cultivation of some of the vibrios, the cylinders should be soaked in a 1 per cent. solution of sodium carbonate for thirty minutes. 4. cut each cylinder obliquely from end to end, forming two wedge-shaped portions. 5. place a small piece of sterilised cotton-wool, moistened with sterile water, at the bottom of a sterile test-tube; insert the potato wedge into the tube so that its base rests upon the cotton-wool. now plug the tube with cotton-wool (fig. 111). 6. sterilise in the steamer at 100° c. for twenty minutes on each of _five_ consecutive days. [illustration: fig. 111.--potato tube.] note.--the cork borer reserved for cutting the potato cylinders should be silver electro-plated both inside and out, and the knife used for dividing the cylinders should be of silver or silver plated. when these precautions are adopted the potato wedges will retain their white color and will not show the discoloration so often observed when steel instruments are employed. ~beer wort.~--wort is chiefly used as a medium for the cultivation of yeasts, moulds, etc., both in its fluid form and also when made solid by the addition of gelatine or agar. the wort is prepared as follows: 1. weigh out 250 grammes crushed malt and place in a 2-litre flask. 2. add 1000 c.c. distilled water, heated to 70° c., and close the flask with a rubber stopper. 3. place the flask in a water-bath regulated to 60°c. and allow the maceration to continue for one hour. 4. strain through butter muslin into a clean flask and heat in the steamer for thirty minutes. 5. filter through swedish filter paper. 6. tube in quantities of 10 c.c. or store in flasks. 7. sterilise in the steamer at 100° c. for twenty minutes on each of three consecutive days. the natural reaction of the wort should _not_ be interfered with. note.--it is sometimes more convenient to obtain "_unhopped_"[6] beer wort direct from the brewery. in this case it is diluted with an equal quantity of distilled water, steamed for an hour, filtered, filled into sterile flasks or tubes, and sterilised by the discontinuous method. ~wort gelatine.~-1. measure out wort (prepared as above), 900 c.c., into a sterile flask. 2. weigh out gelatine, 100 grammes (= 10 per cent.), and add it to the wort in the flask. 3. bubble live steam through the mixture for ten minutes, to dissolve the gelatine. 4. cool to 60°c.; clarify with egg as for nutrient gelatine (_vide_ page 164). 5. filter through papier chardin. 6. tube, and sterilise as for nutrient gelatine. ~wort agar.~-1. measure out wort (as above), 700 c.c., into a sterile flask. 2. weigh out powdered agar, 20 grammes; mix into a smooth paste with 200 c.c. of cold wort and add to the wort in the flask. 3. bubble live steam through the mixture for twenty minutes, to dissolve the agar. 4. cool to 60° c.; clarify with egg as for nutrient agar (_vide_ page 167). 5. filter through papier chardin, using the hot-water funnel. 6. tube, and sterilise as for nutrient agar. ~peptone water (dunham).~-1. weigh out witté's peptone, 10 grammes, and salt, 5 grammes, and emulsify with about 250 c.c. of distilled water previously heated to 60° c. 2. pour the emulsion into a litre flask and make up to 1000 c.c. by the addition of distilled water. 3. heat in the steamer at 100° c. for thirty minutes. 4. filter through swedish filter paper. 5. tube in quantities of 10 c.c. each. 6. sterilise in the steamer at 100° c. for twenty minutes on each of three consecutive days. ~"sugar" or "carbohydrate" media.~-formerly the ability of bacteria to induce hydrolytic changes in carbohydrate substances was observed only in connection with a few well-defined sugars, but of recent years it has been shown that when using litmus as an indicator these so-called "fermentation reactions" facilitate the differentiation of closely allied species, and the list of substances employed in this connection has been considerably extended. the media prepared with them are now no longer regarded as special, but are comprised in the "stock media" of the laboratory. the chief of these substances are the following, arranged in accordance with their chemical constitution: _monosaccharides_ dextrose (glucose), lævulose, galactose, mannose, arabinose, xylose. _disaccharides_ maltose, lactose, saccharose. _trisaccharides_ raffinose (mellitose). _polysaccharides_ dextrin, inulin, starch, glycogen, amidon. _glucosides_ amygdalin, coniferin, salicin, helicin, phlorrhizin. _polyatomic alcohols_ _trihydric_, glycerin. _tetrahydric_, erythrite. _pentahydric_, adonite. _hexahydric_, dulcite, (dulcitol or melampirite), isodulcite (rhamnose), mannite (mannitol), sorbite (sorbitol), inosite. these substances should be obtained from kahlbaum (of berlin); in the pure form, and when possible as large crystals, and the method of preparing a medium containing either of them may be exemplified by describing dextrose solution. ~dextrose solution.~-1. weigh out peptone 20 grammes glucose 10 grammes and grind together in a mortar; then emulsify in 100 c.c. of distilled water heated to 60° c. 2. place in a flask and add distilled water 850 c.c. 3. steam in the steamer at 100° c. for twenty minutes to dissolve the peptone and glucose. 4. add kubel-tiemann litmus solution (kahlbaum) 50 c.c. (the substances enumerated above react acid to phenolphthalein, but variously toward the neutral litmus solution. to such as react acid, add very cautiously n/1 sodium hydrate solution to the medium in bulk until the neutral tint has returned). 5. fill into tubes in which have previously been placed the inverted durham's gas tubes. 6. sterilise in the steamer at 100° c. for _twenty minutes_ on each of three successive days. note.--on no account should these media be sterilised in the autoclave, as temperatures above 100° c. themselves induce hydrolytic changes in the substances in question. it is equally important that the twenty minutes should not be exceeded in sterilisation, as neglect of this precaution may discolour the litmus or lead to the production of yellowish tints when the tubes are subsequently inoculated with acid-forming bacteria. ~neutral litmus solution.~ the most satisfactory is the kubel-tiemann, prepared by kahlbaum. it can however be made in the laboratory as follows: 1. weigh out commercial litmus 50 grammes, and place in a well stoppered 500 c.c. bottle; measure out and add 300 c.c. alcohol 95 per cent. 2. shake well at least once a day for seven days--the alcohol acquires a green colour. 3. decant off the green alcohol and fill a further 300 c.c. 95 per cent. alcohol into the bottle and repeat the shaking. 4. repeat this process until on adding fresh alcohol the fluid only becomes tinged with violet. 5. pour off the alcohol, leaving the litmus as dry as possible. connect up the bottle to an air pump and evaporate off the last traces of alcohol. 6. transfer the dry litmus to a litre flask, measure in 600 c.c. distilled water and allow to remain in contact 24 hours with frequent shakings. 7. filter the solution into a clean flask and add one or two drops of pure concentrated sulphuric acid until the litmus solution is distinctly wine-red in colour. 8. add excess of pure solid baryta and allow to stand until the reaction is again alkaline. 9. filter. 10. bubble co_{2} through the solution until reaction is definitely acid. 11. sterilise in the steamer at 100° c. for thirty minutes on each of three consecutive days. this sterilises the solution and also drives off the carbon dioxide, leaving the solution neutral. ~media for anaerobic cultures.~ in addition to the foregoing media, all of which can be, and are employed in the cultivation of anaerobic bacteria, certain special media containing readily oxidised substances are commonly used for this purpose. the principal of these are as follows: ~bile salt broth (macconkey).~- 1. weigh out witté's peptone, 20 grammes (= 2 per cent.), and emulsify with 200 c.c. distilled water previously warmed to 60°c. 2. weigh out sodium taurocholate (commercial), 5 grammes (= 0.5 per cent.), and glucose, 5 grammes (= 0.5 per cent.), and dissolve in the peptone emulsion. 3. wash the peptone emulsion into a flask with 800 c.c. distilled water, and heat in the steamer at 100° c. for twenty minutes. 4. filter through swedish filter paper into a sterile flask. 5. add sterile litmus solution sufficient to colour the medium to a deep purple, usually 13 per cent. required. 6. fill, in quantities of 10 c.c., into tubes containing small gas tubes (_vide_ fig. 104, page 161). sterilise in the steamer at 100° c. for twenty minutes on each of three consecutive days. ~glucose formate bouillon (kitasato).~- 1. measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1 to 6). 2. weigh out glucose, 20 grammes (= 2 per cent.), sodium formate, 4 grammes (= 0.4 per cent.), and dissolve in the fluid. 3. tube, and sterilise as for bouillon. ~glucose formate gelatine (kitasato).~- 1. prepare nutrient gelatine (_vide_ page 164, sections 1 to 7) and measure out 1000 c.c. 2. weigh out glucose, 20 grammes (= 2 per cent.), and sodium formate, 4 grammes (= 0.4 per cent.), and dissolve in the hot gelatine. 3. filter through papier chardin. 4. tube, and sterilise as for nutrient gelatine. ~glucose formate agar (kitasato).~- 1. prepare nutrient agar (_vide_ page 167, sections 1 to 8). measure out 1000 c.c. 2. weigh out glucose, 20 grammes (= 2 per cent.), sodium formate, 4 grammes (= 0.4 per cent.), and dissolve in the agar. 3. tube, and sterilise as for nutrient agar. ~sulphindigotate bouillon (weyl).~- 1. measure out nutrient bouillon (_vide_ page 163, sections 1 to 6 1000 c.c.). 2. weigh out glucose, 20 grammes (= 2 per cent.), sodium sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in the fluid. 3. tube, and sterilise as for bouillon. ~sulphindigotate gelatine (weyl).~- 1. prepare nutrient gelatine (_vide_ page 164, sections 1 to 7). measure out 1000 c.c. 2. weigh out glucose, 20 grammes (= 2 per cent.), and sodium sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in the hot gelatine. 3. filter through papier chardin. 4. tube, and sterilise as for nutrient gelatine. ~sulphindigotate agar.~- 1. prepare nutrient agar (_vide_ page 167, sections 1 to 8). measure out 1000 c.c. 2. weigh out glucose, 20 grammes (= 2 per cent.), sodium sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in the hot agar. 3. tube, and sterilise as for nutrient agar. note.--the sulphindigotate media are of a blue colour, which during the growth of anaerobic bacteria is oxidised and decolourised to a light yellow. footnotes: [4] this figure is obtained by adding together 1 litre water, 1000 grammes; 10 per cent. gelatine, 100 grammes; 1 per cent. peptone, 10 grammes; 0.5 per cent. salt, 5 grammes; total, 1115 grammes. modifications of the above process, as to quantities and percentages, will require corresponding alterations of the figures. the average weight of a measured litre of 10 per cent. nutrient gelatine when prepared in this way _after filtration_ is 1080 grammes. [5] this figure is obtained by adding together 1 litre of water (meat extract), 1000 grammes; 2 per cent. agar, 20 grammes; 1 per cent. peptone, 10 grammes; 0.5 per cent. salt, 5 grammes--total 1035 grammes. modifications of the process as to quantities or percentages will necessitate corresponding alterations in the calculated medium figure. the average weight of a measured litre of 2 per cent. agar when prepared in this way, _after filtration_, is 1010.5 grammes. [6] "hopped" wort exerts a toxic effect upon many bacteria, including the lactic acid bacteria. xii. special media. in this chapter are collected a number of media which have been elaborated by various workers for special purposes, grouped together under headings which indicate their chief utility. in many instances the name of the originator of the medium is given, but without reference to his original instructions, since these are in many cases inadequate to the requirements of the isolated worker, who would probably fail to reproduce the medium in a form giving the results attributed to it by its author. such modifications have therefore been introduced as make for uniformity between the different batches of media. a considerable number of coloured media, chiefly intended for work with intestinal bacteria, have been included; but beyond the fact that the author's modification of the drigalski-conradi medium has been included amongst the routine media of the laboratory, no comment has been made upon their relative values, since only by observation and practice can the skill necessary to utilise their full value be acquired. the instructions as to sterilisation are rarely given in full; the routine method of exposure in the steam steriliser at 100° c. (without pressure) for twenty minutes on each of three successive days for all fluid media, and thirty minutes on each of three successive days for all liquefiable or solid media must be carried out; and only when these general rules are to be departed from are further details given. _media for the study of the chemical composition of bacteria._ ~asparagin medium (uschinsky).~-1. weigh out and mix asparagin 3.4 grammes ammonium lactate 10.0 grammes sodium chloride 5.0 grammes magnesium sulphate 0.2 gramme calcium chloride 0.1 gramme acid potassium phosphate (kh_{2}po_{4}) 1.0 gramme 2. dissolve the mixture in distilled water 1000 c.c. 3. add glycerine, 40 c.c. 4. tube, and sterilise as for nutrient bouillon. ~asparagin medium (frankel and voges).~-1. weigh out and mix asparagin 4 grammes sodium phosphate, (na_{2}hpo_{4}) 12oh 2 grammes ammonium lactate 6 grammes sodium chloride 5 grammes and dissolve in distilled water 1000 c.c. 2. tube, and sterilise as for nutrient bouillon. note.--either of the above asparagin media, after the addition of 10 per cent. gelatine or 1.5 per cent. agar, may be advantageously employed in the solid condition. ~proteid free broth (uschinsky).~-1. weigh out and mix calcium chloride 0.1 gramme magnesium sulphate 0.2 gramme acid potassium phosphate (kh_{2}po_{4}) 2.0 grammes potassium aspartate 3.0 grammes sodium chloride 5.0 grammes ammonium lactate 6.0 grammes 2. dissolve the mixture in distilled water 1000 c.c. 3. add glycerine 30 c.c. 4. tube and sterilise as for nutrient broth. _media for the study of biochemical reaction._ ~inosite-free media--bouillon (durham).~-1. prepare meat extract, 1000 c.c. (_vide_ page 148), from bullock's heart which has been "hung" for a couple of days. 2. prepare nutrient bouillon (+10), 1000 c.c. (_vide_, page 161), from the meat extract, and store in 1-litre flask. 3. inoculate the bouillon from a pure cultivation of the b. lactis aerogenes, and incubate at 37° c. for forty-eight hours. 4. heat in the steamer at 100° c. for twenty minutes to destroy the bacilli and some of their products. 5. estimate the reaction of the medium and if necessary restore to +10. 6. inoculate the bouillon from a pure cultivation of the b. coli communis and incubate at 37° c. for forty-eight hours. 7. heat in the steamer at 100° c. for twenty minutes. now fill two fermentation tubes with the bouillon, tint with litmus solution, and sterilise; inoculate with b. lactis aerogenes. if no acid or gas is formed, the bouillon is in a sugar-free condition; but if acid or gas is present, again make the bouillon in the flask +10, reinoculate with one or other of the above-mentioned bacteria, and incubate; then test again. repeat this till neither acid nor gas appears in the medium when used for the cultivation of either of the bacilli referred to above. 8. after the final heating, stand the flask in a cool place and allow the growth to sediment. filter the supernatant broth through swedish filter paper. if the filtrate is cloudy, filter through a porcelain filter candle. 9. tube, and sterilise as for bouillon. bouillon prepared in the above-described manner will prove to be absolutely sugar-free; and from it may be prepared nutrient sugar-free gelatine or agar, by dissolving in it the required percentage of gelatine or agar respectively and completing the medium according to directions given on pages 166 and 167. the most important application of inosite-free bouillon is its use in the preparation of sugar bouillons, whether glucose, maltose, lactose, or saccharose, of exact percentage composition. ~sugar (dextrose) bouillon.~-1. measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1 to 6) or sugar-free bouillon (_vide supra_). 2. weigh out glucose (anhydrous), 20 grammes (= 2 per cent.), and dissolve in the fluid. 3. tube, and sterilise as for bouillon. ordinary commercial glucose serves the purpose equally well, but is not recommended, as during the process of sterilisation it causes the medium to gradually deepen in colour. note.--in certain cases a corresponding percentage of lactose, maltose, or saccharose is substituted for glucose. ~sugar gelatine.~-1. prepare nutrient gelatine (_vide_ page 164, sections 1 to 7). measure out 1000 c.c. 2. weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the hot gelatine. 3. filter through papier chardin. 4. tube, and sterilise as for nutrient gelatine. ~sugar agar.~-1. prepare nutrient agar (_vide_ page 167, sections 1 to 8). measure out 1000 c.c. 2. weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the clear agar. 3. tube, and sterilise as for nutrient agar. note.--other "sugar" media are prepared by substituting a corresponding percentage of lactose, maltose (or any other of the substances referred to under "sugar media," page 177) for the glucose. ~iron bouillon.~-1. measure out nutrient bouillon, 1000 c.c. (_vide_ page 141, sections 1 to 6). 2. weigh out ferric tartrate, 1 gramme (= 0.1 per cent.), and dissolve it in the bouillon. 3. tube, and sterilise as for bouillon. note.--the lactate of iron may be substituted for the tartrate. ~lead bouillon.~-1. measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1 to 6). 2. weigh out lead acetate, 1 gramme (= 0.1 per cent.), and dissolve it in the bouillon. 3. tube, and sterilise as for bouillon. ~nitrate bouillon.~-1. measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1 to 6). 2. weigh out potassium nitrate, 5 grammes (= 0.5 per cent.), and dissolve it in the bouillon. 3. tube, and sterilise as for bouillon. note.--the nitrate of sodium or ammonium may be substituted for that of potassium, or the salt may be added in the proportion of from 0.1 to 1 per cent. to meet special requirements. ~iron peptone solution (pakes).~-1. weigh out peptone, 30 grammes, and emulsify it with 200 c.c. tap water, previously heated to about 60°c. 2. wash the emulsion into a litre flask with 800 c.c. tap water. 3. weigh out salt, 5 grammes, and sodium phosphate, 3 grammes, and dissolve in the mixture in the flask. 4. heat the mixture in the steamer at 100° c. for thirty minutes, to complete the solution of the peptone, and filter into a clean flask. 5. fill into tubes in quantities of 10 c.c. each. 6. add to each tube 0.1 c.c. of a 2 per cent. neutral solution of ferric tartrate. (a yellowish-white precipitate forms.) 7. sterilise as for nutrient bouillon. ~lead peptone solution.~-prepare as for iron peptone solution but in step 6 substitute 0.1 c.c. of a 1 per cent. neutral aqueous solution of lead acetate. ~nitrate peptone solution (pakes).~-1. weigh out witté's peptone, 10 grammes, and emulsify it with 200 c.c. ammonia-free distilled water previously heated to 60°c. 2. wash the emulsion into a flask and make up to 1000 c.c., with ammonia-free distilled water. 3. heat in the steamer at 100° c. for twenty minutes. 4. weigh out sodium nitrate, 1 gramme, and dissolve in the contents of the flask. 5. filter through swedish filter paper. 6. tube, and sterilise as for nutrient bouillon. ~litmus bouillon.~-1. measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1 to 6). 2. add sufficient sterile litmus solution to tint the medium a dark lavender colour. (media rendered +10 will usually react very faintly alkaline or occasionally neutral to litmus.) 3. tube, and sterilise as for bouillon. ~rosolic acid peptone solution.~-1. weigh out rosolic acid (corallin), 0.5 gramme, and dissolve it in 80 per cent. alcohol, 100 c.c. keep this as a stock solution. 2. measure out peptone water (dunham), 100 c.c., and rosolic acid solution, 2 c.c., and mix. 3. heat in the steamer at 100° c. for thirty minutes. 4. filter through swedish filter paper. 5. tube, and sterilise as for nutrient bouillon. ~capaldi-proskauer medium, no. i.~-1. weigh out and mix sodium chloride 2.0 grammes magnesium sulphate 0.1 gramme calcium chloride 0.2 gramme monopotassium phosphate 2.0 grammes 2. dissolve in water 1000 c.c. in a 2-litre flask 3. weigh out and mix asparagin 2 grammes mannite 2 grammes and add to contents of flask. 4. measure out 25 c.c. of the solution and titrate it against decinormal sodic hydrate, using litmus as the indicator. control the result and estimate the amount of sodic hydrate necessary to be added to render the remainder of the solution neutral to litmus. add this quantity of sodic hydrate. 5. filter. 6. add litmus solution 47.5 c.c. (= 5 per cent.). 7. tube, and sterilise as for nutrient bouillon. ~capaldi-proskauer medium no. ii.~-1. weigh out and mix peptone 20 grammes mannite 1 gramme 2. dissolve in water 1000 c.c. in a 2-litre flask. 3. neutralise to litmus as in no. i (_vide supra_, step 4). 4. filter. 5. add litmus solution 47.5 c.c. (= 5 per cent.). 6. tube, and sterilise as for nutrient bouillon. ~urine media. bouillon.~-1. collect freshly passed urine in sterile flask. 2. place the flask in the steamer at 100° c. for thirty minutes. 3. filter through two thicknesses of swedish filter paper. 4. tube, and sterilise as for nutrient bouillon. (leave the reaction unaltered.) ~urine gelatine.~-1. collect freshly passed urine in sterile flask. 2. take the specific gravity, and, if above 1010, dilute with sterile water until that gravity is reached. 3. estimate (with control) at the boiling-point, and note the reaction of the urine. 4. weigh out gelatine, 10 per cent., and add to the urine in the flask. 5. heat in the steamer at 100° c. for one hour to dissolve the gelatine. 6. estimate the reaction and add sufficient caustic soda solution to restore the reaction of the medium mass to the equivalent of the original urine. 7. cool to 60° c. and clarify with egg as for nutrient gelatine (_vide_ page 166). 8. filter through papier chardin. 9. tube, and sterilise as for nutrient gelatine. ~urine gelatine (heller).~-1. collect freshly passed urine in sterile flask. 2. filter through animal charcoal to remove part of the colouring matter. 3. take the specific gravity, and if above 1010, dilute with sterile water till this gravity is reached. 4. add witté's peptone, 1 per cent.; salt, 0.5 per cent.; gelatine, 10 per cent. 5. heat in the steamer at 100° c. for one hour, to dissolve the gelatine, etc. 6. add normal caustic soda solution in successive small quantities, and test the reaction from time to time with litmus paper, until the fluid reacts faintly alkaline. 7. cool to 60° c. and clarify with egg as for nutrient gelatine (_vide_ page 166). 8. filter through papier chardin. 9. tube, and sterilise as for nutrient gelatine. ~urine agar.~-1. collect freshly passed urine in sterile flask. 2. take the specific gravity and if above 1010, dilute with sterile water till this gravity is reached. 3. weigh out 1.5 per cent. or 2 per cent. powdered agar, and add it to the urine. 4. heat in the steamer at 100° c. for ninety minutes to dissolve the agar. 5. cool to 60° c. and clarify with egg as for nutrient agar (_vide_ page 168). 6. filter through papier chardin, using the hot-water funnel. 7. tube, and sterilise as for nutrient agar. (leave the reaction unaltered.) ~serum sugar media (hiss).~-in these media the fermentation of carbohydrate substance by bacterial action is indicated by the coagulation of the serum proteids in addition to the production of an acid reaction. ~serum dextrose water (hiss).~-1. measure out into a litre flask serum water (see page 170) 1000 c.c. 2. weigh out dextrose 10 grammes and dissolve in the serum water. 3. filter through swedish filter paper. 4. measure out and add to the medium litmus solution (kahlbaum) 50 c.c. 5. tube in quantities of 10 c.c. and sterilise in the steamer at 100° c. for twenty minutes on each of three successive days. lævulose, galactose, maltose, lactose, etc., can be substituted in similar amounts for dextrose and the medium completed as above. ~omeliansky's nutrient fluid~ (_for cellulose fermenters_).-1. weigh out and mix potassium phosphate 4.0 grammes magnesium sulphate 2.0 grammes ammonium sulphate 4.0 grammes sodium chloride 0.25 gramme 2. dissolve in distilled water 4000 c.c. 3. flask in quantities of 250 c.c. 4. weigh out and add 5 grammes precipitated chalk to each flask. 5. sterilise in the steamer at 100° c. for twenty minutes on each of three successive days. _media for the study of chromogenic bacteria._ ~milk rice (eisenberg).~-1. measure out nutrient bouillon, 70 c.c., and milk, 210 c.c., and mix thoroughly. 2. weigh out rice powder, 100 grammes, and rub it up in a mortar with the milk and broth mixture. 3. fill the paste into sterile capsules, spreading it out so as to form a layer about 0.5 cm. thick, over the bottom of each. 4. heat over a water-bath at 100° c. until the mixture solidifies. 5. replace the lids of the capsules. sterilise in the steamer at 100° c. for thirty minutes on each of three consecutive days. (a solid medium of the colour of _café au lait_ is thus produced.) ~milk rice (soyka).~-1. measure out nutrient bouillon, 50 c.c., and milk, 150 c.c., and mix thoroughly. 2. weigh out rice powder, 100 grammes, and rub it up in a mortar with the milk and broth mixture. 3. fill the paste into sterile capsules, to form a layer over the bottom of each. 4. replace the lids of the capsules. 5. sterilise in the steamer at 100° c. for thirty minutes on each of three consecutive days. (a pure white, opaque medium is thus formed.) _media for the study of phosphorescent and photogenic bacteria._ ~fish bouillon.~-1. weigh out herring, mackerel, or cod, 500 grammes, and place in a large porcelain beaker (or enamelled iron pot). 2. weigh out sodium chloride, 26.5 grammes; potassium chloride, 0.75 gramme; magnesium chloride, 3.25 grammes; and dissolve in 500 c.c. distilled water. add the solution to the fish in the beaker. 3. place the beaker in a water-bath and proceed as in preparing meat extract--i. e., heat gently at 40° c. for twenty minutes, then rapidly raise the temperature to, and maintain at, the boiling-point for ten minutes. 4. strain the mixture through butter muslin into a clean flask. 5. weigh out peptone, 5 grammes, and emulsify with about 200 c.c. of the hot fish water; incorporate thoroughly with the remainder of the fish water in the flask. 6. heat in the steamer at 100° c. for twenty minutes to complete the solution of the peptone. 7. filter through swedish filter paper. 8. when the fish bouillon is cold, if it is to be used as fluid medium, make up to 1000 c.c. by the addition of distilled water. if, however, it is to be used as the basis for agar or gelatine media store it in the "double strength" condition. 9. tube and sterilise as for nutrient bouillon. as an alternative method "marvis" fish food (16 grammes) may be substituted for the 500 grammes of fresh fish. ~fish gelatine.~-1. measure out double strength fish bouillon, 500 c.c., into a "tared" 2-litre flask. 2. add sheet gelatine, 100 grammes, cut into small pieces. 3. bubble live steam through the mixture for fifteen minutes to dissolve the gelatine. 4. weigh the flask and its contents; adjust the weight to the calculated figure for one litre of medium (1135.5 grammes) by the addition of distilled water at 100° c. (_vide_ page 166). 5. cool to below 60°c., and clarify with egg. 6. filter through papier chardin. 7. tube, and sterilise as for nutrient gelatine. shake well after the final sterilisation, to aerate the medium. ~fish gelatine-agar.~-1. weigh out powdered agar, 5 grammes, and emulsify it with 200 c.c. double strength fish bouillon. 2. wash the emulsion into a "tared" 2-litre flask with 300 c.c. fish bouillon. 3. weigh out sheet gelatine, 70 grammes, cut it into small pieces and add it to the contents of the flask. 4. bubble live steam through the mixture to dissolve the gelatine and agar. 5. weigh the flask and contents. adjust the weight to the calculated figure for one litre of medium (1110.5 grammes) by the addition of distilled water at 100° c. (_vide_ page 166). 6. cool to below 60° c. and clarify with egg. 7. filter through papier chardin. 8. tube, and sterilise as for nutrient gelatine. shake well after the final sterilisation, to aerate the medium. _media for the study of yeasts and moulds._ ~pasteur's solution.~-(reaction alkaline). 1. weigh out and mix the ash from 10 grammes of yeast; ammonium tartrate, 10 grammes; cane sugar, 100 grammes. 2. dissolve the mixture in distilled water, 1000 c.c. 3. tube or flask, and sterilise as for nutrient bouillon. ~yeast water (pasteur).~-1. weigh out pressed yeast, 75 grammes; place in a 2-litre flask and add 1000 c.c. distilled water. 2. heat in the steamer at 100° c. for thirty minutes. 3. filter through papier chardin. 4. tube or flask, and sterilise as for nutrient bouillon. ~cohn's solution.~-1. weigh out and mix acid potassium phosphate (kh_{2}po_{4}) 5.0 grammes calcium phosphate 0.5 gramme magnesium sulphate 5.0 grammes ammonium tartrate 10.0 grammes and dissolve in distilled water 1000 c.c. 2. tube, or flask and sterilise as for nutrient bouillon. ~naegeli's solution.~-1. weigh out and mix dibasic potassium phosphate (k_{2}hpo_{4}) 1.0 gramme magnesium sulphate 0.2 gramme calcium chloride 0.1 gramme ammonium tartrate 10.0 grammes and dissolve in distilled water 1000 c.c. 2. tube or flask; sterilise as for nutrient bouillon. ~plaster-of-paris discs.~-1. take large corks, 2.5 cm. diameter, and roll a piece of stiff note-paper round each, so that about a centimetre projects as a ridge above the upper surface of the cork, and secure in position with a pin (fig. 112). 2. mix plaster-of-paris into a stiff paste with distilled water, and fill each of the cork moulds with the paste. 3. when the plaster has set, remove the paper from the corks, and raise the plaster discs. 4. place the plaster discs on a piece of asbestos board and sterilise by exposing in the hot-air oven to 150° c. for half an hour. [illustration: fig. 112.--cork and paper mould for plaster-of-paris disc.] 5. remove the sterile discs from the oven by means of sterile forceps, place each inside a sterile capsule, and moisten with a little sterile water. 6. sterilise in the steamer at 100° c. for thirty minutes on each of three consecutive days. ~gypsum blocks (engel and hansen).~-these are in the form of truncated cones and for their preparation small tin moulds are required, each having a diameter of 5.5 cm. at the base and 4 cm. at the truncated apex. the height (or depth) of a mould is 4.5 to 5 cm. 1. mix powdered calcined gypsum into a stiff paste with distilled water. 2. fill the paste into the moulds and allow it to set and dry by exposure to air. 3. remove the block from the mould and transfer it to a double glass dish of adequate size (7 cm. diameter × 7 cm. high). 4. sterilise block in its dish for one hour in the hot-air oven at 115°c. 5. carefully open the dish and add sterile distilled water to moisten the block and form a layer in the bottom of the dish 1 cm. deep. ~wine must.~--(wine must is obtained from sicily, in hermetically sealed tins, in a highly concentrated form--as a thick syrup--but not sterilised.) 1. weigh out "wine must," 200 grammes, place in a 2-litre flask and add distilled water, 800 c.c. 2. weigh out ammonium tartrate, 5 grammes, and add to the dilute must. 3. place the flask in a water-bath regulated to 60° c. for one hour and incorporate the mixture thoroughly by frequent shaking. 4. filter through papier chardin. 5. tube, and sterilise as for nutrient bouillon. ~wheat bouillon (gasperini).~-1. weigh out and mix wheat flour, 150 grammes; magnesium sulphate, 0.5 gramme; potassium nitrate, 1 gramme; glucose, 15 grammes. 2. dissolve the mixture in 1000 c.c. of water heated to 100°c. 3. filter through papier chardin. 4. tube, and sterilise as for nutrient bouillon. ~bread paste.~-1. grate stale bread finely on a bread-grater. 2. distribute the crumbs in sterile erlenmeyer flasks, sufficient to form a layer about one centimetre thick over the bottom of each. 3. add as much distilled water as the crumbs will soak up, but not enough to cover the bread. 4. plug the flasks and sterilise in the steamer at 100° c. for thirty minutes on each of _four_ consecutive days. _media for the study of parasitic moulds._ ~french proof agar (sabouraud).~-1. weigh out chassaing's peptone, 10 grammes, and emulsify it with 200 c.c. distilled water previously heated to 60°c. 2. weigh out powdered agar, 13 grammes, and emulsify with 200 c.c. cold distilled water. 3. mix the two emulsions and wash into a tared 2-litre flask with 600 c.c. distilled water. 4. bubble live steam through the mixture for twenty minutes, to dissolve the agar. 5. cool to 60° c. and clarify with egg as for nutrient agar (_vide_ page 168). 6. filter through papier chardin, using the hot-water funnel. 7. weigh out _french_ maltose, 40 grammes, and dissolve in the agar. 8. tube, and sterilise as for nutrient agar. ~english proof agar (blaxall).~--substitute witté's peptone for that of chassaing, and proceed as for french proof agar. ~french mannite agar, sabouraud.~--(_for cultivation of favus._) proceed exactly as in preparing french proof agar _vide supra_ substituting mannite (38 grammes) for maltose. _media for the study of milk bacteria._ ~gelatine agar.~--this medium is prepared by adding to nutrient gelatine sufficient agar to ensure the solidity of the medium when incubated at temperatures above 22° c. if it is intended to employ an incubating temperature of 30°c., 10 per cent. gelatine and 0.5 per cent. agar must be dissolved in the meat extract before the addition of the peptone and salt; while for incubating at 37°c., 12 per cent. gelatine and 0.75 per cent. agar must be used. avoid the addition of more agar than is absolutely necessary, otherwise the action upon the medium of such organisms as elaborate a liquefying ferment may be retarded or completely absent. 1. measure out 400 c.c. double strength meat extract into a "tared" 2-litre flask, and add to it gelatine, 100 grammes. 2. weigh out powdered agar, 5 grammes, emulsify with 100 c.c., cold distilled water and add to the contents of the flask. 3. dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes. 4. weigh out peptone, 10 grammes; salt, 5 grammes; emulsify with 100 c.c. double strength meat extract previously heated to 60°c., and add to the contents of the flask. 5. replace in the steamer for fifteen minutes. then adjust the weight to the calculated figure for one litre (in this instance 1120 grammes) by the addition of distilled water at 100°c. 6. estimate the reaction; control the result. then add sufficient caustic soda solution to render the reaction +10. 7. replace in the steamer at 100° c. for twenty minutes. 8. cool to 60° c. clarify with egg as for nutrient agar. 9. filter through papier chardin, using the hot-water funnel. 10. tube, and sterilise as for nutrient agar. ~agar gelatine (guarniari).~-1. measure out double strength meat extract, 400 c.c., into a "tared" 2-litre flask, and add to it gelatine, 50 grammes. 2. weigh out powdered agar, 3 grammes; emulsify with cold distilled water, 50 c.c., and add to the contents of the flask. 3. dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes. 4. weigh out witté's peptone, 25 grammes; salt, 5 grammes, and emulsify with 100 c.c. double strength meat extract previously heated to 60°c., and add to the contents of the flask. 5. replace in the steamer for fifteen minutes. 6. weigh the flask and make up the medium mass to the calculated figure for one litre (1083 grammes) by the addition of distilled water at 100°c. 7. neutralise carefully to litmus paper by the successive additions of small quantities of normal soda solution. 8. replace in the steamer at 100° c. for twenty minutes. 9. cool to 60° c. clarify with egg as for nutrient agar. 10. filter through papier chardin, using the hot-water funnel. 11. tube, and sterilise as for nutrient agar. ~whey gelatine.~-1. curdle fresh milk by warming to 60°c., and adding rennet; filter off the whey into a sterile "tared" flask. 2. estimate and note the reaction of the whey. 3. weigh out gelatine, 10 per cent., and add it to the whey in the flask. 4. bubble live steam through the mixture fifteen minutes to dissolve the gelatine; and weigh. 5. estimate the reaction of the medium mass; then add sufficient caustic soda solution to restore the reaction of the medium mass (i. e., total weight minus weight of flask) to the equivalent of the original whey. 6. cool to 60° c. and clarify with egg as for nutrient gelatine (_vide_ page 166). 7. filter through papier chardin. 8. tube, and sterilise as for nutrient gelatine. ~whey agar.~-1. curdle fresh milk by warming to 60°c., and adding rennet; filter off the whey into a sterile flask. 2. weigh out agar, 1.5 or 2 per cent., and add it to the whey in the flask. 3. bubble live steam through the mixture for twenty minutes, to dissolve the agar. 4. cool to 60°c.; clarify with egg as for nutrient agar (_vide_ page 168). 5. filter through papier chardin, using the hot-water funnel. 6. tube, and sterilise as for nutrient agar. ~litmus whey.~-1. curdle fresh milk by warming to 60° c. and adding rennet. 2. filter off the whey through butter muslin into a sterile flask. 3. neutralise to litmus by the cautious addition of citric acid solution 4 per cent. (do not neutralise with _mineral_ acid.) 4. heat in the steamer at 100° c. for one hour to coagulate all the proteid. (if the whey is cloudy when removed from the steamer allow it to stand for forty-eight hours in the ice chest and then decant off the clear fluid--or filter through a berkefeld filter candle.) 5. filter into a sterile flask. 6. tint the whey with litmus solution to a deep purple red. 7. tube, and sterilise as for milk. ~litmus whey (petruschky).~-1. measure out into a flask fresh milk 1000 c.c. 2. add hydrochloric acid (or glacial acetic acid) 1.5 c.c. and boil. 3. filter off coagulated casein. 4. neutralise to litmus by the addition of n/1 caustic soda solution and boil. whey now cloudy and acid again. 5. again neutralise to litmus by addition of n/10 caustic soda solution. 6. filter. 7. tint the whey with neutral litmus solution to a deep purple colour. 8. tube and sterilise as for milk. ~litmus whey gelatine.~-1. measure out milk 1000 c.c. into a tared 2-litre flask. 2. add hydrochloric acid (or glacial acetic acid) 1.5 c.c. and boil for five minutes. 3. filter off the casein, and make the whey faintly alkaline to litmus. 4. weigh out peptone 10 grammes and emulsify in a few cubic centimeters of the whey and return to the flask. 5. weigh out gelatine 50 grammes add it to the whey in the flask and incorporate the mixture by bubbling through live steam. 6. clear with egg and filter. 7. make the weight of the medium mass to the calculated figure for one litre (1060 grammes) by the addition of distilled water. 8. weigh out dextrose 15 grammes and dissolve in the fluid whey gelatine. 9. add sterile litmus solution to the required tint. 10. tube and sterilise for twenty minutes in steamer at 100°c. on each of five successive days. this medium will remain semi-fluid at the room temperature, and may be used for cultures in the cool or hot incubator. ~litmus whey agar~ is prepared in a similar manner to whey gelatine, with the substitution of 15 grammes of agar for the gelatine. ~malt extract solution (herschell).~-1. measure into a flask distilled water 1000 c.c. 2. weigh out extractum malti (malt extract) 25 grammes and add to distilled water in flask. 3. boil for five minutes, allow to stand, and decant off clear fluid from sediment. 4. tube and sterilise as for nutrient bouillon. _media for the study of earth bacteria, nitrogen fixers._ ~earthy salts agar (lipman and brown).~--(_for the enumeration of soil organisms._) 1. measure out agar 20 grammes. emulsify in 200 c.c. distilled water. 2. wash the agar emulsion into a tared 2-litre flask with 400 c.c. distilled water. 3. weigh out peptone 0.5 gramme. emulsify in 50 c.c. distilled water and add to the contents of the flask. 4. bubble live steam through the mixture for twenty minutes to dissolve the agar. 5. weigh out and mix dextrose 10.0 grammes. potassium phosphate 0.5 gramme. magnesium sulphate 0.2 gramme. potassium nitrate 0.06 gramme. and add to the contents of the flask. 6. adjust the weight of the medium mass to the calculated figure for one litre (1025 grammes) by the addition of distilled water at 100°c. 7. titrate the medium mass and adjust the reaction to +5. 8. cool to 60° c. clarify with egg and filter. 9. tube in quantities of 10 c.c. and sterilise as for nutrient agar. ~beyrinck's solution. i.~--(_for the cultivation of nitrogen fixing organisms._) 1. weigh out and mix 1 gramme potassium hydrogen phosphate, 0.2 gramme magnesium sulphate, and 0.02 gramme sodium chloride. 2. dissolve in water 1000 c.c., in a 2-litre flask. 3. add 1 c.c. of a one per thousand aqueous solution of ferrous sulphate. 4. add 1 c.c. of a one per thousand solution manganese sulphate. 5. weigh out 20 grammes dextrose and add to the contents of the flask (dextrose up to 40 grammes may be used for the different organisms). 6. steam for twenty minutes, filter. 7. tube, and sterilise as for nutrient bouillon. ~beyrinck's solution. ii.~--(_for growth of azobacter._) proceed as in preparing solution no. i, substituting mannite for dextrose in step 5. ~winogradsky's solution (for nitric organisms).~-1. weigh out and mix. potassium phosphate 1.0 gramme magnesium sulphate 0.5 gramme calcium chloride 0.01 gramme sodium chloride 2.0 grammes and dissolve in distilled water 1000 c.c. 2. fill into flasks, in quantities of 20 c.c. and add to each a small quantity of freshly washed magnesium carbonate. 3. sterilise in the steamer at 100° c. for twenty minutes on each of three consecutive days. 4. add to each flask containing 20 c.c. solution, 2 c.c. of a sterile 2 per cent. solution of ammonium sulphate. 5. incubate at 37° c. for forty-eight hours and eliminate any contaminated culture flasks. store the remainder for future use. ~winogradsky's solution (for nitrous organisms).~-1. weigh out and mix ammonium sulphate 1 gramme potassium sulphate 1 gramme and dissolve in distilled water 1000 c.c. 2. add 5 to 10 grammes basic magnesium carbonate, previously sterilised by boiling. 3. fill into flasks and sterilise, etc., as for previous solution. ~silicate jelly (winogradsky).~-1. weigh out and mix ammonium sulphate 0.40 gramme magnesium sulphate 0.05 gramme calcium chloride 0.01 gramme and dissolve in distilled water 50 c.c. label--solution a. 2. weigh out and mix potassium phosphate 0.10 gramme sodium carbonate 0.60 gramme and dissolve in distilled water 50 c.c. label--solution b. 3. weigh out silicic acid 3.4 grammes and dissolve in distilled water 100 c.c. 4. pour the silicic acid solution into a large porcelain basin. 5. mix equal quantities of the solutions a and b; then add successive small quantities of the mixed salts to the silicic acid solution, stirring continuously with a glass rod, until a jelly of sufficiently firm consistence has been formed. 6. spread a layer of this jelly over the bottom of each of several large capsules or "plates." 7. sterilise in the steamer at 100° c. for thirty minutes on each of three consecutive days. _media for the study of water bacteria._ ~naehrstoff agar (hesse and niedner).~--(_for enumeration of water organisms._) 1. weigh out: agar, 12.5 grammes and emulsify in 250 c.c. distilled water. 2. wash the agar emulsion into a tared 2-litre flask with a further 250 c.c. distilled water. 3. dissolve by bubbling live steam through the mixture. 4. emulsify naehrstoff-heyden (albumose) 7.5 grammes in 200 c.c. cold distilled water and add to melted agar. 5. adjust weight of medium mass to the calculated figure for one litre (1020 grammes) by addition of distilled water at 100° c. 6. clarify with white of egg and filter. 7. tube in quantities of 10 c.c. and sterilise in the steamer at 100° c. for twenty minutes on each of three successive days. ~bile salt broth--double strength.~-1. weigh out witté's peptone, 40 grammes, and emulsify with 300 c.c. distilled water previously warmed to 60° c. 2. wash the peptone emulsion into a litre flask with 600 c.c. distilled water. 3. weigh out sodium taurocholate, 10 grammes, and glucose, 10 grammes; dissolve in 100 c.c. distilled water and add to the peptone emulsion in the flask. 4. heat in the steamer at 100° c. for twenty minutes. 5. filter through swedish filter paper into a sterile flask. 6. add sterile neutral litmus solution sufficient to colour the medium to a deep purple. 7. fill into small erlenmeyer flasks in quantities of 25 c.c. 8. sterilise as for nutrient bouillon. _media for the study of plant bacteria._ ~beetroot.~- } ~carrot.~- } are prepared tubes and sterilised in a manner ~turnip.~- } precisely similar to that described for potato. ~parsnip.~- } ~hay infusion.~-1. weigh out dried hay, 10 grammes, chop it up into fine particles and place in a flask. 2. add 1000 c.c. distilled water, heated to 70° c.; close the flask with a solid rubber stopper. 3. macerate in a water-bath at 60° c. for three hours. 4. replace the stopper by a cotton-wool plug, and heat in the steamer at 100° c. for one hour. 5. filter through swedish filter paper. 6. tube, and sterilise as for nutrient bouillon. ~haricot bouillon.~--(_for cultivation of bacteria from tubercles of legumes._) 1. measure out 1000 c.c. distilled water into a 2-litre flask. 2. weigh out 250 grammes haricot beans and add to the water in the flask. 3. weigh out 10 grammes sodium chloride and add to the contents of the flask. 4. add 1 c.c. of a 1 per cent. solution of sodium bicarbonate. 5. place in the steamer at 100° c. for thirty minutes. 6. filter. 7. weigh out 20 grammes saccharose and add to the filtrate. 8. tube, and sterilise as for nutrient bouillon. ~haricot agar.~-1. measure out 400 c.c. distilled water into a "tared" 2-litre flask. 2. weigh out 15 grammes agar and mix into a thick paste with 100 c.c. cold distilled water, and add to the flask. 3. dissolve the agar by bubbling live steam through the mixture as in making nutrient agar. 4. weigh out 250 grammes haricot beans, place in the flask with the agar mixture. 5. add 1 c.c. of 1 per cent. aqueous solution sodium bicarbonate. 6. weigh out 10 grammes sodium chloride and add to the contents of the flask. 7. place in the steamer at 100° c. for thirty minutes. 8. adjust the weight of the medium mass to 1030 grammes (the figure per litre obtained experimentally) by the addition of distilled water at 100° c. 9. cool to 60°c., clarify with egg and filter. 10. weigh out 20 grammes saccharose and add to the contents of the flask. 11. tube, and sterilise as for nutrient agar. ~wood ash agar.~-1. measure 400 c.c. distilled water into a tared 2-litre flask. 2. weigh out 10 grammes agar and make into a thick paste with 100 c.c. cold distilled water. 3. add this agar paste to the distilled water in the flask. 4. dissolve the agar by passing live steam through it, as in preparing nutrient agar. 5. weigh out 5 grammes clean wood ash and place in a second flask containing 200 c.c. distilled water with some sterile glass beads: shake thoroughly in a mechanical shaker for ten minutes. 6. heat in steamer at 100°c., for thirty minutes. 7. after removal from the steamer dry the outside of the flask thoroughly, place it over a bunsen flame and boil for one minute. 8. filter directly into the flask containing the melted agar mixture. 9. weigh out 4 grammes maltose. add to the contents of the flask. 10. adjust the weight of the medium mass to the calculated figure for one litre (1019 grammes) by the addition of distilled water at 100°c. 11. replace the flask in the steamer for twenty minutes, cool to 60°c., and clarify with egg and filter. 12. tube, and sterilise as for nutrient agar. _media for the study of special bacilli._ _b. acnes._ ~oleic acid agar (fleming).~-1. measure out into a sterile stout glass bottle which already contains about 10 sterile glass beads ascitic fluid 250 c.c. 2. weigh out oleic acid 25 grammes and add it to the ascitic fluid in the bottle. 3. emulsify evenly by shaking (either by hand or in a shaking machine) for ten minutes. 4. liquefy and measure out into a flask nutrient agar 750 c.c. then cool to 55°c. 5. mix the oleic acid emulsion with the agar. 6. add 10 c.c. sterile neutral red, 1 per cent. aqueous solution. 7. tube in quantities of 10 c.c., slant, and allow to set. 8. incubate for forty-eight hours at 37° c. and reject any contaminated tubes. store the sterile tubes for future use. _coli-typhoid group._ ~parietti's bouillon.~-1. measure out pure hydrochloric acid, 4 c.c., and add to it carbolic acid solution (5 per cent.), 100 c.c. allow the solution to stand at least a few days before use. 2. this solution is added in quantities of 0.1, 0.2. and 0.3 c.c. (delivered by means of a sterile graduated pipette) to tubes each containing 10 c.c. of previously sterilised nutrient bouillon (_vide_ page 163). 3. incubate at 37° c. for forty-eight hours to eliminate contaminated tubes. store the remainder for future use. ~carbolised bouillon.~-1. prepare nutrient bouillon (_vide_ page 163, sections 1 to 6). measure out 1000 c.c. 2. weigh out carbolic acid, 1 gramme (2.5 or 5 grammes may be needed for special purposes), and dissolve it in the medium. 3. tube, and sterilise as for bouillon. ~carbolised gelatine.~-1. prepare nutrient gelatine (_vide_ page 164, sections 1 to 7). measure out 1000 c.c. 2. weigh out carbolic acid, 5 grammes (= 0.5 per cent.), and dissolve it in the gelatine. 3. filter if necessary through papier chardin. 4. tube, and sterilise as for nutrient gelatine. one or 2.5 grammes of carbolic acid (= 0.1 per cent. or 0.25 per cent.) are occasionally used in place of the 5 grammes to meet special requirements. ~carbolised agar.~-1. prepare nutrient agar (_vide_ page 167, sections 1 to 8). measure out 1000 c.c. 2. weigh out 1 gramme pure phenol and dissolve in the medium. 3. filter if necessary through papier chardin. 4. tube, and sterilise as for nutrient agar. ~litmus gelatine.~-1. prepare nutrient gelatine (_vide_ page 164, sections 1 to 8). 2. add sterile litmus solution, sufficient to tint the medium a deep lavender colour. 3. tube, and sterilise as for nutrient gelatine. ~lactose litmus bouillon (lakmus molke).~-1. weigh out peptone, 4 grammes, and emulsify it with 200 c.c. meat extract (_vide_ page 148), previously heated to 60°c. 2. weigh out salt, 2 grammes, and lactose, 20 grammes, and mix with the emulsion. 3. wash the mixture into a sterile litre flask with 200 c.c. meat extract and add 600 c.c. distilled water. 4. heat in the steamer at 100° c. for thirty minutes, to completely dissolve the peptone, etc. 5. _neutralise carefully to litmus paper_ by the successive additions of small quantities of decinormal soda solution. 6. replace in the steamer for twenty minutes to precipitate phosphates, etc. 7. filter through two thicknesses of swedish filter paper. 8. add sterile litmus solution, sufficient to colour the medium a deep purple. 9. tube, and sterilise as for bouillon. ~lactose litmus gelatine (wurtz).~-1. prepare nutrient gelatine (_vide_ page 164, sections 1 to 4). 2. render the reaction of the medium mass -5. 3. replace in the steamer at 100° c. for twenty minutes. 4. clarify with egg as for gelatine. 5. weigh out lactose, 20 grammes (= 2 per cent.), and dissolve it in the medium. 6. filter through papier chardin. 7. add sufficient sterile litmus solution to colour the medium pale lavender. 8. tube, and sterilise as for nutrient gelatine. ~lactose litmus agar (wurtz).~-1. prepare nutrient agar (_vide_ page 167, sections 1 to 4). 2. render the reaction of the medium mass -5. 3. replace in the steamer at 100° c. for twenty minutes. 4. cool to 60° c. and clarify with egg as for nutrient agar. 5. weigh out lactose, 20 grammes (= 2 per cent.), and dissolve it in the medium. 6. filter through papier chardin, using the hot-water funnel. 7. add sterile litmus solution, sufficient to colour the medium a pale lavender. 8. tube, and sterilise as for nutrient agar. ~glycerine potato bouillon.~-1. take 1 kilo of potatoes, wash thoroughly in water, peel, and grate finely on a bread-grater. 2. weigh the potato gratings, place them in a 2-litre flask, and add distilled water in the proportion of 1 c.c. for every gramme weight of potato. allow the flask to stand in the ice-chest for twelve hours. 3. strain the mixture through butter muslin and filter through swedish filter paper into a graduated cylinder. note the amount of the filtrate. 4. place the filtrate in a flask, add an equal quantity of distilled water, and heat in the steam steriliser for sixty minutes. 5. add glycerine, 4 per cent., mix thoroughly, and again filter. 6. tube and sterilise as for nutrient bouillon. ~potato gelatine (elsner).~-1. take 1 kilo of potatoes, wash thoroughly in water, peel, and finally grate finely on a bread-grater. 2. weigh the potato gratings, place them in a 2-litre flask, and add distilled water in the proportion of 1 c.c. for every gramme weight of potato. allow the flask to stand in the ice-chest for twelve hours. 3. strain the mixture through butter muslin, and filter through swedish filter paper into a graduated cylinder. 4. add 15 per cent. gelatine to the potato decoction and bubble live steam through the mixture for ten minutes. 5. estimate the reaction; adjust the reaction of the medium mass to +25. 6. cool the medium to below 60°c.; clarify with egg as for nutrient gelatine (_vide_ page 166). 7. add 1 per cent. potassium iodide (powdered) to the medium. 8. filter through papier chardin. 9. tube and sterilise as for nutrient gelatine. ~aesculin agar.~--(b. coli and allied organisms give black colonies surrounded by black halo.) 1. measure out 400 c.c. distilled water into a tared 2-litre flask. 2. weigh out agar 15 grammes peptone 10 grammes sodium taurocholate 5 grammes and make into a thick paste with 150 c.c. distilled water. 3. add this paste to the distilled water in the flask. 4. dissolve the ingredients by bubbling live steam through the mixture. 5. weigh out aesculin 1.0 gramme ferric citrate 0.5 gramme and dissolve in a second flask containing 100 c.c. distilled water. 6. mix the contents of the two flasks--adjust the weight to the calculated medium figure (in this case 1031.5 grammes) by the addition of distilled water at 100°c. 7. clarify with egg and filter. 8. tube and sterilise as for nutrient agar. ~bile salt agar (macconkey).~-1. weigh out powdered agar, 15 grammes (= 1.5. per cent.), and emulsify with 200 c.c. _cold tap_ water. 2. weigh out peptone, 20 grammes (= 2 per cent.), and emulsify with 200 c.c. _tap_ water previously warmed to 60°c. 3. mix the peptone and agar emulsions thoroughly. 4. weigh out sodium taurocholate, 5 grammes (= 0.5 per cent.), dissolve it in 300 c.c. _tap_ water, and use the solution to wash the agar-peptone emulsion into a tared 2-litre flask. 5. bubble live steam through the mixture for twenty minutes. 6. adjust the weight of the medium mass to the calculated figure for one litre (1040 grammes). 7. cool to 60° c. and clarify with egg as for nutrient agar (_vide_ page 168). 8. filter through papier chardin, using the hot-water funnel. 9. weigh out lactose, 10 grammes (= 1 per cent.), and dissolve it in the agar. if desired, add 5 c.c. of a 1 per cent. (= 0.5 per cent.) aqueous solution of neutral red. 10. tube, and sterilise as for nutrient agar. ~litmus nutrose agar (drigalski-conradi).~-this medium should be prepared in precisely the same manner as the nutrose agar described on page 172 substituting meat extract for serum water, and increasing the percentage of agar added per litre to 3 per cent. ~fuchsin agar (braun).~-1. liquefy and measure out into a sterile flask: nutrient agar 1000 c.c. 2. weigh out: lactose 10 grammes and dissolve in the fluid agar. 3. adjust the reaction to -5 and filter. 4. measure out and mix thoroughly with agar: fuchsin, alcoholic solution 5 c.c. the fuchsin solution is prepared by mixing: fuchsin (basic) 3 grammes. absolute alcohol 60 c.c. allow to stand twenty-four hours, then centrifugalise thoroughly and decant the supernatant fluid into a well-stoppered bottle. 5. measure out and add to the nutrient agar, sodium sulphite, 10 per cent. aqueous solution, freshly prepared 25 c.c. 6. tube and sterilise as for nutrient agar. 7. store in a dark cupboard. ~fuchsin sulphite agar (endo).~-1. liquefy and measure out into a sterile flask: nutrient agar 1000 c.c. 2. weigh out lactose 10 grammes. and dissolve in the fluid agar. 3. adjust the reaction to +3 and filter. 4. measure out and mix thoroughly with the fluid agar. fuchsin, alcoholic solution (_vide supra_) 5 c.c. 5. measure out and add to the medium sodium sulphite, 10 per cent. aqueous solution 25 c.c. 6. tube and sterilise as for nutrient agar. ~brilliant green agar (conradi).~-1. liquefy and measure out into a sterile flask nutrient agar 1000 c.c. 2. adjust reaction to +30 by the addition of normal phosphoric acid; and filter. 3. measure out and mix thoroughly with the fluid medium brilliant green (hoechst) 1 per thousand aqueous solution 6.5 c.c. 4. measure out and add to the medium picric acid (gruebler), 1 per cent. aqueous solution 6.5 c.c. 5. tube and sterilise as for nutrient agar. ~brilliant green bile salt agar (fawcus).~-1. weigh out agar 20 grammes and emulsify in 100 c.c. cold distilled water. 2. wash the emulsion into a "tared" 2-litre flask with 500 c.c. distilled water. 3. dissolve the agar by bubbling live steam through the flask. 4. cool, clarify with egg and filter. 5. weigh out sodium taurocholate 5 grammes peptone 20 grammes and add to the medium in the flask. 6. weigh out lactose 5 grammes and add to the medium in the flask. 7. adjust reaction to +15 and filter if necessary. 8. measure out brilliant green, 1 per thousand aqueous solution 20 c.c. and mix thoroughly with the fluid agar. 9. measure out and add to the medium picric acid, 1 per cent. aqueous solution 20 c.c. 10. tube and sterilise as for nutrient agar. ~china green agar (werbitski).~-1. liquefy and measure out into a sterile flask nutrient agar 1000 c.c. 2. adjust the reaction accurately to +13 and filter. 3. measure out and mix thoroughly with the fluid agar china green 0.2 per cent. aqueous solution 15 c.c. 4. tube and sterilise as for nutrient agar. ~malachite green agar (loeffler).~-1. liquefy and measure out into a sterile flask nutrient agar 1000 c.c. 2. weigh out dextrose 10 grammes. and dissolve in nutrient agar. 3. adjust the reaction to +3, and filter. 4. measure out and mix thoroughly in the fluid agar malachite green, 0.1 per cent. aqueous solution 16 c.c. for ~"weak"~ medium. _4a._ to the filtered agar add malachite green, 2 per cent. aqueous solution 25 c.c. for ~"strong"~ medium. 5. tube and sterilise as for nutrient agar. ~double sugar agar (russell).~-1. liquefy and measure out into a sterile flask nutrient agar 1000 c.c. 2. add 100 c.c. litmus solution to the fluid agar. 3. weigh out and dissolve in the fluid agar. lactose 10 grammes dextrose 10 grammes. 4. render the reaction of the medium neutral to litmus paper by the cautious addition of normal caustic soda. 5. tube in quantities of 10 c.c. and sterilise in the steamer at 100° c. for twenty minutes on each of three successive days. 6. store for use in a cool dark place. _b. diphtheriæ._ ~glycerine blood-serum.~-1. prepare blood-serum as described, page 168, sections 1 to 4. 2. add 5 per cent. pure glycerine. 3. complete as described above for ordinary blood-serum, sections 5 to 7. note.--different percentages of glycerine--from 4 per cent. to 8 per cent.--are used for special purposes. five per cent. is that usually employed. ~blood-serum (loeffler).~-1. prepare nutrient bouillon (_vide_ page 163), using meat extract made from veal instead of beef. 2. add 1 per cent. glucose to the bouillon, and allow it to dissolve completely. 3. now add 300 c.c. clear blood-serum (_vide_ page 168, sections 1 to 4) to every 100 c.c. of this bouillon. 4. fill into sterile tubes and complete as for ordinary blood-serum. ~blood-serum (lorrain smith).~-1. collect blood-serum (_vide_ page 168, sections 1 to 4), as free from hæmoglobin as possible. 2. weigh out 0.15 per cent. sodium hydrate and dissolve it in the fluid (or add 0.375 c.c. of dekanormal soda solution for every 100 c.c. of serum). 3. tube, and stiffen at 100° c. in the serum inspissator. 4. incubate at 37° c. for forty-eight hours to eliminate any contaminated tubes. store the remainder for future use. ~blood serum (councilman and mallory).~-1. collect blood serum in slaughterhouse, coagulate, remove serum and tube (_vide_ page 168). great care must be taken to avoid the inclusion of air bubbles--indeed if only a few tubes are filled at one time, it is a good plan to stand them upright in the receiver of an air pump and to exhaust as completely as possible before transferring to the serum inspissator. 2. heat the tubes in a slanting position in hot-air steriliser at 90° c. till firmly coagulated, say half an hour. 3. sterilise in steam steriliser at 100° c. for 20 minutes on each of three successive days. resulting medium not translucent, but opaque and firm. _b. tuberculosis._ ~egg medium (lubenau).~-this modification of dorset's egg medium (_quod vide_ page 174) is preferred by some for the growth of the tubercle bacillus of the human type. it consists in the addition of one part of 6 per cent. glycerine in normal saline solution, to the egg mixture between steps 4 and 5. ~glycerine bouillon.~-1. measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1 to 6). 2. measure out glycerine, 60 c.c. (= 6 per cent.), and add to the bouillon. 3. tube, and sterilise as for bouillon. ~glycerine agar.~-1. prepare nutrient agar (_vide_ page 167, sections 1 to 8). measure out 1000 c.c. 2. measure out pure glycerine, 60 c.c. (= 6 per cent.), and add to the agar. 3. tube, and sterilise as for nutrient agar. ~glycerine blood-serum.~-1. prepare blood-serum as described, page 168, sections 1 to 4. 2. add 5 per cent. pure glycerine. 3. complete as described above for ordinary blood-serum, sections 5 to 7. note.--different percentages of glycerine--from 4 per cent. to 8 per cent.--are used for special purposes. five per cent. is that usually employed. ~glycerinated potato.~-1. prepare ordinary potato wedges (_vide_ page 174, sections 1 to 4). 2. soak the wedges in 25 per cent. solution of glycerine for fifteen minutes. 3. moisten the cotton-wool pads at the bottom of the potato tubes with a 25 per cent. solution of glycerine. 4. insert a wedge of potato in each tube and replug the tubes. 5. sterilise in the steamer at 100° c. for twenty minutes on each of _five_ consecutive days. ~animal tissue media (frugoni).~-1. take a number of sterile test-tubes 16 × 3 or 4 cm., plugged with cotton wool, and into each insert a 2 cm. length of stout glass tubing (about 1 cm. diameter); fill in glycerine (6 per cent.) bouillon to the upper level of the piece of glass tubing. sterilise in the steamer at 100° c. for twenty minutes on each of three successive days. 2. kill a small rabbit by means of chloroform vapour. 3. under strictly aseptic precautions remove the lungs, liver and other solid organs and transfer them to a sterile double glass dish. 4. with the help of sterile scissors and forceps divide the organs into roughly rectangular blocks 3 × 1.5 × 1 cm. 5. pour into the dish a sufficient quantity of sterile glycerine solution (6 per cent. in normal saline), cover, and allow to stand for one hour. 6. introduce a block of tissue into each tube so that it rests upon the upper end of the piece of glass tubing. (the surface of the tissue will now be kept moist by capillary attraction and condensation). 7. sterilise in the autoclave at 120° c. for thirty minutes. 8. cap the tubes and store them in the ice chest for future use. tissues obtained at postmortems can also be used after preliminary sterilisation by boiling or autoclaving. _media for the study of special cocci._ _diplococcus gonorrhoeæ._ ~ascitic bouillon (serum bouillon).~-1. collect ascitic fluid (pleuritic fluid, hydrocele fluid, etc.), by aspiration directly into sterile flasks, under strictly aseptic precautions. 2. mix the serum with twice its bulk of sterile nutrient bouillon (_vide_ page 163). 3. if considered necessary (on account of the presence of blood, crystals, etc.), filter the serum bouillon through porcelain filter candle. 4. tube, and sterilise in the water bath at 56° c. for half an hour on each of five consecutive days. 5. incubate at 37° c. for forty-eight hours and eliminate contaminated tubes. store the remainder for future use. ~serum agar (heiman).~-1. prepare nutrient agar (_vide_ page 167), to following formula: agar 2.0 per cent. peptone 1.5 per cent. salt 0.5 per cent. meat extract _quantum sufficit._ 2. make reaction of medium + 10. 3. filter; tube in quantities of 6 c.c. 4. sterilise as for nutrient agar. 5. after the third sterilisation cool the tubes to 42°c., and add to each 3 c.c. of sterile hydrocele fluid, ascitic fluid, or pleuritic effusion (previously sterilised, if necessary, by the fractional method); allow the tubes to solidify in a sloping position. 6. when solid, incubate at 37° c. for forty-eight hours, and eliminate any contaminated tubes. store the remainder for future use. ~serum agar (wertheimer).~-1. prepare nutrient agar (_vide_ page 167), to the following formula: agar 2.0 per cent. peptone 2.0 per cent. salt 0.5 per cent. meat extract _quantum sufficit._ 2. make reaction of medium +10. 3. filter; tube in quantities of 5 c.c. 4. sterilise as for nutrient agar. 5. after the last sterilisation cool to 42°c., then add 5 c.c. sterile blood-serum from human placenta (sterilised, if necessary, by the fractional method) to each tube; slope the tubes. 6. when solid, incubate at 37° c. for forty-eight hours, and eliminate any contaminated tubes. store the remainder for future use. ~serum agar (kanthack and stevens).~-1. collect ascitic, pleuritic, or hydrocele fluid in sterile flasks and allow to stand in the ice-chest for twelve hours to sediment. 2. decant 1000 c.c. of the clear fluid into a measuring cylinder and transfer to sterile litre flask. 3. add 0.5 c.c. dekanormal naoh solution for every 100 c.c. serum (_i. e._, 5.0 c.c.), and mix thoroughly. 4. heat in the steamer for twenty minutes. 5. weigh out 15 grammes agar, emulsify in a separate vessel with 200 c.c. of the alkaline fluid previously cooled to about 20°c., and then add to the remainder of the fluid in the flask. 6. bubble live steam through the mixture for twenty minutes to dissolve the agar. 7. filter through papier chardin, using a hot-water funnel. 8. weigh out glucose 10 grammes (= 1 per cent.), and dissolve it in the clear agar. 8a. if desired, add glycerine, 5 per cent., to the clear agar. 9. tube, and sterilise as for nutrient agar. ~serum agar (libman).~-1. prepare nutrient agar (_vide_, page 167) using, however, 1.5 per cent. peptone (that is 15 grammes per litre instead of 10 grammes). 2. adjust the reaction to 0 (i. e., neutral to phenolphthalein). 3. filter and transfer 1000 c.c. liquefied medium to a sterile flask. 4. weigh out dextrose 20 grammes and dissolve in the fluid agar. 5. tube in quantities of 6 c.c.; and sterilise in the steamer at 100° c. for thirty minutes on each of three consecutive days. 6. after the third sterilisation cool to 42° c. and add to each tube 3 c.c. of sterile hydrocele fluid, ascitic fluid or pleuritic effusion (previously sterilised, if necessary, by the fractional method); allow the tubes to solidify in a sloping position. 7. when solid, incubate at 37° c. for forty-eight hours, and eliminate any contaminated tubes. store the remainder for future use. ~egg-albumen, inspissated.~-1. break several fresh eggs (hens', ducks', or turkeys' eggs), and collect the "whites" in a graduated cylinder, taking care to avoid admixture with the yolks. 2. add 40 per cent. distilled water, and incorporate the mixture thoroughly by the aid of an egg-whisk. 3. weigh out 0.15 per cent. sodium hydrate and dissolve it in the fluid (or add the amount of dekanormal caustic soda solution calculated to yield the required percentage of soda in the total bulk of the fluid--i. e., 0.375 c.c. of dekanormal naoh solution per 100 c.c. of the mixture). _3a._ glucose to the extent of 1 to 2 per cent. may now be added, if desired. 4. strain the mixture through butter muslin and filter through a porcelain filter candle into a sterile filter flask. 5. tube, and stiffen at 100° c. in the serum inspissator. 6. incubate at 37° c. for forty-eight hours and eliminate any contaminated tubes; store the remainder for future use. ~egg-albumen (tarchanoff and kolesnikoff).~-1. place unbroken hens' eggs in dekanormal caustic soda solution for ten days. (after this time the white becomes firm like gelatine.) 2. carefully remove the shell and cut the egg into fine slices. 3. wash for two hours in running water. 4. place the egg slices in a large beaker and sterilise in the steamer at 100° c. for one hour. 5. transfer each slice of egg by means of a pair of sterilised forceps to a petri dish or large capsule. 6. sterilise in the steamer at 100° c. for twenty minutes on each of three consecutive days. ~egg albumin broth (lipschuetz).~-1. weigh out egg albumin (extra fine powder, merck). 4 grammes and place in a 2-litre flask with a number of sterile glass beads. 2. measure out distilled water 200 c.c. into a half-litre flask and warm to 37° c. in the incubator. 3. add the water to the flask containing the albumin and beads and dissolve by shaking. 4. add n/10-naoh, 40 c.c. allow the mixture to stand for thirty minutes with frequent shaking. 5. filter through swedish filter paper. 6. sterilise by boiling two or three times at intervals of two hours. 7. add ordinary nutrient bouillon 600 c.c. 8. fill into small erlenmeyer flasks in quantities of 50 c.c. 9. incubate for forty-eight hours at 37°c.--discard any contaminated flasks and store the remainder for future use. ~egg albumin agar.~-1. prepare egg albumin solution as above 1-6. 2. liquefy and measure out ordinary nutrient agar 600 c.c. and add to the egg albumin solution (in place of the nutrient broth). 3. complete as above 8-9. _diplococcus meningitidis intracellularis._ ~ascitic fluid agar (wassermann)~ _synonym_ ~n-as-gar (mervyn gordon).~ 1. liquefy and measure out into a sterile flask: nutrient agar 600 c.c. 2. measure out into a half litre flask distilled water 210 c.c. and add to it ascitic fluid 90 c.c. nutrose 6 grammes 3. heat over a bunsen flame, shaking constantly until the fluid boils, and the nutrose is dissolved. 4. add the nutrose ascitic solution to the fluid agar. 5. heat in the steamer for thirty minutes, then filter. 6. tube and sterilise as for nutrient agar. note.--the finished medium in this case measures 900 c.c. only since inconvenient fractions would be introduced in making up to one litre exactly. _diplococcus pneumoniæ._ ~blood agar (washbourn).~-1. melt up several tubes of nutrient agar (_vide_ page 167) and allow them to solidify in the oblique position. 2. place the tubes, in the horizontal position, in the "hot" incubator for forty-eight hours, to evaporate off some of the condensation water. 3. kill a small rabbit with chloroform and nail it out on a board (as for a necropsy). moisten the hair thoroughly with 2 per cent. solution of lysol. 4. sterilise several pairs of forceps, scissors, etc., by boiling. 5. reflect the skin over the thorax with sterile instruments. 6. open the thoracic cavity by the aid of a fresh set of sterile instruments. 7. open the pericardium with another set of sterile instruments. 8. sear the surface of the left ventricle with a red-hot iron and remove fluid blood from the heart by means of sterile pipettes (e. g., those shown in fig. 13, c). 9. deliver a small quantity of the blood on the slanted surface of the agar in each of the tubes, and allow it to run over the entire surface of the medium. 10. place the tubes in the slanting position and allow the blood to coagulate. 11. return the "blood agar" to the hot incubator for forty-eight hours and eliminate any contaminated tubes. store the remainder for future use. _media for the study of mouth bacteria generally._ ~potato gelatine (goadby).~-1. prepare glycerine potato broth (see page 203, sections 1 to 5). 2. add 10 per cent. gelatine to the potato decoction and bubble live steam through the mixture for ten minutes. 3. estimate the reaction; adjust the reaction of the medium to +5. 4. cool the medium to below 60°c., clarify with egg as for nutrient gelatine. 5. filter through papier chardin. 6. tube, and sterilise as for nutrient gelatine. _media for the study of protozoa._ ~tissue medium (noguchi).~--_for spirochætes (cultivations must be grown anaerobically)._ 1. plug and sterilise test-tubes 20 × 2 cm. 2. kill a small rabbit with chloroform vapour. open the abdomen with all aseptic precautions, remove kidneys and testicles and transfer to a sterile glass dish. cut up the organs with sterile scissors into small pieces--say 4 millimetre cubes. the four organs should yield from 25 to 30 pieces of tissue. 3. drop a small piece of sterile tissue into the bottom of each sterilised tube. 4. take a flask containing about 400 c.c. nutrient agar (+10 reaction), liquefy the medium by heat and cool in a water bath to 50°c. 5. add 200 c.c. ascitic or hydrocele fluid (horse or sheep serum may be employed, but is not so good) to the liquid agar and mix carefully to avoid formation of air bubbles. 6. fill about 20 c.c. of the ascitic agar into each of the sterilised tubes which already contains a piece of sterile rabbit's tissue, stand all the tubes upright in racks or a jar, and allow agar to set. 7. after solidification pour sterile paraffin oil on the surface of the medium in each tube to the depth of 3 centimetres. 8. incubate tubes at 37° c. for several days and discard any which prove to be contaminated. 9. store such tubes as are sterile for future use. xiii. incubators. [illustration: fig. 113.--incubator.] an incubator (fig. 113) consists essentially of a chamber for the reception of cultivations, etc., surrounded by a water jacket, the walls of which are of metal, usually copper, and outside all an asbestos or felt jacket, or wooden casing. the water in the jacket is heated by gas or electricity and maintained at some constant temperature by a thermo-regulator. the cellular incubator (fig. 114) which was made for me[7] some years ago is of the greatest practical utility. here the central cavity is subdivided by five double-walled partitions (in which water circulates in connection with the water tanks at the top and base of the incubator) and again by iron shelves to form twenty-four pigeon holes. into each of these slides an iron drawer 35 cm. long × 12 cm. wide × 22 cm. high forming a self-contained incubator. the drawer is fitted with a wooden form to which is fixed a handle and a numbered label. the thermo-regulating apparatus is the well-known hearson capsule. [illustration: fig. 114.--cellular incubator.] two incubators at least are required in the laboratory, for the cultivation of bacteria the one regulated to maintain a temperature of 37°c., and known as the "hot" incubator; the other, 20° c. to 22°c., and known as the "cool" or "cold" incubator. two other incubators, regulated to 42° c. and 60°c. respectively, whilst not absolutely, necessary very soon justify their purchase. ~thermo-regulators.~--the thermo-regulator is the most essential portion of the incubator, as upon its efficient working depends the maintenance of a constant temperature in the cultivation chamber. it is also used in the fitting up of water and paraffin baths, and for many other purposes. [illustration: fig. 115.--reichert's thermo-regulator.] of the many forms and varieties of thermo-regulator (other than electrical), two only are of sufficiently general use to need mention. in one of these the flow of gas to the gas-jet is controlled by the expansion or contraction of mercury within a glass bulb; in the other, by alterations in the position of the walls of a metallic capsule containing a fluid, the boiling-point of which corresponds to the temperature at which the incubator is intended to act. they are: (a) _reichert's_ (fig. 115), consists of a bulb containing mercury which is to be suspended in the medium, whether air or water, the temperature of which it is desired to regulate. gas enters at a, and passes out to the jet by b. as the temperature rises the mercury expands and cuts off the main gas supply. as the temperature falls the mercury contracts and reopens the narrow tube c. by means of a thumbscrew d (which mechanically raises or lowers the column of mercury irrespective of the temperature) and the aid of a thermometer the apparatus can be set to keep the incubator at any desired temperature. with this form a special gas burner is required, with separate supply of gas to a pilot jet at the side. (b) _hearson's capsule regulator_ consists of a metal capsule hermetically sealed and filled with a liquid which boils at the required temperature, this is adjusted in the interior of the incubator. soldered to the upper side of the capsule is a thick piece of metal having a central cup to receive the lower end of a rigid rod, through which the movements of the walls of the capsule are transmitted to the gas valve fixed outside the incubator. the gas valve or governor is shown in figure 116. a is the inlet for gas, c the outlet to burner heating the water jacket, b d a lever pivoted to standards at g, and acted upon by the capsule, through the rigid rod which enters the socket below the screw p. [illustration: fig. 116.--capsule thermo-regulator.] the construction of the valve is such that, whenever the short arm of the lever b d presses on the disc below the end b, the main supply of gas is entirely cut off. at such times, however, a very small portion of gas passes from a to c, through an aperture inside the valve, the size of which aperture can be adjusted by the screw needle s, hence the gas flame below the incubator is never extinguished. the expansion of the metal walls of the capsule, which takes place upon the boiling of its contents, provides the motive force, transmitted through the rigid rod to raise the long arm of the lever b d, and as this expansion only takes place at a predetermined temperature, the lever will only be acted upon when the critical temperature is reached, no sensible effect being produced at even 1° c. below that at which the capsule is destined to act. w is a weight sliding on the lever rod d; by increasing the distance between the weight and the fulcrum of the lower increased pressure is brought to bear upon the walls of the capsule with the result that the boiling-point of the liquid in the capsule is slightly raised, and a range of about two degrees can thus be obtained with any particular capsule. footnotes: [7] made by the firm of chas. hearson & co., 235 regent st., london, w. xiv. methods of cultivation. cultivations of micro-organisms are usually prepared in the laboratory in one of three ways: ~tube cultures.~ ~plate cultures.~ ~hanging-drop cultures.~ these may be incubated either ~aerobically~ (i. e., in the presence of oxygen) or ~anaerobically~ (i. e., in the absence of oxygen, or in the presence of an indifferent gas, such as hydrogen, nitrogen, or carbon dioxide). with regard to the temperature at which the cultivations are grown, it may be stated as a general rule that all media rendered solid by the addition of gelatine are incubated at 20°c., or at any rate at a temperature not exceeding 22° c. (that is, in the "cold" incubator); whilst fluid media and all other solid media are incubated at 37° c. (that is, in the "hot" incubator). exceptions to this rule are numerous. for instance, in studying the growth of the psychrophylic bacteria, the yeasts and the moulds, the cold incubator is employed for all media. tube cultivations are usually packed in the incubator in small tin cylinders, such as those in which american cigarettes are sold, or in square tin boxes. beakers or tumblers may be used for the same purpose, but being fragile are not so convenient. metal test-tube racks, long enough to just fit into the interior of the incubator and each accommodating two rows of tubes, are also exceedingly useful. ~aerobic.~ ~the preparation of tube cultivations.~ the preparation of a tube cultivation consists in: (a) inoculating a tube of sterile nutrient medium with a portion of the material to be examined. (b) incubating the inoculated tube at a suitable temperature. the details of the first of these processes must be varied somewhat according to whether the tubes of nutrient media are inoculated or "planted" from-1. pre-existing cultivations. 2. morbid material previously collected (_vide_ page 373). 3. fluids, tissues, etc., or from the animal body direct. the method of preparing tube cultivations from pre-existing cultivations is as follows: [illustration: fig. 117.--inoculating tubes, seen from the front.] ~1. fluid media~ (e. g., nutrient bouillon).-1. flame the cotton-wool plug of the tube containing the cultivation and also that of the tube of sterile bouillon. 2. hold the two tubes, side by side, between the left thumb and the first and third fingers, allowing the sealed ends to rest on the dorsum of the hand, and separating the mouths of the tubes (which are pointed to the right) by the tip of the second finger. keep the tubes as nearly horizontal as is possible without allowing the fluid in the bouillon tube to reach the cotton-wool plug (fig. 117). 3. sterilise the platinum loop and allow it to cool.[8] 4. grasp the plug of the tube containing the cultivation between the little finger and palm of the hand and remove it from the tube. 5. grasp the plug of the bouillon tube between the fourth finger and the ball of the thumb and remove it from the tube. 6. pass the platinum loop into the tube containing the culture--do not allow the loop to touch the sides of the tube, or the handle to touch the medium--and remove a small portion of the growth; withdraw the loop from the tube, keeping the infected side of the loop downward. 7. pass the loop into the bouillon tube almost down to the level of the fluid, reverse the loop so that the infected side faces upward, emulsify the portion of the growth in the moisture adhering to the side of the tube which is uppermost. withdraw the loop. 8. replug both tubes. 9. sterilise the platinum loop. 10. label the bouillon tube with (a) the name of the organism and (b) the date of inoculation. 11. incubate. ~2. solid media.~--solid media are stored in tubes in one of two ways: 1. oblique tube or slanted tube (fig. 118), in which the medium has been allowed to solidify whilst the tube was retained in an inclined position, so forming an extensive surface of medium extending from the bottom of the tube almost to its mouth. this is employed for "streak" or "smear" cultivations (_strichcultur_). 2. straight tube (fig. 119), in which the medium forms a cylindrical mass in the lower portion of the tube and presents an upper surface which is at right angles to the long axis of the tube. this is employed for "stab" or "stick" cultivations (_stichcultur_), or by inoculating the medium whilst fluid, and allowing to solidify in this position, for "shake" cultivations. _streak culture._-1. flame the plugs, sterilise the platinum loop (or spatula). open the tubes and charge the loop as in previous inoculation. 2. pass the infected loop to the bottom of the tube to be inoculated and draw it, as lightly as possible, along the centre of the surface of the medium, terminating the "streak" over the thin layer of medium near the mouth of the tube. 3. replug the tubes, sterilise the platinum loop. 4. label the newly inoculated tube and incubate. _smear culture._--proceed generally as in streak culture, but rub the infected loop all over the surface of the medium, instead of restricting the inoculation to a narrow line. note.--gelatine and agar oblique tubes should be freshly "slanted" before use. _stab culture._-1. flame the plugs, open the tubes, sterilise the platinum needle and charge it with the inoculum as in the previous cultivations. 2. pass the platinum needle into the tube to be inoculated until it touches the centre of the surface of the medium. now thrust it deeply into the substance of the medium, keeping the needle as nearly as possible in the axis of the cylinder of medium. then withdraw the needle. 3. replug the tubes. sterilise the platinum needle. 4. label the newly planted tube and incubate. note.--when gelatine is stored for some time the upper surface of the cylinder becomes concave owing to evaporation. tubes showing this appearance should be liquefied and again allowed to set before use for stab culture, otherwise when the needle enters the medium, the surface tension will cause the gelatine cylinder to split. [illustration: fig. 118.--sloped or slanted medium for streak or smear culture.] [illustration: fig. 119.--straight tube.] _shake culture._-1. liquefy a tube of nutrient gelatine (or agar, or other similar medium), by heating in a water-bath (fig. 121). 2. inoculate the liquefied medium and label it, etc., precisely as if dealing with a tube of bouillon. 3. place the newly planted tube in the upright position (e. g., in a test-tube rack) and allow it to solidify. 4. label the tube; when solid, incubate. _esmarch's roll cultivation._- 1. liquefy three tubes of gelatine by heat. 2. prepare three dilutions of the inoculum (as described for plate cultivations, page 228, steps 4 to 7). 3. roll the tubes, held almost horizontally, in a groove made in a block of ice, until the gelatine has set in a thin film on the inner surface of tube (fig. 120); or under the cold-water tap. [illustration: fig. 120. esmarch's roll culture on block of ice.] in order that the medium may adhere firmly to the glass, the agar used for roll cultivation should have 1 per cent. gelatine or 1 per cent. gum arabic added to it before sterilisation. roll cultivations, which served a most important purpose in the days before the introduction of petri dishes for plate cultivations, are now obsolete in modern laboratories and are merely mentioned for the benefit of students, since examiners who are interested in the academic and historical aspects of bacteriology sometimes expect candidates to be acquainted with the method of preparing them. the preparation of plate cultures. if a small number of bacteria are suspended in liquefied gelatine, agar, or other similar medium, and the infected medium spread out in an even layer over a flat surface and allowed to solidify, each individual micro-organism becomes fixed to a certain spot and its further development is restricted to the vicinity of this spot. after a variable interval the growth of this organism becomes visible to the naked eye as a "colony." this is the principle upon which the method of plate cultivation is based and its practice enables the bacteriologist to study the particular manner of development affected by each species of microbe when growing (a) unrestricted upon the surface of the medium, (b) in the depths of the medium. the method itself is as follows: ~apparatus required.~- 1. tripod levelling stand. 2. large shallow glass dish, with a square sheet of plate glass to cover it. 3. spirit level. 4. case of sterile petri dishes. 5. tubes of sterile nutrient media, gelatine (or agar) previously liquefied by heating in the water-bath and cooled to 42°c., otherwise the heat of the medium would destroy many, if not all, of the bacteria introduced. 6. tube of cultivation to be planted from. 7. platinum loop. 8. bunsen burner. 9. grease pencil. [illustration: fig. 121.--handy form of water-bath for melting tubes of agar and gelatine previous to slanting them; or to making shake cultures or pouring plates.] method of "pouring" plates.-1. place the glass dish on the levelling tripod (figs. 122, 123); if gelatine plates are to be poured fill the dish with ice water--gelatine solidifies so slowly that it is necessary to hasten the process; if agar is to be used fill with water at 50°c.--agar sets almost immediately at the room temperature and by slightly retarding the process lumpiness is avoided; cover the dish with the square sheet of glass. 2. place the spirit level on the sheet of glass and by means of the levelling screws adjust the surface of the glass to the horizontal. this leveling is an important matter since the development of a colony is to some extent proportionate to the supply of medium available for its nutrition. thus in a "smear" on sloped tube culture, the colonies at the upper part of the medium are stunted and small but increase in size and luxuriance of growth the nearer they approach to the bottom of the tube, where there is the greatest depth of medium. [illustration: fig. 122.--plate-levelling stand.] 3. place three sterile petri dishes in a row on the surface of the glass plate and number them 1, 2, and 3, from left to right. [illustration: fig. 123.--plate-levelling stand, side view.] 4. number the previously liquefied tubes of nutrient media 1, 2, and 3. flame the plugs and see that each plug can be readily removed from the mouth of its tube. 5. add one loopful of the inoculum to tube no. 1, treating the liquefied medium as bouillon. after replugging, grasp the tube near its mouth by the thumb and first finger of the right hand, and with an even circular movement of the whole arm, diffuse the inoculum throughout the medium; avoid jerky movements, as these cause bubbles of air to form in the medium. [illustration: fig. 124.--mixing emulsion for plates.] the knack of mixing evenly without producing air bubbles, is not always easily acquired, by this method. an alternative plan is to hold the inoculated tube vertically upright between the opposed palms and to rotate it between them by rapid backward and forward movements of the two hands (fig. 124). [illustration: fig. 125.--pouring plates.] 6. sterilise the platinum loop, and add two loopfuls of diluted inoculum to tube no. 2, and mix as before. 7. in a similar manner transfer three loopfuls of liquefied medium from tube no. 2 to tube no. 3, and mix thoroughly. 8. flame the plug of tube no. 1, remove it, then flame the lips of the tube; slightly raise the cover of petri dish no. 1, introduce the mouth of the tube; then, elevating the bottom of the tube, pour the liquefied medium into the petri dish, to form a thin layer. remove the mouth of the tube and close the "plate." if the medium has failed to flow evenly over the bottom of the plate, raise the plate from the levelling platform and by tilting in different directions rectify the fault. 9. pour plates no. 2 and no. 3, in a similar manner, from tubes nos. 2 and 3. 10. label the plates with the distinctive name or number of the inoculum, also the date; the number of the dilution having been previously indicated (step 3). 11. place in the cool incubator for three or more days, as may be necessary. in this way colonies may be obtained quite pure and separate from each other. in plate no. 1, probably, the colonies will be so numerous and crowded, and therefore so small, as to render it useless. in plate no. 2 they will be more widely separated, but usually no. 3 is the plate reserved for careful examination, as in this the colonies are usually widely separated, few in number, and large in size. _agar plates_ are poured in a similar manner, but the agar must be melted in boiling water and then allowed to cool to 45° c. or 42° c. in a carefully regulated water-bath before being inoculated, and the entire process must be carried out very rapidly, otherwise the agar will have solidified before the operation is completed. note.--in pouring plates, since tube no. 1 (for the first dilution) rarely gives a plate that is of any practical value it is frequently replaced by a tube of bouillon or sterile salt solution, and in such case plate no. 1 is not poured. ~surface plates.~-this method of pouring what may be termed "whole" plates (since colonies may appear both on the surface and in the depths of the medium) is essential to the accurate study of the formation of colonies under various conditions, but when the main object of the separation of the bacteria is to obtain subcultivations from a number of individual bacteria, "surface" plates must be prepared, since here colony formation is restricted to the surface of the medium. the method adopted varies slightly according to whether the medium employed is gelatine or agar, or one of the derivatives or variants of the latter. (a) ~gelatine surface plates.~-1. liquefy three tubes of nutrient gelatine. 2. pour each tube into a separate petri dish and allow it to solidify. then turn each plate and its cover upside down. [illustration: fig. 126.--surface plate spreader.] 3. when quite cold raise the bottom of plate 1, revert it and deposit a drop of the inoculum (whether a fluid culture or an emulsion from solid culture) upon the surface of the gelatine with a platinum loop--close to one side of the plate; replace the bottom half of the petri dish in its cover. 4. take a piece of thin glass rod, stout platinum wire or best of all a piece of aluminium wire (say 2 mm. diameter) about 28 cm. long. bend the terminal 4 cm. at right angles to the remainder, making an l-shaped rod (fig. 126). sterilise the short arm and adjacent portion of the long arm, in the bunsen flame, and allow it to cool. 5. now raise the bottom of the petri dish in the left hand, leaving the cover on the laboratory bench, and holding it vertically, smear the drop of inoculum all over the surface of the gelatine with the short arm of the spreader by a rotatory motion, (fig. 127). replace the dish in its cover. 6. raise the bottom of plate 2 and rub the infected spreader all over the surface of the gelatine--then go on in like manner to the third plate in the series. 7. sterilise the spreader. 8. label and incubate the plates. [illustration: fig. 127.--spreading surface plate.] after incubation, plate no. 1 will probably yield an enormous number of colonies; plate 2 will show fewer colonies, since only those bacteria adhering to the rod after rubbing over plate 1 would be deposited on its surface, and by the time the rod reached plate 3 but very few organisms should remain upon it. so that the third plate as a rule will only show a very few scattered colonies, eminently suitable for detailed study. (b) ~agar surface plates.~-1. liquefy three tubes of nutrient agar--nutrose agar or the like. 2. pour each tube into a separate petri dish and allow it to solidify. 3. when quite solid invert each dish, raise the bottom half and rest it obliquely on its inverted cover (fig. 128) and place it in this position in an incubator at 60° c. for forty-five minutes (or in an incubator at 42° c. for two hours). this evaporates the water of condensation and gives the medium a firm, dry surface. 4. on removing the plates from the incubator close each dish and place it--still upside down--on the laboratory bench. [illustration: fig. 128.--drying surface plate of agar.] 5. inoculate the plates in series of three, as described for gelatine surface plates 3-8. hanging-drop cultivation. ~apparatus required.~- hanging-drop slides. cover-slips. section rack (fig. 75). blotting paper. bell glass to cover slides. original culture. tubes of broth, or liquefied gelatine or agar. forceps. platinum loop. bunsen burner. grease pencil. sterile vaseline. lysol. (a) ~fluid media.~-1. prepare first and second dilutions of the inoculum as directed for plate cultivations (_vide_ pages 228-229, sections 4 to 6), substituting tubes of nutrient broth for the liquefied gelatine. 2. sterilise a hanging-drop slide by washing thoroughly in water and drying, then plunging it into a beaker of absolute alcohol, draining off the greater part of the spirit, grasping the slide in a pair of forceps, and burning off the remainder of the alcohol in the flame. 3. place the hanging-drop slide on a piece of blotting paper moistened with 2 per cent. lysol solution and cover it with a small bell glass that has been rinsed out with the same solution and _not dried_. 4. raise the bell glass slightly and smear sterile vaseline around the rim of the metal cell by means of a sterile spatula of stout platinum wire. 5. remove a clean cover-slip from the alcohol pot with sterile forceps and burn off the alcohol; again raise the bell glass and place the sterile cover-slip on the blotting paper by the side of the hanging-drop slide. 6. remove a drop of the broth from the second dilution tube with a large platinum loop; raise the bell glass and deposit the drop on the centre of the cover-slip. sterilise the loop. 7. raise the bell glass sufficiently to allow of the cover-slip being grasped with forceps, inverted, and adjusted over the cell of the hanging-drop slide. remove the bell glass altogether and press the cover-slip firmly on to the cell. 8. either incubate and examine at definite intervals, or observe continuously with the microscope, using a warm stage if necessary (fig. 53). (b) ~solid media.~--observing precisely similar technique, a few drops of liquefied gelatine or agar from the second dilution tube may be run over the surface of the sterile cover-slip and a hanging-drop plate cultivation thereby prepared. this method is extremely useful in connection with the study of yeasts, when the circular cell on the hanging-drop slide should be replaced by a rectangular cell some 38 by 19 mm., and the gelatine spread over a cover-slip of similar size. after sealing down the preparation, the upper surface of the cover-slip may be ruled into squares by the aid of the grease pencil or a writing diamond and numbered to facilitate the subsequent identification of the colonies which are observed to develop from solitary germs. ~hanging-block culture~ (hill).-_apparatus required_: as for hanging-drop cultivation with the addition of a scalpel. carry out the method as far as possible under cover of a bell glass, to avoid aerial contamination. 1. liquefy a tube of nutrient agar (or gelatine) and pour into a petri dish to the depth of about 4 mm. and allow to set. 2. with a sharp scalpel cut out a block some 8 mm. square, from the entire thickness of the agar layer. 3. raise the agar block on the blade of the scalpel and transfer it, under side down, to the centre of a sterile slide. 4. spread a drop of fluid cultivation (or an emulsion of growth from a solid medium) over the upper surface of the agar block as if making a cover-slip film. 5. place the slide and block covered by the bell glass in the incubator at 37° c. for ten minutes to dry slightly. 6. take a clean dry sterile cover-slip in a pair of forceps, and with the help of a second pair of forceps lower it carefully on the inoculated surface of the agar (avoiding air bubbles), so as to leave a clear margin of cover-slip overlapping the agar block. 7. invert the preparation and with the blade of the scalpel remove the slide from the agar block. 8. with a platinum loop run a drop or two of melted agar around the edges of the block. this solidifies at once and seals the block to the cover-slip. 9. prepare a sterile hanging-drop slide, and smear hard vaseline or melted white wax on the rim of the metal cell. 10. invert the cover-slip with the block attached on to the hanging-drop slide, and seal the cover-slip firmly in place. 11. observe as for hanging-drop cultivations. anaerobic cultivations. numerous methods have been devised for the cultivation of anaerobic bacteria, the majority requiring the employment of special apparatus. the principle upon which any method is based and upon which it depends for its success falls under one or another of the following headings: (a) ~exclusion of air~ from the cultivation. (b) ~exhaustion of air~ from the vessel containing the cultivation by means of an air pump--i. e., cultivation _in vacuo_. (c) ~absorption of oxygen~ from the air in contact with the cultivation by means of pyrogallic acid rendered alkaline with caustic soda--i. e., cultivation in an atmosphere of nitrogen. (d) ~displacement of air~ by an indifferent gas, such as hydrogen or coal gas--i. e., cultivation in an atmosphere of hydrogen. (e) a combination of two or more of the above methods. a selection of the simplest and most generally useful methods is given here. whenever possible, the nutrient media that are employed in any of the processes should contain some easily oxidisable substance, such as sodium formate (0.4 per cent.) or sodium sulphindigotate (0.1 per cent.), which will absorb all the available oxygen held in solution by the medium. the further addition of glucose, 2 per cent., favors the growth of anaerobic bacteria (_vide_, pages 189-190). further, it is advisable to seal all joints between india-rubber stoppers and tubulures or the mouths of the tubes with melted paraffin; glass stoppers and taps should be lubricated with resin ointment or a mixture of beeswax 1 part, olive oil 4 parts. (a) ~method i~ (hesse's method).-1. make a stab culture in gelatine or agar, choosing for the purpose a straight tube containing a deep column of medium, and thrusting the inoculating needle to the bottom of the tube. 2. pour a layer of sterilised oil (olive oil, vaseline, or petroleum), 1 or 2 cm. deep, upon the surface of the medium. 3. incubate. ~method ii.~--this method is only available when dealing with pure cultivations. 1. liquefy a tube of gelatine (or agar) by heat, pour it into a petri dish, and allow it to solidify. 2. inoculate the surface of the medium in one spot only. 3. remove a cover-slip from the pot of absolute alcohol with sterile forceps; burn off the alcohol in the gas flame. 4. lower the now sterile cover-slip carefully on to the inoculated surface of the medium, carefully excluding air bubbles, and press it down firmly with the points of the forceps. (a sterile disc of mica may be substituted for the cover-slip.) 5. incubate. ~method iii~ (roux's physical method).-1. prepare tube cultures of fluid media (or solid media rendered fluid by heat) in the usual way. 2. aspirate some of the inoculated media into capillary pipettes. 3. seal both ends of each pipette in the blowpipe flame. 4. incubate. ~method iv~ (roux's biological method).-1. plant a deep stab, as in method i. 2. pour a layer, 1 or 2 cm. deep, of broth cultivation of a vigourous aerobe--e. g., b. aquatilis sulcatus or b. prodigiosus--upon the surface of the medium; or an equal depth of liquefied gelatine, which is then inoculated with the aerobic organism. 3. incubate. the growth of the aerobe will use up all the oxygen that reaches it and will not allow any to pass through to the medium below, which will consequently remain in an anaerobic condition. (b) ~method v.~-1. prepare tube or flask cultivations in the usual way. 2. replace the cotton-wool plug by an india-rubber stopper perforated with one hole and fitted with a length of glass tubing which has a constriction about 3 cm. above the stopper and is then bent at right angles (fig. 129). the stopper and glass tubing are sterilised by being boiled in a beaker of water for five minutes. [illustration: fig. 129.--vacuum culture.] 3. connect the tube leading from the culture vessel with a water or air pump, interposing a wulff's bottle fitted as a wash-bottle and containing sulphuric acid. 4. exhaust the air from the culture vessel. 5. before disconnecting the apparatus, seal the glass tube from the culture vessel at the constriction, using the blowpipe flame. 6. incubate. (c) ~method vi~ (buchner's method). ~apparatus and solutions required.~- buchner's tube (a stout glass test-tube 23 cm. long and 4 cm. in diameter, fitted with india-rubber stopper, fig. 130). pyrogallic acid in compressed tablets each containing 1 gram. dekanormal solution of caustic soda. method.-1. prepare the tube cultivation in the usual way. 2. moisten the india-rubber stopper of the buchner's tube with water and see that it fits the mouth of the tube accurately. 3. remove the stopper from the caustic soda bottle. 4. drop one of the pyrogallic acid tablets[9] into the buchner's tube (roughly, use 1 gramme pyrogallic acid for every 100 c.c. air capacity of the receiving vessel). 5. add about 1 c.c. of the soda solution. 6. place the inoculated tube inside the buchner's tube. the pyrogallic tablet acts as a buffer and prevents damage to either the inoculated tube or the buchner's tube even should it be slipped in hurriedly. 7. fit the india-rubber stopper tightly into the mouth of the buchner's tube. [illustration: fig. 130.--buchner's tube.] the pyrogallic acid tablet dissolves slowly in the soda solution and its oxidation proceeds very slowly at first so that ample time is available when this method is adopted. 8. restopper the caustic soda bottle. 9. place buchner's tube in a wire support, and incubate. ~method vii~ (wright's method).-1. prepare tube cultivation in the usual way. 2. cut off that portion of the cotton-wool plug projecting above the mouth of the tube with scissors, then push the plug into the tube for a distance of 2 or 3 cm. 3. by means of a pipette drop about 1 c.c. of pyrogallic acid 10 per cent. aqueous solution on to the plug. it will immediately be absorbed by the cotton-wool. 4. with another pipette run in an equal quantity of the caustic soda solution. 5. quickly close the mouth of the tube with a tightly fitting india-rubber stopper. 6. incubate. [illustration: fig. 131.--mcleod's anaerobic plate base with half petri dish inverted _in situ_] ~method viii~ (mcleod's method).-~apparatus and solutions required.~- mcleod's plate base (a hollow glazed earthenware disc 9 cm. in diameter and 2 cm. deep: the upper surface is pierced by a central hole, 2 cm. in diameter, giving access to the interior, the lower part of which is divided into two by a low partition. a shallow groove encircles the upper surface near to the edge). plasticine. pyrogallic acid (1 gramme) compressed tablets. sodic hydroxide (0.4 gramme) compressed tablets. wash bottle of distilled water. surface plates of one or other agar medium (in petri dishes of 8 cm. diameter). surface plate spreader. method.-1. roll out a long cylinder of plasticine and fit it into the groove on the upper surface of the earthenware base. 2. place a tablet of pyrogallic acid in one division of the interior of the plate base, and two tablets of sodic hydroxide in the other. 3. prepare surface plate culture of the organism to be cultivated. 4. run a few cubic centimetres of distilled water into that division of the plate base containing the sodic hydroxide. 5. invert the bottom half of the surface plate over the plate base and press its edges firmly down into the plasticine filling the groove. 6. label and incubate. (d) ~method ix.~-~apparatus required.~- small ruffer's or woodhead's flask (fig. 33). sterile india-rubber stopper. india-rubber tubing. glass tubing. metal screw clips. cylinder of compressed hydrogen; or hydrogen gas apparatus method.-1. sterilise a glass vessel, shaped as in a ruffer's or woodhead's flask, in the hot-air oven. (the tubulure and the side tubes are plugged with cotton-wool.) after sterilisation, fix a short piece of rubber tubing occluded by a metal clip to each side tube. 2. inoculate a large quantity (e. g., 200 c.c.) of the medium. where solid media are employed they must first be liquefied by heat. 3. remove the cotton-wool plug from the tubulure and pour the inoculated medium into the glass vessel. 4. close the tubulure by means of an india-rubber stopper previously sterilised by boiling in a beaker of water. [illustration: fig. 132.--kipp's hydrogen apparatus, (a) connected up to two washing bottles containing (b) lead acetate 10 per cent. solution, to remove h_{2}s and (c) silver nitrate solution to remove ash_{3}. a third washing bottle containing pyrogallic acid 10 per cent. solution, rendered alkaline, to remove any trace of oxygen, is sometimes introduced.] [illustration: fig. 133.--improved gas apparatus; the metal is contained in a perforated glass tube which is submerged in acid when the triangular bottle is upright (a), but is above the level of the liquid when the bottle is turned on its side (b).] 5. connect up the india-rubber tubing on one of the side tubes with a cylinder of compressed hydrogen (or the delivery tube of a kipp's fig. 132 or other hydrogen apparatus, fig. 133), interposing a short piece of glass tubing; and in like manner connect a long piece of rubber tubing which should be led into a basin of water, to the opposite side tube. 6. open both metal clips and pass hydrogen through the vessel until the atmospheric air is replaced by hydrogen. this is determined by collecting some of the gas which bubbles through the water in the basin in a test-tube and testing it by means of a lighted taper. 7. close the metal clip on the tube through which the gas is entering; close the clip on the exit tube. 8. disconnect the gas apparatus. 9. incubate. ~method x~ (botkin's method).-~apparatus required.~- large glass dish 20 cm. diameter and 8 cm. deep. flat leaden cross slightly shorter than the internal diameter of the glass dish. bell glass about 15 cm. diameter and 20 to 25 cm. high. metal frame for plate cultivations. _or_, glass battery jar for tube cultivations. cylinder of compressed hydrogen. rubber tubing. two pieces of ~u~-shaped glass tubing (each arm 8 cm. in length). half a litre of glycerine (or metallic mercury). method.-1. place the leaden cross inside the glass dish, resting on the bottom. 2. prepare the cultivations in the usual way. 3. place the tube cultivations in a glass battery jar (or the plate cultivations on a metal frame), resting on the centre of the leaden cross. 4. cover the cultivations with the bell jar. 5. adjust the u-shaped pieces of glass tubing in a vertical position on opposite sides of the bell jar, one arm of each inside the jar, the other outside. these tubes are best held in position by embedding the u-shaped bends in two lumps of plasterine stuck on the bottom of the glass dish. fix a short length of rubber tubing clamped with a metal clip to each of the outside arms (fig. 134). 6. fill the glass dish with glycerine or metallic mercury to a depth of about 5 cm. [illustration: fig. 134.--botkin's apparatus.] 7. connect up one u-shaped tube with the hydrogen cylinder (or gas apparatus) by means of rubber tubing. replace the atmospheric air by hydrogen, as in method ix. 8. clamp the tubes and disconnect the gas apparatus. 9. incubate. ~method xi~ (novy's method).-~apparatus required.~- jar for plate cultivations (fig. 135). _or_, jar for tube cultivations (fig. 136). lubricant for stopper of jar. rubber tubing. cylinder of compressed hydrogen. method.-1. prepare cultivations in the usual way. 2. place these inside the jar. 3. lubricate the stopper and insert it in the mouth of the jar, with the handle in a line with the two side tubes. 4. connect up the delivery tube a with the hydrogen gas supply by means of rubber tubing. [illustration: fig. 135.--novy's jar for plate cultivations.] [illustration: fig. 136.--novy's jar for tube cultivations.] 5. attach a piece of rubber tubing to the exit tube b and collect samples of the issuing gas (over water) and test from time to time. 6. when the air is completely displaced by hydrogen, turn the handle of the stopper at right angles to the line of entry and exit tubes; this seals the orifice of both tubes. 7. disconnect the gas apparatus and incubate. (e) ~method xii~ (bulloch's method).-~apparatus required.~- bulloch's jar. pot of resin ointment. small glass dish 14 cm. diameter by 5 cm. deep. vessel for tube cultures or metal rack for plate cultures. pyrogallic acid tablets. cylinder of compressed hydrogen. geryk or other air pump. rubber pressure tubing. 10 c.c. pipette. glass tubing. dry granulated caustic soda or compressed tablets each, containing 0.4 grammes sodic hydroxide. small beaker of water. method.-1. prepare the cultivations in the usual way. 2. place the glass dish in the centre of the glass slab, and stand the cultivations inside this. 3. place a sufficient number of pyrogallic acid tablets at one side of the glass dish (i. e., 1 tablet for each 100 cubic centimeters air capacity of the bell jar). place a small heap of dry granulated soda (or half a dozen tablets of sodic hydroxide) by the side of the pyro tablets. 4. smear the flange of the bell jar with resin ointment and apply the jar firmly to the glass slab, covering the cultivations--so arranged that the long tube passes with its lower end into the glass dish at a point directly opposite to the pyrogallic acid tablets. lubricate the two stop-cocks with resin ointment (fig. 137). 5. connect up the short tube a with the gas-supply by means of rubber pressure tubing and open both stop-cocks. 6. connect a long, straight piece of glass tubing to the long tube b by means of a piece of rubber tubing interposing a screw clamp: and collect samples of the issuing gas from time to time and test. 7. when the air is displaced, shut off the stop-cock of the entry tube, then that of the exit tube b. screw down the clamp and remove the glass tube from the rubber connection and connect up the short tube a to the air pump by means of pressure tubing. 8. open the stop-cock of tube a and with two or three strokes of the air pump, aspirate a small quantity of gas, so creating a slight vacuum. then shut off the stop-cock and disconnect the air pump. 9. fill the 10 c.c. bulb pipette with water; insert its point into the rubber tubing on the long tube b as far as the screw clamp. open the screw clamp and run in water until stopped by the internal pressure. shut off stop-cock. (the water dissolves the soda and pyrogallic acid converting the latter into alkaline pyro. and so bringing its latent capacity for oxygen into action). [illustration: fig. 137.--bulloch's jar.] 10. reverse the tubes from the tubulures so that they meet, out of harm's way, over the top of the bell glass; again see that all joints are tight and transfer the apparatus to the incubator. this last method is the most satisfactory for anaerobic cultivations, as by its means complete anaerobiosis can be obtained with the least expenditure of time and trouble. footnotes: [8] see also method of opening and closing culture tubes, pages 74-76. [9] if compressed tablets of pyrogallic acid cannot be obtained make up a stock "acid pyro" solution pyrogallic acid, 10 grammes hydrochloric acid, 1.5 c.c. distilled water, 100 c.c. and at step 4, run in 10 c.c. of the solution. xv. methods of isolation. the work in the preceding sections, arranged to demonstrate the chief biological characters of bacteria in general, is intended to be carried out by means of cultivations of various organisms previously isolated and identified and supplied to the student in a state of purity. a cultivation which comprises the progeny of a single cell is termed a "pure culture"; one which contains representatives of two or more species of bacteria is spoken of as an "impure," or "mixed" "cultivation," and it now becomes necessary to indicate the chief methods by which one or more organisms may be isolated in a state of purity from a mixture; whether that mixture exists as an impure laboratory cultivation, or is contained in pus and other morbid exudations, infected tissues, or water or food-stuffs. [illustration: fig. 138.--hæmatocytometer cell, showing, a, section through the centre of the cell, and b, a magnified image of the cell rulings.] before the introduction of solid media the only method of obtaining pure cultivations was by "dilution"--by no means a reliable method. "dilution" consisted in estimating approximately the number of bacteria present in a given volume of fluid (by means of a graduated-celled slide resembling a hæmatocytometer, fig. 138), and diluting the fluid by the addition of sterile water or bouillon until a given volume (usually 1 c.c.) of the dilution contained but one organism. by planting this volume of the fluid into several tubes or flasks of nutrient media, it occasionally happened that the resulting growth was the product of one individual microbe. a method so uncertain is now fortunately replaced by many others, more reliable and convenient, and in those methods selected for description here, the segregation and isolation of the required bacteria may be effected-a. ~by mechanical separation.~ 1. by surface plate cultivation: (a) gelatine. (b) agar. (c) serum agar. (d) blood agar. (e) hanging-drop or block. [2. by esmarch's roll cultivation: this archaic method (see page 226) is no longer employed for the isolation of bacteria.] 3. by serial cultivation. b. ~by biological differentiation.~ 4. by differential media. (a) selective. (b) deterrent. 5. by differential incubation. 6. by differential sterilisation. 7. by differential atmosphere cultivation. 8. by animal inoculation. the selection of the method to be employed in any specific instance will depend upon a variety of circumstances, and often a combination of two or more will ensure a quicker and more reliable result than a rigid adherence to any one method. experience is the only reliable guide, but as a general rule the use of either the first or the third method will be found most convenient, affording as each of them does an opportunity for the simultaneous isolation of several or all of the varieties of bacteria present in a mixture. ~1. surface plate cultivations.~-(a) _gelatine_ (_vide_ page 164). (b) _agar_ (_vide_ page 167). (c) _alkaline serum agar_ (_vide_ page 211). these plates are prepared in a manner precisely similar to that adopted for nutrient gelatine and agar surface plates (_vide_ pages 231-233). (d) _serum agar._-1. melt three tubes of nutrient agar, label them 1, 2, and 3, and place them, with three tubes of sterile fluid serum, also labelled 1a, 2a, and 3a, in a water-bath regulated at 45° c.; allow sufficient time to elapse for the temperature of the contents of each tube to reach that of the water-bath. 2. take serum tube no. 1a and agar tube no. 1. flame the plugs and remove them from the tubes (retaining the plug of the agar tube in the hand); flame the mouths of the tubes, pour the serum into the tube of liquefied agar and replace the plug of the agar tube. 3. mix thoroughly and pour plate no. 1 _secundum artem_. 4. treat the remaining tube of agar and serum in a similar fashion, and pour plates nos. 2 and 3. 5. dry the serum agar plates in the incubator running at 60° c. for one hour (see page 232). 6. inoculate the plates in series as described for gelatine surface plates (page 231). (e) _blood agar, human._-1. melt a tube of sterile agar and pour it into a sterile plate; let it set. 2. collect a few drops of human blood, under all aseptic conditions, in a sterile capillary teat pipette. 3. raise the cover of the petri dish very slightly, insert the extremity of the capillary pipette, and deposit the blood on the centre of the agar surface. close the dish. 4. charge a platinum loop with a small quantity of the inoculum. raise the cover of the plate, introduce the loop, mix its contents with the drop of blood, remove the loop, close the dish and sterilise the loop. 5. finally smear the mixture over the surface of the agar with a sterilised l-shaped rod. 6. label and incubate. (if considered necessary, two, three, or more similar plates may be inoculated in series.) (f) _blood agar, animal._-when preparing citrated blood agar (page 171) it is always advisable to pour several blood agar tubes into plates, which can be stored in the ice chest ready for use at any moment for surface plate cultures. (g) hanging-drop or block culture, (_vide_ page 233). ~3. serial cultivations.~--these are usually made upon agar or blood-serum, although gelatine may also be used. the method is as follows: 1. take at least four "slanted" tubes of media and number them consecutively. 2. flame all the plugs and see that each can be readily removed. 3. charge the platinum loop with a small quantity of the inoculum, observing the usual routine, and plant tube no. 1, smearing thoroughly all over the surface. if any water of condensation has collected at the bottom of the tube, use this as a diluent before smearing the contents of the loop over the surface of the medium. 4. without sterilising or recharging the loop, inoculate tube no. 2, by making three parallel streaks from end to end of the slanted surface. 5. plant the remainder of the tubes in the series as "smears" like tube no. 1. 6. label with distinctive name or number, and date; incubate. the growth that ensues in the first two or three tubes of the series will probably be so crowded as to be useless. toward the end of the series, however, discrete colonies will be found, each of which can be transferred to a fresh tube of nutrient medium without risk of contamination from the neighbouring colonies. ~"working" up plates.~-having succeeded in obtaining a plate (or tube cultivation) in which the colonies are well grown and sufficiently separated from each other, the process of "working up," "pricking out," or "fishing" the colonies in order to obtain subcultures in a state of purity from each of the different bacteria present must now be proceeded with. occasionally it happens that this is quite a simple matter. for example, the original mixed cultivation when examined microscopically was found to contain a gram positive micrococcus, a gram positive straight bacillus and a gram negative short bacillus. the third gelatine plate prepared from this mixture, on inspection after four day's incubation, showed twenty-five colonies--seven moist yellow colonies, each sinking into a shallow pit of liquefied gelatine, fourteen flat irridescent filmy colonies, and four raised white slimy colonies. a film preparation (stained gram) from each variety examined microscopically showed that the yellow liquefying colony was composed of gram positive micrococci; the flat colony of gram positive bacilli and the white colony of gram negative bacilli. one of each of these varieties of colonies would be transferred by means of the sterilised loop to a fresh gelatine culture tube, and after incubation the growth in each subculture would correspond culturally and microscopically with that of the plate colony from which it was derived,--the object aimed at would therefore be achieved. usually, however, the colonies cannot be thus readily differentiated, and unless they are "worked up" in an orderly and systematic manner much labour will be vainly expended and valuable time wasted. the following method minimises the difficulties involved. (a) inspection. a. without opening the plate carefully study the various colonies with the naked eye, with the assistance of a watchmaker's lens or by inverting the plate on the stage of the microscope and viewing with the 1-inch objective through the bottom of the plate and the layer of medium. b. if gross differences can be detected mark a small circle on the bottom of the plate around the site of each of the selected colonies, with the grease pencil. c. if no obvious differences can be made out choose nine colonies haphazard and indicate their positions by pencil marks on the bottom of the plate. (b) fishing colonies.-a. take a sterile petri dish and invert it upon the laboratory bench. rule two parallel lines on the bottom of the dish with a grease pencil, and two more parallel lines at right angles to the first pair--so dividing the area of the dish into nine portions. number the top right-hand portion 1, and the central bottom portion 8 (fig. 139). revert the dish. the numbers 1 and 8 can be readily recognised through the glass and by their positions enable any of the other divisions to be localised by number. this is the stock dish. b. slightly raise the cover of the dish, and with a sterile teat-pipette deposit a small drop of sterile water in the centre of each of the nine divisions. c. with the sterilised platinum spatula raise one of the marked colonies from the "plate 3" and transfer it to the first division in the ruled plate and emulsify it in the drop of water awaiting it. repeat this process with the remaining colonies, emulsifying a separate colony in each drop of water. (c) preliminary differentiation of bacteria.-a. prepare a cover-slip film preparation from each drop of emulsion in the "stock dish" and number to correspond to the division from which it was taken. stain by gram's method. b. examine microscopically, using the oil immersion lens and note the numbers of those cover-slips which morphologically and by gram results appear to be composed of different species of bacteria. [illustration: fig. 139.--diagram for stock plate.] (d) preparing isolation subcultures.-a. inoculate an agar slope and a broth tube from the emulsion in the stock dish corresponding to each of these specially selected numbers. b. ascertain whether the cover-slips from the nine emulsions in the stock dish include all the varieties represented in the cover-slip film preparation made from the original mixture before plating. c. if some varieties are missing prepare a second stock dish from other colonies on plate 3, and repeat the process until each morphological form or tinctorial variety has been secured in subculture. _d._ place the stock dishes in the ice chest to await the results of incubation. (if any of the subcultures fail, further material can be obtained from the corresponding emulsion; or if it has dried, by moistening it with a further drop of sterile distilled water.) _e._ incubate all the subcultures and identify the organisms picked out. 4. differential media.-(a) _selective._--some varieties of media are specially suitable for certain species of bacteria and enable them to overgrow and finally choke out other varieties; e. g., wort is the most suitable medium-base for the growth of torulæ and yeasts and should be employed when pouring plates for the isolation of these organisms. to obtain a pure cultivation of yeast from a mixture containing bacteria as well, it is often sufficient to inoculate wort from the mixture and incubate at 37° c. for twenty-four hours. plant a fresh tube of wort from the resulting growth and incubate. repeat the process once more, and from the growth in this third tube plant a streak on wort gelatine, and incubate at 20°c. the resulting growth will almost certainly be a pure culture of the yeast. (b) _deterrent._--the converse of the above also obtains. certain media possess the power of inhibiting the growth of a greater or less number of species. for instance, media containing carbolic acid to the amount of 1 per cent. will inhibit the growth of practically everything but the bacillus coli communis. ~5. differential incubation.~-in isolating certain bacteria, advantage is taken of the fact that different species vary in their optimum temperature. a mixture containing the bacillus typhosus and the bacillus aquatilis sulcatus, for example, may be planted on two slanted agar tubes, the one incubated at 40°c., and the other at 12° c. after twenty-four hours' incubation the first will show a pure cultivation of the bacillus typhosus, whilst the second will be an almost pure culture of the bacillus aquatilis. 6. differential sterilisation.-(a) _non-sporing bacteria._--similarly, advantage may be taken of the varying thermal death-points of bacteria. from a mixture of two organisms whose thermal death-points differ by, say, 4°c.--e. g., bacillus pyocyaneus, thermal death-point 55°c., and bacillus mesentericus vulgatus, thermal death-point 60°c.--a pure cultivation of the latter may be obtained by heating the mixture in a water-bath to 58° c. and keeping it at that point for ten minutes. the mixture is then planted on to fresh media and incubated, when the resulting growth will be found to consist entirely of the b. mesentericus. (b) _sporing bacteria._--this method finds its chief practical application in the differentiation of a spore-bearing organism from one which does not form spores. in this case the mixture is heated in a water-bath at 80° c. for fifteen to twenty minutes. at the end of this time the non-sporing bacteria are dead, and cultivations made from the mixture will yield a growth resulting from the germination of the spores only. differential sterilisation at 80° c. is most conveniently carried out in a water-bath of special construction, designed by balfour stewart (fig. 140). it consists of a double-walled copper vessel mounted on legs, and provided with a tubulure communicating with the space between the walls. this space is nearly filled with benzole (boiling-point 80°c.; pure benzole, free from thiophene must be employed for the purpose, otherwise the boiling-point gradually and perceptibly rises in the course of time), and to the tubulure is fitted a long glass tube, some 2 metres long and about 0.75 cm. diameter, serving as a condensing tube (a tube half this length if provided with a condensing bulb at the centre will be equally efficient). the interior of the vessel is partly filled with water and covered with a lid which is perforated for a thermometer. this latter dips into the water and records its temperature. a very small bunsen flame under the apparatus suffices to keep the benzole boiling and the water within at a constant temperature of 80° c. the bath is thus always ready for use. method.--to use the apparatus. 1. place some of the mixture itself, if fluid, containing the spores, or an emulsion of the same if derived from solid material, in a test-tube. 2. immerse the test-tube in the water contained in the benzole bath, taking care that the upper level of the liquid in the tube is at least 2 cm. beneath the surface of the water in the copper vessel. 3. the temperature of the water, of course, falls a few degrees after opening the bath and introducing a tube of colder liquid, but after a few minutes the temperature will have again reached 80°c. 4. when the thermometer again records 80°c., note the time, and fifteen minutes later remove the tube containing the mixture from the bath. 5. make cultures upon suitable media; incubate. [illustration: fig. 140.--benzole bath.] 7. differential atmosphere cultivation.-(a) by adapting the atmospheric conditions to the particular organism it is desired to isolate, it is comparatively easy to separate a strict aerobe from a strict anaerobe, and _vice versa_. in the first case, however, it is important that the cultivations should be made upon solid media, for if carried out in fluid media the aerobes multiplying in the upper layers of fluid render the depths completely anaerobic, and under these conditions the growth of the anaerobes will continue unchecked. (b) when it is desired to separate a facultative anaerobe from a strict anaerobe, it is generally sufficient to plant the mixture upon the sloped surface agar, incubate aerobically at 37°c., and examine carefully at frequent intervals. at the first sign of growth, subcultivations must be prepared and treated in a similar manner. as a result of these rapid subcultures, the facultative anaerobe will be secured in pure culture at about the third or fourth generation. (c) if, on the other hand, the strict anaerobe is the organism required from a mixture of facultative and strict anaerobes, pour plates of glucose formate agar (or gelatine) in the usual manner, place them in a bulloch's or novy's jar, and incubate at a suitable temperature. pick off the colonies of the required organism when the growth appears, and transfer to tubes of the various media. incubate under suitable conditions as to temperature and atmosphere. ~8. animal inoculation.~-finally, when dealing with pathogenic organisms, it is often advisable to inoculate some of the impure culture (or even some of the original _materies morbi_) into an animal specially chosen on account of its susceptibility to the particular pathogenic organism it is desired to inoculate. indeed, with some of the more sensitive and strictly parasitic bacteria this method of animal inoculation is practically the only method that will yield a satisfactory result. xvi. methods of identification and study. in order to identify an organism after isolation, tube, plate, and other cultivations must be prepared, incubated under suitable conditions as to temperature and environment, and examined from time to time (a) ~macroscopically~, (b) by ~microscopical methods~, (c) by ~chemical methods~, (d) by ~physical methods~, (e) by ~inoculation methods~, and the results of these examinations duly recorded. it must be stated definitely that no micro-organism can be identified by any _one_ character or property, whether microscopical, biological or chemical, but that on the contrary its entire life history must be carefully studied and then its identity established from a consideration of the sum total of these observations. in order to give to the recorded results their maximum value it is essential that they should be exact and systematic, therefore some such scheme as the following should be adhered to; and especially is this necessary in describing an organism not previously isolated and studied. scheme of study. designation: originally isolated by (_observer's name_) in (_date_), from (_source of organism_). ~1. cultural characters.~--(_vide_ macroscopical examination of cultivation, page 261.) gelatine plates, } gelatine streak, } at 20°c. gelatine stab, } gelatine shake, } agar plates, } agar streak or smear, } agar stab, } inspissated blood-serum, } at 20° c. and 37°c. bouillon, } litmus milk, } potato, } special media for the purpose of demonstrating characteristic appearances. ~2. morphology~.--(_vide_ microscopical examination of cultivations, page 272.) vegetative forms: shape. size. motility. flagella (if present). capsule (if present). involution forms. pleomorphism (if observed). sporing forms (if observed). of which class? staining reactions. ~3. chemical products of growth.~--(_vide_ chemical examination of cultivations, page 276.) chromogenesis. photogenesis. enzyme formation. fermentation of carbohydrates: acid formation. alkali formation. indol formation. phenol formation. reducing and oxidising substances. gas formation. ~4. biology.~--(_vide_ physical examination of cultures, page 295.) atmosphere. temperature. reaction of nutrient media. resistance to lethal agents: physical: desiccation. light. colours. chemical germicides. vitality. ~5. pathogenicity:~ susceptible animals, subsequently arranged in order of susceptibility. immune animals. experimental inoculation, symptoms of disease. post-mortem appearances. virulence: length of time maintained. optimum medium? minimal lethal dose. exaltation and attenuation of virulence? toxin formation. macroscopical examination of cultivations. in describing the naked-eye and low-power appearances of the bacterial growth the descriptive terms introduced by chester (and included in the following scheme) should be employed. solid media. ~plate cultures.~-_gelatine._--note the presence or absence of liquefaction of the surrounding medium. if liquefaction is present, note shape and character (_vide_ page 269, "stab" cultures). _agar._--no liquefaction takes place in this medium. the liquid found on the surface of the agar (or at the bottom of the tube in agar tube cultures) is merely water which has been expressed during the rapid solidification of the medium and has subsequently condensed. _gelatine and agar._--examine the colonies at intervals of twenty-four hours. (a) with the naked eye. (b) with a hand lens or watchmaker's glass. (c) under a low power (1 inch) of the microscope, or by means of a small dissecting microscope. distinguish superficial from deep colonies and note the characters of the individual colonies. (a) ~size.~--the diameter in millimetres, at the various ages. (b) ~shape.~-punctiform: dimensions too slight for defining form by naked eye; minute, raised, hemispherical. round: of a more or less circular outline. elliptical: of a more or less oval outline. irregular: outlines not conforming to any recognised shape. fusiform: spindle-shaped, tapering at each end. cochleate: spiral or twisted like a snail shell (fig. 141, a). [illustration: fig. 141.--types of colonies: a, cochleate; b, amoeboid; c, mycelioid.] amoeboid: very irregular, streaming (fig. 141, b). mycelioid: a filamentous colony, with the radiate character of a mould (fig. 141, c). filamentous: an irregular mass of loosely woven filaments (fig. 142, a). floccose: of a dense woolly structure. rhizoid: of an irregular, branched, root-like character (fig. 142, b). conglomerate: an aggregate of colonies of similar size and form (fig. 142, c). toruloid: an aggregate of colonies, like the budding of the yeast plant (fig. 142, d). rosulate: shaped like a rosette. [illustration: fig. 142.--types of colonies: a, filamentous; b, rhizoid; c, conglomerate; d, toruloid.] (c) ~surface elevation.~-1. _general character of surface as a whole_: flat: thin, leafy, spreading over the surface (fig. 143, a). effused: spread over the surface as a thin, veily layer, more delicate than the preceding. raised: growth thick, with abrupt terraced edges (fig. 143, b). convex: surface the segment of a circle, but very flatly convex (fig. 143, c). pulvinate: surface the segment of a circle, but decidedly convex (fig. 143, d). capitate: surface hemispherical (fig. 143, e). umbilicate: having a central pit or depression (fig. 143, f). conical: cone with rounded apex (fig. 143, g). umbonate: having a central convex nipple-like elevation (fig. 143, h). 2. _detailed characters of surface_: smooth: surface even, without any of the following distinctive characters. alveolate: marked by depressions separated by thin walls so as to resemble a honeycomb (fig. 144). punctate: dotted with punctures like pin-pricks. bullate: like a blistered surface, rising in convex prominences, rather coarse. vesicular: more or less covered with minute vesicles due to gas formation; more minute than bullate. [illustration: fig. 143.--surface elevation of colonies: a, flat; b, raised; c, convex; d, pulvinate; e, capitate; f, umbilicate; g, conical; h, umbonate.] [illustration: fig. 144.--types of colonies--alveolate.] verrucose: wart-like, bearing wart-like prominences. squamose: scaly, covered with scales. echinate: beset with pointed prominences. papillate: beset with nipple or mamma-like processes. rugose: short irregular folds, due to shrinkage of surface growth. corrugated: in long folds, due to shrinkage. contoured: an irregular but smoothly undulating surface, resembling the surface of a relief map. rimose: abounding in chinks, clefts, or cracks. (d) ~internal structure of colony~ (_microscopical_).-refraction weak: outline and surface of relief not strongly defined. refraction strong: outline and surface of relief strongly defined; dense, not filamentous colonies. [illustration: fig. 145.--types of colonies: a, grumose; b, moruloid; c, clouded.] 1. _general_: amorphous: without any definite structure, such as is specified below. hyaline: clear and colourless. homogeneous: structure uniform throughout all parts of the colony. homochromous: colour uniform throughout. 2. _granulations or blotchings_: finely granular. coarsely granular. grumose: coarser than the preceding, with a clotted appearance, and particles in clustered grains (fig. 145, a). moruloid: having the character of a mulberry, segmented, by which the colony is divided in more or less regular segments (fig. 145, b). clouded: having a pale ground, with ill-defined patches of a deeper tint (fig. 145, c). [illustration: fig. 146.--types of colonies: a, reticulate; b, gyrose; c, marmorated.] 3. _colony marking or striping_: reticulate: in the form of a network, like the veins of a leaf (fig. 146, a). areolate: divided into rather irregular, or angular, spaces by more or less definite boundaries. gyrose: marked by wavy lines, indefinitely placed (fig. 146, b). marmorated: showing faint, irregular stripes, or traversed by vein-like markings, as in marble (fig. 146, c). rivulose: marked by lines like the rivers of a map. rimose: showing chinks, cracks, or clefts. [illustration: fig. 147.--types of colonies--curled.] 4. _filamentous colonies:_ filamentous: as already defined. floccose: composed of filaments, densely placed. curled: filaments in parallel strands, like locks or ringlets (fig. 147). (e) ~edges of colonies.~-entire: without toothing or division (fig. 148, a). undulate: wavy (fig. 148, b). repand: like the border of an open umbrella (fig. 148, c). erose: as if gnawed, irregularly toothed (fig. 148, d). [illustration: fig. 148.--edges of colonies: a, entire; b, undulate; c, repand; d, erose.] lobate. lobulate: minutely lobate (fig. 149, e). auriculate: with ear-like lobes (fig. 149, f). lacerate: irregularly cleft, as if torn (fig. 149, g). fimbriate: fringed (fig. 149, h). ciliate: hair-like extensions, radiately placed (fig. 149, j). tufted. filamentous: as already defined. curled: as already defined. [illustration: fig. 149.--edges of colonies: e, lobar-lobulate; f, auriculate; g, lacerate; h, fimbriate; i, ciliate.] (f) ~optical characters~ (after shuttleworth).-1. _general characters_: transparent: transmitting light. vitreous: transparent and colourless. oleaginous: transparent and yellow; olive to linseed-oil coloured. resinous: transparent and brown, varnish or resin-coloured. translucent: faintly transparent. porcelaneous: translucent and white. opalescent: translucent; greyish-white by reflected light. nacreous: translucent, greyish-white, with pearly lustre. sebaceous: translucent, yellowish or greyish-white. butyrous: translucent and yellow. ceraceous: translucent and wax-coloured. opaque. cretaceous: opaque and white, chalky. dull: without lustre. glistening: shining. fluorescent. iridescent. 2. _chromogenicity_: colour of pigment. pigment restricted to colonies. pigment restricted to medium surrounding colonies. pigment present in colonies and in medium. ~streak or smear cultures.~-_gelatine and agar._--note general points as indicated under plate cultivations. _inspissated blood-serum._--note the presence or absence of liquefaction of the medium. (the presence of condensation water at the bottom of the tube must not be confounded with liquefaction of the medium.) _all oblique tube cultures._-1. colonies discrete: size, shape, etc., as for plate cultivations (_vide_ page 261). 2. colonies confluent: surface elevation and character of edge, as for plate cultivations (_vide_ page 263). chromogenicity: as for plate cultures. ~gelatine stab cultures.~-(a) _surface growth._--as for individual colonies in plate cultures (_vide_ page 261). [illustration: fig. 150.--stab cultivations--types of growth: a, filiform; b, beaded; c, echinate; d, villous; e, arborescent.] (b) _line of puncture._-filiform: uniform growth, without special characters (fig. 150, a). nodose: consisting of closely aggregated colonies. beaded: consisting of loosely placed or disjointed colonies (fig. 150, b). papillate: beset with papillate extensions. echinate: beset with acicular extensions (fig. 150, c). villous: beset with short, undivided, hair-like extensions (fig. 150, d). plumose: a delicate feathery growth. [illustration: fig. 151.--stab cultivations--types of growth: f, crateriform; g, saccate; h, infundibuliform; j, napiform; k, fusiform; l, stratiform.] arborescent: branched or tree-like, beset with branched hair-like extensions (fig. 150, e). (c) _area of liquefaction_ (if present).-crateriform: a saucer-shaped liquefaction of the gelatine (fig. 151, f). saccate: shape of an elongated sack, tubular cylindrical (fig. 151, g). infundibuliform: shape of a funnel, conical (fig. 151, h). napiform: shape of a turnip (fig. 151, j). fusiform: outline of a parsnip, narrow at either end, broadest below the surface (fig. 151, k). stratiform: liquefaction extending to the walls of the tube and downward horizontally (fig. 151, l). (d) _character of the liquefied gelatine._-1. pellicle on surface. 2. uniformly turbid. 3. granular. 4. mainly clear, but containing flocculi. 5. deposit at apex of liquefied portion. (e) _production of gas bubbles._ ~shake cultures.~-1. presence or absence of liquefaction. 2. production of gas bubbles. 3. bulk of growth at the surface--aerobic. 4. bulk of growth in depths--anaerobic. ~fluid media.~ ~1. surface of the liquid.~-presence or absence of froth due to gas bubbles. presence or absence of pellicle formation. character of pellicle. ~2. body of the liquid.~-uniformly turbid. flocculi in suspension. granules in suspension. clear, with precipitate at bottom of tube. colouration of fluid, presence or absence of. ~3. precipitate.~-character. amount. colour. ~carbohydrate media.~-growth. reaction. gas formation. coagulation or not of serum albumen (when serum water media are employed). ~litmus milk cultivations.~- {unaltered. 1. reaction: {acid. {alkaline. 2. odour. 3. formation of gas. {unaltered. 4. consistency: {peptonised (character of solution). {coagulated. {hard: solid. 5. clot: character {soft: floculent. {ragged and broken up by gas {bubbles. (a) coagulum undissolved. (b) coagulum finally peptonised, completely: incompletely. resulting solution, clear: turbid. {abundant. {scanty. 6. whey: {clear. {turbid. {coagulated by boiling, or not. ~by microscopical methods.~ as a council of perfection preparations must be made from pure cultivations 4, 6, 8, 12, 18, and 24 hours; and subsequently at intervals of, say, twenty-four hours, during the entire period they are under observation, and examined-(a) ~living.--1.~ in ~hanging drop~, to determine _motility_ or _non-motility_. in this connection it must be remembered that under certain conditions as to environment (e. g., when examined in an unsuitable medium, atmosphere, temperature, etc.) motile bacilli may fail to exhibit activity. no organism, therefore, should be recorded as non-motile from one observation only; a series of observations at different ages and under varying conditions should form the basis of an opinion as to the absence of true locomotion. _size._--in the case of non-motile or sluggishly motile organisms, endeavour to measure several individuals in each hanging drop by means of the eyepiece micrometer or the eikonometer (_vide_ page 63), and average the results. if the organism is one which forms spores, observe-(a) _spore formation._--prepare hanging-drop cultivations (_vide_ page 78) from vegetative forms of the organism, adding a trace of magenta solution (0.5 per cent.) or other intra vitam stain (see page 77) to the drop, on the point of the platinum needle, to facilitate the observation of the phenomenon by rendering the bacilli more distinct. place the preparation on the stage of the microscope; if necessary, using a warm stage. arrange illumination, etc., and select a solitary bacillus for observation, by the help of the 1/6-inch lens. substitute the 1/12-inch oil-immersion lens for the sixth, and observe the formation of the spore; if possible, measure any alteration in size which may occur by means of the ramsden micrometer. (b) _spore germination._--prepare hanging-drop cultivations from old cultivations in which no living vegetative forms are present, and observe the process of germination in a similar manner. the comfort of the microscopist is largely enhanced in those cases where the period of observation is at all lengthy, by use of some form of eye screen before the unemployed eye, such as is figured on page 58 (fig. 49). if it is impossible to carry out the method suggested above, proceed as follows: (a) _spore formation._--plant the organism in broth and incubate under optimum conditions. at regular intervals, say every thirty minutes, remove a loopful of the cultivation and prepare a cover-slip film preparation. fix, while still wet, in the corrosive sublimate fixing solution. stain with aniline gentian violet, and partially decolourise with 2 per cent. acetic acid. mount and number consecutively; then examine. (b) _spore germination._--expose a thick emulsion of the spores to a temperature of 80° c. for ten minutes in the differential steriliser (_vide_ page 257). transfer the emulsion to a tube of sterile nutrient broth and incubate. remove specimens from the tube culture at intervals of, say, five minutes. fix, stain, etc., wet, as under (a), and examine. (b) ~fixed.--2.~ in ~stained preparations~. (a) to determine points in _morphology_: _shape_ (_vide_ classification, page 131). _size_: (a) prepare cover-slip film preparations at the various ages, and fix by exposure to a temperature of 115° c. for twenty minutes in hot-air oven. (b) stain the preparations by gram's method (if applicable) or with dilute carbol-fuchsin, and mount in the usual way. (c) measure (_vide_ page 66) some twenty-five individuals in each film by means of the ramsden's or the stage micrometer and average the result. _pleomorphism_; if noted, record- the predominant character of the variant forms. on what medium or media they are observed. at what period of development. (b) to demonstrate details of _structure_: _flagella_: if noted, record- method of staining (_vide_ page 101). position and arrangement (_vide_ page 136). number. _spores_: if noted, record- method of staining. shape. size. position within the parent cell. condition, as to shape, of the parent cell (_vide_ page 139). optimum medium and temperature. age of cultivation. conditions of environment as to temperature, atmosphere. method of germination (_vide_ page 140). _involution forms_: if noted, record- method of staining. character (e. g., if living or dead). shape. on what medium they are observed. age of medium. environment. _metachromatic granules_: if noted, record- method of staining. character of granules. number of granules. colour of granules. ~3. staining reactions.~-1. _gram's method._--positive or negative. 2. _neisser's method._--if granules are noted, record- 1. position. 2. number. 3. _ziehl-neelsen's method._--acid-fast or decolourised. 4. _simple aniline dyes._--(noting those giving the best results, with details of staining processes.) methylene-blue } fuchsin } and their modifications. gentian violet } thionine blue } by biochemical methods. test cultivations of the organism for the presence of-soluble enzymes--proteolytic, diastatic, invertase. organic acids--(a) quantitatively--i. e., estimate the total acid production; (b) qualitatively for formic, acetic, propionic, butyric, lactic. ammonia. neutral volatile substances--ethyl alcohol, aldehyde, acetone. aromatic products--indol, phenol. soluble pigments. test the power of reducing (a) colouring matters, (b) nitrates to nitrites. investigate the gas production--h_{2}s, co_{2}, h_{2}. estimate the ratio between the last two gases. prepare all cultivations for these methods of examination under _optimum_ conditions, previously determined for each of the organisms it is intended to investigate, as to (a) reaction of medium; (b) incubation temperature; (c) atmospheric environment; and keep careful records of these points, and also of the age of the cultivation used in the final examination. examine the cultivations for the various products of bacterial metabolism after forty-eight hours' growth, and ~never omit to examine "control" (uninoculated) tube or flask of medium from the same batch, kept for a similar period under identical conditions~. if the results are negative, test further cultivations at three days, five days, and ten days. ~1. enzyme production.~-(a) _proteolytic enzymes._--(convert proteins into proteose, peptone and further products of hydrolysis; e. g., b. pyocyaneus.) _media required_: blood-serum and milk-serum which have been carefully filtered through a porcelain candle. _reagents required_: ammonium sulphate. thirty per cent. caustic soda solution. copper sulphate, 0.5 per cent. aqueous solution. one per cent. acetic acid solution. millon's reagent. glyoxylic acid solution. concentrated sulphuric acid. method.-1. prepare cultivations in bulk (50 c.c.) in a flask and incubate. 2. make the liquid faintly acid with acetic acid, then boil. (this precipitates the unaltered proteins.) 3. filter. 4. take 10 c.c. of the filtrate in a test-tube and add 1 c.c. of the caustic soda, then add the copper sulphate drop by drop. pink colour which becomes violet with more copper sulphate = proteose and peptone. 5. saturate the rest of the filtrate with ammonium sulphate. precipitate = proteose. 6. filter and divide the filtrate into three parts a, b and c. a. repeat the copper sulphate test, using excess of caustic soda to displace the ammonia from the ammonium sulphate. pink colour = peptone. b. boil with millon's reagent. red colour = tyrosine. c. add glyoxylic acid solution and run in concentrated sulphuric acid. violet ring at upper level of acid = tryptophane. both the tyrosine and tryptophane may be either in the free state or in combination as polypeptid or peptone. (b) _diastase._--(converts starch into sugar; e. g., b. subtilis.) _medium required_: inosite-free bouillon. _reagents required_: starch. thymol. fehling's solution. method.-1. prepare tube cultivation and incubate. 2. prepare a thin starch paste and add 2 per cent. thymol to it. 3. mix equal parts of the cultivation to be tested and the starch paste, and place in the incubator at 37°c. for six to eight hours. 4. filter. test the filtrate for sugar. boil some of the fehling's solution in a test-tube. add the filtrate drop by drop until, if necessary, a quantity has been added equal in amount to the fehling's solution employed, keeping the mixture at the boiling-point during the process. yellow or orange precipitate = sugar. (c) _invertase._--(convert saccharose into a mixture of dextrose and lævulose e. g., b. fluorescens liquefaciens.) _medium required_: inosite-free bouillon. _reagents required_: cane sugar, 2 per cent. aqueous solution. carbolic acid. method.-1. prepare tube cultivations and incubate. 2. add 2 per cent. of carbolic acid to the sugar solution. 3. mix equal quantities of the carbolised sugar solution and the cultivation in a test-tube; allow the mixture to stand for several hours. 4. filter. test the filtrate for reducing sugar as in the preceding section. (d) _rennin and "lab" enzymes._--(coagulate milk independently of the action of acids; e. g., b. prodigiosus.) _media required_: inosite-free bouillon. litmus milk. method.-1. prepare tube cultivations and incubate. 2. after incubation heat the cultivation to 55° c. for half an hour, to sterilise. 3. by means of a sterile pipette run 5 c.c. of the cultivation into each of three tubes of litmus milk. 4. place in the cold incubator at 22° c. and examine each day for ten days. absence of coagulation at the end of that period will indicate absence of rennin ferment formation. fermentation reactions. as tested upon carbohydrate substances and organic salts. _media required_: peptone water containing various percentages (generally 2 per cent.) of each of the substances referred to under "sugar" media (page 177), also tubes of peptone water containing 1 per cent. respectively of each of the following: organic salts: sodium citrate, formate, lactate, malate, tartrate. method.-1. prepare tube cultivations in each of the above media. 2. observe from day to day up to the expiration of ten days if necessary. 3. note growth, reaction, gas production. 2. acid production. (a) _quantitative._- _medium required_: sugar (glucose) bouillon of known "optimum" reaction. _apparatus and reagents required_: as for estimating reaction of media (_vide_ page 150). method.-1. prepare cultivation in bulk (100 c.c.) in a flask; also "control" flask of medium from same batch. 2. after suitable incubation, heat both flasks in the steamer at 100° c. for thirty minutes to sterilise. 3. determine the _titre_ of the medium in "inoculated" and "control" flasks as described in the preparation of nutrient media (_vide_ page 151). 4. the difference between the titre of the medium in the two flasks gives the total acid production of the bacterium under observation in terms of normal naoh. note.--if the growth is very heavy it may be a difficult matter to determine the end-point. the cultivation should then be filtered through a berkefeld filter candle previous to step 2, and the filtrate employed in the titration. (b) _qualitative_ (of all the organic acids present).- _medium required_: sugar (glucose or lactose) bouillon as in quantitative examination. _reagents required_: hydrochloric acid, concentrated. hydrochloric acid, 25 per cent. sulphuric acid, concentrated (pure). phosphoric acid, concentrated solution. ammonia. ammonium sulphate. baryta water. sodium carbonate, saturated aqueous solution. absolute alcohol. ether. calcium chloride. calcium chloride solution. zinc carbonate. copper sulphate saturated aqueous solution. alcoholic thiophene solution (0.15 c.c. in 100 c.c.). animal charcoal. five per cent. sodium nitroprusside solution. potassium bichromate. schiff's reagent. arsenious oxide. ferric chloride, 4 per cent. aqueous solution. silver nitrate, 1 per cent. aqueous solution. lugol's iodine. ten per cent. caustic soda solution. hard paraffin wax (melting-point about 52° c.). method.-1. prepare cultivation in bulk (500 c.c.) in a litre flask and add sterilised precipitated chalk, 10 grammes. incubate at the optimum temperature. 2. after incubation throw a piece of paraffin wax (about a centimetre cube) into the cultivation and connect up the flask with a condenser. the paraffin, which liquefies and forms a thin layer on the surface of the fluid, is necessary to prevent the cultivation frothing up and running unaltered through the condenser during the subsequent process of distillation. 3. distill over 200 to 300 c.c. use a rose-top burner to minimise the danger of cracking the flask; and to the same end, well agitate the contents of the flask to prevent the chalk settling. the distillate "a" will contain alcohol, etc. (_vide_ page 285); the residue "a" will contain the volatile and fixed acids. 4. disconnect the flask and filter. the residue "a" then = filtrate b and residue b. [illustration: fig. 152.--arrangement of distillation apparatus for acids, etc.] 5. residue b. wash the residue from the filter paper, dissolve by heating with dilute hydrochloric acid, and add calcium chloride solution and ammonia until alkaline. white precipitate insoluble in acetic acid = oxalic acid. 6. make up filtrate b to 500 c.c. with distilled water and divide into two parts. 7. acidify 250 c.c. with 20 c.c. concentrated phosphoric acid (this liberates the volatile acids) and distil to small bulk. the distillate "b" may contain formic, acetic, propionic, butyric and benzoic acids. distillate "b." (volatile acids.) ¦ ¦ 1. add baryta water till alkaline, and evaporate to dryness. 2. add 50 c.c. absolute alcohol and allow to stand, with frequent stirring, for two to three hours. 3. filter and wash with alcohol. ¦ ¦ ¦---------------------------------------¦ ¦ ¦ ¦ ¦ filtrate residue ¦ ¦ ¦ ¦ may contain barium propionate, may contain barium acetate, barium butyrate. barium formate, barium benzoate. ¦ ¦ ¦ ¦ 1. evaporate to dryness. 1. evaporate off alcohol and dissolve up the residue on 2. dissolve residue in 150 the filter in hot water and c.c. water. neutralise. 3. acidify with phosphoric 2. divide the solution into acid and distil. four portions: 4. saturate distillate with (a) add ferric chloride solution. calcium chloride and distill over a few c.c. ~brown~ colour = _acetic_ or _formic_ acids. 5. test distillate for butyric acid: ~buff ppt.~ = _benzoic_ acid (see ether soluble acids). add 3 c.c. alcohol and 4 drops concentrated sulphuric acid. (b) add silver nitrate solution; then add one drop ~smell of pineapple~ = _butyric_ ammonia water, and boil. acid. ~black~ precipitate of metallic propionic acid in small silver = _formic_ acid. quantities cannot be distinguished from butyric (c) evaporate to dryness; mix acid by tests within the with equal quantity of scope of the bacteriological arsenious oxide and heat laboratory. on platinum foil. unpleasant ~smell of cacodyl~ = _acetic_ acid. (d) add a few drops of mercuric chloride solution in test-tube, and heat to 70° c. ~precipitate~ of mercurous chloride which is slowly reduced to mercury = _formic_ acid. 8. if the distillation of "b" is continued as long as acid comes over (distilled water being occasionally added to the distilling flask) the distillate can be measured and 50 c.c. used for titration. this will give the amount of volatile acid formation. 9. the second part of the filtrate "b" (see page 282) should be examined for lactic, oxalic, succinic, benzoic, salicylic, gallic and tannic acids, as follows: ~ether soluble acids.~-1. evaporate to a thin syrup, acidify strongly with phosphoric acid. 2. extract with five times its volume of ether by agitation in a separatory funnel. 3. evaporate the ethereal extract to a thin syrup. 4. add 100 c.c. water and mix thoroughly. 5. to a small portion of this solution add slight excess of sodium carbonate, evaporate to dryness on the water-bath, dissolve in 5-10 c.c. pure sulphuric acid, add 2 drops saturated copper sulphate solution, place in a test-tube and heat in a boiling water-bath for 2 minutes, cool, add 2 or 3 drops of the alcoholic thiophene and warm gently. cherry red colour = lactic acid. if a brown colour is produced on the addition of sulphuric acid, another sample should be taken and boiled with animal charcoal before evaporating. 6. if lactic acid is definitely present, prepare zinc lactate by boiling part of the solution of the ether extract with excess of zinc carbonate, filtering and evaporating to crystallise. the crystals so obtained have a characteristic form, and if dried at 110° c, should contain 26.87 per cent. of zinc. 7. test a portion of the rest of the solution of the ether extract for oxalic acid (page 282, step 5). carefully neutralise the remainder and add ferric chloride solution. red brown gelatinous precipitate = succinic acid. buff precipitate = benzoic acid, and other acids related to benzoic acid. violet colour = salicylic acid. inky black colour or precipitate = gallic acid or tannic acid. for further identification the melting-points of the crystalline acids, and the percentage of silver in their silver salts should be determined. ~3. ammonia production.~- _medium required_: nutrient bouillon. _reagent required_: nessler reagent. method.-1. prepare cultivation in bulk (100 c.c.) in a 250 c.c. flask and incubate together with a control flask. test the cultivation and the control for ammonia in the following manner: 2. to each flask add 2 grammes of calcined magnesia, then connect up with condensers and distil. 3. collect 50 c.c. distillate, from each, in a nessler glass. 4. add 1 c.c. nessler reagent to each glass by means of a clean pipette. yellow colour = ammonia. the depth of colour is proportionate to the amount present. ~4. alcohol, etc., production.~--divide the distillate "a" obtained in the course of a previous experiment (_vide_ page 282, step 3) into four portions and test for the production of alcohol, acetaldehyde, acetone. 1. add lugol's iodine, then a little naoh solution, and stir with a glass rod till the colour of the iodine disappears. pale-yellow crystalline precipitate of iodoform, with its characteristic smell, appearing in the cold, indicates acetaldehyde, or acetone; appearing only on warming indicates alcohol. the precipitate may be absent even when the odour is pronounced. 2. add schiff's reagent. violet or red colour = aldehyde. 3. to 10 c.c. of solution add 2.5 c.c., 25 per cent. sulphuric acid, and a crystal or two of potassium bichromate and distil. reduction of the bichromate to a green colour and a distillate, which smells of acetaldehyde and reacts with schiff's reagent, shows the presence of alcohol in the original liquid. 4. add a few drops of sodium nitroprusside solution, make alkaline with ammonia, then saturate with ammonium sulphate crystals. acetone gives little colour on the addition of ammonia, but after the addition of ammonium sulphate a deep permanganate colour, which takes ten minutes to reach its full intensity. aldehyde gives a carmine red unaltered by ammonium sulphate. ~5. indol production.~- _media required_: inosite-free bouillon (_vide_ page 183). or peptone water (_vide_ page 177). _reagents required_: potassium persulphate, saturated aqueous solution. paradimethylamino-benzaldehyde solution. this is prepared by mixing: paradimethylamino-benzaldehyde 4 grammes absolute alcohol 380 c.c. hydrochloric acid, concentrated 80 c.c. method.-prepare several test-tube cultivations of the organism to be tested, and incubate. test for indol by means of the rosindol reaction in the following manner. (if the culture has been incubated at 37°c., it must be allowed to cool to the room temperature before applying the test.) 1. remove 2 c.c. of the cultivation by means of a sterile pipette and transfer to a clean tube, then, 2. add 2 c.c. paradimethylamino-benzaldehyde solution. 3. add 2 c.c. potassium persulphate solution. the presence of indol is indicated by the appearance of a delicate rose-pink colour throughout the mixture which deepens slightly on standing. indol is tested for in many laboratories by the ordinary nitrosoindol reaction which, however, is not so delicate a method as that above described. the test is carried out as follows: 1. remove the cotton-wool plug from the tube, and run in 1 c.c. pure concentrated sulphuric acid down the side of the tube by means of a sterile pipette. place the tube upright in a rack, and allow it to stand, if necessary, for ten minutes. a rose-pink or red colour at the junction of the two liquids = indol (_plus a nitrite_). 2. if the colour of the medium remains unaltered, add 2 c.c. of a 0.01 per cent. aqueous solution sodium nitrite, and again allow the culture to stand for ten minutes. red colouration = indol. note.--in place of performing the test in two stages as given above, 2 c.c. concentrated _commercial_ sulphuric, hydrochloric, or nitric acid (all of which hold a trace of nitrite in solution), may be run into the cultivation. the development of a red colour within twenty minutes will indicate the presence of indol. ~5a. phenol production.~- _medium required_: nutrient bouillon. _reagents required_: hydrochloric acid, concentrated. millon's reagent. ferric chloride, 1 per cent. aqueous solution. method.-1. prepare cultivation in a bohemian flask containing at least 50 c.c. of medium, and incubate. test for phenol in the following manner: 2. add 5 c.c., 25 per cent. sulphuric acid to the cultivation and connect up the flask with a condenser. 3. distil over 15 to 20 c.c. divide the distillate into three portions a, b and c. 4. add to (a) 0.5 c.c. millon's reagent and boil. red colour = phenol. 5. add to (b) about 0.5 c.c. ferric chloride solution. violet colour = phenol. (if the distillate be acid the reaction will be negative.) 6. add to (c) bromine water. crystalline white ppt. of tribromo-phenol = phenol. note.--if both indol and phenol appear to be present in cultivations of the same organism, it is well to separate them before testing. this may be done in the following manner: 1. prepare inosite-free bouillon cultivation, say 200 or 300 c.c., in a flask as before. 2. render definitely acid by the addition of acetic acid and connect up the flask with a condenser. 3. distil over 50 to 70 c.c. distillate will contain both indol and phenol. 4. render the distillate strongly alkaline with caustic potash and redistil. distillate will contain indol; residue will contain phenol. 5. test the distillate for indol (_vide ante_). 6. saturate the residue, when cold, with carbon dioxide and redistil. 7. test this distillate for phenol (_vide ante_). ~6. pigment production.~-1. prepare tube cultivations upon the various media and incubate under varying conditions as to temperature (at 37° c. and at 20°c.), atmosphere (aerobic and anaerobic), and light (exposure to and protection from). note the conditions most favorable to pigment formation. 2. note the solubility of the pigment in various solvents, such as water (hot and cold), alcohol, ether, chloroform, benzol, carbon bisulphide. 3. note the effect of acids and alkalies respectively upon the pigmented cultivation, or upon solutions of the pigment. 4. note spectroscopic reactions. ~7. reducing agent formation.~-(a) _colour destruction._-1. prepare tube cultivations in nutrient bouillon tinted with litmus, rosolic acid, neutral red, and incubate. 2. examine the cultures each day and note whether any colour change occurs. (b) _nitrates to nitrites._- _medium required_: nitrate bouillon (_vide_ page 185). or nitrate peptone solution (_vide_ page 186). _reagents required_: sulphuric acid (25 per cent.). metaphenylene diamine, 5 per cent. aqueous solution. method.-1. prepare tube cultivations and incubate together with control tubes (i. e., uninoculated tubes of the same medium, placed under identical conditions as to environment). this precaution is necessary as the medium is liable to take up nitrites from the atmosphere, and an opinion as to the absence of nitrites in the cultivation is often based upon an equal colouration of the medium in the control tube. test both the culture tube and the control tube for the presence of nitrites. 2. add a few drops of sulphuric acid to the medium in each of the tubes. 3. then run in 2 or 3 c.c. metaphenylene diamine into each tube. brownish-red colour = nitrites. the depth of colour is proportionate to the amount present. ~8. gas production.~-(a) _carbon dioxide and hydrogen._- _apparatus required_: fermentation tubes (_vide_ page 161) containing sugar bouillon (glucose, lactose, etc.). the medium should be prepared from inosite-free bouillon (_vide_ page 183). _reagent required_: n/2 caustic soda. method.-1. inoculate the surface of the medium in the bulb of a fermentation tube and incubate. 2. mark the level of the fluid in the closed branch of the fermentation tube, at intervals of twenty-four hours, and when the evolution of gas has ceased, measure the length of the column of gas with the millimetre scale. express this column of gas as a percentage of the entire length of the closed branch. 3. to analyse the gas and to determine roughly the relative proportions of co_{2} and h_{2}, proceed as follows: fill the bulb of the fermentation tube with caustic soda solution. close the mouth of the bulb with a rubber stopper. alternately invert and revert the tube six or eight times, to bring the soda solution into intimate contact with the gas. return the residual gas to the end of the closed branch, and measure. the loss in volume of gas = carbon dioxide. the residual gas = hydrogen. transfer gas to the bulb of the tube, and explode it by applying a lighted taper. (b) _sulphuretted hydrogen._- _media required_: iron peptone solution (_vide_ page 185). lead peptone solution. 1. inoculate tubes of media, and incubate together with control tubes. 2. examine from day to day, at intervals of twenty-four hours. the liberation of the h_{2}s will cause the yellowish-white precipitate to darken to a brownish-black, or jet black, the depth of the colour being proportionate to the amount of sulphuretted hydrogen present. quantitative: for exact quantitative analyses of the gases produced by bacteria from certain media of definite composition, the methods devised by pakes must be employed, as follows: [illustration: fig. 153.--gas-collecting apparatus.] _apparatus required_: bohemian flask (300 to 1500 c.c. capacity) containing from 100 to 400 c.c. of the medium. the mouth of the flask is fitted with a perforated rubber stopper, carrying an l-shaped piece of glass tubing (the short arm passing just through the stopper). to the long arm of the tube is attached a piece of pressure tubing some 8 cm. in length, plugged at its free end with a piece of cotton-wool. measure accurately the total capacity of the flask and exit tube, also the amount of medium contained. note the difference. gas receiver. this is a bell jar of stout glass, 14 cm. high and 9 cm. in diameter. at its apex a glass tube is fused in. this rises vertically 5 cm., and is then bent at right angles, the horizontal arm being 10 cm. in length. a three-way tap is let horizontally into the vertical tube just above its junction with the bell jar. an iron cylinder just large enough to contain the bell jar. about 15 kilos of metallic mercury. melted paraffin. an orsat-lunge working with mercury instead of water, provided with two gas tubes of extra length (capacity 120 and 60 c.c. respectively and graduated throughout, both being water-jacketed) or other gas analysis apparatus, capable of dealing with co_{2}, o_{2}, h_{2}, and n_{2}. method.-1. inoculate the medium in the flask in the usual manner, by means of a platinum needle, taking care that the neck of the flask and the rubber stopper are thoroughly flamed before and after the operation. [illustration: fig. 154.--orsat-lunge gas analysis apparatus.] 2. fill the iron cylinder with mercury. 3. place the bell jar mouth downward in the mercury--first seeing that there is free communication between the interior of the jar and the external air--and suck up the mercury into the tap; then shut off the tap. 4. plug the open end of the three-way tap with melted wax. 5. connect up the horizontal arm of the culture flask with that of the gas receiver by means of the pressure tubing (after removing the cotton-wool plug from the rubber tube), as shown in fig. 153. 6. give the three-way tap half turn to open communication between flask and receiver, and seal _all_ joints by coating with a film of melted wax. when the tap is turned, the mercury in the receiver will naturally fall. 7. place the entire apparatus in the incubator. (two hours later, by which time the temperature of the apparatus is that of the incubator, mark the height of the mercury on the receiver.) 8. examine the apparatus from day to day and mark the level of the mercury in the receiver at intervals of twenty-four hours. 9. when the evolution of gas has ceased, remove the apparatus from the incubator; clear out the wax from the nozzle of the three-way tap (first adjusting the tap so that no escape of gas shall take place) and connect it with the orsat. 10. remove, say, 100 c.c. of gas from the receiver, reverse the tap and force it into the culture flask. remove 100 c.c. of mixed gases from the culture flask and replace in the receiver. repeat these processes three or four times to ensure thorough admixture of the contents of flask and receiver. 11. now withdraw a sample of the mixed gases into the orsat and analyse. in calculating the results be careful to allow for the volume of air contained in the flask at the commencement of the experiment. for the collection of gases formed under anaerobic conditions a slightly different procedure is adopted: 1. fix a culture flask (500 c.c. capacity) with a perforated rubber stopper carrying an ~l~-shaped piece of manometer tubing, each arm 5 cm. in length. 2. prepare a second ~l~-shaped piece of tubing, the short arm 5 cm. and the long arm 20 cm., and connect its short arm to the horizontal arm of the tube in the culture flask by means of a length of pressure tubing, provided with a screw clamp. 3. fill the culture flask completely with boiling medium and pass the long piece of tubing through the plug of an erlenmeyer flask (150 c.c. capacity) which contains 100 c.c. of the same medium. 4. sterilise these coupled flasks by the discontinuous method, in the usual manner. immediately the last sterilisation is completed, screw up the clamp on the pressure tubing which connects them, and allow them to cool. as the fluid cools and contracts it leaves a vacuum in the neck of the flask below the rubber stopper. 5. to inoculate the culture flask, withdraw the long arm of the bent tube from the erlenmeyer flask and pass it to the bottom of a test-tube containing a young cultivation (in a fluid medium similar to that contained in the culture flask) of the organism it is desired to investigate. 6. slightly release the clamp on the pressure tubing to allow 4 or 5 c.c. of the culture to enter the flask. 7. clamp the rubber tube tightly; remove the bent glass tube from the culture tube and plunge it into a flask containing recently boiled and quickly cooled distilled water. 8. release the clamp again and wash in the remains of the cultivation until the culture flask and tubing are completely filled with water. 9. clamp the rubber tubing tightly and take away the long-armed glass tubing. 10. prepare the gas receiver as in the previous method (in this case, however, the mercury should be warmed slightly) and fill the horizontal arm of the receiver with hot water. 11. connect up the culture flask with the horizontal arm of the gas receiver. 12. remove the screw clamp from the rubber tubing, adjust the three-way tap, seal all joints with melted wax, and incubate. 13. complete the investigation as described for the previous method. by physical methods. examine cultivations of the organism with reference to its growth and development under the following headings: atmosphere: (a) in the presence of oxygen. (b) in the absence of oxygen. (c) in the presence of gases other than oxygen. temperature: (a) range. (b) optimum. (c) thermal death-point: moist: vegetative forms. spores. dry: vegetative forms. spores. reaction of medium. resistance to lethal agents: (a) desiccation. (b) light: diffuse. direct. primary colours. (c) heat. (d) chemical antiseptics and disinfectants. vitality in artificial cultures. ~i. atmosphere.~--the question as to whether the organism under observation is (a) an obligate aerobe, (b) a facultative anaerobe, or (c) an obligate anaerobe is roughly decided by the appearance of cultivations in the fermentation tubes. obvious growth in the closed branch as well as in the bulb or in the inverted gas tube as well as in the bulk of the medium will indicate that it is a facultative anaerobe; whilst growth only occurring in the bulb or in the closed branch shows that it is an obligate aerobe or anaerobe respectively. this method, however, is not sufficiently accurate for the present purpose, and the examination of an organism with respect to its behaviour in the absence of oxygen is carried out as follows: _apparatus required:_ buchner's tubes. bulloch's apparatus. exhaust pump. pyrogallic acid. dekanormal caustic soda. _media required:_ glucose formate agar. glucose formate gelatine. glucose formate bouillon. method.-1. prepare four sets of cultivations: (a) sloped glucose formate agar, and incubate aerobically at 37° c. sloped glucose formate gelatine, and incubate aerobically at 20° c. (b) sloped glucose agar to incubate anaerobically at 37° c. sloped glucose formate gelatine to incubate anaerobically at 20° c. (c) sloped glucose formate agar to incubate anaerobically at 37° c. glucose formate bouillon to incubate anaerobically at 37° c. (d) sloped glucose formate gelatine to incubate anaerobically at 20° c. glucose formate bouillon to incubate anaerobically at 20° c. 2. seal the cultures forming set b in buchner's tubes (_vide_ page 239). 3. seal the cultures forming set c in bulloch's apparatus; exhaust the air by means of a vacuum pump, and provide for the absorption of any residual oxygen by the introduction of pyrogallic acid and caustic soda in solution (_vide_ page 245). treat set d in the same way. 4. observe the cultivations macroscopically and microscopically at intervals of twenty-four hours until the completion, if necessary, of seven days' incubation. 5. control these results. _gases other than oxygen._-_apparatus required:_ bulloch's apparatus. sterile gas filter (_vide_ page 40). gasometer containing the gas it is desired to test (so_{2}, n_{2}o, no, co_{2}, etc.) or gas generator for its production. method.-1. prepare at least seven tube cultivations upon solid media and deposit them in bulloch's apparatus. 2. connect up the inlet tube of the bulloch's jar with the sterile gas filter, and this again with the delivery tube of the gasometer or gas generator. 3. open both stop-cocks of the bulloch's apparatus and pass the gas through until it has completely replaced the air in the bell jar as shown by the result of analyses of samples collected from the exit tube. 4. incubate under optimum conditions as to temperature. 5. examine the cultivations at intervals of twenty-four hours, until the completion of seven days. 6. remove one tube from the interior of the apparatus each day. if no growth is visible, incubate the tube under optimum conditions as to temperature _and_ atmosphere, and in this way determine the length of exposure to the action of the gas necessary to kill the organisms under observation. 7. control these results. ~ii. temperature.~-(a) _range._-1. prepare a series of ten tube cultivations, in fluid media, of optimum reaction. 2. arrange a series of incubators at fixed temperatures, varying 5° c. and including temperatures between 5° c. and 50° c. (in the absence of a sufficient number of incubators utilise the water-bath employed in testing the thermal death-point of vegetative forms.) 3. incubate one tube cultivation of the organism aerobically or anaerobically, as may be necessary, in each incubator, and examine at half-hour intervals for from five to eighteen hours. 4. note that temperature at which growth is first observed macroscopically (optimum temperature). 5. continue the incubation until the completion of seven days. note the extremes of temperature at which growth takes place (range of temperature). 6. control these results--if considered necessary arranging the series of incubators to include each degree centigrade for five degrees beyond each of the extremes previously noted. (b) _optimum._-1. prepare a second series of ten tube cultivations under similar conditions as to reaction of medium. 2. incubate in a series of incubators in which the temperature is regulated at intervals of 1° c. for five degrees on either side of optimum temperature observed in the previous experiment (a, step 4). 3. observe again at half-hour intervals and note that temperature at which growth is first visible to the naked eye = optimum temperature. (c) _thermal death-point (t. d. p.)_-moist--vegetative forms: the _t. d. p._ here is that ~temperature~ which with certainty kills a watery suspension of the organisms in question after an exposure of ~10 minutes~. [illustration: fig. 155.--hearson's water-bath.] _apparatus required:_ water-bath. for the purpose of observing the thermal death-point a special water-bath is necessary. the temperature of this piece of apparatus is controlled by means of a capsule regulator that can be adjusted for intervals of half a degree centigrade through a range of 30°, from 50° c. to 80° c. by means of a spring, actuated by the handle a, which increases the pressure in the interior of the capsule. a hole is provided for the reception of the nozzle of a blast pump, so that a current of air may be blown through the water while the bath is in use, and thus ensure a uniform temperature of its contents. through a second hole is suspended a certified centigrade thermometer, the bulb of which although completely immersed in the water is raised at least 2 cm. above the floor of the bath. sterile glass capsules. flask containing 250 c.c. sterile normal saline solution. case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre). special platinum loop. test-tubes, 18 by 1.5 cm., of thin german glass. case of sterile petri dishes. tubes of agar or gelatine. method.-1. prepare tube cultivations on solid media of optimum reaction; incubate forty-eight hours under optimum conditions as to temperature and atmosphere. 2. examine preparations from the cultivation microscopically to determine the absence of spores. 3. pipette 5 c.c. salt solution into each of twelve capsules. 4. suspend three loopfuls of the surface growth (using a special platinum loop, _vide_ page 316) in the normal saline solution by emulcifying evenly against the moist walls of each capsule. 5. transfer emulsion from each capsule to sterile 250 c.c. flask, and mix. 6. pipette 5 c.c. emulsion into each of twelve sterile test-tubes numbered consecutively. 7. adjust the first tube in the water-bath, regulated at 40° c, by means of two rubber rings around the tube, one above and the other below the perforated top of the bath, so that the upper level of the fluid in the tube is about 4 cm. below the surface of the water in the bath, and the bottom of the tube is a similar distance above the bottom of the bath. 8. arrange a control test-tube containing 5 c.c. sterile saline solution under similar conditions. plug the tube with cotton-wool and pass a thermometer through the plug so that its bulb is immersed in the water. 9. close the unoccupied perforations in the lid of the water-bath by means of glass balls. 10. watch the thermometer in the test-tube until it records a temperature of 40° c. note the time. ten minutes later remove the tube containing the suspension, and cool rapidly by immersing its lower end in a stream of running water. 11. pour three gelatine (or agar) plates containing respectively 0.2, 0.3, and 0.5 c.c. of the suspension, and incubate. 12. pipette the remaining 4 c.c. of the suspension into a culture flask containing 250 c.c. of nutrient bouillon, and incubate. 13. observe these cultivations from day to day. "no growth" must not be recorded as final until after the completion of seven days' incubation. 14. extend these observations to the remaining tubes of the series, but varying the conditions so that each tube is exposed to a temperature 2° c. higher than the immediately preceding one--i. e., 42° c., 44° c., 46° c., and so on. 15. note that temperature, after exposure to which no growth takes place up to the end of seven days' incubation, = the thermal death-point. 16. if greater accuracy is desired, a second series of tubes may be prepared and exposed for ten minutes to fixed temperatures varying only 0.5° c., through a range of 5° c. on either side of the previously observed death-point. moist--spores: the thermal death-point in the case of spores is that ~time exposure~ to a ~fixed temperature of 100° c.~ necessary to effect the death of all the spores present in a suspension. note.--if it is desired to retain the ~time constant 10 minutes~ and investigate the temperature necessary to destroy the spores, varying amounts of calcium chloride must be added to the water in the bath, when the boiling-point will be raised above 100° c. according to the percentage of calcium in solution. in such case use the bath figured on page 227; the bath figured on page 299 can only be used if the capsule is first removed. it is determined in the following manner _apparatus required:_ steam-can fitted with a delivery tube and a large bore safety-valve tube. water-bath at 100° c. erlenmeyer flask, 500 c.c. capacity, containing 140 c.c. sterile normal saline solution and fitted with rubber stopper perforated with four holes. the rubber stopper is fitted as follows: (a) thermometer to 120° c., its bulb immersed in the normal saline. (b) straight entry tube, reaching to the bottom of the flask, the upper end plugged with cotton-wool. (c) bent syphon tube, with pipette nozzle attached by means of rubber tubing and fitted with pinch-cock. the nozzle is protected from accidental contamination by passing it through the cotton-wool plug of a small test-tube. (d) a sickle-shaped piece of glass tubing passing just through the stopper, plugged with cotton-wool, to act as a vent for the steam. sterile plates. sterile pipettes. sterile test-tubes graduated to contain 5 c.c. _media required:_ gelatine or agar. culture flasks containing 200 c.c. nutrient bouillon. [illustration: fig. 156.--apparatus arranged for the determination of the death-point of spores.] method.-1. prepare twelve tube cultivations upon the surface (or two cultures in large flat culture bottles--_vide_ page 5) of nutrient agar and incubate under the optimum conditions (previously determined), for the formation of spores. examine preparations from the cultures microscopically to determine the presence of spores. 2. pipette 5 c.c. sterile normal saline into each culture tube or 30 c.c. into each bottle and by means of a sterile platinum spatula emulsify the entire surface growth with the solution. 3. add the 60 c.c. emulsion to 140 c.c. normal saline contained in the fitted erlenmeyer flask. 4. place the flask in the water-bath of boiling water. 5. connect up the straight tube, after removing the cotton-wool plug, with the delivery tube of the steam can; remove the plug from the vent tube. 6. when the thermometer reaches 100° c., open the spring clip on the _syphon_, discard the first cubic centimeter of suspension that syphons over (i. e., the contents of the syphon tube); collect the next 5 c.c. of the suspension in the sterile graduated test-tube and pour plates and prepare flask cultures therefrom as in the previous experiments. 7. repeat this process at intervals of twenty-five minutes' steaming. 8. observe the inoculated plates and flasks up to the completion, if necessary, of seven days' incubation. 9. control these experiments, but in this instance syphon off portions of the suspension at intervals of one-half to one minute during the five or ten minutes preceding the previously determined death-point. _thermal death-point._-dry--vegetative forms: the thermal death-point in this case is that ~temperature~ which with certainty kills a thin film of the organism in question after a time exposure of ~ten minutes~. _apparatus required:_ hot-air oven, provided with thermo-regulator. sterile cover-slips. flask containing 250 c.c. sterile normal saline solution. case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre). case of sterile capsules. crucible tongs. method.-1. prepare an emulsion with three loopfuls from an optimum cultivation in 5 c.c. normal saline in a sterile capsule and examine microscopically to determine the absence of spore forms. 2. make twelve cover-slip films on sterile cover-slips; place each in a sterile capsule to dry. 3. expose each capsule in turn in the hot-air oven for ten minutes to a different fixed temperature, varying 5° c. between 60° c. and 120° c. 4. remove each capsule from the oven with crucible tongs immediately after the ten minutes are completed; remove the cover-glass from its interior with a sterile pair of forceps. 5. deposit the film in a flask containing 200 c.c. nutrient bouillon. 6. prepare subcultivations from such flasks as show evidence of growth, to determine that no accidental contamination has taken place but that the organism originally spread on the film is responsible for the growth. 7. control the result of these experiments. dry--spores: the thermal death-point in this case is that ~temperature~ which with certainty kills the spores of the organism in question when present in a thin film after a time exposure of ~10 minutes~. _apparatus required:_ as for vegetative forms. method.-1. prepare a sloped agar tube cultivation and incubate under optimum conditions as to spore formations. 2. pipette 5 c.c. sterile normal saline into the culture tube and emulsify the entire surface growth in it. examine microscopically to determine the presence of spores in large numbers. 3. spread thin even films on twelve sterile cover-slips and place each cover-slip in a separate sterile capsule. 4. expose each capsule in turn for ten minutes to a different fixed temperature, varying 5°c, between 100° c. and 160°c. 5. complete the examination as for vegetative forms. ~iii. reaction of medium.~ (a) _range._-1. prepare a bouillon culture of the organism and incubate, under optimum conditions as to temperature and atmosphere, for twenty-four hours. 2. pipette 0.1 c.c. of the cultivation into a sterile capsule; add 9.9 c.c. sterile bouillon and mix thoroughly. 3. prepare a series of tubes of nutrient bouillon of varying reactions, from +25 to -30 (_vide_ page 155), viz.: +25, +20, +15, +10, +5, neutral, -5, -10, -15, -20, -25, -30. 4. inoculate each of the bouillon tubes with 0.1 c.c. of the diluted cultivation by means of a sterile graduated pipette and incubate under optimum conditions. 5. observe the cultures at half-hourly intervals from the third to the twelfth hours. note the reaction of the tube or tubes in which growth is first visible macroscopically (probably optimum reaction). 6. continue the incubation until the completion, if necessary, of seven days. note the extremes of acidity and alkalinity in which macroscopical growth has developed (range of reaction). 7. control the result of these observations. (b) _optimum reaction._--the optimum reaction has already been roughly determined whilst observing the range. it can be fixed within narrower limits by inoculating in a similar manner a series of tubes of bouillon which represent smaller variations in reaction than those previously employed (say, 1 instead of 5) for five points on either side of the previously observed optimum. for example, the optimum reaction observed in the set of experiments to determine the range was +10. now plant tubes having reactions of +15, +14, +13, +12, +11, +10, +9, +8, +7, + 6, +5, and observe as before. ~iv. resistance to lethal agents.~-(a) _desiccation._- _apparatus required:_ mueller's desiccator. this consists of a bell glass fitted with an exhaust tube and stop-cock (d), which can be secured to a plate-glass base (c) by means of wax or grease. it contains a cylindrical vessel of porous clay (a) into the top of which pure sulphuric acid is poured whilst the material to be dried is placed within its walls on a glass shelf (b). the air is exhausted from the interior and the acid rapidly converts the clay vessel into a large absorbing surface (fig. 157). exhaust pump. pure concentrated sulphuric acid. sterile cover-slips. sterile forceps. culture flask containing 200 c.c. nutrient bouillon. sterile ventilated petri dish. this is prepared by bending three short pieces of aluminium wire into v shape and hanging these on the edge of the lower dish and resting the lid upon them (fig. 158). method.-1. prepare a surface cultivation on nutrient agar in a culture bottle and incubate under optimum conditions for forty-eight hours. 2. examine preparations from the cultivation, microscopically, to determine the absence of spores. 3. pipette 5 c.c. sterile normal saline solution into the flask and suspend the entire growth in it. 4. spread the suspension in thin, even films on sterile cover-slips and deposit inside sterile "plates" to dry. 5. as soon as dry, transfer the cover-slip films to the ventilated petri dish by means of sterile forceps. [illustration: fig. 157.--mueller's desiccator.] 6. place the petri dish inside the mueller's desiccator; fill the upper chamber with pure sulphuric acid, cover with the bell jar, and exhaust the air from its interior. ten minutes later connect up the desiccator to a sulphuric acid wash-bottle interposing an air filter so that only dry sterile air enters. [illustration: fig. 158.--petri dish for drying cultivations.] 7. at intervals of five hours open the apparatus, remove one of the cover-slip films from the petri dish, and transfer it to the interior of a culture flask, with every precaution against contamination. reseal the desiccator and again exhaust, and subsequently admit dry sterile air as before. 8. incubate the culture flask under optimum conditions until the completion of seven days, if necessary; and determine the time exposure at which death occurs. 9. pour plates from those culture flasks which grow, to determine the absence of contamination. 10. repeat these observations at hourly intervals for the five hours preceding and succeeding the death time, as determined in the first set of experiments. (b) _light._-(a) diffuse daylight: 1. prepare a tube cultivation in nutrient bouillon, and incubate under optimum conditions, for forty-eight hours. [illustration: fig. 159.--plate with star for testing effect of light.] 2. pour twenty plate cultivations, ten of nutrient gelatine and ten of nutrient agar, each containing 0.1 c.c. of the bouillon culture. 3. place one agar plate and one gelatine plate into the hot and cold incubators, respectively, as _controls_. 4. fasten a piece of black paper, cut the shape of a cross or star, on the centre of the cover of each of the remaining plates (fig. 159). 5. expose these plates to the action of diffuse daylight (not direct sunlight) in the laboratory for one, two, three, four, five, six, eight, ten, twelve hours. 6. after exposure to light, incubate under optimum conditions. 7. examine the plate cultivations after twenty-four and forty-eight hours' incubation, and compare with the two controls. record results. if growth is absent from that portion of the plate unprotected by the black paper, continue the incubation and daily observation until the end of seven days. 8. control the results. (b) direct sunlight: 1. prepare plate cultivations precisely as in the former experiments and place the two controls in the incubators. 2. arrange the remaining plates upon a platform in the direct rays of the sun. 3. on the top of each plate stand a small glass dish 14 cm. in diameter and 5 cm. deep. 4. fill a solution of potash alum (2 per cent. in distilled water) into each dish to the depth of 2 cm. to absorb the heat of the sun's rays and so eliminate possible effects of temperature on the cultivations. 5. after exposures for periods similar to those employed in the preceding experiment, incubate and complete the observation as above. (c) primary colours: each colour--violet, blue, green and red--must be tested separately. 1. prepare plate cultivations, as in the previous "light" experiments, and incubate controls. 2. fasten a strip of black paper, 3 cm. wide, across one diameter of the cover of each plate. 3. coat the remainder of the surface of the cover with a film of pure photographic collodion which contains 2 per cent. of either of the following aniline dyes, as may be necessary: chrysoidin (for red). malachite green (for green). eosin, bluish (for blue). methyl violet (for violet). 4. expose the plates, thus prepared, to bright daylight (but not direct sunlight) for varying periods, and complete the observations as in the preceding experiments. the bactericidal action of light appears to depend upon the more refrangible rays of the violet end of the spectrum and is noted whether the red yellow rays are transmitted or not. 5. control the results. note.--the ultra-violet rays obtained from a quartz mercury vapour lamp destroy bacterial life with great rapidity under laboratory conditions. (c) _heat._--(_vide_ thermal death-point, page 298.) (d) _antiseptics and disinfectants._--the resistance exhibited by any given bacterium toward any specified disinfectant or germicide should be investigated with reference to the following points: (a) ~inhibition coefficient~--i. e., that _percentage of the disinfectant_ present in the nutrient medium which is sufficient to prevent the growth and multiplication of the bacterium. (b) ~inferior lethal coefficient~--i. e., the _time exposure_ necessary to kill _vegetative forms_ of the bacterium suspended in water at 20° to 25° c, in which the disinfectant is present in _medium_ concentration (concentration insufficient to cause plasmolysis). and if the bacterium is one which forms spores, (c) ~superior lethal coefficient~--i. e., the _time exposure_ necessary to kill the _spores_ of the bacterium under conditions similar to those obtaining in b. the example here detailed only specifically refers to certain of the disinfectants: viz:--bichloride of mercury; formaldehyde; carbolic acid; investigated with regard to b. anthracis, but the technique is practically similar for all other chemical disinfectants. ~inhibition coefficient.~- _apparatus required:_ case of sterile pipettes, 10 c.c. (in tenths). case of sterile pipettes, 1 c.c. (in tenths). sterile tubes or capsules for dilutions. tubes of nutrient bouillon each containing a measured 10 c.c. of medium. twenty-four-hour-old agar culture of a recently isolated b. anthracis. _germicides:_ 1. five per cent. aqueous solution of carbolic acid. 2. one per cent. aqueous solution of perchloride of mercury. 3. one-tenth per cent. aqueous solution of formaldehyde. method.-1. number six bouillon tubes consecutively 1 to 6. inoculate each from the stock cultivation of b. anthracis and at once add varying quantities[10] of the carbolic acid solution, viz.: to tube 1 add 2.0 c.c. (= 1:100) to tube 2 add 1.0 c.c. (= 1:200) to tube 3 add 0.6 c.c. (= 1:300) to tube 4 add 0.5 c.c. (= 1:400) to tube 5 add 0.4 c.c. (= 1:500) to tube 6 add 0.2 c.c. (= 1:1,000) 2. prepare a similar series of tube cultivations numbered consecutively 7 to 12 and add varying quantities of the mercuric perchloride solution, viz.: to tube 7 add 0.1 (= 1:1,000) to tube 8 add 0.05 (= 1:2,000) to tube 9 add 0.03 (= 1:3,000) to tube 10 add 0.025 (= 1:4,000) to tube 11 add 0.02 (= 1:5,000) to tube 12 add 0.01 (= 1:10,000) 3. prepare a similar series of tube cultivations numbered consecutively 13 to 18 and add varying quantities of the formaldehyde solution, viz.: to tube no. 13 add 1.0 c.c. (= 1:1,000) to tube no. 14 add 0.4 c.c. (= 1:2,500) to tube no. 15 add 0.2 c.c. (= 1:5,000) to tube no. 16 add 0.1 c.c. (= 1:10,000) to tube no. 17 add 0.075 c.c. (= 1:15,000) to tube no. 18 add 0.05 c.c. (= 1:20,000) 4. incubate all three sets of cultivations under optimum conditions as to temperature and atmosphere. 5. examine each of the culture tubes from day to day, until the completion of seven days, and note those tubes, if any, in which growth takes place. 6. from such tubes as show growth prepare subcultivations upon suitable media, and ascertain that the organism causing the growth is the one originally employed in the test and not an accidental contamination. ~inferior lethal coefficient.~- _apparatus required:_ highly concentrated solutions of the disinfectants. sterile test-tubes in which to make dilutions from the concentrated solutions of the disinfectants. hanging-drop slides. cover-slips. erlenmeyer flask containing 100 c.c. sterile distilled water. case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre). case of sterile pipettes, 1 c.c. (in tenths of a cubic centimetre). method.-1. prepare a surface cultivation of the "test" organism b. anthracis upon nutrient agar in a culture bottle and incubate under optimum conditions for twenty-four hours; then examine the cultivation microscopically to determine the absence of spores. 2. prepare solutions of different percentages of each disinfectant. 3. make a series of hanging-drop preparations from the agar culture, using a loopful of disinfectant solution of the different percentages to prepare the emulsion on each cover-slip. 4. examine microscopically and note the strongest solution which does not cause plasmolysis and the weakest solution which does plasmolyse the organism. 5. make control preparations of these two solutions and determine the percentage to be tested. 6. pipette 10 c.c. sterile water into the culture bottle and suspend the entire surface growth in it. 7. transfer the suspension to the erlenmeyer flask and mix it with the 90 c.c. of sterile water remaining in the flask. 8. pipette 10 c.c. of the diluted suspension into each of ten sterile test-tubes. 9. label one of the tubes "control" and place it in the incubator at 18° c. 10. add to each of the remaining tubes a sufficient quantity[11] of a concentrated solution of the disinfectant to produce the percentage previously determined upon (_vide_ step 5). 11. incubate the tubes at 18° c. to 20° c. 12. at hourly intervals remove the control tube and one of the tubes with added disinfectant from the incubator. 13. make a subcultivation from both the control and the test suspension, upon the surface of nutrient agar; incubate under optimum conditions. 14. observe these culture tubes from day to day until the completion of seven days, and determine the shortest exposure necessary to cause the death of vegetative forms. ~superior lethal coefficient.~-1. prepare surface cultivations of the "test" organisms upon nutrient agar in a culture bottle, and incubate under optimum conditions, for three days, for the formation of their spores. 2. transfer the emulsion to a sterile test-tube and heat in the differential steriliser for ten minutes at 80° c. to destroy all vegetative forms. 3. employing that percentage solution of the disinfectant determined in the previous experiment, and complete the investigations as detailed therein, steps 7 to 14, increasing the interval between planting the subcultivations to two, three, or five hours if considered advisable. note.--where it is necessary to leave the organisms in contact with a strong solution of the disinfectant for lengthy periods, some means must be adopted to remove every trace of the disinfectant from the bacteria before transferring them to fresh culture media; otherwise, although not actually killed, the presence of the disinfectant may prevent their development, and so give rise to an erroneous conclusion. consequently it is essential in all germicidal experiments to determine first of all the inhibition coefficient of the germicide employed. under the circumstances referred to above it is usually sufficient to prepare the subcultures in such a volume of fluid nutrient medium as would suffice to reduce the concentration of the germicide to about one hundredth of the inhibition percentage, assuming that the entire bulk of inoculum was made up of that strength of germicide employed in the test. in some cases it is a simple matter to neutralise the germicide and render it inert by washing the organisms in some non-germicidal solution (such for example as ammonium sulphide when using mercurial salts as the germicide). when, however, it is desired to remove the last traces of germicide proceed as follows: 1. transfer the suspension of bacteria to sterile centrifugal tubes; add the required amount of disinfectant, and allow it to remain in contact with the bacteria for the necessary period. 2. centrifugalise thoroughly, pipette off the supernatant fluid; fill the tube with sterile water and distribute the deposit evenly throughout the fluid. 3. centrifugalise again, pipette off the supernatant fluid; fill the tube with sterile water; distribute the deposit evenly throughout the fluid, and transfer the suspension to a litre flask. 4. make up to a litre by the addition of sterile water; filter the suspension through a sterile porcelain candle. 5. emulsify the bacterial residue with 5 c.c. sterile bouillon. 6. prepare the necessary subcultivations from this emulsion. pathogenesis. _living bacteria._-(a) psychrophilic bacteria: when the organism will only grow at or below 18° to 20° c., 1. prepare cultivations in nutrient broth and incubate under optimum conditions. 2. after seven days' incubation inject that amount of the culture corresponding to 1 per cent. of the body-weight of a healthy frog, into the reptile's dorsal lymph sac. 3. observe until death takes place, or, in the event of a negative result, until the completion of twenty-eight days (_vide_ chapter xviii). 4. if, and when, death occurs, make a careful post-mortem examination (_vide_ chapter xix). (b) mesophilic bacteria: when the organism grows at 35° to 37° c., 1. prepare cultivations in nutrient broth and incubate under optimum conditions for forty-eight hours. 2. select two white mice, as nearly as possible of the same age, size, and weight. 3. inoculate the first mouse, subcutaneously at the root of the tail, with an amount of cultivation equivalent to 1 per cent. of its body-weight. 4. inoculate the second mouse intraperitoneally with a similar dose. 5. observe carefully until death occurs, or until the lapse of twenty-eight days. 6. if the inoculated animals succumb, make complete post-mortem examination. if death follows shortly after the injection of cultivations of bacteria, the inoculation experiments should be repeated two or three times. then, if the organism under observation invariably exhibits pathogenic effects, steps should be taken to ascertain, if possible, the minimal lethal dose (_vide infra_) of the growth upon solid media for the frog or white mouse respectively. other experimental animals--_e. g._, the white rat, guinea-pig, and rabbit--should next be tested in a similar manner. 7. if the inoculated mice are unaffected, test the action of the organism in question upon white rats, guinea-pigs, rabbits, etc. _minimal lethal dose_ (_m. l. d._); if the purpose of the inoculation is to determine the minimal lethal dose, a slightly different procedure must be followed. for this and other exact experiments a special platinum loop is manufactured, some 2.5 mm. by 0.75 mm., with parallel sides, and calibrated by careful weighing, to determine approximately the amount of moist bacterial growth, the loop will hold when filled. 1. the cultivation must be prepared on a solid medium of the optimum reaction, incubated at the optimum temperature, and injected at the period of greatest activity and vigour, of the particular organism it is desired to test. 2. arrange four sterile capsules in a row and label them i, ii, iii, and iv. into the first deliver 10 c.c. sterile bouillon by means of a sterile graduated pipette; and into each of the remaining three, 9.9 c.c. 3. remove one loopful of the bacterial growth from the surface of the medium in the culture tube, observing the usual precautions against contamination, and emulsify it evenly with the bouillon in the first capsule. each cubic centimetre of the emulsion will now contain one-tenth of the organisms contained in the original loopful (written shortly 0.1 loop). 4. remove 0.1 c.c. of the emulsion in the first capsule by means of a sterile graduated pipette and transfer it to the second capsule and mix thoroughly. drop the infected pipette into a jar of lysol solution. this makes up the bulk of the fluid in the second capsule to 10 c.c., and therefore every cubic centimetre of bouillon in capsule ii contains 0.001 loop. 5. similarly, 0.1 c.c. of the mixture is transferred from capsule ii to capsule iii (1 c.c. of bouillon in capsule iii contains 0.00001 loop), and then from capsule iii to capsule iv (1 c.c. of bouillon in capsule iv contains 0.0000001 loop). the dilutions thus prepared may be summarised in a table; capsule i = 1 loopful + 10 c.c. water [.'.] 1 c.c.=0.1 loop. capsule ii = 0.1 c.c. capsule i + 9.9 c.c. water [.'.] 1 c.c.=0.001 loop. capsule iii = 0.1 c.c. capsule ii + 9.9 c.c. water [.'.] 1 c.c.=0.00001 loop. capsule iv = 0.1 c.c. capsule iii + 9.9 c.c. water [.'.] 1 c.c. = 0.0000001 loop. 6. with sterile graduated pipettes remove the necessary quantity of bouillon corresponding to the various divisors of ten of the loop from the respective capsules, and transfer each "dose" to a separate sterile capsule and label; and to such doses as are small in bulk, add the necessary quantity of sterile bouillon to make up to 1 c.c. 7. multiples of the loop are prepared by emulsifying 1, 2, 5, or 10 loops each with 1 c.c. sterile bouillon in separate sterile capsules. 8. inoculate a series of animals with these measured doses, filling the syringe first from that capsule containing the smallest dose, then from the capsule containing the next smallest, and so on. if care is taken, it will not be found necessary to sterilise the syringe during the series of inoculations. 9. plant tubes of gelatine or agar, liquefied by heat, from each of the higher dilutions, say from 0.0000001 loop to 0.01 loop; pour plates and incubate. when growth is visible enumerate the number of organisms present in each, average up and calculate the number of bacteria present in one loopful of the inoculum. 10. the smallest dose which causes the infection and death of the inoculated animal is noted as the minimal lethal dose. _toxins._-prepare flask cultivations of the organism under observation in glucose formate broth, and incubate for fourteen days under optimum conditions. (a) intracellular or insoluble toxins: 1. heat the fluid culture in a water-bath at 60° c. for thirty minutes. (the resulting sterile, turbid fluid is often spoken of as "killed" culture,) 2. inoculate a tube of sterile bouillon with a similar quantity, and incubate under optimum conditions. this "control" then serves to demonstrate the freedom of the toxin from living bacteria. [illustration: fig. 160.--apparatus arrange for toxin filtration.] 3. inject intraveneously that amount of the cultivation corresponding to 1 per cent. of the body-weight of the selected animal, usually one of the small rodents. 4. observe during life or until the completion of twenty-eight days, and in the event of death occurring during that period, make a complete post-mortem examination. 5. repeat the experiment at least once. in the event of a positive result estimate the minimal lethal dose of "killed" culture for each of the species of animals experimented upon. (b) extracellular or soluble toxins: 1. filter the cultivation through a porcelain filter candle (berkefeld) into a sterile filter flask, arranging the apparatus as in the accompanying figure (fig. 160). 2. inoculate mice, rats, guinea-pigs, and rabbits subcutaneously with that quantity of toxin corresponding to 1 per cent. of the body-weight of each respectively, and observe, if necessary, until the completion of one month. 3. inoculate a "control" tube of bouillon with a similar quantity and incubate, to determine the freedom of the filtered toxin from living bacteria. 4. in the event of a fatal termination make complete and careful post-mortem examinations. 5. repeat the experiments and, if the results are positive, ascertain the minimal lethal dose of toxin for each of the susceptible animals. the estimation of the _m. l. d._ of a toxin is carried out on lines similar to those laid down for living bacteria (_vide_ page 316) merely substituting 1 c.c. of toxin as the unit in place of the unit "loopful" of living culture. it frequently happens, during the course of casual investigations that a bouillon-tube culture is available for a toxin test whilst a flask cultivation is not. in such cases, martin's small filter candle and tube (fig. 161) specially designed for the filtration of small quantities of fluid, is invaluable. this consists of a narrow filter flask just large enough to accommodate an ordinary 18 × 2 cm. test-tube. the mouth of the tubular chamberland candle 15 × 1.5 cm. is closed by a perforated rubber cork into which fits the end of the stem of a thistle headed funnel, whilst immediately below the butt of the funnel is situated a rubber cork to close the mouth of the filter flask. when the apparatus is fixed in position and connected to an exhaust pump, the cultivation is poured into the head of the funnel and owing to the relatively large filtering surface the germ free filtrate is rapidly drawn through into the test-tube receiver. ~raising the virulence of an organism.~--if it is desired to raise or "exalt" the virulence of a feebly pathogenic organism, special methods of inoculation are necessary, carefully adjusted to the exigencies of each individual case. among the most important are the following: 1. _passage of virus._--the inoculation of pure cultivations of the organism into highly susceptible animals, and passing it as rapidly as possible from animal to animal, always selecting that method of inoculation-e. g., intraperitoneal--which places the organism under the most favorable conditions for its growth and multiplication. [illustration: fig. 161--martin's filtering apparatus for small quantities of fluid.] 2. _virus plus virulent organisms._--the inoculation of pure cultivations of the organism together with pure cultivations of some other microbe which in itself is sufficiently virulent to ensure the death of the experimental animal, either into the same situation or into some other part of the body. by this association the organism of low virulence will frequently acquire a higher degree of virulence, which may be still further raised by means of "passages" (_vide supra_). 3. _virus plus toxins._--the inoculation of pure cultivations of the organism into some selected situation, together with the subcutaneous, intraperitoneal, or intravenous injection of a toxin--e. g., one of those elaborated by the proteus group--either simultaneously with, before, or immediately after, the injection of the feeble virus. by this means the natural resistance of the animal is lowered, and the organism inoculated is enabled to multiply and produce its pathogenic effect, its virulence being subsequently exalted by means of "passages." ~attenuating the virulence of an organism.~--attenuating or lowering the virulence of a pathogenic microbe is usually attained with much less difficulty than the exaltation of its virulence, and is generally effected by varying the environment of the cultivations, as for example: 1. cultivating in such media as are unsuitable by reason of their (a) composition or (b) reaction. 2. cultivating in suitable media, but at an unsuitable temperature. 3. cultivating in suitable media, but in an unsuitable atmosphere. 4. cultivation in suitable media, but under unfavorable conditions as to light, motion, etc. attenuation of the virus can also be secured by 5. passage through naturally resistant animals. 6. exposure to desiccation. 7. exposure to gaseous disinfectants. 8. by a combination of two or more of the above methods. immunisation. the further study of the pathogenetic powers of any particular bacterium involves the active immunisation of one or more previously normal animals. this end may be attained by various means; but it must be remembered that immunisation is not carried out by any hard and fast rule or by one method alone, but usually by a combination of methods adapted to the exigencies of each particular case. the ordinary methods include: a. active immunisation. i. by inoculation with dead bacteria (i. e., bacteria killed by heat; the action of ultra-violet rays, of chemical germicides, or by autolysis). ii. by the inoculation of attenuated strains of bacteria. iii. by the inoculation of living virulent bacteria (exalted in virulence if necessary). b. combined active and passive immunisation: iv. by the inoculation of toxin-antitoxin mixtures. active immunisation. the immunisation of the rabbit against the diplococcus pneumoniæ may be instanced as an example of the general methods of immunisation of laboratory animals. 1. take a full grown rabbit weighing not less than 1200 to 1500 grammes (large rabbits of 2000 grammes and over are the most suitable for immunising experiments). observe weight and temperature carefully during the few days occupied in the following steps. 2. inoculate a small rabbit intraperitoneally with one or two loopfuls of a twenty-four-hour-old blood agar cultivation of a _virulent_ strain of diplococcus pneumoniæ. death should follow within twenty-four hours, and in any case will not be delayed beyond forty-eight hours. 3. under aseptic precautions, at the post-mortem, transfer a loopful of heart blood to an erlenmeyer flask containing 50 c.c. sterile nutrient broth. incubate at 37° c. for twenty-four hours. 4. prepare also several blood agar cultures from the heart blood of the rabbit, label them all o.c. (original culture). after twenty-four hours incubation at 37° c. place an india-rubber cap over the plugged mouth of the tube of all but one of these cultures and paint the cap with canada balsam or shellac varnish, dry, and replace in the hot incubator. this will prevent evaporation, and cultures thus sealed will remain unaltered in virulence for a considerable time. 5. make a fresh subcultivation on blood agar from the uncapped o.c. cultivation and after twenty-four hours incubation at 37° c. determine the minimal lethal dose of this strain upon a series of mice (see page 316). 6. suspend the flask containing the twenty-four-hour-old broth culture (step 3) in the water-bath at 60° c. for one hour. cool the flask rapidly under a stream of cold water. 7. determine the sterility of this (?) killed cultivation by transferring one cubic centimetre to each of several tubes of nutrient broth, and incubate at 37° c. for twenty-four hours. if growth of diplococcus pneumoniæ occurs, again heat culture in water-bath at 60° c. for one hour and again test for sterility. 8. inject the selected rabbit intravenously (see page 363) with 2 c.c. of the killed cultivation, and inject a further 10 c.c. into the peritoneal cavity. during the next few days the animal will lose some weight and perhaps show a certain amount of pyrexia. 9. when the temperature and weight have again returned to normal--generally about seven days after the inoculation--again inject killed cultivation, this time giving a dose of 5 c.c. intravenously and 20 c.c. intraperitoneally. a temperature and weight reaction similar to, but less marked than that following the first injection will probably be observed, but after about a week's interval the animal will be ready for the next injection. 10. when ready to give the third injection prepare a fresh blood agar subculture from another o.c. tube and after twenty-four hours incubation prepare a minimal lethal dose (as determined in 5) and inject it subcutaneously into the rabbit's abdominal wall. a slight local reaction will probably be observed as well as the weight and temperature reactions. 11. a week to ten days later inject a similar minimal lethal dose into the peritoneal cavity. 12. observe the weight and temperature of the rabbit very carefully, and regulating the dates of inoculation by the animal's general condition, continue to inject living cultivations of the pneumococcus into the peritoneal cavity, gradually increasing the dose by multiples of ten. 13. at intervals of two months samples of blood may be collected from the posterior auricular vein and the serum tested for specific antibodies. 14. under favourable conditions it will be found after some six months steady work that the rabbit may be injected intraperitoneally with an entire blood agar cultivation without any ill effects being apparent; and this characteristic--resistance to the lethal effects of large doses of the virus--is the sole criterion of _immunity_. further, the serum separated from blood withdrawn from the animal about a week after an injection, if used in doses of .01 c.c., will protect a mouse against the lethal effects of at least ten minimal lethal doses of living pneumococci. in the foregoing illustration it has been assumed that complete acquired active immunity has been conferred upon the experimental rabbit in consequence of the formation of antibody, specific to the diplococcus pneumoniac, sufficient in amount to ensure the destruction of enormous doses of the living cocci--the _antigen_ (that is the substance injected in response to which _antibody_ has been elaborated) in this particular case being the bacterial protoplasm of the pneumococcus with its endo-toxins. but provided death does not immediately follow the injection of the antigen, specific antibody is always formed in greater or lesser amount; and in experimental work a sufficient amount of any required antibody can often be obtained without carrying the process of immunisation to its logical termination. for instance, if the immunisation of a rabbit toward bacillus typhosus is commenced on the lines already set out it will often be found, after a few injections of "killed" cultivation that the blood serum of the animal (even when diluted with several hundred times its volume of normal saline) contains specific agglutinin for b. typhosus--and if the sole object of the experiment has been the preparation of agglutinin the inoculations may well be stopped at this point, although the animal is not yet immune in the strict meaning of the word. again, antibodies may be formed in response to antigens other than infective particles--thus the injection into suitable animals of foreign proteins such as egg albumin, heterologous blood sera or red blood discs from a different species of animal, will result in the formation of specific antibodies possessing definite affinities for their respective antigens. the most important antibody of this latter type is hæmolysin, a substance that makes its appearance in the blood serum of an animal previously injected with washed blood cells from an animal of a different species. the serum from such an animal possesses the power of disintegrating red blood discs of the variety employed as antigen and causing the discharge of their contained hæmoglobin, and is specific in its action to the extent of failing to exert any injurious effect upon the red blood cells of any other species of animal. the action of this serum is due to the presence of two distinct bodies, complement and hæmolysin. _complement_ (or alexine) is a thermo-labile readily oxidised body present in variable but unalterable amount in the normal serum of every animal. it is a substance which exerts a lytic effect upon all foreign matter introduced into the blood or tissues; but by itself is a comparatively inert body, and is only capable of exerting its maximum lytic effect in the presence of and in combination with a specific antibody, or immune body. complement is obtained (unmixed with antibody) by collecting fresh blood serum from any healthy normal (that is uninoculated) animal. guinea-pigs' serum is that most frequently employed for experimental work. _hæmolysin_ (immune body, copula, sensitising body, amboceptor) is a _thermostable_ antibody formed in response to the injection of red cells which although in itself inert is capable of linking up complement present in the normal serum to the red cells of the variety used as antigen--a combination resulting in hæmolysis. hæmolysin is obtained by collecting fresh blood serum from a suitably inoculated animal and exposing it to a temperature of 56° c. (to destroy the thermo-labile complement) for 15 to 30 minutes before use. it is then referred to as _inactivated_, and is _reactivated_ by the addition of fresh normal serum--that is serum containing complement. hæmolysin is of importance academically owing to the fact that many of the problems of immunity have been elucidated by its aid; but its present practical importance lies in the application of the _hæmolytic system_ (that is hæmolysin, corresponding erythrocyte solution and complement) to certain laboratory methods having for their object either the identification of the infective entity or the diagnosis of the existence of infection. for use in these laboratory methods of diagnosis it is most convenient to prepare hæmolytic serum specific for human blood--whether the laboratory is isolated or attached to a large hospital. ox blood, sheep blood or goat blood if readily obtainable, may however be used instead, and although the following method is directed to the preparation of human hæmolysin the same procedure serves in all cases. the preparation of hæmolytic serum. _apparatus required:_ small centrifuge, preferably electrically driven, with two receptacles for tubes, and enclosed in a safety shield (fig. 162). sterile centrifuge tubes (10 c.c. capacity), fig. 163. sterile pipettes (10 c.c. graduated) in case. sterile glass capsules (in case). sterile test-tubes. sterile all glass syringe (5 c.c. or 10 c.c. capacity) and needle. [illustration: fig. 162.--small electrical centrifuge.] [illustration: fig. 163.--centrifuge tube.] _reagents required:_ normal saline solution. 10 per cent. sodium citrate solution in normal saline. human blood (_vide infra_). method.-1. select a healthy full-grown rabbit of not less than 2500 grammes weight in accordance with the directions already given (page 322) and prepare it for intraperitoneal inoculation. 2. measure out 2 c.c. citrated human blood (collected at a surgical operation or a venesection, or withdrawn by venipuncture from the median basilic or median cephalic vein of a normal adult) into a centrifuge tube and centrifugalise thoroughly. 3. wash with three changes of normal saline (_vide_ also page 388). 4. transfer the washed cells to a sterile capsule by means of a sterile pipette. add 5 c.c. of normal saline and mix thoroughly. 5. take up the mixture of cells and saline in the all-glass syringe and inject into the peritoneal cavity of the rabbit. 6. seven days later inject intraperitoneally the washed cells from 5 c.c. human blood mixed with 5 c.c. normal saline. 7. seven days later inject the washed cells from 10 c.c. human blood mixed with 5 c.c. normal saline. 8. after a further interval of seven days repeat the injection of washed cells from 10 c.c. human blood mixed with 5 c.c. normal saline. note.--better results are obtained if the second and subsequent injections are made intravenously, even when smaller quantities of washed red cells are employed. if, however, the intravenous route is selected exceeding great care must be exercised to avoid the introduction of air into the vein--an accident which is followed, within a few minutes, by the death of the rabbit from pulmonary embolism. 9. allow five days to elapse, then collect a preliminary sample of blood, say about 2 c.c., from the rabbit's ear. allow it to clot, separate off the serum and transfer to a sterile test-tube. place the test-tube in a water-bath at 56° c. for fifteen minutes (to inactivate) and test the serum quantitatively for hæmolytic properties in the following manner: the titration of hæmolytic serum. _apparatus required:_ electrical centrifuge. sterile centrifuge tubes. water-bath regulated at 56°c. sterilised pipettes 10 c.c. graduated in tenths. sterilised pipettes 1 c.c. graduated in tenths. sterile test-tubes, 16 × 2 cm. small sterile test-tubes, 9 × 1 cm. small test-tube rack, or roll of plasticine. capillary teat pipettes. stout rubber band or length of small rubber tubing. _reagents required and method of preparation:_ 1. normal saline solution. 2. hæmolytic serum inactivated by preliminary heating to 56° c. for 15 minutes (_vide supra_) in test-tube labelled h. s. 3. complement. fresh guinea-pig serum in test-tube labelled c. kill a normal guinea-pig with chloroform vapour. open the thorax with all aseptic precautions, and collect as much blood as possible from the heart with a sterile pasteur pipette. transfer it to a sterile centrifuge tube and place the tube in the incubator at 37° c. two hours later separate the clot from the sides of the tube, and centrifugalise thoroughly. pipette off the clear serum to a clean sterilised test-tube. 4. erythrocyte solution, in test-tube labelled e. collect and wash human red blood cells (see page 388, 1-8). measure the volume of red cells available and prepare a 2 per cent. suspension in normal saline solution. method.-1. take two test-tubes and number them 1 and 2, and pipette into each 9 c.c. of normal saline solution. 2. add 1 c.c. of hæmolytic rabbit serum to tube no. 1 and mix thoroughly: take up 1 c.c. of the mixture and add it to tube no. 2; mix thoroughly. 3. set up ten small test-tubes in test-tube rack or in roll of plasticine, and number 1 to 10. 4. pipette into tube no. 1 0.5 c.c. = 0.5 c.c.} hæmolytic serum } from tube pipette into tube no. 2 0.1 c.c. = 0.1 c.c. } h. s. hæmolytic serum } pipette into tube no. 3 0.5 c.c. = 0.05 c.c. } hæmolytic serum } pipette into tube no. 4 0.3 c.c. = 0.03 c.c. } hæmolytic serum } from pipette into tube no. 5 0.2 c.c. = 0.02 c.c. } tube 1. hæmolytic serum } pipette into tube no. 6 0.1 c.c. = 0.01 c.c. } hæmolytic serum } pipette into tube no. 7 0.5 c.c. = 0.005 c.c. } hæmolytic serum } pipette into tube no. 8 0.3 c.c. = 0.003 c.c. } hæmolytic serum } from pipette into tube no. 9 0.2 c.c. = 0.002 c.c. } tube 2. hæmolytic serum } pipette into tube no. 10 0.1 c.c. = 0.001 c.c. } hæmolytic serum } 5. to each tube add 1 c.c. of erythrocyte solution. 6. when necessary (that is to say in tubes 2, 4, 5, 6, 8, 9 and 10) add normal saline solution to the mixture in the test-tubes till the column of fluid in each reaches to the same level. 7. shake each tube in turn, so as to thoroughly mix its contents. plug the mouth of each tube with cotton wool, and place entire set in the incubator at 37°c. for one hour. 8. remove the tubes from the incubator and into each tube pipette 0.1 c.c. complement (guinea-pig's serum) and replace tubes in incubator at 37° c. for further period of one hour. 9. remove the tubes from the incubator, and if complete hæmolysis has not taken place in every tube, stand on one side, preferably in the ice chest, for an hour. 10. then examine the tubes. complete hæmolysis is indicated by a clear red solution, with no deposit of red cells at the bottom of the test-tube. absence of hæmolysis is indicated by a clear or turbid colourless fluid, with a deposit of red cells at the bottom of the test-tubes. the smallest amount of hæmolytic serum that has caused complete hæmolysis is known as the minimal hæmolytic dose (_m. h. d._) and if hæmolysis has occurred in all the tubes down to no. 7--the m. h. d. of this particular serum is .005 c.c. = 200 minimal hæmolytic doses per cubic centimetre. such a serum is strong enough for experimental work; indeed, for many purposes, complete hæmolysis down to tube 6 will indicate a serum sufficiently strong(= 100 m. h. d. per cubic centimetre). if, however, only the first one or two tubes are completely hæmolysed, this is an indication that the rabbit should receive further injections in order to raise the hæmolytic power to a sufficiently high level. storage of hæmolysin. if, and when the hæmolysin content of the rabbit's serum is found to be sufficient, destroy the animal by chloroform vapour. remove as much of its blood as possible from the heart under aseptic precautions into sterilized centrifuge tubes. transfer the tubes of blood to the incubator at 37° c. for two hours--then centrifugalize thoroughly. pipette off the clear serum, and fill in quantities of 1 c.c., into small glass ampoules or pipettes, and hermetically seal in the blowpipe flame, care being taken to avoid scorching the serum. place the ampoules when filled with serum and sealed, in a water-bath at 56° c. for 30 minutes. this destroys the complement, i. e., inactivates the serum, and at the same time, provided the various operations have been carried out under aseptic precautions, ensures its sterility. a longer exposure reduces the hæmolytic power. place the ampoules in a closed metal box and store in the ice chest for future use. footnotes: [10] the quantities here given are not absolutely correct. if exactitude is essential the student must calculate the amount required by the aid of the percentage formula, appendix, page 496. [11] see percentage formula, appendix, page 496. xvii. experimental inoculation of animals. the use of living animals for inoculation experiments may become a necessary procedure in the bacteriological laboratory for some one or more of the following reasons: a. ~determination of pathogenetic properties of bacteria already isolated in pure culture~ (see page 315). the exact study of the conditions influencing the virulence (including its maintenance, exaltation and attenuation) of an organism, and precise observations upon the pathogenic effects produced by its entrance into, and multiplication within the body tissues can obviously only be carried out by means of experimental inoculation; whilst many points relating to vitality, longevity, etc., can be most readily elucidated by such experiments. b. ~isolation of pathogenetic bacteria.~ certain highly parasitic bacteria (which grow with difficulty upon the artificial media of the laboratory) can only be isolated with considerable difficulty from associated saprophytic bacteria when cultural methods alone are employed; but if the mixture of parasite and saprophytes is injected into an animal susceptible to the action of the former, the pathogenic organism can readily be isolated from the tissues of the infected animal. the pneumococcus for example occurs in the sputum of patients suffering from acute lobar pneumonia, but usually in association with various saprophytes derived from the mouth and pharynx. the optimum medium for the growth of the pneumococcus, blood agar, is also an excellent pabulum for the saprophytes of the mouth, and plate cultures are rapidly overgrown by them to the destruction of the more delicate pneumococcus. but inoculate some of the sputum under the skin of a mouse and three or four days later the pneumococcus will have entered the blood stream (leaving the saprophytes at the seat of inoculation) and killed the animal. cultivations made at the post-mortem (see page 398) from the mouse's heart blood will yield a pure growth of the pneumococcus. c. ~identification of pathogenetic bacteria.~ the resemblances, morphological and cultural, existing between certain pathogenetic bacteria are in some cases so great as to completely overwhelm the differences; again the same bacterium may under varying conditions assume appearances so different from those regarded as typical or normal as to throw doubt on its identity. in each case a simple inoculation experiment may decide the point at once. as a concrete example may be instanced an autopsy on an animal dead from an unknown infection. cultivations from the heart blood gave a pure growth of a typical (capsulated) pneumococcus. cultivations from the liver gave a pure growth of what appeared to be a typical (non-capsulated) streptococcus pyogenes longus. the latter inoculated into a rabbit caused the death of the animal from pneumococcic septicæmia, and cultures from the rabbit's blood gave a pure growth of a typical (capsulated) pneumococcus. ~d. study of the problems of immunity.~ it is only by a careful and elaborate study of the behaviour of the animal cell and the body fluids vis-à-vis with the infecting bacterium that it becomes possible to throw light upon the complex problem whereby the cell opposes successful resistance to the diffusion of the invading microbe, or succeeds in driving out the microbe subsequently to the occurrence of that diffusion. at the moment, however, our attention is directed to the first of these broad headings, for it is by the application of the knowledge acquired in its pursuit that we are able to deal with problems arising under any of the remainder. for whatever purpose the inoculation is performed, it is essential that the experiment should be planned to secure the maximum amount of information and the minimum of discomfort to the animal used. every care therefore must be taken to ensure that the virus is introduced into the exact tissue or organ selected; and the operation itself must be carried out with skill and expedition, and under strictly aseptic conditions. in the course of inoculation studies many instances of natural immunity, both racial and individual, will be met with; but it must be recollected that natural immunity is relative only and never absolute, and care be taken not to label an organism as _non-pathogenic_ until many different methods of inoculation have been performed upon different species of animals, combined when necessary with various procedures calculated to overcome any apparent immunity, and have invariably given negative results. in some countries experiments upon animals are only permitted under direct license from the government, and then only within premises specially licensed for the purpose. in england this license is in the grant of the home secretary, and confers the permission to experiment upon animals under general anæsthesia, provided that after the experiment is completed the animal must be destroyed before regaining consciousness. if it is intended to carry out simple hypodermic inoculations and superficial venesections, certificate a, granting this specific permission and dispensing with the necessity for general anæsthesia must be obtained _in addition to the license_; whilst if the inoculation entails more extensive operative procedures, and it is necessary to observe the subsequent course of the infection, should such occur, the license must be _coupled with certificate b_--since this certificate removes the compulsion to destroy the animal whilst under the anæsthetic. further special certificates and combinations of certificates are required if cats, dogs, horses, asses or cattle are to be the subjects of experiment. under every certificate it is expressly stipulated that if the animal shows signs of pain it must be destroyed immediately. the animals generally employed in the study of the pathogenic properties of the various micro-organisms are: _cold blooded._ _warm blooded._ _hot blooded._ frog. mouse. fowl. toad. rat. pigeon. lizard. guinea pig. rabbit. monkey. ~preparation.~--before inoculation, the experimental animals should be carefully examined, to avoid the risk of employing such as are already diseased: since it must be remembered that in a state of nature, as well as in captivity, the animals employed for laboratory inoculations are subject to infection by various animal and vegetable parasites, and in some instances such infection presents no symptoms which are obvious to the casual examination; the sex should be noted, the weight recorded, and the rectal temperature taken. the remaining items of importance are the time of the inoculation, the material that is inoculated, and the method of inoculation, and finally under what authority the experiment is performed. in the author's laboratory these data are entered upon a pink card which forms part of a card index system. the card further provides space for notes on the course of the resulting infection, and carries on the reverse the weight and temperature chart (figs. 164 and 165). [illustration: fig. 164.--front of inoculation card.] ~preliminary inspection and examination.~--the preliminary examination should comprise observation of the animal at rest and in motion; the appearance of the fur, feathers or scales, inspection of the eyes, and of external orifices of the body; tactile examination of the body and limbs, and palpation of the groins and abdomen; and in many cases the microscopical examination of fresh and stained blood-films. some of the commoner forms of naturally acquired infection may be briefly mentioned, without however touching upon the various fleas, lice and ticks which at times infect the ordinary laboratory animals. [illustration: fig. 165.--back of inoculation card.] ~the rabbit~, particularly in captivity, is subject to attacks of psoric acari, and the infection is readily transmitted to rabbits in neighbouring cages and also to guinea pigs, but not to rats and mice. one species (_sarcoptes minor_ var. _cuniculi_) gives rise to the ordinary mange. the infection first shows itself as thick yellowish scales and crusts around the nose, mouth and eyes, spreads to the bases and outer surfaces of the ears (never to the inside of the concha), to the fore and hind legs and into the groins and around the genitals. the acari can be readily demonstrated microscopically in scrapings of the skin, treated with liquor potassæ. another form of scabies (due to psoroptes _communis cuniculi_) commences at the bottom of the concha, which is filled with whitish-yellow masses consisting of dried crusts, scales, fæces, and dead acari. the base of the ear is hard and swollen, and lifting the animal by the ears--as is usually done--gives rise to considerable pain; indeed this symptom may be the one which first attracts attention to an infection, which causes progressive wasting and terminates in death. a mixed infection--sarcoptic plus psorotic acariasis--is sometimes seen. if it is decided to try and save animals suffering from infection by these parasites, they must be segregated, the scabs carefully cleaned from the infected areas and the denuded surfaces washed with 5 per cent. solution of potassium persulphate (a few drops being allowed to run into the concha), or with a preparation containing equal parts of soft paraffin and vaseline with a few drops of lysol. this treatment should be repeated daily until the acarus is destroyed and the animal has regained its normal condition. the cages should be disinfected and all neighbouring animals carefully examined, and any which show signs of infection should be treated in a similar manner. favus also attacks the rabbit, and the typical spots are first noted around the base of the ear. infection by _coccidium oviforme_ is very common, without however presenting any symptoms by which the infection may be recognised. usually the condition is only noted post-mortem, when the liver is found to be studded with numerous cascating tubercles, which on examination prove to be cystic areas crowded with coccidia. sometimes too the liver of a rabbit dead from some intentional or accidental bacterial infection is found at the post-mortem to be marked by fine yellowish streaks and small tubercles due to the embryos of _tænia serrata_, while the cystic form (_cysticercus pisiformis_) is often noted free in the peritoneal cavity, or invading the mesentery. abscess formation from infection with ordinary pyogenic bacteria occurs naturally in the rabbit, and frequently the animal house of a laboratory is decimated by an infective septicæmia due to _b. cuniculicida_. the ~mouse~ and ~rat~ suffer from septicæmia, and from the cysticercus form of _tænia murina_; the cystic form (_cysticercus fasciolaris_) of _t. crassicollis_ has its habitat in their livers. these small rodents are frequently infected with scabies, but if freely provided with clean straw will clean themselves by rubbing through it. the mouse is also attacked by favus, and the rat is often infected with _trypanosoma lewisi_. the ~guinea pig~, like the rabbit, suffers from scabies and coccidiosis. in addition it is often naturally infected with _b. tuberculosis_, and it is a wise precaution to test animals as soon as they reach the laboratory by injecting koch's old tuberculin--0.5 c.c. causing death in the tuberculous cavy within 48 hours. the ~monkey~ is naturally prone to tuberculosis, and should be injected with 1 c.c. old tuberculin on arrival in the laboratory. the tissues of the monkey also serve as the habitat for a nematode worm parasitic in cattle (_oesophagostoma inflatum_) resembling the anchylostomum, and this parasite frequently bores through the intestinal wall, and provokes the formation of small cysts in the immediately adjacent mesentery. the presence of these cysts may give rise to considerable speculation at the post-mortem. the ~pigeon~ may be infected by _hæmosporidia_, and its blood show the presence of halteridia. this bird may also be the subject of a bacterial infection known as pigeon diphtheria; while the fowl may be subject to scabies and ringworm, or suffer from fowl cholera or fowl septicæmia--infections due to members of the hæmorrhagic septicæmia group. ~weighing.~--the larger animals are most conveniently weighed in a decimal scale provided with a metal cage for their reception instead of the ordinary pan (fig. 166). mice and rats are weighed in a modification of the letter balance, weighing to 250 grammes, which has a conical wire cage, (carefully counterpoised) substituted for its original pan (fig. 167). [illustration: fig. 166.--rabbit scales.] ~temperature.~--to take the rectal temperature of any of the laboratory animals, the animal should be carefully and firmly held by an assistant. introduce the bulb of an ordinary clinical thermometer, well greased with vaseline, just within the sphincter ani. allow it to remain in this position for a few seconds, and then push it on gently and steadily until the entire bulb and part of the stem, as far as the constriction, have passed into the rectum. three to five minutes later, the time varying of course with the sensibility of the thermometer used, withdraw the instrument and take the reading. the thermometers employed for recording temperature should be verified from time to time by comparison with a standard kew certified thermometer kept in the laboratory for that purpose. [illustration: fig. 167.--mouse scales] ~cages.~--during the period which elapses between inoculation and death, or complete recovery, the experimental animals must be kept in suitable receptacles which can easily be kept clean and readily disinfected. the _mouse_ is usually stored in a glass jar (fig. 168) 11 cm. high and 11 cm. in diameter, closed by a wire gauze cover which is weighted with lead or fastened to the mouth of the jar by a bayonet catch. a small oblong label, 5 cm. by 2.5 cm., sand-blasted on the side of the cylinder, is a very convenient device as notes made upon this with an ordinary lead pencil show up well and only require the use of a damp cloth to remove them (fig. 168). the _rat_ is kept under observation in a glass jar similar, but larger, to that used for the mouse. [illustration: fig. 168.--mouse jar.] [illustration: fig. 169.--tripod.] a layer of sawdust at the bottom of the jar absorbs any moisture and cotton-wool or paper shavings should be provided for bedding. the food should consist of bran and oats with an occasional feed of bread-and-milk sop. the use of a metal tripod, on the platform of which are soldered two small cups for the reception of the food, inside the cage, prevents waste of food or its contamination with excreta (fig. 169). after use the jars and tripods are sterilised either by chemical reagents or by autoclaving. the _rabbit_ and the _guinea-pig_ are confined in cages of suitable size, made entirely of metal (fig. 170). the sides and top and bottom are of woven wire work; beneath the cage is a movable metal tray filled with sawdust, for the reception of the excreta. the cage as a whole is raised from the ground on short legs. the sides, etc., are generally hinged so that the cage packs up flat, for convenience of storing and also of sterilising. the ordinary rat cage, a rectangular wire-work box, 30 cm. from front to back, 20 cm. wide, and 14 cm. high, makes an excellent cage for guinea-pigs if fitted with a shallow zinc tray, 35 cm. by 24 cm., for it to stand upon. [illustration: fig. 170.--metal rabbit rage.] a plentiful supply of straw should be provided for bedding and the food should consist of fresh vegetables, cabbage leaves, carrot and turnip tops and the like for the morning meal and broken animal biscuits for the evening meal. occasionally a little water may be placed in the cage in an earthenware dish. the tray which receives the dejecta should be cleaned out and supplied with fresh sawdust each day, and the soiled sawdust, remains of food, etc., should be cremated. these cages are sterilised after use either by autoclaving or spraying with formalin. as ~animal inoculation~ is purely a surgical operation, the necessary instruments will be similar to those employed by the surgeon, and, like them, must be sterile. in the performance of the inoculation strict attention must be paid to asepsis, and suitable precautions adopted to guard against accidental contamination of the material to be introduced into the animal. in addition, the hands of the operator should be carefully disinfected. the list of apparatus used in animal inoculations given below comprises practically everything needed for any inoculation. needless to remark, all the apparatus will never be required for any one inoculation. [illustration: fig. 171.--hypodermic syringe with finger rests.] apparatus required for animal inoculation: 1. water steriliser (_vide_ page 33). it is also convenient to have a second water steriliser, similar but smaller (23 by 7 by 5 cm.), for the sterilisation of the syringes. 2. injection syringe. the best form is one of the ordinary hypodermic pattern, 1 c.c. capacity graduated in twentieths of a cubic centimeter (0.05 c.c.), fitted with finger rests, but with the leather washers and the packing of the piston replaced by those made of asbestos (fig. 171). the instrument must be easily taken to pieces, and spare parts should be kept on hand to replace accidental breakage or loss. other useful syringes are those of 2 c.c., 5 c.c., 10 c.c., and 20 c.c. capacity. a good supply of needles must be kept on hand, both sharp-pointed and with blunt ends. to sterilise the syringe, fill it with water, loosen the packing of the piston and all the screw joints, place it in the steriliser and boil for at least five minutes. disinfect the syringe _after use_, in a similar manner. the needles, which are exceedingly apt to rust after being boiled, should be stored in a pot of absolute alcohol when not in use. 3. operating table. 4. surgical instruments. sterilise these before use by boiling, and disinfect them _after use_ by the same means. wipe perfectly dry immediately after the disinfection is completed. scissors, probe and sharp-pointed. dissecting forceps of various patterns. pressure forceps. retractors (small self retaining fig. 172). aneurism needles, sharp and blunt. scalpels, } keratomes, } with metal handles. trephines, } michel's steel clips and special forceps for applying the same. these small steel clips enable the operator to easily and rapidly close skin incisions and are most satisfactory for animal operations. surgical needles. needle holder. soft rubber catheters, various sizes. gum elastic oesophageal bougies with connection to fit syringe. [illustration: fig. 172. small self retaining retractors.] 5. anæsthetic. (a) general: the safest general anæsthetic for animals is an a. c. e. mixture, freshly prepared, containing by volume alcohol 1 part, chloroform 2 parts, ether 6 parts, and should be administered on a "cone" formed by twisting up one corner of a towel and placing a wad of cotton-wool inside it, or from a saturated cotton-wool pad packed into the bottom of a small beaker. (b) local: 1. cocaine hydrochloride, 2 per cent. in adrenalin 1 per mille solution. 2. beta-eucaine, 2 per cent. in adrenalin, 1 per mille solution. 3. ethyl chloride jet. 6. sterile glass capsules of various sizes. 7. cases of sterile pipettes { 10 c.c. (in tenths of a cubic centimetre). { 1 c.c. (in hundredths of a cubic centimetre). 8. flasks (75 c.c.) containing sterilised normal saline solution (or sterile bouillon). 9. sterilised cotton-wool. cotton-wool (absorbent) is packed loosely in a copper cylinder similar to that used for storing capsules, and sterilised in the hot-air oven. 10. sterilised gauze. gauze is sterilised in the same way as cotton-wool. 11. sterilised silk and catgut for sutures. these are sterilised, as required, by boiling for some ten minutes in the water steriliser. 12. flexible collodion (or compound tincture of benzoin). 13. grease pencil. 14. tie-on celluloid labels, to affix to the cages. 15. razor. 16. small pot of warm water. 17. liquid soap. liquid soap is prepared as follows: measure out 100 grammes of soft soap and add to 500 c.c. of 2 per cent. lysol solution in a large glass beaker; dissolve by heating in a water-bath at about 90° c. bottle and label "liquid soap." 18. in place of the liquid soap and razor it is sometimes convenient to use a depilatory powder. barium sulphide 1 part rice starch 3 parts dust the powder thickly over the area to be denuded of hair, sprinkle with water and mix into a thin paste _in situ_; allow the paste to act for three minutes, then scrape off with a bone spatula--the hair comes away with the paste and leaves a perfectly bare patch. this process is preferably carried out, the day previous to the operation. ~material utilised for inoculation.~--the material inoculated may be either-1. cultures of bacteria--grown in fluid media, or on solid media. 2. metabolic products of bacterial activity--e. g., toxins in solution. 3. pathological products (fluid secretions and excretions, solid tissues). ~the preparation of the inoculum.~-(a) _cultivations in fluid media._-1. flame the plug of the culture tube. 2. remove the plug and flame the mouth of the tube. 3. slightly raise the lid of a sterile capsule, insert the mouth of the culture tube into the aperture and pour some of the cultivation into the capsule. 4. remove the mouth of the culture tube from the capsule, replace the lid of the latter, flame the mouth of the tube, and replug. 5. remove the syringe from the steriliser, squirt out the water from its interior, and allow to cool. 6. raise the lid of the capsule sufficiently to admit the needle of the syringe and draw the required amount of the cultivation into the barrel of the syringe. (or, remove a definite measured quantity of the cultivation directly from the tube or flask by means of a sterile graduated pipette, discharge the measured amount into a sterile capsule, and fill into the syringe; or take up the required quantity of the cultivation directly into the graduated syringe from the tube or flask.) [illustration: fig. 173.--conical separatory funnel, fitted for injection of fluid cultivations.] if it is necessary to introduce a large bulk of fluid into the animal, the cultivation should be transferred with aseptic precautions, to a sterile separatory funnel, preferably of the shape shown in figure 173, and graduated if necessary. this is supported on a retort stand and raised sufficiently above the level of the animal to be injected, so as to secure a good "fall." a piece of sterilised rubber tubing of suitable length, fitted with an injection needle and provided with a screw clamp, is now attached to the nozzle of the funnel and the operation completed according to the requirements of the particular case. this method is quite satisfactory when the injection is made into the pleural or abdominal cavities or directly into a vein but if the injection has to be made into the subcutaneous tissue the "fall" may not be sufficient to force the fluid in. in this case it will be necessary to transfer the culture to a sterile wash-bottle and fasten a rubber hand bellows to the air inlet tube (interposing an air filter) and attach the tubing with the injection needle to the outlet tube (fig. 174). by careful use sufficient force can be obtained to drive the injection in. (b) _cultivations on solid media (e. g., sloped agar)._-1. by means of a sterile graduated pipette introduce a suitable small quantity of sterile bouillon (or sterile normal saline solution) into the culture tube. [illustration: fig. 174.--arrangement of pressure injection apparatus.] 2. with a sterile platinum loop or spatula scrape the bacterial growth off the surface of the medium, and emulsify it with the bouillon. it then becomes to all intents and purposes a fluid inoculum. 3. pour the emulsion into a sterile capsule and fill the syringe therefrom. (c) _toxins._--prepared by previously described methods (_vide_ page 318), are manipulated in a similar manner to cultivations in fluid media. (d) _pathological products._--fluid secretions, excretions, etc., such as serous exudation, pus, blood, etc., are treated as fluid cultivations; but if the material is very thick or viscous, a small quantity of sterile bouillon or normal saline solution may be used to dilute it, and thorough incorporation effected by the help of a sterile platinum rod. solid tissues, such as spleen, lymph glands, etc., may be divided into small pieces by sterile instruments and rubbed up in a sterilised agate mortar (using an agate pestle), with a small quantity of sterile bouillon, and the syringe filled from the resulting emulsion. [illustration: fig. 175.--holding rabbit for shaving.] if it is desired to inoculate tissue _en masse_, remove from the material a small cube of 1 or 2 mm. and introduce it into a wound made by sterile instruments in a suitable situation, and occlude the wound by means of michel's steel clips and a sealed dressing. ~method of securing animals during inoculation.~-for the majority of inoculations, especially when no anæsthetic is administered, it is customary to employ an assistant to hold the animal (see fig. 175). if working single handed voge's holder for guinea-pigs, is a useful piece of apparatus the method of using which is readily seen from the accompanying figures (figs. 176, 177). the instrument itself consists of a hollow copper cylinder, one end of which is turned over a ring of stout copper wire, and from this open end a slot is cut extending about half way along one side of the cylinder. the opposite end is closed by a "pull-off" cap and is perforated around its edge by a row of ventilating holes, which correspond with holes cut in the rim of the cap. in the event of the animal resisting attempts to remove it from the holder backwards, this cap is taken off and the holder placed on the table and the guinea-pig allowed to walk out. [illustration: fig. 176.--taking guinea-pig's temperature.] to provide for different-sized animals, two sizes of this holder will be found useful: 1. length, 16 cm.; breadth, 6 cm.; size of slot, 8 cm. by 2.5 cm. 2. length, 20 cm.; breadth, 8 cm.; size of slot, 10 cm. by 2.5 cm. a convenient holder for mice and even small rats is shown in figure 178, the tail being securely held by the spring clip. needless to say, the holder should be entirely of metal, and the wire cage detachable and easily renewed. [illustration: fig. 177.--voge's holder.] when the animal is anæsthetised, it is more convenient to secure it firmly to some simple form of operating table, such as tatin's (fig. 179), which will accommodate rabbits, guinea-pigs, and rats: or to the more elaborate table devised by the author (fig. 180). [illustration: fig. 178.--mouse holder.] [illustration: fig. 179.--tatin's operation table.] ~operation table.~--this is a table of the "aseptic" type, composed of steel tubing, nickel-plated or enamelled. the table-top frame is sufficiently large to accommodate rabbits, dogs and monkeys; and is supported upon telescopic uprights, so that it is adjustable as to height; in its long axis it can be inclined (at either end) to 45° from the horizontal. further it can be completely rotated about its long axis. the table-top itself is composed of a sheet of copper wire gauze loosely suspended from the long sides of the tubular frame. the slackness of the gauze bed permits of an india rubber hot water bottle, or an electrotherm being placed under the animal, and if during the course of an experiment it is necessary to reverse the animal, the table-top frame is completely rotated, the device adopted for suspending the gauze is detached and the gauze reversed also, so that it again supports the animal from below. [illustration: fig. 180.--author's operating table[12].] methods of inoculation. the following methods of inoculation apply more particularly to the rabbit, but from them it will readily be seen what modifications in technique, if any, are necessary in the case of the other experimental animals. ~1. cutaneous inoculation.~--(_anæsthetic, none._) 1. have the animal firmly held by an assistant (or secured to the operating table). 2. apply the liquid soap to the fur, over the area selected for inoculation, with a wad of cotton-wool, and lather freely by the aid of warm water; shave carefully and thoroughly; or apply the depilatory powder. 3. wash the denuded area of skin thoroughly with 2 per cent. lysol solution. 4. wash off the lysol with ether and allow the latter to evaporate. 5. make numerous short, parallel, superficial incisions with the point of a sterile scalpel. 6. when the oozing from the incisions has ceased, rub the inoculum into the scarifications by means of the flat of a scalpel blade, or a sterile platinum spatula. 7. cover the inoculated area with a pad of sterile gauze secured _in situ_ by strips of adhesive plaster or by sealing down the edges of the gauze with collodion. 8. release the animal, place it in its cage, and affix a label upon which is written: (a) distinctive name or number of the animal. (b) its weight. (c) particulars as to source and dose of inoculum. (d) date of inoculation. ~2. subcutaneous inoculation.~-(a) _fluid inoculum._--(_anæsthetic, none._) steps 1-4. as for cutaneous inoculation. 5. pinch up a fold of skin between the forefinger and thumb of the left hand; take the charged hypodermic syringe in the right hand, enter the needle into a ridge of skin raised by the left finger and thumb, and push it steadily onward until about 2 cm. of the needle are lying in the subcutaneous tissue. now release the grasp of the left hand and slowly inject the fluid contained in the syringe. 6. withdraw the needle, and at the same moment close the puncture with a wad of cotton wool, to prevent the escape of any of the inoculum. the injected fluid, unless large in amount, will be absorbed within a very short time. 7. label, etc. (b) _solid inoculum.--(anæsthetic, none; or ethyl chloride spray.)_ steps 1-4. as for cutaneous inoculation. 5. raise a small fold of skin in a pair of forceps, and make a small incision through the skin with a pair of sharp-pointed scissors or with the point of a scalpel. 6. insert a probe through the opening and push it steadily onward in the subcutaneous tissue, and by lateral movements separate the skin from the underlying muscles to form a funnel-shaped pocket with its apex toward the point of entrance. 7. by means of a pair of fine-pointed forceps introduce a small piece of the inoculum into this pocket and deposit it as far as possible from the point of entrance. [illustration: fig. 181.--glass tube syringe for subcutaneous "solid" inoculation.] or, improvise a syringe by sliding a piece of glass rod (to serve as a piston) into the lumen of a slightly shorter length of glass tubing and secure in position by a band of rubber tubing. sterilise by boiling. withdraw the rod a few millimetres and deposit the piece of tissue within the orifice of the tube, by means of sterile forceps. now pass the tube into the depths of the "pocket," push on the glass rod till it projects beyond the end of the tube, and withdraw the apparatus, leaving the tissue behind in the wound. 8. close the wound in the skin with michel's clips and a dressing of gauze sealed with collodion (or tinct. benzoin). 9. label, etc. ~3. intramuscular.~-(a) _fluid inoculum.--(anæsthetic, none.)_ steps 1-4. as for cutaneous inoculation. 5. steady the skin over the selected muscle or muscles with the slightly separated left forefinger and thumb. 6. thrust the needle of the injecting syringe boldly into the muscular tissue and inject the inoculum slowly. 7. label, etc. (b) _solid inoculum.--(anæsthetic, a. c. e.)_ 1. secure the animal to the operation table and anæsthetise. 2. shave and disinfect the skin at the seat of operation. 3. surround the field of operation by strips of gauze wrung out in 2 per cent. lysol solution. 4. incise skin, aponeurosis, and muscle in turn. 5. deposit the inoculum in the depths of the incision. 6. close the wound in the muscle with buried sutures and the cutaneous wound with either continuous or interrupted sutures or with michel's steel clips. 7. apply a sealed dressing of gauze and collodion. 8. remove the animal from the operating table. 9. label, etc. ~4. intraperitoneal.~-(a) _fluid inoculum.--(anæsthetic, none.)_ steps 1-4. as for cutaneous inoculation. shave a fairly broad transverse area, stretching from flank to flank. 5. place the left forefinger on one flank and the thumb on the opposite, and pinch up the entire thickness of the abdominal parietes in a triangular fold. now, by slipping the peritoneal surfaces (which are in apposition) one over the other, ascertain that no coils of intestine are included in the fold. 6. take the syringe in the right hand and with the needle transfix the fold near its base (fig. 182). 7. now release the fold, but hold the syringe steady; as the parietes flatten out, the point of the needle is left free in the peritoneal cavity (see fig. 183). [illustration: fig. 182.--intraperitoneal inoculation--fluid.] 8. inject the fluid from the syringe. 9. label, etc. [illustration: fig. 183.--section of abdominal wall, etc., showing point of needle lying free in the peritoneal cavity above the coils of intestine.] second method: steps 1-4. as in the first method. 5. anæsthetise a small selected area of skin by spraying it with ethyl chloride. 6. heat platinum searing wire (0.5 mm. wire, twisted to the shape indicated in figure 184, mounted in an aluminium handle) to redness, and with it burn a hole through the anæsthetic area of skin and abdominal muscle down to, but not through, the visceral peritoneum. 7. fix a blunt-ended needle on to the charged syringe, and by pressing the rounded end firmly against the peritoneum it can easily be pushed through into the peritoneal cavity. 8. inject the fluid from the syringe. 9. label, etc. this method is especially useful when it is desired to collect samples of the peritoneal fluid from time to time during the period of observation, as fluid can be removed from the peritoneal cavity, at intervals, through this aperture in the abdominal parietes, by means of a sterile capillary pipette. [illustration: fig. 184.--platinum wire for burning hole through parietes.] (b) _solid inoculum_ (or the implantation of capsules containing fluid cultivations).--(_anæsthetic, a. c. e._) 1. anæsthetise the animal and secure it to the operating table. 2. shave a large area of the abdominal parietes. 3. make an incision through the skin in the middle line about 2 cm. in length, midway between the lower end of the sternum and the pubes. 4. divide the aponeuroses between the recti upon a director. 5. divide the peritoneum upon a director. 6. introduce the inoculum into the peritoneal cavity. 7. close the peritoneal cavity with lembert's sutures. 8. close the skin and aponeurosis incisions together with interrupted sutures or michel's steel clips, and apply a sealed dressing. 9. release the animal from the operating table. 10. label, etc. suitable sacs may be readily prepared by either of the following methods: a. ~collodion sacs.~ 1. dip a small test-tube (5 by 0.5 cm.), bottom downward, into a beaker of collodion, and dry in the air; repeat this process three or four times. 2. dip the tube, with its coating of collodion, alternately into a beaker of alcohol and one of water. this loosens the collodion and allows it to be peeled off in the shape of a small test-tube. 3. take a 20 cm. length of glass tubing, of about the diameter of the test-tube used in forming the sac, and insert one end into the open mouth of the sac. 4. suspend the glass tube with attached sac, inside a larger test-tube, by packing cotton-wool in the mouth of the test-tube around the glass tubing, and place in the incubator at 37° c. for twenty-four hours. when removed from the incubator, the sac will be firmly adherent to the extremity of the glass tubing. 5. plug the open end of the glass tubing with cotton-wool, and sterilise the test-tube and its contents in the hot-air oven. to use the sac, remove the plug from the glass tubing, partly fill the sac with cultivation to be inoculated, by means of a sterile capillary pipette, and replug the tubing. when the abdominal cavity has been opened, remove the tubing and attached sac from the protecting test-tube, close the sac by tying a sterilised silk thread tightly around it a little below the end of the glass tubing, and separate it from the tubing by cutting through the collodion above the ligature, and the sac is ready for insertion in the peritoneal cavity. b. ~celloidin sacs~ (_harris_). _materials required._ quill glass tubing. gelatine capsules such as pharmacists prepare for the exhibition of bulky powders. various grades of celloidin, thick and thin, in wide-mouthed bottles. 1. take a piece of quill glass tubing some 4 cm. long by 5 mm. diameter; heat one end in the bunsen flame. 2. thrust the heated end of the tube just through one end of a gelatine capsule and allow it to cool (fig. 185). 3. remove any gelatine from the lumen of the tube with a heated platinum needle; paint the joint between capsule and tube with moderately thick celloidin and allow to dry. [illustration: fig. 185.--making celloidin capsules.] 4. dip the capsule into a beaker containing thin celloidin, beyond the junction with the glass and after removal rotate it in front of the blowpipe air blast to dry it evenly. repeat these manoeuvres until a sufficiently thick coating is obtained. 5. apply thick celloidin to the tube-capsule joint, the opposite end of the capsule, and the line of junction of the capsule with its cap; dry thoroughly. 6. with a teat pipette fill the capsule (through the attached tube) with hot water, and stand the capsule in a beaker of boiling water for a few minutes to melt the gelatine. 7. remove the solution of gelatine from the interior of the celloidin case with a pipette. 8. fill the sac with nutrient broth and place it, _glass tube downward_, in a tube containing sufficient sterile nutrient broth to cover the sac to the depth of 1 cm. plug the tube and sterilise in the steamer in the usual manner. 9. to prepare the sac for use, empty it out of the broth tube into a sterile glass dish. 10. grasp the tube near its junction with the sac in the jaws of sterile forceps, and with a teat pipette remove sufficient of the contained broth to leave a small space in the sac. introduce the inoculum in the form of an emulsion by means of another pipette. 11. still holding the tube in the forceps, draw it out and seal off near the sac in the blowpipe flame. 12. when cool wash the sac in sterile water, then transfer to a tube of nutrient broth and incubate over night to determine its impermeability to bacteria. 13. if the broth outside the sac remains sterile, insert the sac in the peritoneal cavity of the experimental animal. ~5. intracranial.~--(_anæsthetic, a. c. e._) [illustration: fig. 186.--guarded trephine.] _trephines and surgical engine._--the most useful instrument for intracranial operations upon animals is the small nasal trephine (curtis) having a tooth cutting circle of 7 mm. the addition of an adjustable collar guard--secured by a screw--prevents accidental laceration of the dura mater or brain substance[13] (fig. 186). this size is suitable for monkeys, dogs, cats and large rabbits. other smaller sizes which will be found useful for guinea pigs and other small animals cut circles of 6 and 4 mm.; for very small animals--young guinea pigs and rats--a small dental drill or screw will make a sufficiently large hole to admit the syringe needle. the trephine can be set in ordinary metal handles and rotated by hand, but a surgical engine of some kind is much preferable on the score of rapidity and safety to the animal. the guy's electrical dental engine[14] (fig. 187) which can be connected to a lamp socket or wall plug, and is operated by a foot switch, although inexpensive is eminently satisfactory. note.--a fine dental drill attached to the dental engine renders the manufacture of aluminium handles needles (see page 71) quite an easy matter. (a) _subdural._ 1. anæsthetise the animal and secure it to the operating table, dorsum uppermost. 2. shave a portion of the scalp immediately in front of the ears. [illustration: fig. 187.--guy's electrical dental engine.] 3. mark out with a sharp scalpel a crescentic flap of skin muscle, etc., convexity forward, commencing 0.5 cm. in front of the root of one ear and terminating at a similar spot in front of the other ear. reflect the marked flap. 4. make a corresponding incision through the periosteum and raise it with a blunt dissector. 5. with a small trephine (diameter 6 mm.) remove a circular piece of bone from the parietal segment. the centre of the trephine hole should be at the intersection of the median line and a line joining the posterior canthi (fig. 188). 6. introduce the inoculum by means of a hypodermic syringe, perforating the dura mater with the needle and depositing the material immediately below this membrane, at the same time taking care to avoid injuring the sinuses. 7. turn back the flap of skin and secure it in position with michel's steel clips. 8. dress with sterile gauze and wool and seal the dressing with collodion. 9. label, etc. (b) _intracerebral._--this inoculation is performed precisely as for subdural save in step 6 the needle after perforating the dura mater is pushed onward into the substance of one or other cerebral hemispheres before the contents are ejected. [illustration: fig. 188.--intracranial inoculation of rabbit. the circle indicates the situation of the trephine hole.] ~6. intraocular.~-(a) _fluid inoculum._--(_anæsthetic, cocaine._) 1. instil a few drops of a sterile solution of cocaine, and repeat the instillation in two minutes. 2. five minutes later have the animal firmly held by an assistant as in intravenous injection (see fig. 189), the head being steadied by the assistant's hands. 3. select two needles to accurately fit the same syringe and sterilise. 4. attach one needle to the syringe and take up the required dose of inoculum and remove the needle. 5. steady the eye with fixation forceps; then pierce the cornea with the other syringe needle and allow the aqueous to escape through the needle. 6. without removing the needle from the cornea attach the syringe and make the injection into the anterior chamber. 7. irrigate the conjunctival sac with sterile saline solution. 8. label, etc. (b) _solid inoculum._--(_anæsthetic, a. c. e._) 1. anæsthetise the animal and secure it firmly to the operating table. 2. irrigate the conjunctival sac thoroughly with sterile saline solution. 3. make an incision through the upper quadrant of the cornea into the anterior chamber by means of a triangular keratome. 4. separate the lips of the corneal wound with a flexible silver spatula; seize the solid inoculum in a pair of iris forceps, introduce it through the corneal wound, and deposit it on the anterior surface of the iris; withdraw the forceps. 5. again irrigate the sac and the surface of the cornea. 6. release the animal from the operating table. 7. label, etc. ~7. intrapulmonary.~-_fluid inoculum._--(_anæsthetic, none._) 1. have the animal firmly held by an assistant. (in this case the foreleg of the selected side is drawn up by the assistant and held with the ear of that side.) 2. shave carefully in the axillary line and disinfect the denuded skin. 3. thrust the needle of the syringe boldly through the fifth or sixth intercostal space into the lung tissue. 4. inject the contents of the syringe slowly. 5. label, etc. ~8. intravenous.~-_fluid inoculum._--(_anæsthetic, none._) the site selected for the injection in the rabbit is the posterior auricular vein (see fig. 192). although this is smaller than the median vein, it is firmly bound down to the cartilage of the ear by dense connective tissue, and is therefore more readily accessible. (in the guinea-pig the jugular vein must be utilised, and in order to perform the inoculation satisfactorily a general anæsthetic must be administered to the animal. in the monkey or the dog, the internal saphenous vein is the most convenient and before puncturing should be distended or rendered prominent by compressing the vein above the selected site.) _preparation of the inoculum._--care must be taken in preparing the inoculum, as the injection of even small fragments may cause fatal embolism. to obviate this risk the fluid should, if possible, be filtered through sterile filter paper before filling into the syringe. air bubbles, when injected into a vein, frequently cause immediate death. to prevent this, the syringe after being filled should be held in the vertical position, needle uppermost. a piece of sterile filter paper is then impaled on the needle and the piston of the syringe pressed upward until all the air is expelled from the barrel and needle. should any drops of the inoculum be forced out, they will fall on the filter paper, which should be immediately burned. 1. have the animal firmly held by an assistant. the selected ear is grasped at its root and stretched forward toward the operator. 2. shave the posterior border of the dorsum of the ear. 3. disinfect the skin over the vein, rubbing it vigourously with cotton-wool soaked in lysol. the friction will make the vein more conspicuous. wash the lysol off with ether and allow the latter to evaporate. 4. direct the assistant to compress the vein at the root of the ear. this will cause its peripheral portion to swell up and increase in calibre. 5. hold the syringe as one would a pen and thrust the point of the needle through the skin and the wall of the vein till it enters the lumen of the vein (fig. 189). now press it onward in the direction of the blood stream--i. e., toward the body of the animal. 6. direct the assistant to cease compressing the root of the ear, and _slowly_ inject the inoculum. (if the fluid is being forced into the subcutaneous tissue, a condition which is at once indicated by the swelling that occurs, the injection must be stopped and another attempt made at a spot closer to the root of the ear or at some point on the corresponding vein on the opposite ear.) 7. withdraw the needle and press a pledget of cotton-wool over the puncture to ensure closure of the aperture in the vein wall. 8. label, etc. [illustration: fig. 189.--intravenous inoculation.] ~9. inhalation.~-(a) _fluid inoculum._--(_anæsthetic, none._) 1. place the animal in a closed metal box. 2. through a hole in one side introduce the nozzle of some simple spraying apparatus, such as is used for nasal medicaments. 3. fill the reservoir of the instrument (previously sterilised) with the fluid inoculum, and having attached the bellows, spray the inoculum into the interior of the box. 4. on the completion of the spraying, open the box, spray the animal thoroughly with a 10 per cent. solution of formaldehyde (to destroy any of the virus that may be adhering to fur or feathers). 5. transfer the animal to its cage. 6. label, etc. 7. thoroughly disinfect the inhalation chamber. (b) _fluid or powdered inoculum._--_anæsthetic, a. c. e._ 1. anæsthetise the animal and secure it firmly to the operating table. [illustration: fig. 190.--gag for rabbits.] 2. prop open the mouth by means of some form of gag; seize the tongue with a pair of forceps and draw it forward. the most convenient form of gag for the rabbit or cat is that shown in fig. 190. it is simply a strip of hard wood shaped at the middle and provided with a square orifice through which a tracheal or oesophageal tube can be passed. 3. pass a previously sterilised glass tube (17 cm. long, 0.5 cm. diameter, with its terminal 2 cm. slightly curved) down through the larynx into the trachea. 4. connect the straight portion of a ~y~-shaped piece of tubing to the upper end of the sterilised tube and couple one branch of the ~y~ to a separatory funnel containing the fluid inoculum, or insufflator containing the powdered inoculum, and the other to a hand bellows. 5. allow the fluid inoculum to run into the lungs by gravity, or blow in the powdered inoculum by means of a rubber-ball bellows. 6. remove the intratracheal tube; release the animal from the table. 7. label, etc. as an alternative method in the case of fairly large animals, such as rabbits, etc., a sterile piece of glass tubing of suitable diameter may be passed through the larynx down the trachea almost to its bifurcation. fluid cultivations may then be literally poured into the lungs, or cultivations, dried and powdered, may be blown into the lung by the aid of a small hand bellows or even a teat pipette. ~10. intragastric inoculation.~--_fluid or semi-fluid inoculum. (anæsthetic none.)_ the method of performing the operation is varied slightly according to the size of the experimental animal. _a. monkey, rabbit, guinea-pig._ 1. secure the animal to the operating table ventral surface uppermost. 2. prop the mouth open with a gag; draw the tongue forward with forceps. 3. sterilise a soft rubber catheter (no. 10 or 8 english scale, or no. 18 or 15 french) and lubricate it with sterile glycerine. 4. pass it to the back of the pharynx, keeping the end in the middle line. 5. gently assist the progress of the catheter down the oesophagus until it passes the cardiac orifice of the stomach. do not use any force. 6. take up the required dose of inoculum into a sterilised pipette. insert the point of the pipette into the open end of the catheter and allow the fluid to run down into the stomach. remove the pipette and drop it into a jar of lysol. 7. with another sterile pipette run one cubic centimetre of sterile saline solution through the catheter to wash out the last traces of the inoculum. 8. withdraw the catheter. 9. label, etc. _b. rats and mice (mark's method)._ 1. secure the animal in the vertical position. (a) _rat._--take a pair of catch sinus forceps about 22 cm. in length and seize the animal by the loose skin of the head as far forward as possible--fix the forceps, and holding the instrument vertically upward, transfer to the left hand of an assistant who secures the animal's tail between the fingers grasping the handle of the forceps. (see fig. 191.) [illustration: fig. 191.--intragastric inoculation of rat.] (b) _mouse._--an assistant grasps the loose skin between the ears as far forwards as possible between the forefinger and thumb of the left hand. he now grasps the tail with the right hand, draws the mouse straight and passes the tail between the fourth and little fingers of the left hand and secures it there. 2. the assistant takes a closed pair of thin-bladed forceps in his right hand, passes the ends into the animal's mouth, then allows the blades to separate. this opens the animal's jaw and serves as a gag. 3. moisten the sterilised oesophageal tube with sterile water. (this tube is of silk rubber, 6.5 cm. in length, with the distal end rounded, the proximal end mounted in a syringe needle head, which fits the nozzles of the two sterile syringes to be used.) 4. grasp the tube about its middle and pass it into the animal's mouth, downwards and a little to one side or the other until its length is lost in the digestive tract and mouth. gentle guidance is alone necessary. do not use any force. 5. take up the required dose of inoculum into the syringe; insert the nozzle of the syringe into the needle-mount, and force the piston down. 6. steadying the needle-mount with the left hand, detach the syringe. 7. draw up some sterile water in the second (sterile) syringe, and inserting its nozzle into the needle-mount force a few drops of water through the tube to wash it out. 8. with one quick upward movement remove the tube from the animal's mouth. 9. label, etc. one other method of inoculation remains to be described, which does not require operative interference. ~11. feeding.~-1. _fluid inoculum._--small pieces of sterilised bread or sop (sterilised in the steamer at 100° c.) are soaked in the fluid inoculum and offered to the animals in a sterile petri dish or capsule. 2. _solid inoculum._--small pieces of tissue are placed in sterile vessels and offered to the animals. footnotes: [12] this table is made by messrs. down bros., st. thomas's street, london, s. e. [13] this modification is made for the author by messrs. down bros., st. thomas's street, london, s. e. [14] manufactured by messrs. francis lepper, 56, great marlborough street, london, w. xviii. the study of experimental infections during life. the possession of pathogenetic properties by an organism under study is indicated by the "infection" of the experimental animal--a term which is employed to summarise the condition resulting from the successful invasion of the tissues of the experimental animal by the micro-organisms inoculated and by their multiplication therein. infection is considered to have taken place: 1. when the death of the animal is produced as a direct consequence of the inoculation. 2. when without necessarily producing death the inoculation causes local or general changes of a pathological character. 3. when either with or without death, or local or general changes occurring, certain substances make their appearance in the body fluids, which can be shown (_in vitro_ or _in vivo_) to exert some profound and specific effect when brought into contact with subcultivations of the organism originally inoculated. the important factors in the production of infection are: a. seed. virulence of organism. dose of organism. b. soil. resistance offered by the cells of the experimental animal. the first two factors, although variable, are to a certain extent under the control of the experimenter. thus by suitable means the virulence of an organism can be exalted or attenuated, whilst the size of the dose may be increased or diminished. the third factor also varies, not only amongst different species of animals, but also amongst different individuals of the same species. the essential causes of this variation are not so obvious, so that beyond selecting the animals intended for similar experiments with regard to such points as age, size or sex, but little can be done to standardise cell resistance. immediately an animal has been inoculated a period of clinical observation must be entered upon, which should only terminate with the death of the animal. the general observations should at first and if the infection is an acute one, be made daily--later, and if the animal appears to be unaffected or if the infection is chronic, both general and special observations should be carried out at weekly intervals. if the animal appears to be still unaffected, it should be killed with chloroform vapour at the end of two or three months and a complete post-mortem carried out. a. the ~general observations~ should take cognisance of: 1. _general appearance._ the experimental animal should be inspected daily, not only with a view to detecting symptoms due to the experimental infection, but also to prevent any intercurrent infection, naturally acquired, from escaping notice (_vide_ page 337). 2. _the weight_ of the inoculated animal should be observed and recorded each day during the course of an experimental infection at precisely the same hour, preferably just before the morning feed. 3. _the temperature_ should similarly be recorded daily, if not more frequently, during the whole period the animal is under observation, and carefully charted--individual variations will at once become apparent. it should be borne in mind that the temperature regarded as normal for man (37.5° c.) is not the normal average temperature of any of the lower animals save the rat and mouse. the accompanying table of normal averages for the animals usually employed in bacteriological research may be of use in preventing the erroneous assumption that pyrexia is present in an animal, which merely shows its own normal temperature. normal averages. --------------------------------------------------- | rectal | pulse. | respirations. animal. | temp. °c.|----------------------- | | rate per minute. --------------------------------------------------- | | | frog | 8.9-17.2 | 80 | 12 mouse | 37.4 | 120 | ... rat | 37.5 | ... | 210 guinea pig | 38.6 | 150 | 80 rabbit | 38.7 | 135 | 55 cat | 38.7 | 130 | 24 dog | 38.6 | 95 | 15 goat | 40.0 | 75 | 16 ox | 38.8 | 45 | .. horse | 37.9 | 38 | 11 monkey (rhesus) | 38.4 | 100 | 19 pigeon | 40.9 | 136 | 30 fowl | 41.6 | 140 | 12 | | | ---------------------------------------------------b. ~special observations~ comprise some or all of the following, according to the method of inoculation and the character of the virus. 1. _the site of inoculation_ should be minutely examined at least at weekly intervals, and the neighbouring lymphatic glands palpated. 2. any _local reaction_ at the site of inoculation and any other readily accessible lesion should be carefully investigated. any suppurative process which may occur, whether in the subcutaneous tissues or in joints, should be explored and the pus carefully examined both microscopically and culturally. fluid secretions and excretions, such as pus or serous exudates when accessible are collected direct from the body in sterile capillary pipettes (_vide_ fig. 13a,) in the following manner: 1. open the case containing the pipettes, grasp one by the plugged end, remove it from the case, and replace the lid of the latter. 2. attach a rubber teat (_vide_ page 10) to the plugged end of the pipette and use the teat as the handle of the pipette. 3. pass the entire length of the pipette twice or thrice through the flame of the bunsen burner. 4. snap off the sealed end of the pipette with a pair of sterile forceps. 5. compress the india-rubber teat, thrust the point of the pipette into the secretion; now relax the pressure on the teat and allow the pipette to fill. 6. remove the point of the pipette from the secretion, allow the fluid to run a short distance up the capillary stem and seal the point of the pipette in the flame. (if using a pipette with a constriction below the plugged mouthpiece (fig 13b), this portion of the pipette may also be sealed in the flame.) when ready to examine the morbid material snap off the sealed end of the pipette with sterile forceps and eject the contents of the pipette into a sterile capsule. the material can now be utilized for cover-slip preparations, cultivations and inoculation experiment. 3. _the peripheral blood_ should be examined from time to time for from this tissue is often obtained the fullest information as to the course and progress of an infection. a. the ~histological examination of the blood~ should be directed chiefly to observations on the number and kind of white cells; and since but few bacteriologists are at the same time expert comparative hæmatologists, some notes on the normal characters of the blood of the commoner laboratory animals, contrasted with those of man, are inserted for reference. these have been very kindly compiled for me by my friend and one time colleague dr. cecil price jones. comparative hæmocytology of laboratory animals. ------------------------------------------------------------------- | totals | percentages |-----------------------------------------------------------animal | | | hb, |lympho-|large |poly|eosin-| mast |red cells |white | per | cytes,|monos,|morph,| oph, |cells, | | cells|cent.| per | per | per | per | per | | | | cent. | cent.| cent.|cent. |cent. -------------------------------------------------------------------frog | 490,000| 8,000| 58 | 40 | 10.0 | 22.0 |15 | 13 mouse | 8,700,000| 8,000| 78 | 60 | 21.5 | 17.0 | 1.4 | 0.1 rat | 9,000,000| 9,000| 85 | 54 | 7.0 | 37.5 | 1.3 | 0.2 guinea-| | | | | | | | pig | 5,700,000|10,000| 99 | 55 | 9.0 | 32.8 | 3.0 | 0.2 rabbit | 6,000,000| 7,000| 70 | 50 | 2.0 | 46.0 | 0.6 | 1.4 rhesus | 4,500,000|13,000| 77 | 43 | 5.0 | 50.0 | 1.3 | 0.7 goat |14,600,000|15,000| 58 | 35 | 6.3 | 56.7 | 1.25 | 0.75 fowl | 3,500,000|30,000| 100 | 49 | 3.0 | 42.0 | 1.0 | 5.0 pigeon | 3,500,000|20,000| 101 | 43 | 9.0 | 43.0 | 3.0 | 2.0 -------------------------------------------------------------------man | | | | | | | | (adult)| 5,000,000| 7,500| 100 | 25 | 5.5 | 65 | 4.0 | 0.5 normal | (4.5-5) | (7-9)|(95|(20-30)| (4-8)|(55 |(3-5) |(0.5-2) limits.| millions.| thou-| 101)| | | 68) | | | |sands.| | | | | | -------------------------------------------------------------------the above table represents in each case the average of a large number of counts. remarks. _frog._--the _red cells_ are large oval nucleated (20-25µ by 12-15µ) discs, the nucleus relatively small and irregularly elongated or oval, about 10µ in length. many primitive and developing forms are usually observed--also free nuclei and many cells in various stages of degeneration. hæmoglobin estimation is difficult owing to turbidity of the blood after dilution with water. the _polymorphonuclear_ leucocytes are large cells, about 20µ; no definite granules can be observed. the _eosinophile_ cells contain large deeply staining coccal-shaped granules. _mouse._--the granules of the _polymorphonuclear_ leucocytes are usually not stained, or only very faintly so. the nucleus of the _eosinophile cell_ is ring-shaped or much divided, and the granules are coccal and stain oxyphile. the remarkable character of the blood is the high percentage of large _mononuclear_ cells. _rat._--the fine rod-shaped granules of the _polymorphonuclear_ leucocytes are usually very faintly stained. the granules of _eosinophile_ cells are well stained and coccal-shaped, the nucleus is often ring shaped. the _basophile_ granular cells are few--but the granules are large, and stain deeply basophile. _guinea-pig._--polychromasia and punctate basophilia of _red cells_ are very commonly observed--nucleated red cells are also frequent. the large _mononuclear_ cells often contain vacuoles--"kurlow cells"--possibly of a parasitic nature. _rabbit._--it is not uncommon to find nucleated _red cells_ in films from quite healthy animals. the granules of the _polymorphonuclear_ leucocytes stain oxyphile. the coarse granules of the _eosinophile_ cells appear to stain less deeply oxyphile, probably owing to the basophile staining of the cytoplasm. _rhesus monkey._--the blood cells resemble those met with in human blood. the minute neutrophile granules of the _polymorphonuclear_ leucocytes are often very scanty, and sometimes apparently absent. the _eosinophile_ cells are not so densely packed with coarse oxpyhile granules as in the human eosinophile, and the nuclei of these cells are usually much divided, or polymorphous. _goat._--the _red cells_ are small, nonnucleated discs, only about 4.5µ diameter, not much more than half that of the human red cell. the _polymorphonuclear_ leucocytes have only a few very minute coccal-shaped oxyphile granules, the nucleus is polymorphous. the _eosinophile_ cells are large cells up to 20µ, the cytoplasm is basophile and contains coarse coccal-shaped oxyphile granules, and the nucleus is often much divided. _fowl._--the _red cells_ are oval nucleated discs about 12µ by 6µ, the nucleus being relatively small (about 4µ long), irregularly elongated or oval; round, more deeply stained cells with round or diffuse nuclei, also free nuclei and degenerated forms of red cells are often present. the granules of the cells corresponding to the _polymorphonuclear_ leucocytes are rod-shaped, often beaded or with clubbed ends. the nucleus is not polymorphous, but usually divided into two, though it may be single. the cells probably corresponding to _eosinophile_ leucocytes have fine coccal-shaped granules, faintly staining eosinophile or neutrophile. the basophile granules of the "mast" cells are coccal-shaped, of various size--often quite powdery. _pigeon._--_red cells_ resemble those of the fowl, and similar varieties of appearance may be noted. the granules of those cells which correspond to _polymorphonuclear_ leucocytes are rod-shaped, but smaller and finer than in the fowl, and do not show clubbed appearances. the nucleus is not polymorphous, and only occasionally divided. the coccal-shaped granules of the _eosinophile_ cells are stained more deeply oxyphile than those of the corresponding cells of the fowl. _the preparation of dried films_ for this histological examination of the blood is carried out as follows: 1. small samples of blood for the preparation of blood films are most conveniently obtained from the veins of the ear in most of the ordinary laboratory animals, viz., monkey, goat, dog, cat, rabbit, guinea-pig; in the pigeon and fowl the axillary vein should be punctured; in the rat and mouse either a vein in the ear or preferably by wounding the tip of the tail; in the frog, the web of the foot should be selected. 2. puncture the selected vein with a sharp needle. a flat hagedorn needle (size no. 8) with a cutting edge is the most useful for this purpose. if the vein cannot be distended by proximal compression, vigourous friction with a piece of dry lint may have the desired effect--or a test-tube full of water at about 40°c. may be placed close to the vein. failing these methods, a drop or two of xylol may be dropped on the skin just over the vein, left on for a few seconds and then wiped off with a piece of dry lint. 3. one of the short ends of a 3 by 1 glass slip is brought into contact with the exuding drop of blood, so that it picks up a small drop. 4. the slide is then lowered transversely on to the surface of a second 3 by 1 slip, which rests on the bench near to one end at an angle of about 45°, and retained in this position for a few seconds, while the drop of blood spreads along the whole of the line of contact (see also fig. 69). 5. draw the first slide firmly and evenly along the entire length of the lower slide, leaving a thin regular film which will probably show the blood cells only one layer thick. 6. allow the film to dry in the air. 7. stain with one of the polychrome blood stains (see page 97). 8. examine microscopically. b. the ~bacteriological examination of the blood~ is directed solely to the demonstration of the presence in the circulating blood of the organisms previously injected into the animal. for this purpose several cubic centimetres of blood should be taken in an all-glass syringe from an accessible vein corresponding to one of those suggested as the site of intravenous inoculation--and under similar aseptic precautions. 1. sterilise an all-glass syringe of suitable size, and when cool draw into the syringe some sterile sodium citrate solution and moisten the whole of the interior of the barrel; then eject all the citrate solution if less than 5 c.c. blood are to be withdrawn; if more than 5 c.c. are required retain about half a cubic centimetre of the fluid in the syringe. this prevents coagulation of the blood. the sodium citrate solution is prepared by dissolving: sodium citrate 10 gramme. sodium chloride 0.75 grammes. in distilled water 100 c.c. sterilise by boiling. 2. prepare the animal as for intravenous inoculation (see page 363) and introduce the syringe needle into the lumen of the selected vein. 3. slowly withdraw the piston of the syringe. when sufficient blood has been collected direct the assistant to release the proximal compression of the vein; and withdraw the needle. 4. remove the needle from the nozzle of the syringe and deliver the citrated blood into a small ehlenmeyer flask containing about 250 c.c. of nutrient broth. 5. label, incubate and examine daily until growth occurs or until the expiration of ten days. c. the ~serological examination of the blood~ is directed to the demonstration of the presence of certain specific antibodies in the sera of experimentally infected animals, and within certain limits to an estimation of their amounts. the chief of these bodies are: antitoxin. agglutinin. precipitin. opsonin. immune body or bacteriolysin. none of these substances are capable of isolation in a state of purity apart from the blood serum, consequently special methods have been elaborated to permit of their recognition. in every instance the behaviour of serum from the experimental animal, which may be termed "specific" serum, is studied in comparison with that of serum from an uninoculated animal of the same species, and which is termed "normal" serum. in view of minor differences in constitution exhibited by the serum of various individuals of the same series, it is usual to employ a mixture of sera obtained from several different normal animals of the same species as the inoculated animal, under the term "pooled serum." the method of collecting blood (e. g., from the rabbit) for serological tests is as follows: ~collection of serum.~ _apparatus required:_ razor. liquid soap. cotton-wool. lysol 2 per cent. solution, in drop bottle. ether in drop bottle. flat hagedorn needles. blood pipettes (fig. 16, page 12). centrifugal machine. centrifuge tubes. glass cutting knife. bunsen flame. writing diamond or grease pencil. method. 1. shave the dorsal surface of the ear over the course of the posterior auricular vein (see fig. 192). 2. sterilise the skin by washing with lysol. the lysol should be applied with sterile cotton-wool and the ear vigourously rubbed, not only to remove superficial scales of epithelium, but also to render the ear hyperæmic and the vein prominent. 3. remove the lysol with ether dropped from a drop bottle, and allow the ether to evaporate. 4. puncture the vein with a sterile hagedorn needle. 5. take a small blood-collecting pipette (fig. 161) and hold it at an angle to the ear, one end touching the issuing drop of blood, the other depressed. the blood will now enter the pipette at first by capillarity; afterward gravity will also come into play and the pipette can be two-thirds filled without difficulty. 6. hold the tube by the end containing the blood, the clean end pointing obliquely upward--warm this end at the bunsen flame to expel some of the contained air; then seal the clean point in the flame. [illustration: fig. 192.--collecting blood from rabbit.] 7. place the pipette down on a cool surface (e. g., a glass slide). the rapid cooling of the air in the clean end of the pipette creates a negative pressure, and the blood is sucked back into the pipette, leaving the soiled end free from blood. seal this end in the bunsen flame. 8. mark the distinctive title of the specimen (e. g., animal's number) upon the pipette with a writing diamond or grease pencil. 9. when the sealed ends are cold and the blood has clotted, place the pipette on the centrifuge, clean end downward; counterpoise and centrifugalise thoroughly. on removing the pipette from the centrifuge, the red cells will be collected in a firm mass at one end, and above them will appear the clear serum. 10. by marking the blood pipette above the level of the serum with the glass cutting knife and snapping the tube at that point, the blood-serum becomes readily accessible for testing purposes. if larger quantities of blood are required, the animal, after puncturing the vein, should be inverted, an assistant holding it up by the legs. blood to the volume of several cubic centimetres will now drop from the punctured vein, and should be caught in a tapering centrifuge tube, the tube transferred to the incubator at 37° c. for two hours, then placed in the centrifugal machine, counterpoised and centrifugalised thoroughly. the three most important of the antibodies referred to which can be demonstrated with a certain amount of facility are agglutinin, opsonin and bacteriolysin; and the methods of testing for these bodies will now be considered. agglutinin. agglutinin is the name given to a substance present in the blood-serum of an animal that has successfully resisted inoculation with a certain micro-organism. this substance possesses the power of collecting together in clumps and masses, or agglutinating watery suspensions of that particular microbe. ~dilution of the specific serum~: _apparatus required_: sterile graduated capillary pipettes to contain 10 c. mm. (fig. 17). sterile graduated capillary pipettes to contain 90 c. mm. (fig. 17). small sterile test-tubes 5 × 0.5 cm. normal saline solution in flask or test-tube. pipette of specific serum. glass cutting knife, or three-square file. glass capsule, nearly full of dry silver sand, or roll of plasticine. grease pencil. method.-1. take three sterile test-tubes and number them 1, 2 and 3. 2. pipette 0.9 c.c. sterile normal saline solution into each tube, and stand tubes upright in the sand in the capsule, or in the plasticine block. 3. make a scratch with the glass cutting knife on the blood pipette above the upper level of the clear serum, and snap off and discard the empty portion of the tube. 4. remove 0.1 c.c. of the serum from the blood pipette tube, and mix it thoroughly with the fluid in tube no. 1; and label ~s.s.~, (specific serum), 10 per cent. 5. remove 0.1 c.c. of the solution from tube no. 1 by means of a fresh pipette, and mix it with the contents of tube no. 2; and label ~s.s.~, 1 per cent. 6. remove 0.1 c.c. of the solution from tube no. 2 by means of a fresh pipette, and mix it with the contents of tube no. 3; and label ~s.s.~, 0.1 per cent. when the yield of serum from the specimen of blood which has been collected, or is available, is small, the above method of diluting is not practicable, and the dilution should be carried out by wright's method in a capillary teat pipette. ~dilution of serum by means of a teat pipette.~ _materials required:_ blood pipette containing sample of specific serum after centrifugalisation. capsule of diluting fluid--normal saline solution. supply of pasteur pipettes (fig. 13a). india-rubber teats. small test-tubes. a block of plasticine to act as a test-tube stand. grease pencil. method: 1. mark three small test-tubes 10 per cent., 1 per cent. and 0.1 per cent. respectively, and stand them upright in the plasticine block. 2. take a pasteur pipette, nick the capillary stem just above the sealed end with a glass cutting knife, and snap off the sealed end with a quick movement so that the fracture is clean cut and at right angles to the long axis of the capillary stem--cut "square", in fact. prepare several, say a dozen, in this manner. 3. fit a rubber teat to the barrel of each of the pipettes. 4. make a mark with the grease pencil on the stem of one of the pipettes about 2 or 3 cm. from the open extremity. [illustration: fig. 193.--filling the capillary teat pipette.] 5. compress the teat between the finger and thumb (fig. 193) to such an extent as to drive out the greater part of the contained air. 6. maintaining the pressure on the teat pass the stem of the pipette into the capsule holding the saline solution, until the open end of the pipette is below the level of the fluid. 7. now cautiously relax the pressure on the teat and let the fluid enter the pipette and rise in the stem until it reaches the level of the grease pencil mark. as soon as this point is reached, check the movement of the column of fluid by maintaining the pressure on the teat, neither relaxing nor increasing it. 8. withdraw the point of the pipette clear of the fluid, and again relax the pressure on the teat very slightly. the column of saline solution rises higher in the stem, and a column of air will now enter the pipette and serve as an index to separate the first volume of fluid drawn into the stem from the next succeeding one. 9. again introduce the end of the pipette into the fluid and draw up a second volume of saline to the level of the grease pencil mark, and follow this with a second air index. 10. in like manner take up seven more equal volumes of saline solution and their following air bubbles. there are now nine equal volumes of normal saline in the pipette. 11. now pass the point of the pipette into the blood tube and dip the open end below the surface of the serum. proceeding as before, aspirate a volume of serum into the capillary stem up to the level of the pencil mark. 12. eject the contents of the pipette into the small tube marked 10 per cent. by compressing the rubber teat between thumb and finger. 13. mix the one volume of serum with the nine volumes of saline solution very thoroughly by repeatedly drawing up the whole of the fluid into the pipette and driving it out again into the test-tube. 14. now take a clean pipette and proceed precisely as before, 4 to 10. 15. having aspirated nine equal volumes of saline into this second pipette, now take up one similar volume of the fluid in the "10 per cent. tube." 16. eject the contents of this pipette into the second tube marked 1 per cent. and mix thoroughly as before. 17. in similar fashion make the 0.1 per cent. solution and transfer to the third tube. 18. further dilutions in multiples of ten can be prepared in the same way, and by varying the number of volumes of diluting fluid or serum any required dilution can be made (see appendix, dilution tables). note.--the saline diluting fluid _must always_ be taken into the pipette first, otherwise if the serum contains a very large amount of agglutinin the traces of this serum added to the saline solution may be sufficient to entirely vitiate the subsequent observations--whilst if more than one sample of serum is diluted from the same saline solution serious errors may be introduced into the experiments. ~the microscopical reaction:~ _apparatus required:_ five hanging-drop slides (or preferably two slide), with two cells mounted side by side on each (fig. 62, a), and one slide with one cell only. vaseline. cover-slips. platinum loop. grease pencil. eighteen to twenty-four-hour-old bouillon cultivation of the organism to be tested (e. g., bacillus typhi abdominalis) pipette end with the remainder of the specific serum labelled ~s.s.~ tubes containing the three solutions of the specific serum, 10, 1, and 0.1 per cent. respectively. pipette end with pooled normal serum labelled ~p.s.~ method.-1. make five hanging-drop preparations, thus: (a) one loopful of bouillon cultivation + one loopful pooled serum; label "control." (b) one loopful culture + one loopful undiluted specific serum; label 50 per cent. mount these two cover-slips on a double-celled slide. (c) one loopful bouillon culture + one loopful 10 per cent. serum; label 5 per cent. mount this on single-cell slide. (d) one loopful bouillon culture + one loopful 1 per cent. serum; label 0.5 per cent. (e) one loopful bouillon culture + one loopful 0.1 per cent. serum; label 0.05 per cent. mount these two cover-slips on a double-celled slide. 2. note the time: examine the control to determine that the bacilli are motile and uniformly scattered over the field--not collected into masses. 3. next examine the 50 per cent. serum preparation. if agglutinin is present and the test is giving a positive reaction, the bacilli _will_ be collected in large clumps. if the test is giving a negative reaction, the bacilli _may_ be collected in large clumps owing to the viscosity of the concentrated serum. 4. observe the 5 per cent. preparation microscopically. if the bacilli are aggregated into clumps, positive reaction. if the bacilli are _not_ aggregated into clumps, observe until thirty minutes from the time of preparation before recording a negative reaction. 5. examine the 0.5 and 0.05 per cent. preparations. these may or may not show agglutination when the result of the examination of the 5 per cent. preparation is positive, according to the potency of the specific serum; and by the examination of a series of dilutions a quantitative comparison of the valency of specific sera from different sources, or of serum from the same animal at different periods during the course of active immunisation may be obtained. note.--the graduated pipettes supplied with thoma's hæmatocytometer (intended for the collection of the specimen of blood required for the enumeration of leucocytes), giving a dilution of 1 in 10--i. e., 10 per cent.--may be substituted for the graduated capillary pipettes referred to above, if the vessel in which the serum has been separated is of sufficiently large diameter to permit of their use. ~the macroscopical reaction:~ sterile graduated capillary pipettes to contain 90 c. mm. eighteen to twenty-four-hours-old bouillon cultivation of the organism to be tested. three test-tubes containing the 10, 1, and 0.1 per cent. solutions of specific serum (about 90 c. mm. remaining in each). tube containing 50 per cent. solution of pooled serum. sedimentation pipettes (_vide_ page 17) or teat pipettes. method. 1. pipette 90 c. mm. of the bouillon culture into each of the tubes containing the diluted serum; and the same quantity into the tube containing the pooled serum. 2. fill a sedimentation tube (by aspirating) or a teat pipette from the contents of each tube. seal off the lower ends of the sedimentation tubes in the bunsen flame. 3. label each tube with the dilution of serum that it contains--viz., 5, 0.5, and 0.05 per cent. 4. place the pipettes in a vertical position, in a beaker, in the incubator at 37°c., for one or two hours. 5. observe the granular precipitate which is thrown down when the reaction is positive, and the uniform turbidity of the negative reaction as compared with the appearances in the control pooled serum. opsonin. opsonin is the term applied by wright to a substance, present in the serum of an inoculated animal, which is able to act upon or sensitise bacteria of the species originally injected, so as to render them an easy prey to the phagocytic activity of polymorphonuclear leucocytes. in the method for demonstrating opsonin about to be described, a comparison is made between the opsonic "power" of the pooled serum and the specific serum. _apparatus:_ small centrifuge and tubes for same (made from the barrels of broken capillary pipettes by sealing the conical ends in the bunsen flame). capillary pasteur pipettes. india-rubber teats. grease pencil. bunsen burner with peep flame. electrical signal clock (see page 39) stop watch, or watch. rectangular glass box or tray to hold pipettes. incubator regulated at 37°c. 3 × 1 slides. piece of light rubber tubing. rectangular block of plasticine. flask of normal saline solution. flask of sodium citrate (1.5 per cent.) in normal saline solution. _materials required_, and their preparation: small tube of "washed cells" (red blood discs and leucocytes); human cells are used in estimating the opsonising power of the serum of experimental animals. small tube of emulsion of bacteria of the species responsible for the infection of the experimental animal. blood pipette containing specific serum. blood pipette containing "pooled" serum. _washed cells._-1. take a small centrifuge tube and half fill it with sodium citrate solution. mark with the grease pencil the upper limit of the fluid. 2. cleanse the skin of the distal phalanx of the second finger of the left hand above the root of the nail with lint and ether. wind the rubber tubing tightly round the second phalanx; puncture with a sterile hagedorn needle through the cleansed area of skin. 3. take up a sufficiency of the issuing blood (more or less according to the number of tests to be performed) with a teat pipette, transfer it to the tube of citrate solution and mix thoroughly. make a second mark on the tube at the upper level of the mixed citrate solution and blood. 4. place the tube in the centrifuge, counterpoise accurately and centrifugalise until the blood cells are thrown down in a compact mass occupying approximately the same volume as is included between the two pencil marks. the column of fluid in the tube now shows clear supernatant fluid (citrate solution and blood plasma) separated from the sharp cut upper surface of the red deposit of corpuscles by a narrow greyish layer of leucocytes. 5. remove the supernatant column of citrate solution by means of a teat pipette, fill normal saline solution into the tube up to the upper pencil mark, and distribute the blood cells throughout the saline by means of the teat pipette. centrifugalise as before. 6. again remove the supernatant fluid and fill in a fresh supply of saline solution and centrifugalise once more. 7. remove the supernatant saline solution as nearly down to the level of the leucocytes as can be safely done without removing any of the leucocytes. 8. next distribute the leucocytes evenly throughout the mass of red cells by rotating the tube between the palms of the hands--just as is done with a tube of liquefied medium prior to pouring a plate. 9. set the tube upright in the plasticine block near to one end. _bacterial emulsion._-1. take an 18to 24-hour culture of the required bacterium (e. g., diplococcus pneumoniæ) grown upon sloped blood agar at 37° c. pour over the surface of the medium some 5 c.c. of normal saline solution. 2. with a platinum loop emulsify the growth from the surface of the medium as evenly as possible in the saline solution. 3. allow the tube to stand for a few minutes so that the large masses of growth may settle down; transfer the upper portion of the saline suspension to a centrifuge tube and centrifugalise thoroughly. 4. examine a drop of the supernatant opalescent emulsion microscopically to determine its freedom from clumps and masses. if unsatisfactory prepare another emulsion, this time scraping up the surface growth with a platinum spatula, transferring it to an agate mortar and grinding it up with successive small quantities of normal saline. if satisfactory insert the tube in the plasticine block next to that containing the washed cells. ~specific serum.~-~pooled serum.~-these sera are collected and treated as already described (see page 379), and the portions of the blood pipettes containing them are arranged in the remaining space in plasticine block. [illustration: fig. 194.--plasticine block with materials arranged for opsonin estimations.] the plasticine block now presents the appearances shown in fig. 194. method for determining the opsonic index.-1. take a capillary pipette fitted with a teat, cut the distal end _square_ and make a pencil mark about 2 cm. from the end. 2. aspirate into the pipette one volume of washed cells, air index, one volume of bacterial emulsion, air index, and one volume of specific serum (see fig. 195). [illustration: fig. 195. opsonin pipette.] 3. mix thoroughly on a 3 by 1 slide by compressing the teat and ejecting the contents of the pipette on to the surface of the slide, relaxing the pressure and so drawing the fluid up into the pipette again. these two processes should be repeated several times; finally take up the mixture in an unbroken column to the central portion of the capillary stem. 4. seal the point of the pipette in the peep flame of the bunsen burner and remove teat. 5. mark the pipette (with the grease pencil) with the distinctive number of the serum and place it in the glass box or tray. 6. take another similarly prepared pipette and aspirate into it equal volumes of washed cells, bacterial emulsion and pooled serum. treat precisely as in 3 and 4, label it "control" or "n.s." (normal serum) and place in the box by the side of the specific serum preparation. 7. place the box with the pipettes in the incubator and set the signal clock to ring at 15 minutes (or start the stop watch). 8. at the expiration of the incubation time remove the pipettes from the incubator. 9. cut off the sealed end of the specific serum preparation. mix its contents thoroughly as in step 3, and then divide the mixture between two 3 by 1 slips and carefully spread a blood film (_vide_ page 376) on each in such a way that only one-half of the surface of each slide is covered with blood--the free edge of the blood film approximating to the longitudinal axis of the slide. allow films to dry and label the slides with writing diamond. 10. treat the contents of the control pipette in similar fashion. 11. select the better film from each pair for fixing and staining. 12. fixing and staining must be carried out under strictly comparable conditions, and to this end the slides are best handled by placing in a glass staining rack which can be lowered in turn into each of a series of glass troughs containing the various reagents (fig. 196). place the rack in the first trough which contains the alcoholic solution of leishman's stain for two minutes to fix. transfer to the second trough containing the diluted stain for ten minutes. transfer to the third trough containing distilled water, and holding the trough over a sink, run in a stream of distilled water until washing is complete. remove slides from the rack and dry. leishman's stain is the best for routine work for all bacteria other than b. tuberculosis. films containing tubercle bacilli must of course be stained by the ziehl neelsen method. [illustration: fig. 196. glass staining trough for blood films.] 13. examine specific serum slide microscopically with 1/12 inch oil immersion. find the edge of the blood film--along this the bulk of the leucocytes will be collected. starting at one end of the film move the slide slowly across the microscope stage and as each leucocyte comes into view count and record the number of ingested bacteria. the sum of the contents of the first 50 consecutive polymorphonuclears that are encountered is marked down. (the _average_ number of bacilli ingested per leucocyte = the "_phagocytic index_.") 14. in precisely similar manner enumerate the bacteria present in the first 50 cells of the control preparation. this number is recorded as the denominator of a vulgar fraction of which the numerator is the number recorded for the specific serum. this fraction, expressed as a percentage of unity = the _opsonic index_. immune body. immune body or amboceptor is the name given to a substance present in the serum of an infected animal that has successfully resisted inoculation with some particular micro-organism, and which possesses the power of linking the complement normally present in the serum to bacteria of the species used as antigen in such a manner that the micro-organisms are rendered innocuous, and ultimately destroyed. the presence of the immune body in the serum can be demonstrated _in vitro_ by the reaction elaborated by bordet and gengou, known as the complement fixation test, the existence or the absence of the phenomenon of complement fixation being rendered obvious macroscopically by the absence or presence of hæmolysis on the subsequent addition of "sensitised" red blood corpuscles, (e. g., a mixture of crythrocyte solution and the appropriate hæmolysin--two of the three essentials in the hæmolytic system, _vide_ page 326). _apparatus required:_ sterile pipettes 1 c.c., (graduated in tenths). 16 × 2 cm. test-tubes. 9 × 1 cm. test-tubes. test-tube racks for each size of test-tube. _reagents required:_ normal saline solution. erythrocyte solution (human red cells, page 329) = e. hæmolytic serum (for human cells) = h.s. complement (fresh guinea-pig serum) = c. specific serum from inoculated animal, inactivated = s.s. control pooled serum from normal animals of same species, inactivated = p.s. _antigen_ (cultivation upon solid medium of the organism (e. g., b. typhosus) which has already served as antigen in the inoculation of the experimental animal) = a. to prepare the antigen for use, emulsify the whole of the bacterial growth in 5 c.c. normal saline solution. shake the emulsion in a test-tube with some sterilised glass beads to ensure a homogenous emulsion, and sterilise by heating to 60° c. in a water-bath for one hour. method.-1. take five small test-tubes, and number them 1 to 5 with a grease pencil. 2. into tubes nos. 1, 3, 4 and 5 pipette 0.1 c.c. of complement. 3. into tubes nos. 1 and 2 pipette 0.2 c.c. of the serum to be tested. 4. into tube no. 4 pipette 0.2 c.c. of control serum. 5. into tubes nos. 1, 2, 3 and 4 pipette 1 c.c. of the bacterial emulsion which forms the antigen. 6. place the whole set of tubes in the incubator at 37° c. for a period of one hour. 7. remove the tubes from the incubator and pipette 1 c.c. erythrocyte solution and 4 minimal hæmolytic doses of the corresponding hæmolysin into each tube. 8. mix thoroughly and return the tubes to the incubator at 37° c. for further period of one hour. 9. at the expiration of that time transfer the tubes to the ice chest, and allow them to stand for three hours. 10. examine the tubes. tubes 3, 4 and 5 should show complete hæmolysis; tube 2 should give no evidence whatever of hæmolysis. these tubes form the controls to the first tube, which contains the serum to be tested. in tube no. 1 the absence of hæmolysis would indicate the presence in the serum of the inoculated animal of a specific antibody to the micro-organism used in the inoculations; since it shows that the complement has been bound by the immune body to the bacterial antigen, and none has been left free to enter into the hæmolytic system; on the other hand the presence of hæmolysis would show that no appreciable amount of antibody has yet been formed in response to the inoculations. in other words, there is an absence of infection, since the complement remained unfixed at the time of the addition of the erythrocyte solution and hæmolytic serum, and was ready to combine with those reagents to complete the hæmolytic system. the method may be shown diagramatically as under using the symbols already indicated test-tubes. 1 2 3 4 5 0.1 c.c. c. ........ 0.1 c.c. c. 0.1 c.c. c. 0.1 c.c. c. 0.2 c.c. s.s. 0.2 c.c. s.s. ......... 0.2 c.c. p.s. ........ a. a. a. a. ........ ------------------------------------------------------------------------- incubate at 37° c. for one hour. -------------------------------------------------------------------------1 c.c. e. 1. c.c. e. 1 c.c. e. 1 c.c. e. 1 c.c. e. h.s.^{4} h.s.^{4} h.s.^{4} h.s.^{4} h.s.^{4} ------------------------------------------------------------------------- incubate at 37° c. for one hour. -------------------------------------------------------------------------(?) no hæmolysis. |__________________________________| hæmolysis. note.--it is sometimes more convenient to _sensitise_ the erythrocytes just before they are needed. this is done forty-five minutes after the experiment has been started (page 394, step 6), that is to say, before the completion of the first period of incubation, thus: 1. measure out into a sterile test-tube (or flask) five c.c. of erythrocyte solution. 2. measure out twenty minimal hæmolytic doses of hæmolysin, add to the erythrocyte solution on the test-tube. 3. allow the erythrocyte and hæmolysin to remain in contact for fifteen minutes at room temperature. the red cells are then sensitised and ready for use. 4. when the tubes are removed from the incubator at the end of the first hour (i. e., step 7) add 1 c.c. sensitised red cells to each tube by means of a graduated pipette. 5. mix thoroughly, return the tubes to the incubator at 37°c. and complete the experiment as previously described (steps 8 onward). xix. post-mortem examinations of experimental animals. the post-mortem examination should be carried out as soon as possible after the death of the animal, for it must be remembered that even in cold weather the tissues are rapidly invaded by numerous bacteria derived from the alimentary tract or the cavities of the body, and from external sources. the following outlines refer to a complete and exhaustive necropsy, and in routine work the examination will rarely need to be carried out in its entirety. note.--throughout the autopsy the searing irons must be freely employed, and it must be recollected that one instrument is only to be employed to seize or cut one structure. this done, it must be regarded as contaminated and a fresh instrument taken for the next step. ~apparatus required~: water steriliser. { scalpels. surgical instruments: { scissors. { forceps. { bone forceps. spear-headed platinum spatula (fig. 199). searing irons (fig. 198). tubes of media--bouillon and sloped agar. surface plates in petri dishes (of agar or one of its derivatives). platinum loop. aluminium "spreader." grease pencil. sterile capillary pipettes (fig. 13, a). sterile glass capsules, large and small. cover-slips or slides. bottles of fixing fluid (_vide_ page 114) for pieces of tissue intended for sectioning. 1. place the various instruments, forceps, scissors, scalpels, etc., needed for the autopsy inside the steriliser and sterilise by boiling for ten minutes; then open the steriliser, raise the tray from the interior and rest it crosswise on the edges. 2. heat the searing irons to redness in a separate gas stove. [illustration: fig. 197.--apparatus for post-mortem examination, animal on board.] 3. drench the fur (or feathers) with lysol solution, 2 per cent. this serves the twofold purpose of preventing the hairs from flying about and entering the body cavities during the autopsy, and of rendering innocuous any vermin that may be present on the animal. [illustration: fig. 198.--searing iron.] 4. examine the cadaver carefully. recollect that laboratory animals are not always hardy; death may be due to exposure to heat or cold, to starvation or overor improper feeding or to the attack of rats--and not to the bacterial infection. 5. fasten the body of the animal, ventral surface upward (unless there is some special reason for having the dorsum exposed), out on a board by means of copper nails driven through the extremities. 6. with sterile forceps and scalpel incise the skin in the middle line from the top of the sternum to the pubes. make other incisions at right angles to the first out to the axillæ and groins, and reflect the skin in two lateral flaps. (place the now infected instruments on the board by the side of the body or support them on a porcelain knife rest.) ~seat of inoculation.~-7. inspect the seat of inoculation. if any local lesion is visible, sear its exposed surface and with the platinum loop, remove material from the deeper parts to make tube and surface plate cultivations and cover-slip preparations. collect specimens of pus or other exudation in capillary pipettes for subsequent examination. 8. inspect the neighbouring lymphatic glands and endeavour to trace the path of the virus. 9. sear the whole of the exposed surface of the thorax with the searing irons. ~pleural cavity.~-10. divide the ribs on either side of the sternum and remove a rectangular portion of the anterior chest wall with sterile scissors and a fresh pair of forceps, exposing the heart. place the infected instruments by the side of the first set. 11. observe the condition of the anterior mediastinal glands, the thymus and the lungs. collect a quantity of pleuritic effusion, if such is present, in a pipette for further examination later. 12. raise the pericardial sac in a fresh pair of forceps and burn through this structure with a searing iron. collect a sample of pericardial fluid in a pipette for microscopical and cultural examination. 13. grasp the apex of the heart in the forceps and sear the surface of the right ventricle. 14. plunge the open point of a capillary pipette through the seared area into the ventricle and fill with blood. make cultivations and cover-slip preparations of the heart blood. 15. collect a further sample of blood or serum for subsequent investigation as to the presence of antibodies. ~peritoneal cavity.~-16. sear a broad track in the middle line of the abdominal wall; open the peritoneal cavity by an incision in the centre of the seared line. observe the condition of the omentum, the mesentery, the viscera and the peritoneal surface of the intestines. 17. collect a specimen of the peritoneal fluid (or pus, if present) in a capillary pipette. make cultivations, tube and surface plate, and cover-slip preparations from this situation. 18. collect a specimen of the urine from the distended bladder in a large pipette (in the manner indicated for heart blood), for further examination, by cultivations, microscopical preparations, and chemical analysis. 19. collect a specimen of bile from the gall bladder in similar manner. 20. excise the spleen and place it in a sterile capsule. later, sear the surface of this organ; plunge the spear-headed spatula through the centre of the seared area, twist it round between the finger and thumb, and remove it from the organ. sufficient material will be brought away in the eye in its head to make cultivations. a repetition of the process will afford material for cover-slip preparations. 21. seize one end of the spleen with sterile forceps. sear a narrow band of tissue, right around the organ and divide the spleen in this situation with a pair of scissors. holding the piece of spleen in the forceps, dab the cut surface on to a surface plate in a number of different spots. 22. in like manner examine the other organs--liver, lungs, kidneys, lymphatic glands (mesenteric, hepatic, lumbar, etc), etc. prepare cultivations and cover-slip preparations. 23. dissect out a long bone from one upper and one lower limb and one of the largest ribs. prepare cultures from the bone marrow in each case. set aside these bones for the subsequent preparation of marrow films. 24. film preparations of bone marrow are best made by the price-jones method. seize the bone in a pair of pliers and squeeze out some of the marrow; receive it in a platinum loop, and transfer to a watch glass of dissociating fluid and emulsify. the dissociating fluid is a neutral 10 per cent. solution of glycerine prepared as follows:- measure out 10 c.c. price's best glycerine and 90 c.c. sterile ammonia-free distilled water. mix. titrate against n/10 sodic hydrate solution using phenolphthalein as the indicator. the initial reaction is usually + 0.1 to + 0.5; add the calculated amount of n/10 sodic hydrate solution to neutralise. 25. place a loopful of fresh desiccating fluid on a 3 × 1 glass slide; add a similar loopful of the marrow emulsion, and spread very gently over the surface of the slip. 26. allow film to dry in the air (protected from dust) without heating. 27. stain with jenner's polychrome stain (page 97) for two and a half minutes. 28. wash with ammonia-free distilled water, dry thoroughly and mount in xylol balsam. ~cranial and spinal cavities.~-29. in some instances it may be necessary (e. g., experimental inoculation of rabies) to examine the cranial cavity or to remove the spinal cord. return the viscera to the abdominal cavity; draw the flaps of skin together and secure with michel's steel clips. draw the copper nails securing the limbs to the board, reverse the animal and again nail the limbs down--the body now being dorsum uppermost. 30. make a longitudinal incision in the mesial line from snout to root of tail, and four transverse incisions--one joining the roots of the two ears, one across the body at the level of the spinis of the scapulæ, another at the level of the costal margin and the last across the upper level of the pelvis. reflect these flaps of skin. 31. with forceps and scalpel dissect out the muscles lying in the furrow on either side of the spinal processes. 32. cut through the bases of the transverse processes with bone forceps. cut away the vault of the skull, cut through the roots of the nerves and remove the brain and spinal cord, place in a large glass dish for examination. prepare cultivations from the cerebro-spinal fluid. the removal of the brain and cord is a tedious process and during the dissection it is difficult to avoid injury to these structures. the operation is, however, carried out very expeditiously and neatly with the aid of the surgical engine (_vide_ page 361). a small circular saw is fitted to the hand piece. the bones of the skull are cut through and the whole of the vault removed, exposing the entire vertex of the brain. similarly all the spinous processes can be removed in one string by running the saw down first one side of the spinal column and then the other. in this way ample space for the removal of the nervous tissues is obtained with a minimum of labour. 33. having completed the preparation of cultures remove small portions of various organs at leisure and place each in separate bottles of fixing fluid for future sectioning. affix to each bottle a label bearing all necessary details as to its contents. 34. if necessary, remove portions of the organs for preservation and display as museum specimens (_vide_ page 404). 35. gather up all the infected instruments, return them to the steriliser, and disinfect by boiling for ten minutes. [illustration: fig. 199.--spear-headed platinum spatula (actual size.)] 36. sprinkle dry sawdust into the exposed body cavities to absorb blood and fluid. cover the body with blotting or filter paper, moistened with 2 per cent. lysol solution. place in a galvanised iron pail, provided with a lid, ready for transport to the crematorium. 37. cremate the cadaver together with the board upon which it is fixed. 38. stain the cover-slip preparations by suitable methods and examine microscopically. 39. incubate the cultivations and examine carefully from day to day. 40. make full notes of the condition of the various body cavities and of the viscera immediately the autopsy is completed; and add the result of the microscopical and cultural investigation when available. as part of the card index system in use in the author's laboratory already referred to (_vide_ page 335) there is a special yellow card for p-m notes. on the face of the card are printed headings for various data--some of which are sometimes unintentionally omitted--and on the reverse is a schematic figure which can be utilised for indicating the position of the chief lesions in the cadaver of any of the laboratory animals. autopsy card laboratory no. _________ date ________ animal ______ no. in series ______ [symbols: male female] weight ________ +------------------------------------------------------------------------+ died (or killed) _____ o'clock ____ m. autopsy made _____ o'clock ____ m. +------------------------------------------------------------------------+ notes on post mortem examinations. _general._ a. seat of inoculation. b. thoracic cavity. c. abdominal cavity. d. cranial cavity. +-------------------+----------------+--------------------------+ _bacteriological_ | _histological_ | _organs preserved._ | _examination._ | _examination._ | | a. | | | | | | b. | | | | | | c. | | | | | | d. | | | [illustration: fig. 200.--front of post-mortem card.] 41. finally, the results of the action of the organism or organisms isolated may be correlated with the symptoms observed during life and the observations summarised under the following headings: tissue changes: 1. local--i. e., produced in the neighbourhood of the bacteria. position: (a) at primary lesion. (b) at secondary foci. character: (a) vascular changes and tissue } acute reactions. } or (b) degeneration and necrosis. } chronic. 2. general (i. e., produced at a distance from the bacteria, by absorption of toxins): (a) in special tissues--e. g., nerve cells and fibres, secreting cells, vessel walls, etc. (b) general effects of malnutrition, etc. symptoms: (a) associated with known tissue changes. (b) without known tissue changes. [illustration: fig. 201.--back of post-mortem card.] ~permanent preparations--museum specimens.~-_i. tissues._--the naked-eye appearances of morbid tissues may be preserved by the following method: 1. remove the tissue or organ from the cadaver as soon after death as possible, using great care to avoid distortion or injury. 2. place it in a wide-mouthed stoppered jar, large enough to hold it conveniently, resting on a pad of cotton-wool, and arrange it in the position it is intended to occupy (but if it is intended to show a section of the tissue or organ, do not incise it yet). 3. cover with the kaiserling fixing solution, and stopper the jar; allow the tissues to remain in this solution for from forty-eight hours to seven days (according to size) to fix. make any necessary sections. kaiserling modified solution is prepared as follows: weigh out potassium acetate 30 grammes. potassium nitrate 15 grammes. and dissolve in distilled water 1000 c.c. then add formalin 150 c.c. filter. this fixing solution can be used repeatedly so long as it remains clear. even when it has become turbid, if simple filtration is sufficient to render it clear, the filtrate may be used again. 4. transfer the tissue to a bath of methylated spirit (95 per cent.) for thirty minutes to one hour. 5. remove to a fresh bath of spirit and watch carefully. when the natural colours show in their original tints, average time three to six hours, remove the tissues from the spirit bath, dry off the spirit from the cut surfaces by mopping with a soft cloth, then transfer to the mounting solution. jore's mounting solution (modified) consists of glycerine 500 c.c. distilled water 750 c.c. formalin 2 c.c. equally good but much cheaper is frost's mounting solution: potassium acetate 160 grammes. sodium fluoride 80 grammes. chloral hydrate 80 grammes. cane sugar (tate's cubes) 3,500 grammes. saturated thymol water 8,000 c.c. 6. after twenty-four hours in this solution, or as soon as the tissue sinks, transfer to a museum jar, fill with fresh mounting solution, and seal. _6a._ or transfer to museum jar and fill with liquefied gelatine, to which has been added 1 per cent. formalin. cover the jar and allow the gelatine to set. when solid, seal the cover of the jar in place. 7. to seal the museum preparation first warm the glass plate which forms the cover. this is most conveniently done by placing the cleaned and polished cover-plate upon a piece of asbestos millboard over a bunsen flame turned low. 8. smear an even layer of hot cement over the flange of the jar. the cement is prepared as follows: weigh out and mix in an iron ladle gutta percha (pure) 4 parts. asphaltum 5 parts. and melt together over a bunsen flame, stirring with an iron rod until solution is complete. 9. invert the glass plate over the jar and press down firmly into the cement. place a piece of asbestos board on the top and on that rest a suitable weight until the cement is cold and has thoroughly set. 10. trim off any projecting pieces of cement with an old knife, burr over the joint between jar and cover-plate with a hot smooth piece of metal (e. g., the searing iron). 11. paint a narrow band of japan black to finish off, round the joint, overlapping on to the cover-plate. _ii. tube cultivations of bacteria._--when showing typical appearances these may be preserved, if not permanently, at least for many years, as museum specimens, by the following method: 1. take a large glass jar 25 cm. high by 18 cm. diameter, with a firm base and a broad flange, carefully ground, around the mouth. the jar must be fitted with a disc of plate glass ground on one side, to serve as a lid. 2. smear a thick layer of resin ointment (b.p.) on the flange around the mouth of the jar. 3. cover the bottom of the jar with a layer of cotton-wool and saturate it with formalin. 4. remove the cotton-wool plug from the culture tubes and place them, mouth upward, inside the jar. (if water of condensation is present in any of the culture tubes, it should be removed by means of a capillary pipette before placing the tubes in the formalin chamber.) 5. adjust the glass disc, ground side downward, over the mouth of the jar and secure it by pressing it firmly down into the ointment, with a rotary movement. 6. remove the tubes from the formalin chamber after the lapse of a week, and dry the exterior of each. [illustration: fig. 202.--bulloch's tubes.] 7. seal the open mouth of each tube in the blowpipe flame and label. if the cultivations are intended for museum purposes when they are first planted, it is more convenient to employ bulloch's tubes. these are slightly longer than the ordinary tubes, and are provided with a constriction some 2 cm. below the mouth (fig. 202)--a feature which renders sealing in the blowpipe flame an easy matter. xx. the study of the pathogenic bacteria. the student, who has conscientiously worked out the methods, etc., previously dealt with, is in a position to make accurate observations and to write precise descriptions of the results of such observations. he is, therefore, now entrusted with pure cultivations of the various pathogenic bacteria, in order that he may study the life-history of each and record the results of his own observations--to be subsequently corrected or amplified by the demonstrator. in this way he is rendered independent of text-book descriptions, the statements in which he is otherwise too liable to take for granted, without personally attempting to verify their accuracy. during the course of this work attention must also be directed, as occasion arises, to such other bacteria, pathogenic or saprophytic, as are allied to the particular organisms under observation, or so resemble them as to become possible sources of error, by working them through on parallel lines--in other words the various bacteria should be studied in "groups." in the following pages the grouping in use in the author's elementary classes for medical and dental students and for candidates for the public health service is adopted, since a fairly long experience has completely vindicated the value and utility of this arrangement, and by its means a fund of information is obtained with regard to the resemblances and differences, morphological and cultural, of a large number of bacteria. the fact that some bacteria appear in more than one of these groups, so far from being a disadvantage, is a positive gain to the student, since with repetition alone will the necessary familiarity with the cultural characters of important bacteria be acquired. the study of the various groups will of course vary in detail with individual demonstrators, and with the student's requirements--the general line it should take is indicated briefly in connection with the first group only (pages 410-411). this section should be carefully worked through before the student proceeds to the study of bacterioscopical analysis. it is customary to commence the study of the pathogenic bacteria with the organisms of suppuration. this is a large group, for all the pathogenic bacteria possess the power, under certain conditions, of initiating purely pyogenic processes in place of or in addition to their specific lesions, (e. g., bacillus tuberculosis, streptococcus lanceolatus, bacillus typhosus, etc.). there are, however, a certain few organisms which commonly express their pathogenicity in the formation of pus. these are usually grouped together under the title of "pyogenic bacteria," as distinct from those which only occasionally exercise a pyogenic rôle. the organisms included in this group are: 1. staphylococcus pyogenes albus. 2. staphylococcus pyogenes aureus. 3. staphylococcus pyogenes citreus. 4. streptococcus pyogenes longus. 5. micrococcus tetragenus. 6. bacillus pyocyaneus. 7. bacillus pneumoniæ. and in certain special tissues 8. micrococcus gonorrhoeæ. 9. micrococcus intracellularis meningitidis (meningococcus). 10. micrococcus catarrhalis. 11. bacillus ægypticus (koch-weeks bacillus). the group may with advantage be subdivided as indicated in the following pages: i. _pyogenic cocci._ staphylococcus pyogenes albus. staphylococcus pyogenes aureus. staphylococcus pyogenes citreus. to contrast with micrococcus candicans. micrococcus agilis. 1. prepare subcultivations from each: bouillon, } agar streak, } blood serum, } litmus milk. } and incubate at 37°c. agar streak, } gelatine stab, } potato. } and incubate at 20°c. compare the naked-eye appearances of the cultures from day to day. note m. agilis refuses to grow at 37°c. 2. make hanging-drop preparations from the bouillon and agar cultivations after twenty-four hours' incubation. examine microscopically and compare. note the locomotive activity of m. agilis and the brownian movement of the remaining micrococci. 3. prepare cover-slip films from the agar cultures, after twenty-four hours' incubation. stain for flagella by the modified pitfield's method. note m. agilis is the only micrococcus showing flagella. 4. make microscopical preparations of each from all the various media after twenty-four and forty-eight hours and three days' incubation. stain carbolic methylene-blue, carbolic fuchsin, and gram's method. examine the films microscopically and compare. note in the gram preparation, the gram negative character of certain individual cocci in each film prepared from the three days' growth--such cocci are dead. 5. stain section of kidney tissue provided (showing abscess formation by staphylococcus aureus) by gram's method, and counterstain with cosin. 6. stain film preparation of pus from an abscess (containing staphylococcus pyogenes aureus) with carbolic methylene-blue and also by gram's method, counterstained with cosin. 7. inoculate[15] a white mouse subcutaneously with three loopfuls of a forty-eight-hour agar cultivation of the staphylococcus aureus, emulsified with 0.2 c.c. sterile broth. observe carefully during life, and when death occurs make a careful post-mortem examination. ii. _pyogenic cocci._ micrococcus gonorrhoeæ. micrococcus intracellularis meningitidis (meningococcus). micrococcus catarrhalis. micrococcus tetragenus. micrococcus paratetragenus. iii. _pyogenic cocci._ streptococcus pyogenes longus. streptococcus of bovine mastitis. streptococcus lanceolatus (diplococcus pneumoniæ or pneumococcus). to contrast with streptococcus brevis. streptococcus lebensis. iv. _pyogenic bacilli._ bacillus pneumoniæ (friedlaender). bacillus rhinoscleromatis. bacillus lactis aerogenes. v. _pyogenic bacilli._ bacillus pyocyaneus. to contrast with bacillus fluorescens liquefaciens. bacillus fluorescens non-liquefaciens. vi. _pneumonia group._ streptococcus lanceolatus (pneumococcus). bacillus pneumoniæ (friedlaender). streptococcus pyogenes longus. vii. _diphtheroid group._ bacillus diphtheriæ (klebs-loeffler). bacillus hoffmanni. bacillus xerosis. bacillus septus. viii. _coli-typhoid group._ b. typhi abdominalis (b. typhosus). b. coli communis. b. enteritidis (gaertner). to contrast with b. aquatilis sulcatus. ix. _escherich group._ b. coli communis (escherich). b. coli communior. b. lactis aerogenes. b. cloacæ. x. _gaertner group._ bacillus enteritidis (gaertner). b. paratyphosus a. b. paratyphosus b. bacillus choleræ suum (hog cholera). b. psittacosis. xi. _eberth group._ b. typhosus (eberth). b. dysenteriæ (shiga). b. dysenteriæ (flexner). b. fæcalis alcaligines. xii. _spirillum group._ vibrio choleræ. vibrio metschnikovi. to contrast with vibrio proteus (finkler and prior). spirillum rubrum. spirillum rugula. xiii. _anthrax group._ bacillus anthracis. to contrast with bacillus subtilis. bacillus mycoides. bacillus mesentericus fuscus. xiv. _acid fast group._ bacillus tuberculosis (human). " " (bovine). " " (avian). " " (fish). to contrast with bacillus phlei (timothy grass bacillus). butter bacillus of rabinowitch. xv. _plague group._ bacillus pestis. b. septicæmiæ hæmorrhagicæ. b. suipestifer. xvi. _influenzæ group._ b. influenzæ. bacillus ægypticus (koch-weeks). bacillus pertussis. xvii. _miscellaneous._ bacillus lepræ. bacillus mallei. micrococcus melitensis. xviii. _streptothrix group._ streptothrix actinomycotica. streptothrix maduræ. to contrast with cladothrix nivea. xix. _tetanus group._ bacillus tetani. bacillus oedematis maligni. bacillus chauvei (symptomatic anthrax). xx. _enteritidis sporogenes group._ bacillus enteritidis sporogenes. b. botulinus. b. butyricus. b. cadaveris. footnotes: [15] see note on vivisection license, page 334. xxi. bacteriological analyses. each bacteriological or bacterioscopical analysis of air, earth, sewage, various food-stuffs, etc., includes, as a general rule, two distinct investigations yielding results of very unequal value: 1. quantitative. 2. qualitative. the first is purely quantitative and as such is of minor importance as it aims simply at enumerating (approximately) the total number of bacteria present in any given unit of volume irrespective of the nature and character of individual organisms. the second and more important is both qualitative and quantitative in character since it seeks to accurately identify such pathogenic bacteria as may be present while, incidentally, the methods advocated are calculated to indicate, with a fair degree of accuracy, the numerical frequency of such bacteria, in the sample under examination. the general principles underlying the bacteriological analyses of water, sewage, air and dust, soil, milk, ice cream, meat, and other tinned stuffs, as exemplified by the methods used by the author, are indicated in the following pages, together with the methods of testing filters and chemical germicides; and the technique there set out will be found to be capable of expansion and adaptation to any circumstance or set of circumstances which may confront the student. ~controls.~--the necessity for the existence of adequate controls in all experimental work cannot be too urgently insisted upon. every batch of plates that is poured should include at least one of the presumably "sterile" medium; plate or tube cultures should be made from the various diluting fluids; every tube of carbohydrate medium that is inoculated should go into the incubator in company with a similar but uninoculated tube, and so on. bacteriological examination of water. the bacteria present in the water may comprise not only varieties which have their normal habitat in the water and will consequently develop at 20° c., but also if the water has been contaminated with excremental matter, varieties which have been derived from, or are pathogenic for, the animal body, and which will only develop well at a temperature of 37° c. in order to demonstrate the presence of each of these classes it will be necessary to incubate the various cultivations at each of these temperatures. further, the sample of water may contain moulds, yeasts, or torulæ, and the development of these will be best secured by plating in wort gelatine and incubating at 20° c. ~1. quantitative.~-_collection of the sample._--the most suitable vessels for the reception of the water sample are small glass bottles, 60 c.c. capacity, with narrow necks and overhanging glass stoppers (to prevent contamination of the bottle necks by falling dust). these must be carefully sterilised in the hot-air steriliser (_vide_ page 31). (a) if the sample is obtained from a ~tap~ or ~pipe~, turn on the water and allow it to run for a few minutes. remove the stopper from the bottle and retain it in the hand whilst the water is allowed to run into the bottle and three parts fill it. replace the stopper and tie it down, but _do not seal it_. (b) if the sample is obtained from a ~stream~, ~tank~, or ~reservoir~, fasten a piece of stout wire around the neck of the bottle, remove the stopper, and retain it in the hand. then, using the wire as a handle, plunge the bottle into the water, mouth downward, until it is well beneath the surface; then reverse it, allow it to fill, and withdraw it from the water. pour out a few cubic centimetres of water from the bottle, replace the stopper, and tie it down. [illustration: fig. 203.--esmarch's collecting bottle for water samples.] (c) if the sample is obtained from a ~lake~, ~river~ or the ~sea~; or when it is desired to compare samples taken at varying depths, the apparatus designed by v. esmarch (fig. 203) is employed. in this the sterilised bottle is enclosed in a weighted metal cage which can be lowered, by means of a graduated line, until the required depth is reached. at this point the bottle is opened by a thin wire cord attached to the stopper; when the bottle is full (as judged by the air bubbles ceasing to rise) the pull on the cord is released and the tension of the spiral spring above the stopper again forces it into the neck of the bottle. when the apparatus is taken out of the water, the small bottles are filled from it, and packed in the ice-box mentioned below. an inexpensive substitute for esmarch's bottle can be made in the laboratory thus: select a wide-mouthed glass stoppered bottle of about 500 c.c. capacity (about 20 cm. high and 8 cm. in diameter). remove the glass stopper and insert a rubber cork with two perforations in its place. through one perforation pass a piece of glass tubing about 5 cm. long and through the other a piece 22 cm. long, reaching to near the bottom of the bottle, each tube projecting about 2.5 cm. above the rubber stopper. plug the open ends of the tubes with cotton wool. secure the stopper in place with thin copper wire. [illustration: fig. 204.--thresh's deep water sampling bottle.] sterilise the fitted bottle in the autoclave. remove the cotton wool plugs and connect the projecting tubes by a piece of loosely fitting stout rubber pressure tubing about 5 cm. long, previously sterilised by boiling. take a piece of stout rubber cord about 33 cm. long, and of 10 mm. diameter (such as is used for door springs) thread a steel split ring upon it and secure the free ends tightly to the neck of the bottle by cord or catgut. attach the cord used for lowering the bottle into the water to the split ring on the rubber suspender. the best material for this purpose is cotton insulated electric wire knotted at every metre. connect the split ring also with the short piece of rubber tubing uniting the two glass tubes by a piece of catgut (or thin copper wire) of such length that when the bottle is suspended there is no pull upon the rubber tube, but which, however, will be easily jerked off when a sharp pull is given to the suspending cord. now wind heavy lead tubing about 1 cm. diameter around the upper part of the bottle, starting at the neck just above the shoulder. this ensures the sinking of the bottle in the vertical position (fig. 204). the apparatus being arranged is lowered to the required depth, a sharp jerk is then given to the suspending cord, which detaches the rubber tube and so opens the two glass tubes. water enters through the longer tube and the air is expelled through the shorter tube. the bubbles of air can be seen or heard rising through the water, until the bottle is nearly full, a small volume of compressed air remaining in the neck of the bottle. as the apparatus is raised, the air thus imprisoned expands, and prevents the entry of more water from nearer the surface. [illustration: fig. 205.--ice-box for transmission of water samples, etc.] _transport of sample._--if the examination of the sample cannot be commenced immediately, steps must be taken to prevent the multiplication of the bacteria contained in the water during the interval occupied in transit from the place of collection to the laboratory. to this end an ice-box such as that shown (in fig. 205) is essential. it consists of a double-walled metal cylinder into which slides a cylindrical chamber of sufficient capacity to accommodate four of the 60 c.c. bottles; this in turn is covered by a metal disc--the three portions being bolted together by thumb screws through the overhanging flanges. when in use, place the bottles, rolled in cotton-wool, in the central chamber, pack the space between the walls with pounded ice, securely close the metal box by screwing down the fly nuts, and place it in a felt-lined wooden case. (it has been shown that whilst bacteria will survive exposure to the temperature of melting ice, practically none will multiply at this temperature.) on reaching the laboratory, the method of examination consists in adding measured quantities of the water sample to several tubes of nutrient media previously liquefied by heat, pouring plate cultivations from each of these tubes, incubating at a suitable temperature, and finally counting the colonies which make their appearance on the plates. _apparatus required_: plate-levelling stand. case of sterile plates. case of sterile pipettes, 1 c.c. (in tenths of a cubic centimetre). case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre). case of sterile capsules, 25 c.c. capacity. tubes of nutrient gelatine. tubes of nutrient agar. tubes of wort gelatine. one 250 c.c. flask of sterile distilled water. tall cylinder containing 2 per cent. lysol solution. bunsen burner. grease pencil. water-bath regulated at 42° c. method.-1. arrange the plate-levelling platform with its water compartment filled with water, at 45° c. 2. number the agar tubes, consecutively, 1 to 6; the gelatine tubes, consecutively, 1 to 6, and the wort tubes, 1, 2, and 3. flame the plugs and see that they are not adherent to the lips of the tubes. 3. place the agar tubes in boiling water until the medium is melted, then transfer them to the water-bath regulated at 42° c. liquefy the nutrient gelatine and wort gelatine tubes by immersing them in the same water-bath. 4. remove the bottle containing the water sample from the ice-box, distribute the bacterial contents evenly throughout the water by shaking, cut the string securing the stopper, and loosen the stopper, but do not take it out. [illustration: fig. 206.--withdrawing water from water sample bottle.] 5. remove one of the 1 c.c. pipettes from the case, holding it by the plain portion of the tube. pass the graduated portion twice through the bunsen flame. tilt the bottle containing the water sample on the bench holding the neck between the middle and ring fingers of the left hand; grasp the head of the stopper between the forefinger and thumb, and remove it from the bottle. 6. pass the pipette into the mouth of the bottle, holding its point well below the surface of the water (fig. 206). suck up rather more than 1 c.c. into the pipette and allow the pipette to empty; this moistens the interior of the pipette and renders accurate measurement possible. now draw up exactly 1 c.c. into the pipette. withdraw the pipette from the bottle, replace the stopper, and stand the bottle upright. 7. take the first melted agar tube in the left hand, remove the cotton-wool plug, and add to its contents 0.5 c.c. of the water sample from the pipette; replug the tube and replace it in the water-bath. in a similar manner add 0.3 c.c. water to the contents of the second tube, and 0.2 c.c. to the contents of the third. 8. in a similar manner add 1 c.c. of the sample to the contents of the fourth tube. 9. similarly, add 0.5 c.c. and 0.1 c.c. respectively to the contents of the fifth and sixth tubes. 10. drop the pipette into the cylinder containing lysol solution. 11. mix the water sample with the medium in each tube in the manner described under plate cultivations; pour a plate from each tube. label each plate with (a) the distinctive number of the sample, (b) the quantity of water sample it contains, and (c) the date. 12. pour the contents of a tube of liquefied agar--not inoculated--into a petri dish to act as a control to demonstrate the sterility of the batch of agar employed. 13. allow the plates to set, and incubate at 37° c. 14. empty the water chamber of the levelling apparatus and refill it with ice-water. 15. by means of the sterile 10 c.c. pipette deliver 9.9 c.c. sterile distilled water into a sterile glass capsule. 16. add 0.1 c.c. of the water sample to the 9.9 c.c. sterile water in the capsule. this will give a dilution of 1 in 100. 17. plant the six tubes of nutrient gelatine in the following manner: to the first tube add 0.5 c.c. of the water sample direct from the bottle; to the second, 0.3 c.c.; and to the third, 0.2 c.c.; and pour a plate of each tube. to the fourth tube add 0.5 c.c. of the diluted water sample from the capsule; to the fifth, 0.3 c.c.; and to the sixth, 0.2 c.c.; and pour a plate from each. 18. label each plate with the quantity of the water sample it contains--that is, 0.5 c.c., 0.3 c.c., 0.2 c.c., 0.005 c.c., 0.003 c.c., and 0.002 c.c. 19. pour a control (uninoculated) gelatine plate. 20. allow the plates to set, and incubate at 20°c. 21. to the first tube of liquefied wort gelatine add 0.5 c.c. water sample; to the second, 0.3 c.c.; and to the third, 0.2 c.c. 22. label the plates, allow them to set, and incubate at 20° c. 23. count and record the number of colonies that have developed upon the agar at 37° c. after forty-eight hours' incubation. 24. note the number of colonies present on each of the gelatine and wort gelatine plates after forty-eight hours' incubation. 25. replace the gelatine and wort plates in the incubator; observe again at three days, four days, and five days. 26. calculate and record the number of organisms present per cubic centimetre of the original water from the average of the six gelatine plates at the latest date possible up to seven days--the presence of liquefying bacteria may render the calculation necessary at an earlier date, hence the importance of daily observations. _method of counting._--the most accurate method of counting the colonies on each of the plates is by means of either jeffery's or pakes' counting disc. each of these discs consists of a piece of paper, upon which is printed a dead black disc, subdivided by concentric circles and radii, printed in white. in jeffery's counter (fig. 207), each subdivision has an area of 1 square centimetre; in pakes' counter (fig. 208), radii divide the circle into sixteen equal sectors, and counting is facilitated by concentric circles equidistant from the centre. [illustration: fig. 207.--jeffery's disc, reduced.] [illustration: fig. 208.--pakes' disc, reduced.] (a) in the final counting of each plate, place the plate over the counting disc, and centre it, if possible, making its periphery coincide with one or other of the concentric circles. (b) remove the cover of the plate, and by means of a hand lens count the colonies appearing in each of the sectors in turn. make a note of the number present in each. (c) if the colonies present are fewer than 500, the entire plate should be counted. if, however, they exceed this number, enumerate one-half, or one-quarter of the plate, or count a sector here and there, and from these figures estimate the number of colonies present on the entire plate. in practice it will be found that pakes' disc is more suitable for the former class of plate; jeffery's disc for the latter. it should be recollected however that unless the plates have been carefully leveled and the medium is of equal thickness all over it is useless to try and average from small areas--since where the medium is thick all the bacteria will develop, where the layer is a thin one, only a few bacteria will find sufficient pabulum for the production of visible colonies. it will be noted that the quantities of water selected for addition to each set of tubes of nutrient media have been carefully chosen in order to yield workable results even when dealing with widely differing samples. plates prepared in agar with 0.1 c.c. and in gelatin with 0.02 c.c. can be counted even when large numbers of bacteria are present in the sample; whereas if micro-organisms are relatively few, agar plate 4 and gelatine plate 1 will give the most reliable counts. again the counts of the plates in a measure control each other; for example, the second and third plates of each gelatine series should together contain as many colonies as the first, and the second should contain about half as many more than the third and so on. 2. qualitative examination.-_collection of sample._--the water sample required for the routine examination, which it will be convenient to consider first, amounts to about 110 c.c. it is collected in the manner previously described (_vide_ page 416); similar bottles are used, and if four are filled the combined contents, amounting to about 240 c.c., will provide ample material for both the qualitative and quantitative examinations. unless the examination is to be commenced at once, the ice-box must be employed, otherwise water bacteria and other saprophytes will probably multiply at the expense of the microbes indicative of pollution, and so increase the difficulties of the investigation. in the routine examination of water supplies it is customary to limit the qualitative examination to a search for a. b. coli and its near allies. b. streptococci, organisms which are frequently spoken of as microbes of indication, as their presence is held to be evidence of pollution of the water by material derived from the mammalian alimentary canal, and so to constitute a danger signal. c. some observers still attach importance to the presence of b. enteritidis sporogenes, but as the search for this bacterium, (relatively scarce in water) necessitates the collection of a fairly large quantity of water it is not usually included in the routine examination. in the case of water samples examined during the progress of an epidemic, of new supplies and of unknown waters the search is extended to embrace other members of the coli-typhoid group; and on occasion the question of the presence or absence of vibrio choleræ or (more rarely) such bacteria as b. anthracis or b. tetani, may need investigation. when pathogenic or excremental bacteria are present in water, their numbers are relatively few, owing to the dilution they have undergone, and it is usual in commencing the examination, to adopt one or other of the following methods: a. _enrichment_, in which the harmless non-pathogenic bacteria may be destroyed or their growth inhibited, whilst the growth of the parasitic bacteria is encouraged. this is attained by so arranging the environment, (i. e., media, incubation temperature, and atmosphere) as to favor the growth of the pathogenic organisms at the expense of the harmless saprophytes. b. _concentration_, whereby all the bacteria present in the sample of water, pathogenic or otherwise, are concentrated in a small bulk of fluid. this is usually effected by filtration of the water sample through a porcelain filter candle, and the subsequent emulsion of the bacterial residue remaining on the walls of the candle with a small measured quantity of sterile bouillon. a. ~enrichment method.~ (dealing with the demonstration of bacteria of intestinal origin.) _apparatus required_ (_preliminary stage_): incubator running at 42° c. case of sterile pipettes, 1 c.c. graduated in tenths. case of sterile pipettes, 10 c.c. graduated in c.c. case of sterile pipettes, graduated to deliver 25 c.c. tubes of bile salt broth (_vide_ page 180). flask of double strength bile salt broth (_vide_ page 199). tubes of litmus silk. sterile flasks, 250 c.c. capacity. buchner's tubes. tabloids pyrogallic acid. tabloids sodium hydrate. bunsen burner. grease pencil. (_later stage_): incubator running at 37° c. surface plates of nutrose agar (see page 232). aluminium spreader. tubes of various media, including carbohydrate media. agglutinating sera, etc. method.-1. number a set of bile salt broth, tubes 1-5, and a duplicate set 1a-5a. 2. number one flask 7 and another 8. 3. to tubes no. 1 and 1a add 0.1 c.c. water sample. to tubes no. 2 and 2a add 1 c.c. water sample. to tubes no. 3 and 3a add 2 c.c. water sample. to tubes no. 4 and 4a add 5 c.c. water sample. to tubes no. 5 and 5a add 10 c.c. water sample. 4. put up all the tubes in buchner's tubes and incubate anaerobically at 42°c. note.--the bile salt medium is particularly suitable for the cultivation of bacteria of intestinal origin, and at the same time inhibits the growth of bacteria derived from other sources. the anaerobic conditions likewise favor the multiplication of intestinal bacteria, and also their fermentative activity. the temperature 42° c. destroys ordinary water bacteria and inhibits the growth of many ordinary mesophilic bacteria. 5. pipette 25 c.c. of double strength bile salt broth into flask 6, and 50 c.c. double strength bile salt broth into flask 7. 6. pipette 25 c.c. water sample into flask 6, and 50 c.c. water sample into flask 7. 7. incubate the two flasks aerobically at 42°c. 8. after twenty-four hours incubation note in each culture: a. the presence or absence of visible growth. b. the reaction of the medium as indicated by the colour change, if any, the litmus has undergone. c. the presence or absence of gas formation, as indicated by a froth on the surface of the medium, and the collection of gas in the inner "gas" tube. 9. replace those tubes which show no signs of growth in the incubator. examine after another period of twenty-four hours (total forty-eight hours incubation) with reference to the same points. 10. remove culture tubes which show visible growth from the buchner's tubes, whether acid production and gas formation are present or not. 11. examine all tubes which show growth by hanging-drop preparations. note such as show the presence of chains of cocci. 12. prepare surface plate cultivations upon nutrose agar from each tube that shows growth either macroscopically or microscopically, and incubate for twenty-four hours aerobically at 37° c. 13. examine the growth on the plates either with the naked eye or with the help of a small hand lens. practice will facilitate the recognition of colonies of the coli group, the typhoid group and the paratyphoid group; also those due to the growth of streptococci. the investigation from this stage proceeds along two divergent lines of enquiry--the first being concerned with the identity of the bacilli--typhoid bacilli, the second with that of the cocci. a. _b. coli and its allies._ 14. pick off coliform or typhiform colonies; make streak or smear subcultivations upon nutrient agar; incubate aerobically for twenty-four hours at 37° c. 15. examine the growth in each tube carefully both macroscopically and microscopically. if the growth is impure, replate on nutrose agar, pick off colonies and subcultivate again. when the growth in a tube is pure, add 5 c.c. sterile normal saline solution or sterile broth, and emulsify the entire surface growth with it. 16. utilise the emulsion for the preparation of a series of subcultivations upon the media enumerated below, using the ordinary loop to make the subcultures upon solid media, but adding one-tenth of a cubic centimetre of the emulsion to each of the fluid media by means of a sterile pipette. gelatine streak. agar streak. potato. nutrient broth. litmus milk. dextrose peptone solution. lævulose peptone solution. galactose peptone solution. maltose peptone solution. lactose peptone solution. saccharose peptone solution. raffinose peptone solution. dulcite peptone solution. mannite peptone solution. glycerin peptone solution. inulin peptone solution. dextrin peptone solution. 17. differentiate the bacilli after isolation by means of their cultural reactions and biological characters into members of: i. the escherich group. b. coli communis. b. coli communior. b. lactis aerogenes. b. cloacæ. ii. the gærtner group. bacillus enteritidis (of gærtner). b. paratyphosus a. b. paratyphosus b. bacillus choleræ suum. iii. the eberth group. b. typhosus. b. dysenteriæ (shiga). b. dysenteriæ (flexner). b. fæcalis alcaligines. 18. confirm these results by testing the organisms isolated against specific agglutinating sera obtained from experimentally inoculated animals. if a positive result is obtained when using this method, it only needs a simple calculation to determine the smallest quantity (down to 0.1 c.c.) of the sample that contains at least one of the microbes of indication. for instance, if growth occurs in all the tubes from 4 to 10, and that growth is subsequently proved to be due to the multiplication of b. coli, then it follows that at least one colon bacillus is present in every 10 c.c. of the water sample, but not in every 5 c.c. if, on the other hand, the presence of the b. coli can only be proved in flask no. 7, then the average number of colon bacilli present in the sample is at least one in every 50 c.c. (i. e., twenty per litre), but not one in every 25 c.c. and so on. the general outline of the method of identifying the members of the coli-typhoid group is given in the form of an analytical schema--whilst the full differential details are set out in tabular form. analytical scheme for isolation of members of the coli and typhoid groups. nutrose agar. | ---------------------------------- | | red colonies. blue colonies. escherich group. gaertner and eberth groups. || | ====================-------------- || lactose peptone solution. || ====================-------------- || | gas. no gas. || | b. coli communis and its allies. | || gaertner and eberth groups. acid and gas in glucose peptone solution. | acid and coagulation in milk. | general turbidity and indol in bouillon. glucose peptone solution. | ==================================| || | || | gas. no gas. || | gaertner group. eberth group. || | =================== --------------- || || | | || || | | litmus milk. peptone solution. litmus milk. peptone solution. || || | | acid at first. general turbidity. acid. general turbidity. alkaline later. no indol. no coagulation. no indol. no coagulation. serum reaction. serum reaction. _b. streptococci._ 19. pick off streptococcus colonies and subcultivate upon nutrient agar exactly as directed in steps 14, 15 and 16. 20. differentiate the streptococci isolated into members of the saprophytic group of short-chained cocci, or members of the parasitic (pathogenic) group of long-chained cocci, by means of their cultural characters, and record their numerical frequency in the manner indicated for the members of the coli-typhoid group. differential table of coli-typhoid group transcriber's note: table split to fit 80 spaces. +-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+ | | m | d | l | g | m | l | s | r | d | |a = acid reaction | o | e | æ | a | a | a | a | a | e | |g = gas formation | t | x | v | l | l | c | c | f | t | | | i | t | u | a | t | t | c | f | r | | | l | r | l | c | o | o | h | i | i | | | i | o | o | t | s | s | a | n | n | | | t | s | s | o | e | e | r | o | | | | y | e | e | s | | | o | s | | | | | | | e | | | s | e | | | | | | | | | | e | | | | | +-----+-----+-----+-----+-----+-----+-----+-----+ | | | a g | a g | a g | a g | a g | a g | a g | a g | +-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+ |_the escherich group._ | | | | | | | | | | | b. coli communis | + | + + | + + | + + | + + | + + | o | + + | + + | | b. coli communior | + | + + | + + | + + | + + | + + | + + | + + | + + | | b. lactis aerogenes | | + + | + + | + + | + + | + + | o | o | + + | | b. acidi lactici | | + + | + + | + + | + + | + + | o | o | o | | b. pneumoniæ | | + + | + + | + + | + + | + + | + + | + + | + + | | b cloaceæ(a) | + | + + | + + | + + | + + | + + | + + | + + | + + | | | | | | | | | | | | |_the gærtner group._ | | | | | | | | | | | b. enteritidis | + | + + | + + | + + | + + | o | o | o | o | | b. paratyphosus a | + | + + | + + | + + | + + | o | o | o | o | | b. paratyphosus b | + | + + | + + | + + | + + | o | o | o | o | | b. choleræ suum | + | + + | + + | + + | + + | o | o | | o | | b. suipestifer | + | + + | + + | + + | + + | o | o | | o | | | | | | | | | | | | |_the eberth group._ | | | | | | | | | | | b. typhosus | + | + | + | + | + | o | o | o | + | | b. dysenteriæ (shiga) | | + | + | + | o | o | o | o | o | | b. dysenteriæ (flexner) | | + | + | + | + | o | o | ± | o | | b. fæcalis alkaligines | + | o | o | o | o | o | o | o | o | | | | | | | | | | | | +-------------------------+---+-----+-----+-----+-----+-----+-----+-----+-----+ | table notes: |(b)| (c) | +-------------------------+---+-----------------------------------------------+ +-------------------------+-----+-----+-----+-----+-----+-----+---+-----------+ | | i | s | g | d | m | s | i |litmus milk| |a=acid reaction | n | a | l | u | a | o | n | | |g=gas formation | u | l | y | l | n | r | d | | | | l | i | c | c | n | b | o +-----+-----+ | | i | c | e | i | i | i | l |early|late | | | n | i | r | t | t | t | | | | | | | n | i | e | e | e | | | | | | | | n | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |-----+-----+-----+-----+-----+-----+ | | | | | a g | a g | a g | a g | a g | a g | | | | +-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+ |_the escherich group_ | | | | | | | | | | | b. coli communis | o | o | + + | + + | + + | + + | + | + | + c | | b. coli communior | o | o | + + | + + | + + | + + | + | + | + c | | b. lactis aerogenes | o | o | o | o | + + | + + | | + | + c | | b. acidi lactici | o | o | o | + + | + + | + + | + | + | + c | | b. pneumoniæ | o | o | + + | + + | + + | + + | | + | + c | | b cloaceæ[a] | o | o | + + | o | + + | + | + | + | + c | | | | | | | | | | | | |_the gærtner group._ | | | | | | | | | | | b. enteritidis | o | o | o | + + | + + | + + | | ± | | | b. paratyphosus a | o | ± | o | + + | + + | + + | | + | o | | b. paratyphosus b | o | o | o | + + | + + | + + | | + | | | b. choleræ suum | o | o | o | o | o | + + | ± | + | | | b. suipestifer | o | o | o | + + | + + | + + | | + | | | | | | | | | | | | | |_the eberth group._ | | | | | | | | | | | b. typhosus | o | o | o | o | + | + | | + | + | | b. dysenteriæ (shiga) | o | o | o | o | o | o | | + | | | b. dysenteriæ (flexner) | o | o | o | o | + | o | ± | + | | | b. fæcalis alkaligines | o | o | o | o | o | o | | | | | | | | | | | | | | | +-------------------------+-----+-----+-----+-----+-----+-----+---+-----+-----+ | table notes: | |(d)| (e) | +-------------------------+-----------------------------------+---+-----------+ table notes: (a) * liquefies gelatine. (b) + = motile. = non-motile. (c) + = acid or gas production. ± = slight acid production. o = no change. (d) + = indol production. ± = slight indol production. = no indol formed. (e) + = acid production. = alkali production. o = no change in reaction. c = clot. 21. determine the pathogenicity for mice (subcutaneous inoculation) and rabbits (intravenous inoculation) of the streptococci isolated. on the facing insert page is reproduced a blank from the author's laboratory water analysis book, by means of which an exact record can be kept, with a minimum of labour, of every sample examined. b. ~concentration method.~ the remaining organisms referred to on page 426 are more conveniently sought for by the concentration method. _collection of the sample._--the quantity of water required for this method of examination is about 2000 c.c., and the vessel usually chosen for its reception is an ordinary blue glass winchester quart bottle, sterilised in the hot-air oven, and over this a paper or parchment cap fastened with string. the bottle may be packed in a wooden box or in an ordinary wicker case. the method of collecting the sample is identical with that described under the heading of quantitative examination; there is, however, not the same imperative necessity to pack the sample in ice for transmission to the laboratory. _apparatus required_: sterile chamberland or doulton "white" porcelain open mouth filter candle, fitted with rubber washer. rubber cork to fit mouth of the filter candle, perforated with one hole. kitasato serum flask, 2500 c.c. capacity. geryk air pump or water force pump. wulff's bottle, fitted as wash-bottle, and containing sulphuric acid (to act as a safety valve between filter and pump). pressure tubing, clamps, pinch-cock. retort stand, with ring and clamp. rubber cork for the neck of winchester quart, perforated with two holes and fitted with one 6 cm. length of straight glass tubing, and one v-shaped piece of glass tubing, one arm 32 cm. in length, the other 52 cm., the shorter arm being plugged with cotton-wool. the rubber stopper must be sterilised by boiling and the glass tubing by hot air, before use. flask containing 250 c.c. sterile broth. test-tube brush to fit the lumen of the candle, enclosed in a sterile test-tube (and previously sterilised by dry heat or by boiling). case of sterile pipettes, 10 c.c. in tenths. case of sterile pipettes, 1 c.c. in tenths. case of sterile pipettes, 1 c.c. in hundredths. tubes of various nutrient media (according to requirements). twelve buchner's tubes with rubber stoppers. pyrogallic acid tablets. caustic soda tablets. [illustration: sample form]] [illustration: fig. 209.--water filtering apparatus. that portion of the figure to the left of the vertical line is drawn to a larger scale than that on the right, in order to show details of sprengel's pump.] method.-1. fit up the filtering apparatus as in the accompanying diagram (fig. 209), interposing the wash-bottle with sulphuric acid between the filter flask and the force-pump (in the position occupied in the diagram by the central vertical line), and placing another screw clamp on the rubber tubing connecting the lateral arm of the filter flask with the wash-bottle. [illustration: fig. 210. sterile test-tube brush.] 2. filter the entire 2000 c.c. of water through the filter candle. 3. when the nitration is completed, screw up the clamps and so occlude the two pieces of pressure tubing. 4. reverse the position of the glass tubes in the wulff's bottle so that the one nearest the air pump now dips into the sulphuric acid. 5. slowly open the metal clamps and allow air to gradually pass through the acid, and enter filter flask, and so restore the pressure. 6. unship the apparatus, remove the cork from the mouth of the candle. 7. pipette 10 c.c. of sterile broth into the interior of the candle, and by means of the sterile test-tube brush (fig. 210) emulsify the slimy residue which lines the candle, with the broth. practically all the bacteria contained in the original 2000 c.c. of water are now suspended in 10 c.c. of broth, so that 1 c.c. of the suspension is equivalent, so far as the contained organisms are concerned, to 200 c.c. of the original water. (some bacteria will of course be left behind on the walls of the filter and in its pores.) up to this point the method is identical, irrespective of the particular organism whose presence it is desired to demonstrate; but from this point onward the methods must be specially adapted to the isolation of definite groups of organisms or of individual bacteria. the coli-typhoid group.-1. number nine tubes of bile salt broth (_vide_ page 180), consecutively from 1 to 9. 2. to no 1 add 1 c.c. } of the original water sample 2 add 2 c.c. } before the nitration is commenced. 3 add 5 c.c. } 3. to the remaining tubes of bile salt broth add varying quantities of the suspension by means of suitably graduated sterile pipettes, as follows: no. 4 0.05 c.c. (equivalent to 10 c.c. of the original water sample). no. 5 0.125 c.c. (equivalent to 25 c.c. of the original water sample). no. 6 0.25 c.c. (equivalent to 50 c.c. of the original water sample). no. 7 0.5 c.c. (equivalent to 100 c.c. of the original water sample). no. 8 1.0 c.c. (equivalent to 200 c.c. of the original water sample). no. 9 2.5 c.c. (equivalent to 500 c.c. of the original water sample). 4. put up each tube anaerobically in a buchner's tube and incubate at 42° c. 5. the subsequent steps are identical with those described under the enrichment method (see page 428 to 431; steps 8 to 18). _alternative methods._- a few of the older methods for the isolation of the members of the coli-typhoid groups are referred to but they are distinctly inferior to those already described. (a) the carbolic method: 1. take ten tubes of carbolised bouillon (_vide_ page 202) and number them consecutively from 1 to 10. 2. inoculate each tube with a different amount of the water sample or suspension, as in the previous method. 3. incubate aerobically at 37° c. 4. examine the culture tubes after twenty-four hours' incubation. 5. from those tubes which shows signs of growth, pour plates in the usual manner, using carbolised gelatine (_vide_ page 202) in place of the ordinary gelatine, and incubate at 20° c. for three, four, or five days as may be necessary. 6. subcultivate from any colonies that make their appearance, and determine their identity on the lines laid down in the previous method. (b) parietti's method: 1. take nine tubes of parietti's bouillon (_vide_ page 202)--i. e., three each of those containing 0.1 c.c., 0.2 c.c., and 0.5 c.c. of parietti's solution respectively. mark plainly on the outside of each tube the quantity of parietti's solution it contains. 2. to each tube add a different amount of the original water, or of the suspension, and incubate at 37° c. 3. examine the culture tubes after twenty-four and forty-eight hours' incubation, and plate in nutrient carbolised or potato gelatine from such as have grown. 4. pick off suspicious colonies, if any such appear on the plates, subcultivate them upon the various media, and identify them. (c) elsner's method: this method simply consists in substituting elsner's potato gelatine (_vide_ page 204) for ordinary nutrient gelatine in any of the previously mentioned methods. (d) cambier's candle method: treat a large volume of the water sample by the concentration method (_vide_ page 434). 1. remove the rubber stopper from the mouth of the filter candle, introduce 10 c.c. sterile bouillon into its interior, and emulsify the bacterial sediment; replug the mouth of the candle with a wad of sterile cotton-wool. 2. remove the filter candle from the filter flask and insert it into the mouth of a flask or a glass cylinder containing sterile bouillon sufficient to reach nearly up to the rubber washer on the candle. 3. incubate for twenty-four to thirty-six hours at 37° c. 4. from the now turbid bouillon in the glass cylinder pour gelatine plates and incubate at 20° c. 5. subcultivate and identify any suspicious colonies that appear. (the method depends upon the assumption that members of the typhoid and coli groups find their way through the porcelain filter from the interior to the surrounding bouillon at a quicker rate than the associated bacteria.) b. ~enteritidis sporogenes.~-1. transfer 5 c.c. of the emulsion from the filter candle to a sterile test-tube and plug carefully. 2. place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (_vide_ page 257), and expose to a temperature of 80° c. for twenty minutes. 3. number ten tubes of litmus milk consecutively from 1 to 10. 4. remove the test-tube from the benzole bath and shake well to distribute the spores evenly through the fluid. 5. to each tube of litmus milk add a measured quantity of the suspension corresponding to the amounts employed in isolating the coli group (_vide_ page 437). 6. incubate each tube anaerobically at 37° c. anaerobic conditions can be obtained by putting the cultures up in buchner's tubes or in bulloch's apparatus. if, however, whole milk has been used in making the litmus milk the layer of cream that rises to the surface will be sufficient to ensure anaerobiosis; whilst if separated milk has been employed it will be sufficient to pour a layer of sterile vaseline or liquid paraffin on the surface of the fluid. 7. examine after twenty-four hours' incubation. note (if b. enteritidis sporogenes is present)-(a) acid reaction of the medium as indicated by the colour of the litmus or its complete decolourisation. (b) presence of clotting, and the separation of clear whey. (c) presence of gas, as indicated by fissures and bubbles in the coagulum, and possibly masses of coagulum driven up the tube almost to the plug. 8. replace the tubes which show no signs of growth in the incubator for a further period of twenty-four hours and again examine with reference to the same points. 9. remove those tubes which give evidence of growth from the buchner's tubes and carefully pipette off the whey; examine the whey microscopically. 10. inoculate two guinea-pigs each subcutaneously with 0.5 c.c. of the whey and observe the result. ~vibrio choleræ.~-1. number ten tubes of peptone water consecutively from 1 to 10. 2. to each of the tubes of peptone water add a measured quantity of the suspension, corresponding to those amounts employed in isolating the members of the coli group (_vide_ page 437). 3. incubate aerobically at 37° c. for twenty-four hours. examine the tubes carefully for visible growth, especially delicate pellicle formation, which if present should be examined microscopically for vibrios, both by stained preparations or by fresh specimens with dark ground illumination. 4. inoculate fresh tubes of peptone water from such of the tubes as exhibit pellicle formation--from the pellicle itself--and incubate at 37° c. for twenty-four hours. 5. test the peptone water itself for the presence of indol and nitrite by the addition of pure concentrated h_{2}so_{4}. 5. prepare gelatine and agar plates in the usual way from such of these tubes as show pellicle formation. 6. pick off from the plates any colonies resembling those of the vibrio choleræ and subcultivate upon all the ordinary laboratory media. 7. test the vibrio isolated against the serum of an animal immunised to the vibrio choleræ for agglutination. ~b. anthracis.~-1. transfer 5 c.c. of the emulsion from the filter candle to a sterile test-tube and plug carefully. 2. place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (_vide_ page 257), and expose to a temperature of 80° c. for twenty minutes. 3. inoculate a _young_ white rat subcutaneously (on the inner aspect of one of the hind legs) with 1 c.c. of the emulsion. observe during life, and, if the animal succumbs, make a complete post-mortem examination. 4. melt three tubes of nutrient agar in boiling water and cool to 42° c. 5. number the tubes 1, 2, and 3. to no. 1 add 0.2 c.c., to no. 2 add 0.3 c.c., and to no. 3 add 0.5 c.c. of the suspension, and pour plates therefrom. 6. incubate at 37° c. for twenty-four or forty-eight hours. 7. pick off any colonies resembling those of anthrax and subcultivate on all the ordinary laboratory media. 8. inoculate another young white rat as in 3, using two loopfuls of the agar subcultivation emulsified with 1 c.c. sterile bouillon. observe during life, and if the animal succumbs, make a complete post-mortem examination. ~b. tetani.~-1. proceed as detailed above in steps 1 and 2 for the isolation of the b. anthracis. 2. add 1 c.c. of the suspension to each of three tubes of glucose formate broth, and incubate anaerobically in buchner's tubes at 37° c. 3. from such of the tubes as show visible growth (with or without the production of gas) after twenty-four hours' incubation inoculate guinea-pigs, subcutaneously (under the skin of the abdomen), using 0.1 c.c. of the bouillon cultivation as a dose. observe carefully during life, and, if death occurs, make a complete post-mortem examination. 4. from the same tubes pour agar plates and incubate anaerobically in bulloch's apparatus, at 37° c. 5. subcultivate suspicious colonies on the various media, incubate anaerobically, making control cultivations on glucose formate agar, stab and streak, to incubate aerobically and carry out further inoculation experiments with the resulting growths. examination of milk. "one-cow" or "whole" milk, if taken from the apparently healthy animal (that is, an animal without any obvious lesion of the udder or teats) with ordinary precautions as to cleanliness, avoidance of dust, etc., contains but few organisms. in dealing with one-cow milk, from a suspected, or an obviously diseased animal, a complete analysis should include the examination (both qualitative and quantitative) of samples of (a) fore-milk, (b) mid-milk, (c) strippings, and, if possible, from each quarter of the udder. "mixed" milk, on the other hand, by the time it leaves the retailer's hands, usually contains as many micro-organisms as an equal volume of sewage and indeed during the examination it is treated as such. it is possible however to collect and store mixed milk in so cleanly a manner that its germ content does not exceed 5000 micro-organisms per cubic centimetre. such comparative freedom from extraneous bacteria is usually secured by the purveyor only when he resorts to the process of pasteurisation (heating the milk to 65° c. for twenty minutes or to 77° c. for one minute) or the simpler plan of adding preservatives to the milk. information regarding the employment of these methods for the destruction of bacteria should always be sought in the case of mixed milk samples, and in this connection the following tests will be found useful: 1. _raw milk_ (saul). to 10 c.c. milk in a test tube, add 1 c.c. of a 1 per cent. aqueous solution of ortol (ortho-methyl-amino-phenol sulphate), recently prepared and mix. next add 0.2 c.c. of a 3 per cent. peroxide of hydrogen solution. the appearance of a brick red color within 30 seconds indicates raw milk. milk heated to 74° c. for thirty minutes undergoes no alteration in color; if heated to 75° c. for ten minutes only, the brick red color appears after standing for about two minutes. 2. _boric acid._ evaporate to dryness, 50 c.c. of the milk which has been rendered slightly alkaline to litmus, then incinerate. dissolve in distilled water, add slight excess of dilute hydrochloric acid and again evaporate to dryness. dissolve the residue in a small quantity of hot water and moisten a piece of turmeric paper with the solution. dry the turmeric paper. _rose_ or _cherry-red_ color = borax or boric acid. 3. _formaldehyde_ (hehner). to 10 c.c. milk in a test tube add 5 c.c. concentrated _commercial_ sulphuric acid slowly, so that the two fluids do not mix. hold the tube vertically and agitate very gently. _violet zone_ at the junction of the two liquids = formaldehyde. 4. _hydrogen peroxide._ to 10 c.c. milk (diluted with equal quantities of water) in a test tube add 0.4 c.c. of a 4 per cent. alcoholic solution of benzidine and 0.2 c.c. acetic acid. _blue coloration_ of the mixture = hydrogen peroxide. 5. _salicylic acid._ precipitate the caseinogen by the addition of acetic acid and filter. to the filtrate add a few drops of 1 per cent. aqueous solution of ferric chloride. _purple coloration_ = salicylic acid. 6. _sodium carbonate or bicarbonate._ to 10 c.c. of the milk in a test tube add 10 c.c. of alcohol and 0.3 c.c. of a 1 per cent. alcoholic solution of rosolic acid. _brownish_ color = pure milk; _rose_ color = preserved milk. [illustration: fig. 211.--milk-collecting bottle and dipper in case.] quantitative.-_collection of sample._-the apparatus used for the collection of a retail mixed milk sample consists of a cylindrical copper case, 16 cm. high and 9 cm. in diameter, provided with a "pull-off" lid, containing a milk dipper, also made of copper; and inside this, again, a wide-mouthed, stoppered glass bottle of about 250 c.c. capacity (about 14 cm. high by 7 cm. diameter), having a tablet for notes, sand-blasted on the side. the copper cylinder and its contents, secured from shaking by packing with cotton-wool, are sterilised in the hot-air oven (fig. 26). when collecting a sample, 1. remove the cap from the cylinder. 2. draw out the cotton-wool. 3. lift out the bottle and dipper together. 4. receive the milk in the sterile dipper, and pour it directly into the sterile bottle. 5. enter the particulars necessary for the identification of the specimen, on the tablet, with a lead pencil, or pen and ink. 6. pack the apparatus in the ice-box for transmission to the laboratory in precisely the same manner as an ordinary water sample. "whole" milk may with advantage be collected in the sterile bottle directly since the mouth is sufficiently wide for the milker to direct the stream of milk into it. ~condensed milk~ must be diluted with sterile distilled water in accordance with the directions printed upon the label, then treated as ordinary milk. _apparatus required_: case of sterile capsules (25 c.c. capacity). case of sterile graduated pipettes, 10 c.c. (in tenths of a cubic centimetre). case of sterile graduated pipettes, 1 c.c. (in tenths of a cubic centimetre). flask containing 250 c.c. sterile bouillon. tall cylinder containing 2 per cent. lysol solution. plate-levelling stand. case of sterile plates. tubes nutrient gelatine or gelatine agar. tubes of wort gelatine. tubes of nutrient agar. water-bath regulated at 42° c. bunsen burner. grease pencil. method.-1. arrange four sterile capsules in a row; number them i, ii, iii, and iv. 2. fill 9 c.c. sterile bouillon into the first, and 9.9 c.c. bouillon into each of the three remaining capsules. 3. remove 1 c.c. milk from one of the bottles by means of a sterile pipette and add it to the bouillon in capsule i; mix thoroughly by repeatedly filling and emptying the pipette. 4. remove 0.1 c.c. of the milky bouillon from capsule i, add it to the contents of capsule ii, and mix as before. 5. in like manner add 0.1 c.c. of the contents of capsule ii to capsule iii; and then 0.1 c.c. of the contents of capsule iii to capsule iv. then 1 c.c. of dilution i contains 0.1 c.c. milk sample. 1 c.c. of dilution ii contains 0.001 c.c. milk sample. 1 c.c. of dilution iii contains 0.00001 c.c. milk sample. 1 c.c. of dilution iv contains 0.0000001 c.c. milk sample. 6. melt the gelatine and the agar tubes in boiling water; then transfer to the water-bath and cool them down to 42° c. 7. number the gelatine tubes consecutively 1 to 12. 8. inoculate the tubes with varying quantities of the material as follows: to tube no. 1 add 1.0 c.c. of the milk sample. 2 add 0.1 c.c. of the milk sample. { 3 add 1.0 c.c. from capsule i. { 4 add 0.1 c.c. from capsule i. { 5 add 1.0 c.c. from capsule ii. { 6 add 0.1 c.c. from capsule ii. { 7 add 0.5 c.c. from capsule iii. { 8 add 0.3 c.c. from capsule iii. { 9 add 0.2 c.c. from capsule iii. { 10 add 0.5 c.c. from capsule iv. { 11 add 0.3 c.c. from capsule iv. { 12 add 0.2 c.c. from capsule iv. 9. pour plates from the gelatine tubes; label, and incubate at 20° c. 10. liquefy five wort gelatine tubes and to them add 1.0 c.c. of the milk sample and a similar quantity of the diluted milk from capsules i, ii, and iii and iv respectively. 11. pour plates from the wort gelatine; label, and incubate at 20° c. 12. inoculate the liquefied agar tubes as follows: to tube no. 1 add 0.1 c.c. of the milk sample. 2 add 0.1 c.c. from capsule i. 3 add 0.1 c.c. from capsule ii. 4 add 0.1 c.c. from capsule iii. 5 add 1.0 c.c. from capsule iv. } 6 add 0.1 c.c. from capsule iv. } 13. pour plates from the agar tubes; label, and incubate at 37° c. 14. after twenty-four hours' incubation "inspect," and after forty-eight hours' incubation, "count" the agar plates and estimate the number of "organisms growing at 37° c." present per cubic centimetre of the sample of milk. 15. after three, four, or five days' incubation, "count" the gelatine plates and estimate therefrom the number of "organisms growing at 20° c." present per cubic centimetre of the sample of milk. 16. after a similar interval "count" the wort gelatine plates and estimate the number of moulds and yeasts present per cubic centimetre of the sample of milk. note.--many observers prefer to employ gelatine agar (see page 193) for the quantitative examination. in this case gelatine-agar plates should be poured from tubes containing the quantities of material indicated in step 8, incubated at 28° c. to 30° c. and after five days the "total number of organisms developing at 28° c." recorded. ~qualitative.~--the qualitative bacteriological examination of milk is chiefly directed to the detection of the presence of one or more of the following pathogenic bacteria and when present to the estimation of their numerical frequency. members of the coli-typhoid group. vibrio choleræ. streptococcus pyogenes longus. micrococcus melitensis. staphylococcus pyogenes aureus. bacillus enteritidis sporogenes. bacillus diphtheriæ. bacillus tuberculosis. some of these occur as accidental contaminations, either from the water supply to the cow farm, or from the farm employees, whilst others are derived directly from the cow. in milk, as in water examinations, two methods are available, viz.: enrichment and concentration--the former is used for the demonstration of bacteria of intestinal origin, the latter for the isolation of the micro-organisms of diphtheria and tubercle. the first essential in the latter process is the concentration of the bacterial contents of a large volume of the sample into a small compass; but in the case of milk, thorough centrifugalisation is substituted for filtration. _apparatus required_: a large centrifugal machine. this machine, to be of real service in the bacteriological examination of milk, must conform to the following requirements: 1. the centrifugal machine must be of such size, and should carry tubes or bottles of such capacity, as to enable from 200 to 500 c.c. of milk to be manipulated at one time. 2. the rate of centrifugalisation should be from 2500 to 3000 revolutions per minute. 3. the portion of the machine destined to carry the tubes should be a metal disc, of sufficient weight to ensure good "flank" movement, continuing over a considerable period of time. in other words, the machine should run down very gradually and slowly after the motive power is removed, thus obviating any disturbance of the relative positions of particulate matter in the solution that is being centrifugalised. 4. the machine should preferably be driven by electricity, or by power, but in the case of hand-driven machines- (a) the gearing should be so arranged that the requisite speed is obtained by not more than forty or fifty revolutions of the crank handle per minute, so that it may be maintained for periods of twenty or thirty minutes without undue exertion. (b) the handle employed should be provided with a special fastening (e. g., a clutch similar to that employed for the free wheel of a bicycle), or should be readily detachable so that, on ceasing to turn, the handle should not, by its weight and air resistance, act as a brake and stop the machine too suddenly. one of the few satisfactory machines of this class is shown in figure 212. [illustration: fig. 212.--electrically driven centrifugal machine, with flexible (broken) spindle encircled by the field magnets of the motor.] sterile centrifugal tubes, of some 60-70 c.c. capacity, tapering to a point at the closed end, plugged with cotton-wool. small centrifugal machine to run two tubes of 10 c.c. capacity at 2500 to 3000 revolutions per minute preferably driven by electricity, of the type figured on page 327 (fig. 162). sterile centrifugal tubes of 10 c.c. capacity with the distal extremity contracted to a narrow tube and graduated in hundredths of a cubic centimetre (fig. 213). sterilised cork borer. case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre). case of sterile pipettes, 1 c.c. (in tenths of a cubic centimetre). sterile teat pipettes. flask of sterile normal saline solution. method.-1. fill 50 c.c. of the milk sample into each of four tubes, and replace the cotton-wool plugs by solid rubber stoppers (sterilised by boiling), and fit the tubes in the centrifugal machine. note.--one or two cubic centimetres of paraffinum liquidum introduced into the buckets of the centrifuge before the glass tubes are inserted will obviate any risk of breakage to the latter. [illustration: fig. 213.--milk sedimenting tubes.] [illustration: fig. 214.--milk in centrifuge tube.] 2. centrifugalise the milk sample for thirty minutes at a speed of 2500 revolutions per minute. 3. remove the motive power and allow the machine to slow down gradually. 4. remove the tubes of milk from the centrifuge. each tube will now show (fig. 214): (a) a superficial layer of cream (varying in thickness with different samples) condensed into a semi-solid mass, which can be shown to contain some organisms and a few leucocytes. (b) a central layer of separated milk, thin, watery, and opalescent, and containing extremely few bacteria. (c) a sediment or deposit consisting of the great majority of the contained bacteria and leucocytes, together with adventitious matter, such as dirt, hair, epithelial cells, fæcal débris, etc. 5. withdraw the rubber stopper and remove a central plug of cream from each tube by means of a sterile cork borer; place these masses of cream in two sterile capsules. label c^{1} and c^{2}. 6. remove all but the last one or two c.c. of separated milk from each tube, by means of sterile pipettes. 7. mix the deposits thoroughly with the residual milk, pipette the mixture from each pair of tubes into one sterile 10 c.c. tube (graduated) by means of sterile teat pipettes, then fill to the 10 c.c. mark with sterile normal saline solution and mix together. label d^{1} and d^{2}. 8. place the two tubes of mixed deposit in the centrifuge, adjust by the addition or subtraction of saline solution so that they counterpoise exactly, and centrifugalise for ten minutes. note.--each tube now contains the deposit from 100 c.c. of the milk sample and the amount can be read off in hundredths of a centimetre. the multiplication of this figure by 100 will give the amount of "apparent filth," in "parts per million"--the usual method of recording this quality of milk. 9. pipette off all the supernatant fluid and invert the tube to drain on to a pad of sterilised cotton-wool, contained in a beaker. (this wool is subsequently cremated.) 10. examine both cream (c^{1}) and deposit (d^{1}) microscopically-(a) in hanging-drop preparations. (b) in film preparations stained carbolic methylene-blue, by gram's method, by neisser's method, and by ziehl-neelsen's method. note the presence or absence of altered and unaltered vegetable fibres; pus cells, blood discs; cocci in groups or chains, diphtheroid bacilli, gram negative bacilli or cocci, spores and acid fast bacteria. 11. adapt the final stages of the investigation to the special requirements of each individual sample, thus: ~1. members of the coli-typhoid group.~-1. emulsify the deposit from the second centrifugal tube (d^{2}) with 10 c.c. sterile bouillon and inoculate three tubes of bile salt broth as follows: to tube no. 1 add 2.5 c.c. milk deposit emulsion (=25 c.c. original milk.) to tube no. 2 add 1.0 c.c. milk deposit emulsion (=10 c.c. original milk.) to tube no. 3 add 0.5 c.c. milk deposit emulsion (= 5 c.c. original milk.) 2. inoculate tube of bile salt broth no. 4 with 1 c.c. of the original milk. 3. inoculate further tubes of bile salt broth with previously prepared dilutions (see page 445) as follows: to tube no. 5 add 1.0 c.c. from capsule i. to tube no. 6 add 0.1 c.c. from capsule i. to tube no. 7 add 1.0 c.c. from capsule ii. to tube no. 8 add 0.1 c.c. from capsule ii. to tube no. 9 add 1.0 c.c. from capsule iii. to tube no. 10 add 0.1 c.c. from capsule iii. to tube no. 11 add 1.0 c.c. from capsule iv. to tube no. 12 add 0.1 c.c. from capsule iv. and incubate anaerobically (in buchner's tubes) at 42° c. for a maximum period of forty-eight hours. 4. if growth occurs complete the investigation as detailed under the corresponding section of water examination (see pages 428 to 431). note.--the b. coli communis, derived from the alvine discharges of the cow, is almost universally present in large or small numbers, in retail milk. its detection, therefore, unless in enormous numbers, (when it indicates want of cleanliness), is of little value. ~2. vibrio choleræ.~--inoculate tubes of peptone water by using the same amounts as in the search for members of the coli-typhoid groups (_vide ante_ 1-3); incubate aerobically at 37° c. and complete the examination as detailed under the corresponding section of water examination (see page 439). ~3. b. enteritidis sporogenes.~--inoculate tubes of litmus milk with similar amounts to those used in the previous searches, omitting tube no. 1 (_vide ante_ 1-3) place in the differential steriliser at 80° c. for ten minutes and then incubate anaerobically at 37° c. for a maximum period of forty-eight hours. complete the investigation as detailed under the corresponding section of water examination (see page 438). ~4. b. diphtheriæ.~-(a) 1. plant three sets of serial cultivations, twelve tubes in each set, from (a) cream c^{2}, (b) deposit d^{1} upon oblique inspissated blood-serum, and incubate at 37° c. 2. pick off any suspicious colonies which may have made their appearance twelve hours after incubation, examine microscopically and subcultivate upon blood-serum and place in the incubator; return the original tubes to the incubator. 3. repeat this after eighteen hours' incubation. 4. from the resulting growths make cover-slip preparations and stain carbolic methylene-blue, neisser's method, gram's method. subcultivate such as appear to be composed of diphtheria bacilli in glucose peptone solution. note those in which acid production takes place. 5. inoculate guinea-pigs subcutaneously with one or two cubic centimetres forty-eight-hour-old glucose bouillon cultivation derived from the first subcultivation of each glucose fermenter, and observe the result. 6. if death, apparently from diphtheritic toxæmia, ensues, inoculate two more guinea pigs with a similar quantity of the lethal culture. reserve one animal as a control and into the other inject 1000 units of antidiphtheritic serum. if the control dies and the treated animal survives, the proof of the identity of the organism isolated with the klebs-loeffler bacillus becomes absolute. 7. inoculate guinea-pigs subcutaneously with filtered glucose bouillon cultivations (toxins?) and observe the result. (b) 1. emulsify the remainder of the deposit with 5 c.c. sterile bouillon and inoculate two guinea-pigs, thus: guinea-pig a, subcutaneously with 1 c.c. emulsion; guinea-pig b, subcutaneously with 2 c.c. emulsion; and observe the result. 2. if either or both of the inoculated animals succumb, make complete post-mortem examination and endeavour to isolate the pathogenic organisms from the local lesion. confirm their identity as in a5 and 6 (_vide supra_). ~5. bacillus tuberculosis.~-(a) 1. inoculate each of three guinea-pigs (previously tested with tuberculin, to prove their freedom from spontaneous tuberculosis) subcutaneously at the inner aspect of the bend of the left knee, with 1 c.c. of the deposit emulsion remaining in one or other tube (d^{1} or d^{2}). 2. introduce a small quantity of the cream into a subcutaneous pocket prepared at the inner aspect of the bend of the right knee of each of these three animals. place a sealed dressing on the wound. 3. observe carefully, and weigh accurately each day. 4. kill one guinea-pig at the end of the second week and make a complete post-mortem examination. 5. if the result of the examination is negative or inconclusive, kill a second guinea-pig at the end of the third week and examine carefully. [illustration: fig. 215.--cadaver of guinea-pig experimentally infected with b. tuberculosis.] 6. if still negative or inconclusive, kill the third guinea-pig at the end of the _sixth_ week. make a careful post-mortem examination. examine material from any caseous glands microscopically and inoculate freely on to dorset's egg medium. note.--every post-mortem examination of animals infected with tuberculous material should include the naked eye and microscopical examination of the popliteal, superficial and deep inguinal, iliac, lumbar and axillary glands on each side of the body, also the retrohepatic, bronchial and sternal glands, the spleen, liver and lungs (fig. 215). (b) 1. intimately mix all the available cream and deposit from the milk sample, and transfer to a sterile erlenmeyer flask. 2. treat the mixture by the antiformin method (_vide_ appendix, page 502). 3. inoculate each of two guinea-pigs, intraperitoneally, with half of the emulsion thus obtained. 4. kill one of the guinea-pigs at the end of the first week and examine carefully. 5. kill the second guinea-pig at the end of the second week and examine carefully. 6. utilise the remainder of the deposit for microscopical examination and cultivations upon dorset's egg medium. note.--no value whatever attaches to the result of a microscopical examination for the presence of the b. tuberculosis unless confirmed by the result of inoculation experiments. ~6. streptococcus pyogenes longus.~-(a) 1. spread serial surface plates upon nutrose agar. also plant serial cultivations upon sloped nutrient agar (six tubes in series). 2. if the resulting growth shows colonies which resemble those of the streptococcus, make subcultivations upon agar and in bouillon, in the first instance, and study carefully. (b) 1. plant a large loopful of the deposit d^{2} into each of three tubes of glucose formate bouillon, and incubate anaerobically (in buchner's tubes) for twenty-four hours at 37° c. 2. if the resulting growth resembles that of the streptococcus, make subcultivations upon nutrient agar. 3. prepare subcultivations of any suspicious colonies that appear, upon all the ordinary media, and study carefully. if the streptococcus is successfully isolated, inoculate serum bouillon cultivations into the mouse, guinea-pig, and rabbit, to determine its pathogenicity and virulence. ~7. staphylococcus pyogenes aureus.~-1. examine carefully the growth upon the serial blood serum cultivations prepared to isolate b. diphtheriæ and the serial agar cultivations to isolate streptococci after forty-eight hours' incubation. 2. pick off any suspicious orange coloured colonies, plant on sloped agar, and incubate at 20° c. observe pigment formation. 3. prepare subcultivations from any suspicious growths upon all the ordinary media, study carefully and investigate their pathogenicity. ~8. micrococcus melitensis.~--the milk from an animal infected with m. melitensis usually contains the organisms in large numbers and but few other bacteria. 1. spread several sets of surface plates upon nutrose agar, each from one loopful of the deposit in tube d^{1} or d^{2}. 2. spread several sets of surface plates upon nutrose agar, each from one drop of the original milk sample. 3. incubate aerobically at 37° c. and examine daily up to the end of ten days. 4. pick off suspicious colonies, examine them microscopically and subcultivate upon nutrose agar in tubes; upon glucose agar and in litmus milk. 5. test the subsequent growth against the serum of an experimental animal inoculated against m. melitensis to determine its agglutinability. 6. if apparently m. melitensis, inoculate growth from a nutrose agar culture after three days incubation intracranially into the guinea-pig. ice cream. ~collection of the sample.~-1. remove the sample from the drum in the ladle or spoon with which the vendor retails the ice cream, and place it at once in a sterile copper capsule, similar to that employed for earth samples (_vide_ page 471). 2. pack for transmission in the ice-box. 3. on arrival at the laboratory place the copper capsules containing the ice cream in the incubator at 20° c. for fifteen minutes--that is, until at least some of the ice cream has become liquid. ~qualitative and quantitative examination.~--treat the fluid ice cream as milk and conduct the examination in precisely the same manner as described for milk (_vide_ page 443). examination of cream and butter. ~collection of the sample.~--collect, store, and transmit samples to the laboratory, precisely as is done in the case of ice cream. ~quantitative.~-_apparatus required_: sterile test-tube. sterilised spatula. water-bath regulated at 42° c. case of sterile plates. case of sterile graduated pipettes, 1 c.c. (in hundredths). tubes of gelatine-agar (+10 reaction). plate-levelling stand, with its water chamber filled with water at 42° c. method.-1. transfer a few grammes of the sample to a sterile test-tube by means of the sterilised spatula. 2. place the tube in the water-bath at 42° c. until the contents are liquid. 3. liquefy eight tubes of gelatine-agar and place them in the water-bath at 42° c, and cool down to that temperature. 4. inoculate the gelatine-agar tubes with the following quantities of the sample by the help of a sterile pipette graduated to hundredths of a cubic centimetre--viz., to tube no. 1 add 1 c.c. liquefied butter. 2 add 0.5 c.c. liquefied butter. 3 add 0.3 c.c. liquefied butter. 4 add 0.2 c.c. liquefied butter. 5 add 0.1 c.c. liquefied butter. 6 add 0.05 c.c. liquefied butter. 7 add 0.03 c.c. liquefied butter. 8 add 0.02 c.c. liquefied butter. 9 add 0.01 c.c. liquefied butter. 5. pour a plate cultivation from each of the gelatine-agar tubes and incubate at 28° c. 6. "count" the plates after three days' incubation, and from the figures thus obtained estimate the number of organisms present per cubic centimetre of the sample. ~qualitative.~-_apparatus required_: sterile beaker, its mouth plugged with sterile cotton-wool. counterpoise for beaker. scales and weights. sterilised spatula. water-bath regulated at 42° c. separatory funnel, 250 c.c. capacity, its delivery tube protected against contamination by passing it through a cotton-wool plug into the interior of a small erlenmeyer flask which serves to support the funnel. this piece of apparatus is sterilised _en masse_ in the hot-air oven. large centrifugal machine. sterile tubes (for the centrifuge) closed with solid rubber stoppers. case of sterile pipettes, 10 c.c. case of sterile graduated pipettes, 1 c.c. (in tenths of a cubic centimetre). method.-1. weigh out 100 grammes of the sample in a sterile beaker. 2. plug the mouth of the beaker with sterile cotton-wool and immerse the beaker in a water-bath at 42° c. until the contents are completely liquefied. 3. fill the liquefied butter into the sterile separatory funnel. 4. transfer the funnel to the incubator at 37° c. and allow it to remain there for four days. at the end of this time the contents of the funnel will have separated into two distinct strata. (a) a superficial oily layer, practically free from bacteria. (b) a deep watery layer, turbid and cloudy from the growth of bacteria. 5. draw off the subnatant turbid layer into sterile centrifugal tubes, previously warned to about 42° c., and centrifugalise at once. 6. pipette off the supernatant fluid and fill the tubes with sterile 1 per cent. sodium carbonate solution previously warmed slightly; stopper the tubes and shake vigourously for a few minutes. 7. centrifugalise again. 8. pipette off the supernatant fluid; filling the tubes with warm sterile bouillon, shake well, and again centrifugalise, to wash the deposit. 9. pipette off the supernatant fluid. 10. prepare cover-slip preparations, fix and clear as for milk preparations, stain carbolic methylene-blue, gram's method, ziehl-neelsen's method, and examine microscopically with a 1/12 inch oil-immersion lens. 11. proceed with the examination of the deposit as in the case of milk deposit (see pages 450 _et seq._). examination of unsound meats. (including tinned or potted meats, fish, etc.) the bacterioscopic examination of unsound food is chiefly directed to the detection of those members of the coli-typhoid group--b. enteritidis of gaertner and its allies--which are usually associated with epidemic outbreaks of food poisoning, and such anaerobic bacteria as initiate putrefactive changes in the food which result in the formation of poisonous ptomaines, consequently the quantitative examination pure and simple is frequently omitted. a. cultural examination. quantitative.-_apparatus required_: sterilised tin opener, (if necessary.) erlenmeyer flask (500 c.c. capacity) containing 200 c.c. sterile bouillon and fitted with solid rubber stopper. counterpoise. scissors and forceps. scales and weights. water steriliser. hypodermic syringe. syringe with intragastric tube. rat forceps. case of sterile capsules. filtering apparatus as for water analysis. case of sterile plates. case of sterile graduated pipettes, 10 c.c. (in tenths of a cubic centimetre). case of sterile graduated pipettes, 1 c.c. (in tenths of a cubic centimetre). plate-levelling stand. tubes of nutrient gelatine. tubes of nutrient agar. water-bath regulated at 42° c. bulloch's apparatus. method.-1. place the flask containing 200 c.c. sterile broth on one pan of the scales and counterpoise accurately. 2. mince a portion of the sample by the aid of sterile scissors and forceps, and add the minced sample to the bouillon in the flask to the extent of 20 grammes. 3. make an extract by standing the flask in the incubator running at 42° c. (or in a water-bath regulated to that temperature) for half an hour, shaking its contents from time to time. better results are obtained if an electrical shaker is fitted inside the incubator and the flask kept in motion throughout the entire thirty minutes. now every centimetre contains the bacteria washed out from 0.1 gramme of the original food. 4. inoculate tubes of liquefied gelatine as follows: to tube no. 1 add 1.0 c.c. of the extract. 2 add 0.5 c.c. of the extract. 3 add 0.3 c.c. of the extract. 4 add 0.2 c.c. of the extract. 5 add 0.1 c.c. of the extract. pour plates from these tubes and incubate at 20° c. 5. prepare a precisely similar set of agar plates and incubate at 37° c. 6. pipette 5 c.c. of the extract into a sterile tube, heat in the differential steriliser at 80° c. for ten minutes. 7. from the heated extract prepare duplicate sets of agar and gelatine plates and incubate anaerobically in bulloch's apparatus at 37° c. and 20° c. respectively. 8. after three days' incubation examine the agar plates both aerobic and anaerobic and enumerate the colonies developed from spores (7), and from vegetative forms and spores (5), and calculate and record the numbers of each group per gramme of the original food. 9. after seven days' incubation (or earlier if compelled by the growth of liquefying colonies) enumerate the gelatine plates in the same way. 10. subcultivate from the colonies that make their appearance and identify the various organisms. 11. continue the investigations with reference to the detection of pathogenic organisms as described under water (page 429 _et seq._). qualitative.-i. _cultural._ the micro-organisms sought for during the examination of unsound foods comprise the following: members of the coli-typhoid groups (chiefly those of the gaertner class). b. anthracis. streptococci anaerobic bacteria: b. enteritidis sporogenes. b. botulinus. b. cadaveris. the methods by which these organisms if present may be identified and isolated have already been described under the corresponding section of water examination with the exception of those applicable to b. botulinus, and b. cadaveris. these can only be isolated satisfactorily from the bodies of experimentally inoculated animals. ii _experimental._ _tissue._-1. feed rats and mice on portions of the sample and observe the result. 2. if any of the animals die, make complete post-mortem examinations and endeavour to isolate the pathogenic organisms. _extract._-1. introduce various quantities of the bouillon extract into the stomachs of several rats, mice and guinea-pigs repeatedly over a period of two or three days by the intragastric method of inoculation (see page 367) and observe the result. guinea-pigs and mice are very susceptible to infection by b. botulinus by this method; rabbits less so. 2. inoculate rats, mice, and guinea-pigs subcutaneously into deep pockets, and intraperitoneally with various quantities of the bouillon extract, and observe the result. 3. filter some of the extract through a chamberland candle and incubate the filtrate to determine the presence of soluble toxins. 4. if any of the animals succumb to either of these methods of inoculation, make careful post-mortem examinations and endeavour to isolate the pathogenic organisms. the examination of oysters and other shellfish. on opening the shell of an oyster a certain amount of fluid termed "liquor" is found to be present. this varies in amount from a drop to many cubic centimetres (0.1 c.c. to 10 c.c.)--in the latter case the bulk of the fluid is probably the last quantum of water ingested by the bivalve before closing its shell. in order to obtain a working average of the bacteriological flora of a sample, ten oysters should be taken and the body, gastric juice and liquor should be thoroughly mixed before examination. the examination, as in dealing with other food stuffs, is directed to the search for members of the coli-typhoid group, sewage streptococci and perhaps also b. enteritidis sporogenes. _apparatus required_: two hard nail brushes. liquid soap. sterile water in aspirator jar with delivery nozzle controlled by a spring clip. sterile oyster knives. sterile glass dish, with cover, sufficiently large to accommodate ten oysters. sterile forceps. sterile scissors. sterile towels or large gauze pads. sterile graduated cylinders 1000 c.c. capacity, with either the lid or the bottom of a sterile petri dish inverted over the open mouth as a cover. glass rods. corrosive sublimate solution, 1 per mille. bile salt broth tubes. litmus milk tubes. surface plates of nutrose agar. case of sterile pipettes, 1 c.c. (in tenths of a c.c.) case of sterile pipettes, 10 c.c. (in tenths of a c.c.) case of sterile glass capsules. erlenmeyer flasks, 250 c.c. capacity. double strength bile salt broth. method.-1. thoroughly clean the outside of the oyster shells by scrubbing each in turn with liquid soap and nail brush under a tap of running water. then, holding an oyster shell in a pair of sterile forceps wash every part of the outside of the shell with a stream of sterile water running from an aspirator jar; deposit the oyster inside the sterile glass dish. repeat the process with each of the remaining oysters. 2. before proceeding further, cleanse the hands thoroughly with clean nail brush, soap and water, then plunge them in lysol 2 per cent. solution, and finally in sterile water. 3. spread a sterile towel on the bench. 4. remove one of the oysters from the sterile glass dish and place it, resting on its convex shell, on the towel. turn a corner of the sterile towel over the upper flat shell to give a firmer grip to the left hand, which holds the shell in position. 5. with the sterile oyster knife (in the right hand) open the shell and separate the body of the oyster from the inner surface of the upper flat shell. bend back and separate the flat shell, leaving the body of the oyster in and attached to the concave shell. avoid spilling any of the liquor. (some dexterity in opening oysters should be acquired before undertaking these experiments). 6. cut up the body of the oyster with sterile scissors into small pieces and allow the liquor freed from the body during the process to mix with the liquor previously in the shell. 7. transfer the comminuted oyster and the liquor to the cylinder. 8. treat each of the remaining oysters in similar fashion. 9. mix the contents of the cylinder thoroughly by stirring with a sterile glass rod. the total volume will amount to about 100 c.c. 10. use 0.1 c.c. of the mixed liquor to inseminate each of a series of three nutrose surface plates. 11. inoculate 0.1 c.c. of the mixed liquor into each of three tubes of litmus milk. 12. add sterile distilled water to the contents of the cylinder up to 1000 c.c. and stir thoroughly with a sterile glass rod and allow to settle. the bacterial content of each oyster may be regarded, for all practical purposes, as comprised in 100 c.c. of fluid. 13. arrange four glass capsules in a row and number i, ii, iii, iv. pipette 9 c.c. sterile distilled water into each. 14. to capsule no. i add 1 c.c. of the diluted liquor, etc. from the cylinder, and mix thoroughly. to capsule ii add 1 c.c. of dilution in capsule i and mix thoroughly. carry over 1 c.c. of fluid from capsule ii to capsule iii, afterwards adding 1 c.c. of fluid from capsule iii to capsule iv. 15. label tubes of bile salt broth and inoculate with the following amounts of diluted oysters: no. 6 with 10 c.c. cylinder fluid = 0.1 oyster. no. 5 with 1 c.c. cylinder fluid = 0.01 oyster. no. 4 with 1 c.c. capsule i fluid = 0.001 oyster. no. 3 with 1 c.c. capsule ii fluid = 0.0001 oyster. no. 2 with 1 c.c. capsule iii fluid = 0.00001 oyster. no. 1 with 1 c.c. capsule iv fluid = 0.000001 oyster. 16. transfer 100 c.c. cylinder fluid (= 1 oyster) to an erlenmeyer flask and add 50 c.c. double strength bile salt broth, and label 7. 17. duplicate all the above indicated cultures. 18. put up the tube cultures in buchner's tubes and incubate anaerobically at 42° c. if growth occurs in tube 1 the organism finally isolated, e. g., b. coli, must have been present to the extent of one million per oyster. 19. complete the examination for members of the coli-typhoid group and sewage streptococci, as directed under water examination, page 429 (steps 11-21). 20. inoculate a series of 6 tubes of litmus milk with quantities of the material similar to those indicated in step 15; heat to 80° c. for ten minutes, and incubate under anaerobic conditions at 37° c. examine for the presence of b. enteritidis sporogenes as directed under water examination, page 438 (steps 7-10). examination of sewage and sewage effluents. quantitative.-_collection of the sample._--as only small quantities of material are needed, the samples should be collected in a manner similar to that described under water for quantitative examination and transmitted in the ice apparatus used in packing those samples. _apparatus required._--as for water (_vide_ page 420). method.-1. arrange four sterile capsules in a row and number them i, ii, iii, iv. 2. pipette 9 c.c. sterile bouillon into capsule no. i. 3. pipette 9.9 c.c. sterile bouillon into capsules ii, iii, and iv. 4. add 1 c.c. of the sewage to capsule no. i by means of a sterile pipette, and mix thoroughly. 5. take a fresh sterile pipette and transfer 0.1 c.c. of the mixture from no. i to no. ii and mix thoroughly. 6. in like manner transfer 0.1 c.c. from no. ii to no. iii, and then 0.1 c.c. from no. iii to no. iv. now 1 c.c. of dilution no. i contains 0.1 c.c. of the original sewage. 1 c.c. of dilution no. ii contains 0.001 c.c. of the original sewage. 1 c.c. of dilution no. iii contains 0.00001 c.c. of the original sewage. 1 c.c. of dilution no. iv contains 0.0000001 c.c. of the original sewage. 7. pour a set of gelatine plates from the contents of each capsule, three plates in a set, and containing respectively 0.2, 0.3, and 0.5 c.c. of the dilution. label carefully; incubate at 20° c. for three, four, or five days. 8. enumerate the organisms present in those sets of plates which have not liquefied, probably those from dilution iii or iv, and calculate therefrom the number present per cubic centimetre of the original sample of sewage. qualitative.--the qualitative examination of sewage is concerned with the identification and enumeration of the same bacteria dealt with under the corresponding section of water examination; it is consequently conducted on precisely similar lines to those already indicated (_vide_ pages 426 to 441). examination of air. quantitative.-_apparatus required_: aspirator bottle, 10 litres capacity, fitted with a delivery tube, and having its mouth closed by a perforated rubber stopper, through which passes a short length of glass tubing. erlenmeyer flask, 250 c.c. capacity (having a wide mouth properly plugged with wool), containing 50 c.c. sterile water. rubber stopper to fit the mouth of the flask, perforated with two holes, and fitted as follows: take a 9 cm. length of glass tubing and bend up 3 cm. at one end at right angles to the main length of tubing. pass the long arm of the angle through one of the perforations in the stopper; plug the open end of the short arm with cotton-wool. take a glass funnel 5 or 6 cm. in diameter with a stem 12 cm. in length and bend the stem close up to the apex of the funnel, in a gentle curve through a quarter of a circle; pass the long stem through the other perforation in the rubber stopper. a battery jar or a small water-bath to hold the erlenmeyer flask when packed round with ice. supply of broken ice. rubber tubing. screw clamps and spring clips, for tubing. water steriliser. retort stand and clamps. apparatus for plating (as for enumeration of water organisms, _vide_ page 420). method.-1. fill 10 litres of water into the aspirating bottle and attach a piece of rubber tubing with a screw clamp to the delivery tube. open the taps fully and regulate the screw clamp, by actual experiment, so that the tube delivers 1 c.c. of water every second. the screw clamp is not touched again during the experiment. at this rate the aspirator bottle will empty itself in just under three hours. shut off the tap and make up the contents of the aspirator bottle to 10 litres again. 2. sterilise the fitted rubber cork, with its funnel and tubing, by boiling in the water steriliser for ten minutes. 3. remove the cotton-wool plug from the flask, and replace it by the rubber stopper with its fittings. make sure that the end of the stem of the funnel is immersed in the bouillon. 4. place the flask in a glass or metal vessel and pack it round with pounded ice. arrange the flask with its ice casing just above the neck of the aspirator bottle. [illustration: fig. 216.--arrangement of apparatus for air analysis.] 5. connect up the free end of the glass tube from the flask--after removing the cotton-wool plug--with the air-entry tube in the mouth of the aspirating bottle (fig. 216). 6. open the tap fully, and allow the water to run. replenish the ice from time to time if necessary. (in emptying itself the aspirator bottle will aspirate 10 litres of air slowly through the water in the erlenmeyer flask.) 7. when the aspiration is completed, disconnect the flask and remove it from its ice packing. 8. liquefy three tubes of nutrient gelatine and add to them 0.5 c.c., 0.3 c.c., and 0.2 c.c., respectively, of the water from the flask, by means of a sterile graduated pipette, as in the quantitative examination of water. pour plates. 9. pour a second similar set of gelatine plates. 10. incubate both sets of plates at 20° c. 11. enumerate the colonies present in the two sets of gelatine plates after three, four, or five days and average the results from the numbers so obtained; estimate the number of micro-organisms present in 1 c.c., and then in the 50 c.c. of broth in the flask. 12. the result of air examination is usually expressed as the number of bacteria present per cubic metre (i. e., kilolitre) of air; and as the number of organisms present in the 50 c.c. water only represent those contained in 10 litres of air, the resulting figure must be multiplied by 100. qualitative.-1. proceed exactly as in the quantitative examination of air (_vide supra_), steps 1 to 10. 2. pour plates of wort agar with similar quantities of the air-infected water, and incubate at 37° c. 3. pour plates of nutrient agar with similar quantities of the water and incubate at 37° c. 4. pour similar plates of wort gelatine and incubate at 20° c. 5. pick off the individual colonies that appear in the several plates, subcultivate them on the various media, and identify them. examination of soil. the bacteriological examination of soil yields information of value to the sanitarian during the progress of the process of homogenisation of "made soil" (e. g., a dumping area for the refuse of town) and determines the period at which such an area may with propriety and safety be utilised for building purposes; or to the agriculturalist in informing him of the suitability of any given area for the growth of crops. the surface of the ground, exposed as it is to the bactericidal influence of sunlight and to rapid alternations of heat and cold, rain and wind, contains but few micro-organisms. again, owing to the density of the molecules of deep soil and lack of aeration on the one hand, and the filtering action of the upper layers of soil and bacterial antagonism on the other, bacterial life practically ceases at a depth of about 2 metres. the intermediate stratum of soil, situated from 25 to 50 cm. below the surface, invariably yields the most numerous and the most varied bacterial flora. ~collection of sample.~--a small copper capsule 6 cm. high by 6 cm. diameter, with "pull-off" cap secured by a bayonet catch, previously sterilised in the hot-air oven, is the most convenient receptacle for samples of soil. [illustration: fig. 217.--soil scoop.] the instrument used for the actual removal of the soil from its natural position will vary according to whether we require surface samples or soil from varying depths. (a) for ~surface~ samples, use an iron scoop, shaped like a shoe horn, but provided with a sharp spine (fig. 217). this is wrapped in asbestos cloth and sterilised in the hot-air oven. when removed from the oven, wrap a piece of oiled paper, silk, or gutta-percha tissue over the asbestos cloth, and secure it with string, as a further protection against contamination. on reaching the spot whence the samples are to be taken, the coverings of the scoop are removed, and the asbestos cloth employed to brush away loose stones and débris from the selected area. the surface soil is then broken up with the point of the scoop, scraped up and collected in the body of the scoop, and transferred to the sterile capsule for transmission. [illustration: fig. 218.--fraenkel's borer.] (b) for ~deep~ samples collected at various distances from the surface, an experimental trench may be cut to the required depth and samples collected at the required points on the face of the section. it is, however, preferable to utilise some form of borer, such as that designed by fraenkel (fig. 218). _fraenkel's earth borer._--this instrument consists of a stout hard-steel rod, 150 cm. long, marked in centimetres from the drill-pointed extremity. it is provided with a cross handle (adjustable at any point along the length of the rod by means of a screw nut). the terminal centimeters are thicker than the remainder of the rod, and on one side a vertical cavity about 0.5 cm. deep is cut. this is covered by a flanged sleeve so long as the borer is driven into the soil clockwise, and is opened for the reception of the sample of soil, when the required depth is reached, by reversing the screwing motion, and again closed before withdrawal of the borer from the earth by resuming the original direction of twist. it can be sterilised in a manner similar to that adopted for the scoop, or by repeatedly filling the cavity with ether and burning it off. ~quantitative.~--four distinct investigations are included in the complete quantitative bacteriological examination of the soil: 1. the enumeration of the aerobic organisms. 2. the enumeration of the spores of aerobes. 3. the enumeration of the anaerobic organisms (including the facultative anaerobes). 4. the enumeration of the spores of anaerobes. further, by a combination of the results of the first and second, and of the third and fourth of these, the ratio of spores to vegetative forms is obtained. _apparatus required_: case of sterile capsules (25 c.c. capacity). case of sterile graduated pipettes, 10 c.c. (in tenths of a cubic centimetre). case of sterile graduated pipettes, 1 c.c. (in tenths of a cubic centimetre). flask containing 250 c.c. sterile bouillon. tall cylinder containing 2 per cent. lysol solution. plate-levelling stand. 12 sterile plates. tubes of nutrient gelatine. tubes of wort gelatine. tubes of nutrient agar. tubes of glucose formate gelatine. tubes of glucose formate agar. water-bath regulated at 42° c. bunsen burner. grease pencil. sterile mortar and pestle (agate). sterile wide-mouthed erlenmeyer flask (500 c.c. capacity). sterile metal funnel with short wide bore delivery tube to just fit mouth of flask. solid rubber stopper to fit the flask (sterilised by boiling). pair of scales. counterpoise (fig. 107). sterile metal (nickel) spoon or spatula. fractional steriliser (fig. 140). method.-1. arrange four sterile capsules numbered i, ii, iii, and iv; pipette 9 c.c. sterile bouillon into the first capsule, and 9.9 c.c. into each of the remaining three. 2. pipette 100 c.c. sterile bouillon into the erlenmeyer flask. 3. remove the cotton-wool plug from the flask and replace it by the sterile funnel. 4. place flask and funnel on one pan of the scales, and counterpoise accurately. 5. empty the sample of soil into the mortar and triturate thoroughly. 6. by means of the sterile spatula add 10 grammes of the earth sample to the bouillon in the flask. the final results will be more reliable if steps 2, 3, 4, and 5 are performed under a hood--to protect from falling dust, etc. 7. remove the funnel from the mouth of the flask; replace it by the rubber stopper and shake vigourously; then allow the solid particles to settle for about thirty minutes. one cubic centimetre of the turbid broth contains the washings from 0.1 gramme of soil. 8. pipette off 1 c.c. of the supernatant bouillon, termed the "soil water," and add it to the contents of capsule i; mix thoroughly. 9. remove 0.1 c.c. of the infected bouillon from capsule i and add it to capsule ii, and mix. 10. in like manner add 0.1 c.c. of the contents of capsule ii to capsule iii, and then 0.1 c.c. of the contents of capsule iii to capsule iv. then 1 c.c. fluid from capsule i contains soil water from .01 gm. earth. then 1 c.c. fluid from capsule ii contains soil water from .0001 gm. earth. then 1 c.c. fluid from capsule iii contains soil water from .000001 gm. earth. then 1 c.c. fluid from capsule iv contains soil water from .00000001 gm. earth. (a) _aerobes (vegetative forms and spores)._-11. pour a set of gelatine plates from the contents of each capsule--two plates in a set, and containing respectively 0.1 c.c. and 0.4 c.c. of the diluted soil water. label and incubate. 12. pour similar sets of wort gelatine plates from the contents of capsules ii and iii, label, and incubate at 20° c. 13. pour similar sets of agar plates from the contents of capsules ii and iii; label and incubate at 37° c. 14. weigh out a second sample of soil--10 grammes--dry over a water-bath until of constant weight and calculate the ratio wet soil weight -------------- dry soil weight 15. "count" the plates after incubation for three, four, or five days, and correcting the figures thus obtained by means of the "wet" to "dry" soil ratio estimate-(a) the number of aerobic micro-organisms present per gramme of the soil. (b) the number of yeasts and moulds present per gramme of the soil. (c) the number of aerobic organisms "growing at 37° c." present per gramme of the soil. (b) _anaerobes (vegetative forms and spores)._-16. pour similar sets of plates in glucose formate gelatine and agar and incubate in bulloch's anaerobic apparatus. (c) _aerobes and anaerobes (spores only)._-17. pipette 5 c.c. soil water into a sterile tube. 18. place in the differential steriliser at 80° c. for ten minutes. 19. pour two sets of four gelatine plates containing 0.1, 0.2, 0.5, and 1 c.c. respectively of the soil water; label and incubate at 20° c., one set aerobically, the other anaerobically in bulloch's apparatus. 20. "count" the plates (delay the enumeration as long as possible) and estimate the number of spores of aerobes and anaerobes respectively present per gramme of the soil. 21. calculate the ratio existing between spores and spores + vegetative forms under each of the two groups, aerobic and anaerobic micro-organisms. ~qualitative examination.~--the qualitative examination of soil is usually directed to the detection of one or more of the following: members of the coli-typhoid group. streptococci. bacillus anthracis. bacillus tetani. bacillus oedematis maligni. the nitrous organisms. the nitric organisms. 1. transfer the remainder of the soil water (88 c.c.) to a sterile erlenmeyer flask by means of a sterile syphon. 2. fix up the filtering apparatus as for the qualitative examination of water, and filter the soil water. 3. suspend the bacterial residue in 5 c.c. sterile bouillon (technique similar to that described for the water sample, _vide_ pages 434-436). every cubic centimetre of suspension now contains the soil water from nearly 1 gramme of earth. the methods up to this point are identical no matter which organism or group of organisms it is desired to isolate; but from this stage onward the process is varied slightly for each particular bacterium. ~i. the coli-typhoid group.~-~ii. streptococci.~-~iii. bacillus anthracis.~-~iv. bacillus tetani.~-the methods adopted for the isolation of these organisms are identical with those already described under water (page 437 _et seq._). ~v. bacillus oedematis maligni.~--method precisely similar to that employed for the b. tetani. ~vi. the nitrous organisms.~-1. take ten tubes of winogradsky's solution no i (_vide_ page 198) and number them consecutively from 1 to 10. 2. inoculate each tube with varying quantities of the material as follows: to tube no. 1 add 1.0 c.c. of the soil water. to tube no. 2 add 0.1 c.c. of the soil water. to tube no. 3 add 1.0 c.c. from capsule i. to tube no. 4 add 0.1 c.c. from capsule i. to tube no. 5 add 1.0 c.c. from capsule ii. to tube no. 6 add 0.1 c.c. from capsule ii. to tube no. 7 add 1.0 c.c. from capsule iii. to tube no. 8 add 0.1 c.c. from capsule iii. to tube no. 9 add 1.0 c.c. from capsule iv. to tube no. 10 add 0.1 c.c. from capsule iv. label and incubate at 30° c. ~vii. the nitric organisms.~-3. take ten tubes of winogradsky's solution no ii, number them consecutively from 1 to 10 and inoculate with quantities of soil water similar to those enumerated in section vi step 2. label and incubate at 30° c. 4. examine after twenty-four and forty-eight hours' incubation. from those tubes that show signs of growth make subcultivations in fresh tubes of the same medium and incubate at 30° c. 5. make further subcultivations from such of those tubes as show growth, and again incubate. 6. if growth occurs in these subcultures, make surface smears on plates of winogradsky's silicate jelly (_vide_ page 198). 7. pick off such colonies as make their appearance and subcultivate in each of these two media. testing filters. porcelain filter candles are examined with reference to their power of holding back _all_ the micro-organisms suspended in the fluids which are filtered through them, and permitting only the passage of germ-free filtrates. in order to determine the freedom of the filter from flaws and cracks which would permit the passage of bacteria no matter how perfect the general structure of the candle might be, the candle must first be attached by means of a long piece of pressure tubing, to a powerful pump, such as a foot bicycle pump, fitted with a manometer. the candle is then immersed in a jar of water and held completely submerged whilst the internal pressure is gradually raised to two atmospheres by the action of the pump. any crack or flaw will at once become obvious by reason of the stream of air bubbles issuing from it. the examination for permeability is conducted as follows: _apparatus required_: filtering apparatus: the actual filter candle that is used must be the one it is intended to test and must be previously carefully sterilised; the arrangement of the apparatus will naturally vary with each different form of filter, one or other of those already described (_vide_ pages 42-48). plate-levelling stand. case of sterile plates. case of sterile pipettes, 10 c.c. (in tenths). case of sterile pipettes, 1 c.c. (in tenths). tubes of nutrient gelatine. flask containing sterile normal saline solution. sterile measuring flask, 1000 c.c. capacity. method.-1. prepare surface cultivations, on nutrient agar in a culture bottle, of the bacillus mycoides, and incubate at 20° c., for forty-eight hours. 2. pipette 5 c.c. sterile normal saline into the culture bottle and emulsify the entire surface growth in it. 3. pipette the emulsion into the sterile measuring flask and dilute up to 1000 c.c. by the addition of sterile water. 4. pour the emulsion into the filter reservoir and start the filtration. 5. when the filtration is completed, pour six agar plates each containing 1 c.c. of the filtrate. 6. incubate at 37° c. until, if necessary, the completion of seven days. 7. if the filtrate is not sterile, subcultivate the organism passed and determine its identity with the test bacterium before rejecting the filter--since the filtrate may have been accidentally contaminated. 8. if the filtrate is sterile, resterilise the candle and repeat the test now substituting a cultivation of b. prodigiosus--a bacillus of smaller size. 9. if the second test is satisfactory, test the candle against a cultivation of a very small coccus, e. g., micrococcus melitensis, in a similar manner; in this instance continuing the incubation of cultivations from the filtrate for fourteen days. testing of disinfectants. methods have already been detailed (page 310) for the purpose of studying the vital resistance offered by micro-organisms to the lethal effect of germicides. but it frequently happens that the bacteriologist has to determine the relative efficiency of "disinfectants" from the standpoints of the sanitarian and commercial man rather than from the research worker's point of view. in pursuing this line of investigation, it is convenient to compare the efficiency, under laboratory conditions, of the proposed disinfectant with that of some standard germicide, such as pure phenol. in so doing, and in order that the work of different observers may be compared, conditions as nearly uniform as possible should be aimed at. the method described is one that has been in use by the writer for many years past, modified recently by the adoption of some of the recommendations of the lancet commission on the standardisation of disinfectants--particularly of the calculation for determining the phenol coefficient. this method has many points in common with that modification of the "drop" method known as the rideal-walker test. ~general considerations.~-these may be grouped under three headings: test germ, germicide, and environment. 1. _test germ._--~b. coli.~ as disinfectants are tested for sanitary purposes, it is obvious that a member of the coli-typhoid group should be selected as the test germ. b. coli is selected on account of its relative nonpathogenicity, the ease with which it can be isolated and identified by different observers in various parts of the world, the stability of its fundamental characters, and evenness of its resistance when utilised for these tests; finally since the colon bacillus is an organism which is slightly more resistant to the lethal action of germicides than the more pathogenic members of this group, a margin of safety is introduced into the test which certainly enhances its value. b. coli should be recently isolated from a normal stool, and plated at least twice to ensure the purity of the strain; and a stock agar culture prepared which should be used throughout any particular test. for any particular experiment prepare a smear culture on agar and incubate at 37° c. for 24 hours anaerobically. then emulsify the whole of the surface growth in 10 c.c. of sterile water. transfer the emulsion to a sterile test-tube with some sterile glass beads and shake thoroughly to ensure homogenous emulsion. transfer to a centrifuge tube and centrifugalise the emulsion to throw down any masses of bacteria which may have escaped the disintegrating action of the beads. pipette off the supernatant emulsion for use in the test. _2. germicide._-_a. disinfectant to be tested._-the first essential point is to test the unknown disinfectant, which may be referred to as germicide-x, on the lines set out on page 311 to determine its inhibition coefficient. this constant having been fixed, prepare various solutions of germicide-x with sterilised distilled water by accurate volumetric methods, commencing with a solution somewhat stronger than that representing the inhibition coefficient. the solutions must be prepared in fairly large bulk, not less than 5 c.c. of the disinfectant being utilised for the preparation of any given percentage solution. _b. standard control._--~phenol.~ the standard germicide used for comparison should be one which is not subject to variation in its chemical composition, and the one which has obtained almost universal use is phenol. the following table shows the effect of different percentages of carbolic acid upon b. coli for varying contact times, compiled from an experiment conducted under the standard conditions referred to under environment. the results closely correspond to those recorded by the lancet commission on disinfectants, 1909. ---------------------+---------------------------------- | contact time in minutes. percentage of phenol +------+---+---+---+---+---+---+--- | 2-1/2| 5 |10 | 15| 20| 25| 30| 35 ---------------------+------+---+---+---+---+---+---+---1.20 | | | | | | | | 1.10 | | | | | | | | 1.0 | + | | | | | | | 0.9 | + | | | | | | | 0.85 | + | + | | | | | | 0.80 | + | + | + | | | | | 0.75 | + | + | + | + | + | | | 0.7 | + | + | + | + | + | + | | 0.65 | + | + | + | + | + | + | + | ---------------------+------+---+---+---+---+---+---+---= no growth, i. e., bacteria killed. + = growth, i. e., bacteria still living. from this it will be seen that the following percentage solutions will need to be prepared, namely: 1.1 per cent., 1.0 per cent., 0.9 per cent., 0.75 per cent., 0.7 per cent., as controls for each experiment. prepare solutions of varying percentages by weighing out the quantity of carbolic acid required for each and dissolving in 100 c.c. of pure distilled water in an accurately standardised measuring flask. the solutions must be prepared freshly as required each day. ~environment.~-_a. general._-close the windows and doors of the laboratory in which the investigation is carried out, to avoid draughts. flush over the work bench and adjacent floor with 1:1000 solution of corrosive sublimate. caution the assistant, if one is employed, to avoid unnecessary movement or speech. _b. contact temperature_, ~15-18° c.~-this is the temperature at which contact between the germicide and the test germ takes place, and is of importance, since some germicides (_e. g._, phenol) appear to be more powerful at high temperatures. 18° c.--practically the ordinary room temperature--is a temperature at which the multiplication of b. coli is a comparatively slow process, but variation of a degree above this temperature or of two or three degrees below is of no moment. if the room temperature is below 15° c. when the experiments are in progress, arrange a water-bath regulated at 18° c. for the reception of the tubes containing the mixture of germ and germicide; if above 19° c. immerse the tubes in cold water, to which small pieces of ice are added from time to time to prevent the temperature rising above 18° c. _c. relative proportional bulk of test germ and germicide_, ~50:1.~-five cubic centimetres is a convenient amount of germicidal solution to employ, and to this 0.1 c.c. of the emulsion of test germ should be added. _d. bulk of sample removed from germ + germicide mixture at each of the time periods_, ~0.1 c.c.~-this is sufficient to afford a fair sample of the germ content of the mixture, and at the same time is insufficient to exert any inhibitory action when transferred to the subculture medium. _e. subculture medium._ ~bile salt broth.~-a _fluid_ medium is essential in order to obtain immediate dilution of the germicide carried over; at the same time it is advantageous to employ a selective medium which favours the growth of the test germ to the exclusion of organisms likely to contaminate the preparation, and if possible one which affords characteristic cultural appearances. bile salt broth (page 180) combines these desiderata; it permits only the growth of intestinal bacteria, whilst the formation of an acid reaction and the production of gas in subcultures prepared from the germ-germicide mixture is fairly complete evidence of the presence of living b. coli. the amount of medium present in each test-tube is a matter of importance, since the medium not only provides pabulum for the test germ, but also acts as a diluent to the germicide, to reduce its strength below its inhibition coefficient. for routine work each subculture tube contains 10 c.c. of medium, but it is obvious that if germicide-x possesses an inhibition coefficient of 0.1 per cent. the addition of 0.1 c.c. of a 10 per cent. solution to 10 c.c. of medium would effectually prevent the subsequent growth of the test germ after a contact period insufficient to destroy its vitality. hence the preliminary tests may in some instances indicate the necessity for the presence of 12 c.c., 15 c.c. or more of the fluid medium in the culture tubes. _f. incubation temperature_, ~37° c.~-_g. observation period of the subcultivations_, ~seven days.~-in order to determine whether or no the test germs have been destroyed, observations must always be continued--when growth appears to be absent--up to the end of seven days before recording "no growth." _h. identification of the organisms developing in the subcultivations after contact in the germ + germicide solution._-this is based on the naked eye characters of the growth in the bile salt broth, supplemented where necessary by plating methods, further subcultivations upon carbohydrate media and agglutination experiments. the sign (+) is used to indicate that growth of the test organism occurred in the subcultivations, and the sign (-) to indicate that the test germs have been destroyed and no subsequent growth has taken place. method.- _apparatus required_: sterile test-tubes (narrow, not exceeding 1.3 cm. diameter). test-tube rack (fig. 219). sterile graduated pipettes in case, 1 c.c. (in tenths). sterile graduated pipettes in case, 5 c.c. (in c.c.). circular rubber washers, 2.5 cm. diameter with central hole, sterilised by boiling immediately before use, then transferred to sterilised glass double dish. electric signal clock or stop watch. sterile forceps. sterilised glass beads. shaking machine. grease pencil. _material required_: percentage solutions of germicide-x (_vide_ page 481). percentage solutions of pure phenol (_vide_ page 482). aqueous emulsion of b. coli (_vide_ page 481). tubes of bile salt broth. ~preliminary tests.~-_a. inhibition coefficient._-determine the lowest percentage of germicide-x which inhibits growth of b. coli in the bile salt broth, and the highest percentage which fails to inhibit (page 311). on the result of this experiment determine the bulk of medium required in the subculture tubes and the percentage solutions to be employed in the trial trip. assuming the inhibition coefficient to be 1:1000, it will be quite safe to employ the ordinary culture tubes containing 10 c.c. medium in the subsequent experiments. _b. trial trip._-determine the lethal effect of a series of five solutions of germicide-x (say 1:100, 1:250, 1:300, 1:500, 1:600) at contact times of 2-1/2, 5, 25 and 30 minutes in the following manner: 1. arrange five test-tubes marked a to e in the lower tier of the test-tube rack. 2. into tube a pipette 5 c.c. germicide-x 1:100 solution. into tube b pipette 5 c.c. germicide-x 1:200 solution. into tube c pipette 5 c.c. germicide-x 1:300 solution. into tube d pipette 5 c.c. germicide-x 1:500 solution. into tube e pipette 5 c.c. germicide-x 1:600 solution. 3. arrange 20 tubes of bile salt broth in the upper tier of the test-tube rack in two rows, those in the front row numbered consecutively from left to right 1-10, those in the back row 11-20. 4. place a square wire basket of about 50 tubes capacity close to the left of the test-tube rack, for the reception of the inoculated tubes. 5. take a sterile 1 c.c. pipette from the case, pick up a sterile rubber washer with forceps and push the point of the pipette into the central hole. 6. put down the forceps on the bench with the sterile points projecting over the edge. without taking the tube from the rack remove the cotton-wool plug from tube a, and lower the pipette, with the rubber washer affixed, on to the open mouth of the tube; with the help of the forceps to steady the washer, push the pipette on through the hole until the point of the pipette has reached to within a few millimetres of the bottom of the tube (see fig. 219). 7. adjust in the same way a pipette and a washer in the mouth of each of the other tubes, b, c, d and e. 8. set the electric signal clock to ring for the commencement of the experiment and at subsequent intervals of 2-1/2, 5, 25 and 30 minutes. 9. take up 0.5 c.c. of b. coli emulsion in sterile pipette graduated in tenths of a cubic centimetre and stand by. 10. as soon as the bell rings lift the pipette from tube a with the left hand and from the charged pipette held in the right hand deliver 0.1 c.c. of b. coli emulsion into the 1:100 solution. then replace the pipette and washer. [illustration: fig. 219.--test-tube rack.] 11. raise the tube with the left hand and shake it to mix germ and germicide, whilst returning the delivery pipette in the right hand. 12. repeat the process with tubes b, c, d and e; then drop the infected delivery pipette in the lysol jar. the inoculation of the five tubes can be carried out very expeditiously, but a period of 10 seconds must be allowed for each tube. 13. when the bell rings at 2-1/2 minutes blow through the pipette in tube a (this agitates the germ + germicide mixture and ensures the collection of a fair sample); allow the mixture to enter the pipette, and as the column of fluid extends well above the terminal graduation, the right forefinger adjusted over the butt-end of the pipette before it is lifted will retain more than 0.1 c.c. of the mixture within the bore when the point of the pipette is clear of the fluid in the tube. touch the point of the pipette on the inner wall of the tube, and allow any excess of fluid to escape, only retaining 0.1 c.c. in the pipette. 14. at the same time, with the left hand remove bile salt tube no. 1 from the upper tier of the rack, take out the cotton-wool plug with the hand already holding the pipette (the relative positions of pipette, plug and culture tubes being practically the same as those of platinum loop, plug and culture tube shown in fig. 68, page 74). 15. insert the point of the pipette into the subculture tube, and blow out the mixture into the medium--replug the tube and drop it into the wire basket. replace the washer-pipette in tube a. as soon as the point of the pipette has entered the mouth of tube a it may be released, since it has already been so adjusted that it just clears the bottom of the test-tube, and the elastic washer will prevent any damage to the tube. steps 13, 14 and 15 occupy on an average 10 seconds. 16. repeat steps 13, 14 and 15 with each of the other tubes b, c, d and e. 17. repeat these various steps 13-16 when the bell rings at 5, 25 and 30 minutes. 18. place all the inoculated tubes in the incubator at 37° c. 19. examine the tubes at intervals of 24 hours, and record the results in tabular form as shown in table page 491 (the figures in the squares indicate the number of hours at which the changes in the medium due to the growth of b. coli first appeared). 20. if a consideration of the tabulated results indicates strengths of germicide-x lethal at 2-1/2 and 30 minutes the final test can be arranged, but if this result has not been attained, sufficient evidence will probably be available to enable a second trial test to be planned which will give the required information. ~final test.~-c. _determination of phenol coefficient._-_x-disinfectant._--this comprises two distinct tests, one of the germicide-x, the other of the standard phenol. 1. arrange five test-tubes clearly marked in the lower tier of the rack. 2. pipette into each 5 c.c. respectively of the five percentage solutions of x-disinfectant which the trial run has already shown will include those affording lethal values at 2-1/2 and 30 minutes. 3. arrange 20 tubes of bile salt broth in the upper tier of the test-tube rack in two rows, those in the front row numbered consecutively from left to right 1-10, those in the back row 11-20. 4. arrange further 20 tubes of bile salt broth numbered 21-40 in two rows in a second smaller rack which can be stood on the upper tier of the rack as soon as the first 20 tubes have been inoculated. 5. place a square wire basket of about 50 tube capacity close to the left of the test-tube rack, for the reception of the inoculated tubes. 6. adjust a sterile 1 c.c. pipette in the mouth of each of the tubes, a, b, c, d and e, by means of a washer, as previously described. 7. set the electric signal clock to ring for the commencement of the experiment and subsequently at 2-1/2, 5, 10, 15, 20, 25, 30 and 35 minutes. 8. complete precisely as indicated in trial runs, steps 9-19. _control phenol._-immediately the subculture tube from the 30-minute contact period have been inoculated, carry out a precisely similar experiment, in which five percentage strengths of phenol, (e. g., 1.1, 1.0, 0.9, 0.75, 0.7) are arranged in the lower tier of the test-tube rack in place of the five strengths of germicide-x. calculate the phenol coefficient by the following method: (a) divide the figure representing the percentage strength of the weakest lethal dilution of the carbolic acid control at the 2-1/2-minute contact period by the figure representing the percentage strength of the weakest lethal dilution of the x-disinfectant at the same period. the quotient = phenol coefficient at 2-1/2 minutes. (b) similarly obtain the phenol coefficient at 30 minutes contact period. (c) record the mean of the two coefficients obtained in (a) and (b) as the _mean phenol coefficient_, or simply as the ~phenol coefficient~. the details of the final test of an actual determination are set out in the accompanying table. table 27 organism employed, b. coli communis. culture medium, nutrient agar (+10). age, 24 hrs. temp. of incubation, 37°c. quantities used { culture } emulsion 0.1 c.c. + 5 c.c. germicide. { emulsion } room temperature during experiments, 17°c. germicide strength time of exposure incubation 2-1/2 5 10 15 20 25 30 35 time temp. 1 germicide-x 4% - - - - - - - - 7 days. 37°c. 2 germicide-x 3% 48 - - - - - - - 7 days. 37°c. 3 germicide-x 2% 24 24 24 24 48 72 7 days. 37°c. 4 germicide-x 1% 24 24 24 24 72 24 72 7 days. 37°c. 5 germicide-x 0.5% 24 24 24 24 24 24 24 24 24 hours. 37°c. 1 phenol 1.10% - - - - - - - - 7 days. 37°c. 2 phenol 1.00% 24 7 days. 37°c. 3 phenol 0.75% 24 24 24 24 48 7 days. 37°c. 4 phenol 0.70% 24 24 24 24 24 72 7 days. 37°c. 5 phenol 0.65% 24 24 24 24 24 48 24 24 2 days. 37°c. ((1.10/4.00) + (0.7/2.0)) 0.27 + 0.35 .62 phenol coefficient = -----------------------= ----------= --= 0.31 2 2 2 appendix. metric and imperial systems of weights and measures. the initial unit of the metric system is the metre (_m._) or unit of length, representing one-fourth-millionth part of the circumference of the earth round the poles. the unit of mass is the gramme (_g._), and represents the weight of one cubic centimetre of water at its maximum density (viz. 4° c. and 760 mm. mercury pressure). the unit of the measure of capacity is the litre (_l._), and represents the volume of a kilogramme of distilled water at its maximum density. the decimal subdivisions of each of the units are designated by the latin prefixes _milli_ = 1/1000; _centi_ = 1/100; _deci_ = 1/10; the multiples of each unit by the greek prefixes _deka_ = 10; _hecto_ = 100; _kilo_ = 1000; _myria_ = 10,000. for a comparison of the values of some of the more frequently employed expressions of the metric system and the imperial system, the following may be found convenient for reference: ~length:~ 1 millimetre (= 1 mm.) = 1/25 of an inch. 1 centimetre (= 1 cm.) = 2/5 of an inch. 1 inch (1") = 25 millimetres or 2-1/2 centimetres. ~mass:~ 1 milligramme (= 1 mg.) = 0.01543 grain (or approximately 1/64 grain). 1 gramme (= 1 g.) = 15.4323 grains. 1 "kilo" or kilogramme (= 1 kgm.) = 2 pounds, 3-1/4 ounces avoirdupois. 1 pound avoirdupois (= 1 lb.) = 453.592 grammes. 1 ounce avoirdupois (= 1 oz.) = 28.35 grammes. 1 grain = 0.0648 gramme or 64.8 milligrammes. ~capacity:~ 1 cubic centimetre (= 1 c.c.) = 16.9 minims imperial measure. 1 litre (= 1 _l._) = 35.196 fluid ounces imperial measure. 1 fluid ounce imperial measure (= 1 [symbol: ounce]) = 28.42 cubic centimetres. 1 pint imperial measure (= 1 o.) = 568.34 cubic centimetres. 1 gallon imperial measure (= 1 c.) = 4.546 litres, or 10 pounds avoirdupois, of pure water at 62° f. and under an atmospheric pressure of 30 inches of mercury. factors for converting from one system to the other. to convert grammes into grains × 15.432. to convert grammes into ounces avoirdupois × 0.03527. to convert kilogrammes into pounds × 2.2046. to convert cubic centimetres into fluid ounces imperial × 0.0352. to convert litres into fluid ounces imperial × 35.2. to convert metres into inches × 39.37. to convert grains into grammes × 0.0648. to convert avoirdupois ounces into grammes × 28.35. to convert troy ounces into grammes × 31.104. to convert fluid ounces into cubic centimetres × 28.42. to convert pints into litres × 0.568. to convert inches into metres × 0.0254. table for the conversion of degrees centigrade into degrees fahrenheit. _x.° c. = ((9x/5) + 32)° f._ | cent. | faht. || cent. | faht. || cent. | faht. | | 0 | 32.0 || 34 | 93.2 || 68 | 154.4 | | 1 | 33.8 || 35 | 95.0 || 69 | 156.2 | | 2 | 35.6 || 36 | 96.8 || 70 | 158.0 | | 3 | 37.4 || 37 | 98.6 || 71 | 159.8 | | 4 | 39.2 || 38 | 100.4 || 72 | 161.6 | | 5 | 41.0 || 39 | 102.2 || 73 | 163.4 | | 6 | 42.8 || 40 | 104.0 || 74 | 165.2 | | 7 | 44.6 || 41 | 105.8 || 75 | 167.0 | | 8 | 46.4 || 42 | 107.6 || 76 | 168.8 | | 9 | 48.2 || 43 | 109.4 || 77 | 170.6 | | 10 | 50.0 || 44 | 111.2 || 78 | 172.4 | | 11 | 51.8 || 45 | 113.0 || 79 | 174.2 | | 12 | 53.6 || 46 | 114.8 || 80 | 176.0 | | 13 | 55.4 || 47 | 116.6 || 81 | 177.8 | | 14 | 57.2 || 48 | 118.4 || 82 | 179.6 | | 15 | 59.0 || 49 | 120.2 || 83 | 181.4 | | 16 | 60.8 || 50 | 122.0 || 84 | 183.2 | | 17 | 62.6 || 51 | 123.8 || 85 | 185.0 | | 18 | 64.4 || 52 | 125.6 || 86 | 186.8 | | 19 | 66.2 || 53 | 127.4 || 87 | 188.6 | | 20 | 68.0 || 54 | 129.2 || 88 | 190.4 | | 21 | 69.8 || 55 | 131.0 || 89 | 192.2 | | 22 | 71.6 || 56 | 132.8 || 90 | 194.0 | | 23 | 73.4 || 57 | 134.6 || 91 | 195.8 | | 24 | 75.2 || 58 | 136.4 || 92 | 197.6 | | 25 | 77.0 || 59 | 138.2 || 93 | 199.4 | | 26 | 78.8 || 60 | 140.0 || 94 | 201.2 | | 27 | 80.6 || 61 | 141.8 || 95 | 203.0 | | 28 | 82.4 || 62 | 143.6 || 96 | 204.8 | | 29 | 84.2 || 63 | 145.4 || 97 | 206.6 | | 30 | 86.0 || 64 | 147.2 || 98 | 208.4 | | 31 | 87.8 || 65 | 149.0 || 99 | 210.2 | | 32 | 89.6 || 66 | 150.8 || 100 | 212.0 | | 33 | 91.4 || 67 | 152.6 || | | table for the conversion of degrees fahrenheit into degrees centigrade. _x° f. = (5(x 32))/9° c._ faht.| cent.|| faht.| cent.|| faht.|cent. || faht.| cent.|| faht.| cent. 32 | 0.|| 68 | 20.0 || 104 | 40.0 || 140 | 60.0 || 176 | 80.0 33 | 0.6 || 69 | 20.6 || 105 | 40.6 || 141 | 60.6 || 177 | 80.6 34 | 1.1 || 70 | 21.1 || 106 | 41.1 || 142 | 61.1 || 178 | 81.1 35 | 1.7 || 71 | 21.7 || 107 | 41.7 || 143 | 61.7 || 179 | 81.7 36 | 2.2 || 72 | 22.2 || 108 | 42.2 || 144 | 62.2 || 180 | 82.2 37 | 2.8 || 73 | 22.8 || 109 | 42.8 || 145 | 62.8 || 181 | 82.8 38 | 3.3 || 74 | 23.3 || 110 | 43.3 || 146 | 63.3 || 182 | 83.3 39 | 3.9 || 75 | 23.9 || 111 | 43.9 || 147 | 63.9 || 183 | 83.9 40 | 4.4 || 76 | 24.4 || 112 | 44.4 || 148 | 64.4 || 184 | 84.4 41 | 5.0 || 77 | 25.0 || 113 | 45.0 || 149 | 65.0 || 185 | 85.0 42 | 5.6 || 78 | 25.6 || 114 | 45.6 || 150 | 65.6 || 186 | 85.6 43 | 6.1 || 79 | 26.1 || 115 | 46.1 || 151 | 66.1 || 187 | 86.1 44 | 6.7 || 80 | 26.7 || 116 | 46.7 || 152 | 66.7 || 188 | 86.7 45 | 7.2 || 81 | 27.2 || 117 | 47.2 || 153 | 67.2 || 189 | 87.2 46 | 7.8 || 82 | 27.8 || 118 | 47.8 || 154 | 67.8 || 190 | 87.8 47 | 8.3 || 83 | 28.3 || 119 | 48.3 || 155 | 68.3 || 191 | 88.3 48 | 8.9 || 84 | 28.9 || 120 | 48.9 || 156 | 68.9 || 192 | 88.9 49 | 9.4 || 85 | 29.4 || 121 | 49.4 || 157 | 69.4 || 193 | 89.4 50 | 10.0 || 86 | 30.0 || 122 | 50.0 || 158 | 70.0 || 194 | 90.0 51 | 10.6 || 87 | 30.6 || 123 | 50.6 || 159 | 70.6 || 195 | 90.6 52 | 11.1 || 88 | 31.1 || 124 | 51.1 || 160 | 71.1 || 196 | 91.1 53 | 11.7 || 89 | 31.7 || 125 | 51.7 || 161 | 71.7 || 197 | 91.7 54 | 12.2 || 90 | 32.2 || 126 | 52.2 || 162 | 72.2 || 198 | 92.2 55 | 12.8 || 91 | 32.8 || 127 | 52.8 || 163 | 72.8 || 199 | 92.8 56 | 13.3 || 92 | 33.3 || 128 | 53.3 || 164 | 73.3 || 200 | 93.3 57 | 13.9 || 93 | 33.9 || 129 | 53.9 || 165 | 73.9 || 201 | 93.9 58 | 14.4 || 94 | 34.4 || 130 | 54.4 || 166 | 74.4 || 202 | 94.4 59 | 15.0 || 95 | 35.0 || 131 | 55.0 || 167 | 75.0 || 203 | 95.0 60 | 15.6 || 96 | 35.6 || 132 | 55.6 || 168 | 75.6 || 204 | 95.6 61 | 16.1 || 97 | 36.1 || 133 | 56.1 || 169 | 76.1 || 205 | 96.1 62 | 16.7 || 98 | 36.7 || 134 | 56.7 || 170 | 76.7 || 206 | 96.7 63 | 17.2 || 99 | 37.2 || 135 | 57.2 || 171 | 77.2 || 207 | 97.2 64 | 17.8 || 100 | 37.8 || 136 | 57.8 || 172 | 77.8 || 208 | 97.8 65 | 18.3 || 101 | 38.3 || 137 | 58.3 || 173 | 78.3 || 209 | 98.3 66 | 18.9 || 102 | 38.9 || 138 | 58.9 || 174 | 78.9 || 210 | 98.9 67 | 19.4 || 103 | 39.4 || 139 | 59.4 || 175 | 79.4 || 211 | 99.4 | || | || | || | || 212 |100.0 ~percentage formula~ for addition of salts, etc., to completed media. ~formula for preparing any desired percentage~ of a given salt, etc., in tubed media; e. g., to make 4 per cent. solution of kno_{3} in a series of tubes of broth each containing 10 c.c. of medium, when there is already available a 25 per cent. stock aqueous solution of potassium nitrate. (_n_ + ~x~) _y_ _a_ (~x~) --------------= --------- 100 100 _n_ = number of cubic centimetres contained in each tube. ~x~ = amount of stock solution to be added to each tube. _y_ = percentage required in the medium. _a_ = percentage of stock solution. then (10 + ~x~) 4 25 ~x~ -----------= ----- 100 100 therefore, 40 + 4~x~ = 25~x~. therefore, 21~x~ = 40. ~x~ = 1.9 c.c. this allows for solution added to the original bulk of medium. therefore, 10 c.c. broth + 1.9 c.c. of a 25 per cent. aqueous solution kno_{3} makes 11.9 c.c. medium containing 4 per cent. kno_{3}. ~tables for preparing dilutions~ (of serum, disinfectants or other substances.) in estimating the agglutinin content or _titre_ of a serum, testing disinfectants and for many other purposes, it becomes necessary to prepare a series of dilutions of the material under examination, and in order to avoid unnecessary expenditure of labour it is convenient to adhere to some definite scale of increment, such for example as the following: from dilutions of 1:10 to 1:80 rise by increments of 5. from dilutions of 1:80 to 1:200 rise by increments of 10. from dilutions of 1:200 to 1:400 rise by increments of 25. from dilutions of 1:400 to 1:500 rise by increments of 50. from dilutions of 1:500 to 1:1000 rise by increments of 100. from dilutions of 1: 1000 to 1:5000 rise by increments of 250. from dilutions of 1: 5000 to 1:10,000 rise by increments of 1000. from dilutions of 1:10,000 to 1:100,000 rise by increments of 5000. from dilutions of 1:100,000 to 1:1,000,000 rise by increments of 100,000. when dealing with a substance of unknown powers--and this is especially true with regard to agglutinating sera--it is customary to run a preliminary test, using a few widely separated dilutions such as may be obtained in the following manner: first dilution--i. 1 c.c. serum + 9 c.c. normal saline solution = 10 per cent. solution or 1: 10 dilution (of which 1 c.c. contains 0.1 c.c. of the original serum). when dealing with fluids other than serum the diluent is usually distilled water; whilst if the original substance is a solid the instructions would read: 1 gram o.s. + 10 c.c. distilled water = 10 per cent. solution, etc. second dilution--ii. 1 c.c. first dilution + 9 c.c. normal saline solution = 1 per cent. solution or 1: 100 dilution. third dilution--iii. 1 c.c. second dilution + 9 c.c. normal saline solution = 1 per mille solution or 1: 1000 dilution. fourth dilution--iv. 1 c.c. second dilution + 9 c.c. normal saline solution = 0.1 per mille solution or 1: 10,000 dilution. the following tables showing the secondary dilutions that can readily be prepared from each of these four primary dilutions for use in the subsequent determination of the exact _titre_ will probably be found of service by those who are not ready mathematicians. tables for preparing dilutions. -----------------------------------+--------------------------------- | table i | table ii using 10 % stock solution | using 1% stock solution first } | second } dilution } + diluent | dilution } + diluent | -----------------------------------+--------------------------------- | 1: 10 = 1 c.c. + 0 c.c. | 1: 100 = 1 c.c. + 0 c.c. 1: 15 = 1 c.c. + 0.5 c.c. | 1: 110 = 1 c.c. + 0.1 c.c. 1: 20 = 1 c.c. + 1.0 c.c. | 1: 120 = 1 c.c. + 0.2 c.c. 1: 25 = 1 c.c. + 1.5 c.c. | [1: 125 = 1 c.c. + 0.25 c.c.] 1: 30 = 1 c.c. + 2.0 c.c. | 1: 130 = 1 c.c. + 0.3 c.c. 1: 35 = 1 c.c. + 2.5 c.c. | 1: 140 = 1 c.c. + 0.4 c.c. 1: 40 = 1 c.c. + 3.0 c.c. | 1: 150 = 1 c.c. + 0.5 c.c. 1: 45 = 1 c.c. + 3.5 c.c. | 1: 160 = 1 c.c. + 0.6 c.c. 1: 50 = 1 c.c. + 4.0 c.c. | 1: 170 = 1 c.c. + 0.7 c.c. 1: 55 = 1 c.c. + 4.5 c.c. | [1: 175 = 1 c.c. + 0.75 c.c.] 1: 60 = 1 c.c. + 5.0 c.c. | 1: 180 = 1 c.c. + 0.8 c.c. 1: 65 = 1 c.c. + 5.5 c.c. | 1: 190 = 1 c.c. + 0.9 c.c. 1: 70 = 1 c.c. + 6.0 c.c. | 1: 200 = 1 c.c. + 1.0 c.c. 1: 75 = 1 c.c. + 6.5 c.c. +-------------------------------- 1: 80 = 1 c.c. + 7.0 c.c. | 1: 200 = 1 c.c. + 1.0 c.c. ------------------------------+ 1: 225 = 1 c.c. + 1.25 c.c. 1: 80 = 1 c.c. + 7.0 c.c. | 1: 250 = 1 c.c. + 1.5 c.c. 1: 90 = 1 c.c. + 8.0 c.c. | 1: 275 = 1 c.c. + 1.75 c.c. 1: 100 = 1 c.c. + 9.00 c.c. | 1: 300 = 1 c.c. + 2.0 c.c. 1: 110 = 1 c.c. + 10.0 c.c. | 1: 325 = 1 c.c. + 2.25 c.c. 1: 120 = 1 c.c. + 11.0 c.c. | 1: 350 = 1 c.c. + 2.5 c.c. [1: 125 = 1 c.c. + 11.5 c.c.] | 1: 375 = 1 c.c. + 2.75 c.c. 1: 130 = 1 c.c. + 12.0 c.c. | 1: 400 = 1 c.c. + 3.0 c.c. 1: 140 = 1 c.c. + 13.0 c.c. +-------------------------------- 1: 150 = 1 c.c. + 14.0 c.c. | 1: 400 = 1 c.c. + 3.0 c.c. 1: 160 = 1 c.c. + 15.0 c.c. | 1: 450 = 1 c.c. + 3.5 c.c. 1: 170 = 1 c.c. + 16.0 c.c. | 1: 500 = 1 c.c. + 4.0 c.c. [1: 175 = 1 c.c. +-16.5 c.c.] +-------------------------------- 1: 180 = 1 c.c. + 17.0 c.c. | 1: 500 = 1 c.c. + 4.0 c.c. 1: 190 = 1 c.c. + 18.0 c.c. | 1: 600 = 1 c.c. + 5.0 c.c. 1: 200 = 1 c.c. + 19.0 c.c. | 1: 700 = 1 c.c. + 6.0 c.c. ----------------------------+ [1: 750 = 1 c.c. + 6.5 c.c.] 1: 200 = 1 c.c. + 19.0 c.c. | 1: 800 = 1 c.c. + 7.0 c.c. 1: 225 = 1 c.c. + 21.5 c.c. | 1: 900 = 1 c.c. + 8.0 c.c. 1: 250 = 1 c.c. + 24.0 c.c. | 1: 1000 = 1 c.c. + 9.0 c.c. 1: 275 = 1 c.c. + 26.5 c.c. +------------------------------- 1: 300 = 1 c.c. + 29.0 c.c. | 1: 1000 = 1 c.c. + 9.0 c.c. 1: 325 = 1 c.c. +-31.5 c.c. | 1: 2000 = 1 c.c. + 19.0 c.c. 1: 350 = 1 c.c. + 34.0 c.c. | 1: 3000 = 1 c.c. + 29.0 c.c. 1: 375 = 1 c.c. + 36.5 c.c. | 1: 4000 = 1 c.c. + 39.0 c.c. 1: 400 = 1 c.c. + 39.0 c.c. | 1: 5000 = 1 c.c. + 49.0 c.c. ------------------------------+------------------------------- 1: 400 = 1 c.c. + 39.0 c.c. | 1: 450 = 1 c.c. + 44.5 c.c. | 1: 500 = 1 c.c. + 49.0 c.c. | ---------------------------------+------------------------------ | table iii | table iv using 0.1% stock solution | using 0.01% stock solution third } | fourth } dilution } + diluent | dilution } + diluent | ---------------------------------+------------------------------ | 1: 1000 = 1 c.c. + 0 c.c. | 1: 10,000 = 1 c.c. + 0 c.c. 1: 1250 = 1 c.c. + 0.25 c.c. | 1: 15,000 = 1 c.c. + 0.5 c.c. 1: 1500 = 1 c.c. + 0.5 c.c. | 1: 20,000 = 1 c.c. + 1.0 c.c. 1: 1750 = 1 c.c. + 0.75 c.c. | 1: 25,000 = 1 c.c. + 1.5 c.c. 1: 2000 = 1 c.c. + 1.0 c.c. | 1: 30,000 = 1 c.c. + 2.0 c.c. 1: 2250 = 1 c.c. + 1.25 c.c. | 1: 35,000 = 1 c.c. + 2.5 c.c. 1: 2500 = 1 c.c. + 1.5 c.c. | 1: 40,000 = 1 c.c. + 3.0 c.c. 1: 2750 = 1 c.c. + 1.75 c.c. | 1: 45,000 = 1 c.c. + 3.5 c.c. 1: 3000 = 1 c.c. + 2.0 c.c. | 1: 50,000 = 1 c.c. + 4.0 c.c. 1: 3250 = 1 c.c. + 2.25 c.c. | 1: 55,000 = 1 c.c. + 4.5 c.c. 1: 3500 = 1 c.c. + 2.5 c.c. | 1: 60,000 = 1 c.c. + 5.0 c.c. 1: 3750 = 1 c.c. + 2.75 c.c. | 1: 65,000 = 1 c.c. + 5.5 c.c. 1: 4000 = 1 c.c. + 3.0 c.c. | 1: 70,000 = 1 c.c. + 6.0 c.c. 1: 4250 = 1 c.c. + 3.25 c.c. | 1: 75,000 = 1 c.c. + 6.5 c.c. 1: 4500 = 1 c.c. + 3.5 c.c. | 1: 80,000 = 1 c.c. + 7.0 c.c. 1: 4750 = 1 c.c. + 3.75 c.c. | 1: 85,000 = 1 c.c. + 7.5 c.c. 1: 5000 = 1 c.c. + 4.0 c.c. | 1: 90,000 = 1 c.c. + 8.0 c.c. --------------------------------+ 1: 95,000 = 1 c.c. + 8.5 c.c. 1: 5000 = 1 c.c. + 4.0 c.c. | 1: 100,000 = 1 c.c. + 9.0 c.c. 1: 6000 = 1 c.c. + 5.0 c.c. +---------------------------------- 1: 7000 = 1 c.c. + 6.0 c.c. | 1: 100,000 = 0.1 c.c. + 0.9 c.c. [1: 7500 = 1 c.c. + 6.5 c.c.] | 1: 200,000 = 0.1 c.c. + 1.9 c.c. 1: 8000 = 1 c.c. + 7.0 c.c. | [1: 250,000 = 0.1 c.c. + 2.4 c.c.] 1: 9000 = 1 c.c. + 8.0 c.c. | 1: 300,000 = 0.1 c.c. + 2.9 c.c. 1: 10,000 = 1 c.c. + 9.0 c.c. | 1: 400,000 = 0.1 c.c. + 3.9 c.c. ------------------------------+ 1: 500,000 = 0.1 c.c. + 4.9 c.c. 1: 10,000 = 1 c.c. + 9.0 c.c. +---------------------------------- 1: 15,000 = 1 c.c. + 14.0 c.c. | 1: 500,000 = 0.1 c.c. + 4.9 c.c. 1: 20,000 = 1 c.c. + 19.0 c.c. | 1: 600,000 = 0.1 c.c. + 5.9 c.c. 1: 25,000 = 1 c.c. + 24.0 c.c. | 1: 700,000 = 0.1 c.c. + 6.9 c.c. 1: 30,000 = 1 c.c. + 29.0 c.c. | [1: 750,000 = 0.1 c.c. + 7.4 c.c.] --------------------------------+ 1: 800,000 = 0.1 c.c. + 7.9 c.c. | 1: 900,000 = 0.1 c.c. + 8.9 c.c. | 1:1,000,000 = 0.1 c.c. + 9.9 c.c. -+------------------------------------temperature pressure table. ---------------+--------------+---------------------+------------ temperature | | pounds per sq. in. | centigrade | mm. of hg. | absolute pressure | atmospheres | | | ---------------+--------------+---------------------+------------ | | | 98° | 707.1 | 13.7 | 0.93 99° | 733.1 | 14.2 | 0.96 100° | 760.0 | 14.7 | 1.00 | | | 101° | 787.8 | 15.2 | 1.03 102° | 816.0 | 15.8 | 1.07 103° | 845.2 | 16.3 | 1.11 104° | 875.4 | 16.9 | 1.15 105° | 906.4 | 17.5 | 1.19 | | | 106° | 938.3 | 18.1 | 1.23 107° | 971.1 | 18.8 | 1.27 108° | 1004.9 | 19.4 | 1.32 109° | 1039.6 | 20.1 | 1.36 110° | 1075.3 | 20.8 | 1.41 | | | 111° | 1112.0 | 21.5 | 1.46 112° | 1149.8 | 22.2 | 1.51 113° | 1188.6 | 22.9 | 1.56 114° | 1228.4 | 23.7 | 1.61 115° | 1269.4 | 24.5 | 1.67 | | | 116° | 1311.4 | 25.3 | 1.72 117° | 1354.6 | 26.2 | 1.78 118° | 1399.0 | 27.0 | 1.84 119° | 1444.5 | 27.9 | 1.90 120° | 1491.2 | 28.8 | 1.96 | | | 121° | 1539.2 | 29.7 | 2.02 122° | 1588.4 | 30.7 | 2.09 123° | 1638.9 | 31.7 | 2.15 124° | 1690.7 | 32.7 | 2.22 125° | 1743.8 | 33.7 | 2.29 ---------------+--------------+---------------------+------------table for desiccation at low temperatures in vacuo. +--------------------------+ | temperature | | | centigrade | mm. of hg. | +-------------+------------+ | 21° | 18.4 | | 22° | 19.6 | | 23° | 20.8 | | 24° | 22.1 | | 25° | 23.5 | | | | | 26° | 24.9 | | 27° | 26.4 | | 28° | 28.0 | | 29° | 29.7 | | 30° | 31.5 | | | | | 31° | 33.3 | | 32° | 35.3 | | 33° | 37.3 | | 34° | 39.5 | | 35° | 41.7 | | | | | 36° | 44.1 | | 37° | 46.6 | | 38° | 49.2 | | 39° | 51.9 | | 40° | 54.8 | | | | | 41° | 57.8 | | 42° | 61.0 | | 43° | 64.3 | | 44° | 67.7 | | 45° | 71.3 | | | | | 46° | 75.1 | | 47° | 79.0 | | 48° | 83.1 | | 49° | 87.4 | | 50° | 91.9 | +-------------+------------+ antiformin method for the detection of b. tuberculosis. _antiformin_ was introduced into bacteriological technique by uhlenhuth in 1908 for the purpose of demonstrating tubercle bacilli when present in small numbers, in sputum or other material. it is a powerful oxidising agent and rapidly destroys most bacteria, but tubercle and other acid-fast organisms resist its lethal action for considerable periods, and upon this fact the method is based. _to prepare antiformin_ measure out and mix:-eau de javelle (liquor sodæ chlorinatæ--b.p.) 50 c.c. sodic hydrate 15 per cent. aqueous solution 50 c.c. method. 1. introduce the sputum or other material (e. g. milk deposit and cream; pus; minced gland or other organ; caseous material; broken down foci, etc.) into a sterile tube and then add an equal volume of antiformin. 2. close the tube with a rubber cork and shake vigorously (a sample of antiformin that does not "foam" at this stage is of little use). disintegration of the material at once starts, associated bacteria are destroyed and the mixture rapidly becomes a homogenous but turbid fluid--a process which may be hastened by:-3. placing the tube in the incubator at 37° c. for 30 minutes--shaking from time to time. 4. centrifugalise the fluid thoroughly, at high speed. 5. pipette off the supernatant fluid, fill up with sterile distilled water, cork the tube and shake to distribute the deposit throughout the water. again centrifugalise. 6. repeat steps 4 and 5 twice more. 7. employ one portion of the final deposit to inoculate guinea pigs. 8. plant the remainder of the deposit freely on dorset's egg medium; cap and incubate at 37°c. note.--if only microscopical films are needed, fill up the centrifuge tube with ligroin (a petroleum ether) in place of sterile distilled water in step 5 and prepare the films from the _surface_ of the fluid, to stain by the ziehl-neelsen process. index abbé's condenser, 7 abbott's stain for spores, 107 aberration, chromatic, 56 spherical, 55 absolute alcohol as a fixative, 82 as an antiseptic, 27 absorbent paper for drying cover-slips, 69 a. c. e. mixture, 345 acetic acid for clearing films, 82 achromatic condenser, 54 acid hæmatin, 96 production, analysis table, 283 by bacteria, 145 investigation of, 280 qualitative examination, 283, 284 quantitative examination, 280 acid-fast bacilli in tissues, to stain, 124 action of various gases on bacteria, 295 active immunisation, illustrative example, 322 adjustable water bath, 299 aerobic cultures, 221 aerogenic bacteria, 131 aesculin agar, 204 agar gelatine (guarniari), 194 methods of preparation, 167 surface plates, 232 agar-agar, preparation of, 167 agglutination reaction, macroscopical, 386 microscopical, 385 agglutinin, 381 air, analysis of, 468 filter, 40 pump, geryk, 43 albumin solution, mayer's, 120 alcohol production, test for, 285 alkaline pyro, 239 alum carmine, 96 ammonia production test for, 285 amphitrichous bacteria, 136 anaerobic cultures, 236 botkin's method, 243 buchner's method, 238 bulloch's method, 245 hesse's method, 237 mcleod's method, 240 media, 180 novy's method, 244 anaerobic cultures, roux's biological method, 237 physical method, 237 vacuum method, 238 wright's method, 239 anæsthetics, 345 analysis of air, apparatus for, 469 method of, 468 qualitative bacteriological, 470 quantitative bacteriological, 468 of butter, qualitative bacteriological, 458 quantitative bacteriological, 457 of cream, qualitative bacteriological, 458 quantitative bacteriological, 457 of fish, 460 of ice cream, qualitative bacteriological, 457 of meat, apparatus for, 460 method of, 460 qualitative bacteriological, 462 of milk, apparatus for, 444 collection of samples, 441 method of, 441 qualitative bacteriological, 446. quantitative bacteriological, 444 of oysters, 463 of sewage, qualitative bacteriological, 467 quantitative bacteriological, 466 of shellfish, 463 of soil, apparatus for, 473 collection of samples, 471 method of, 470 qualitative bacteriological, 476 quantitative bacteriological, 473 of water, apparatus for, 420, 427 collection of samples, 416 method of, 416 qualitative bacteriological, 426 analysis of water, quantitative bacteriological, 420 aniline dyes, 83 gentian violet, 95 water, to prepare, 108 animal tissue media (frugoni), 210 animals, natural infections of, 337 antiformin method for b. tuberculosis, 502 antigen, definition of, 324 antiseptics, 27 action of, 310 apparent filth in milk, 450 arnold's steam steriliser, 34 arthrogenous spores, 138 ascitic bouillon, 210 fluid agar (wassermann), 213 ascomycetæ, 128 ascopores, 129 asparagin media (frankel and voges), 183 (uschinsky), 183 aspergillus, 127 atmospheric conditions, 295 attenuating the virulence of organisms, 321 autoclave, 37 to use, 37 automatic pipettes, 13 autopsies, 396 autopsy, card index for, 402 bacilli, morphology of, 132 bacillus anthracis in soil, 477 in water, 440 coli in water, detection of, 429 diphtheriæ in milk, 452 enteritidis in water, 437 sporogenes in milk, 452 in water, 438 oedematis maligni in soil, 477 tetani in soil, 477 in water, 441 tuberculosis in milk, 453 antiformin method, 502 typhosus in water, 441 bacteria, anatomy of, 134 classification of, 131 grouping of, for study, 410 in tissues, demonstration of, 114 influence of environment on, 142 metabolic products of, 143 methods of identification, 259 microscopical examination of, stained, 81 unstained, 74 physiology of, 136 bacteria, simple stains for, 90 bacterial emulsion, preparation of, 389 enzymes, 144, 277 ferments, 144 food stuffs, 142 toxins, 144 bacteriological analyses, general considerations, 415 examination of blood, 377 base of microscope, 50 basidium, 128 beer wort, preparation of, 175 beetroot media, 200 beggiotoa, morphology of, 133 benzole bath, 256 berkefeld filter, 42 beyrinck's solution i, 197 ii, 198 bile salt agar (macconkey), 205 broth, double strength, 199 (macconkey), 180 biochemical examination of cultures, 276 biochemistry of bacteria, 276 biological differentiation of bacteria, 249 bipolar germination, 140 bismarck brown, 94 blastomycetes, morphology of, 129 blood agar, 171, 214 plates, animal, 251 human, 250 (washbourn), 214 bacteriological examination of, 377 cells, washing of, 388 collection of, for serological examination, 379 films, preparations of, 376 staining of, 97 histological examination of, 373 pipettes, 11 serological examination of, 378 stains, 97 blood-serum (councilman and mallory), 208 inspissated, 168 (loeffler), 208 (lorrain smith), 208 blowpipe table, 9 body tube of microscope, 50 bohemian flask, 4 boiling water, 33 bone marrow, films, preparation of, 400 bordet-gengou reaction, 393 boric acid in milk, test for, 442 botkin's anaerobic method, 243 bouillon, preparation of, 163 brain extract, 149 bread paste, 193 brilliant green agar (conradi), 206 bile salt agar (fawcus), 206 brownian movement, 79 buchner's anaerobic method, 238 bulloch's anaerobic method, 245 tubes for permanent preparations, 407 bunge's mordant, 104 burri's chinese ink stain, 77 butter, analysis of, 457 qualitative analysis of, 458 quantitative analysis of, 457 cadaver, preparation of, for autopsy, 397 cages for guinea-pigs, 343 for laboratory animals, 341 for mice, 342 for rabbits, 343 for rats, 342 calculated figure for weight of medium mass, 166, 167 cambier's candle method of isolating coli-typhoid groups, 438 camera lucida, 62 capaldi-proskauer medium, no i, 186 no ii, 187 capillary pipettes, 10 graduated, 13 capitate bacilli, 139 capsule formation, 134 of bacteria, 134 thermo-regulator, 218 capsules, collodion, inoculation of, 357 preparation of, 357 glass, 6 to clean infected, 20 new, 18 to stain, 99 to sterilise, 31 carbohydrate media, preparation of, 177 carbolic acid as a germicide, 27, 481 method of isolating coli-typhoid group, 437 carbolised agar, 202 bouillon, 202 gelatine, 202 carbon dioxide in cultures, test for, 289 card index, 336, 402 carrot media, 200 cedarwood oil for immersion lens, 88 cell wall of bacteria, 134 celloidin sacs, manufacture of, 358 cellular incubator, 216 centrifugal machine for blood and serum work, 327 for milk work, 447 centrifugalised milk, 449 centrigade degrees, conversion of, 494 chemical products of bacteria, 145 china green agar (werbitski), 207 chloroform as an antiseptic, 27 chromatic aberration, 56 chromogenic bacteria, 131 chromoparous bacteria, 144 chromophorous bacteria, 144 citrated blood agar, 191 cladothrix, morphology, 193 classification of bacteria, 131 of fungi, 126 clavate bacilli, 139 clearing films with acetic acid, 82 clostridium, 139 coarse adjustment, 51 cobweb micrometer, 66 cocaine, 345 cocci, morphology of, 131 coccidium infection, 339 coefficient, inferior lethal, 312 of inhibition, 311 phenol, 489 superior lethal, 313 cohn's solution, 191 cold incubator, 217 coli-typhoid group, differential table, 433 in milk, 451 in soil, 477 isolation of, 432 members of, 430 collection of blood for bacteriological examination, 378 for media making, 168 of milk samples, 443 of pathological material during life, 373 of pus, 373 of soil sample, 471 of water samples, 416 collodion capsules, 357 sacs, manufacture of, 357 colonies of bacteria, edges, 267 coloured light, action of, 309 columella, 127 comparative hæmocytology, 374 complement, definition of, 325 fixation test, 393 concentration method in water, analysis, 434 condenser achromatic, 54 dark ground, 60 paraboloid, 60 substage, 54 condidium, 128 continuous sterilisation, 36 contrast stains, 93 corrosive sublimate (lang), 82 cotton-wool filter, 40 counterstaining films, 84 counting plate colonies, 423 cover-slip films, 81 to clean new, 22 used, 24 crates for test-tubes, 31 cream, analysis of, 457 qualitative analysis of, 458 quantitative analysis of, 457 crenothrix morphology, 133 criteria of infection, 370 criterion of immunity, 324 cultural characters, macroscopical examination, 261 culture flask, guy's, 5 kolle, 4 roux, 5 cuneate bacilli, 139 cutaneous inoculation, 352 dark ground condenser, 60 illumination, 87 daughter cells, 129 daylight, diffuse, action of, 308 decimal scales, 340 decolourising agents, 84 definition of objective, 56 depilatory powder, 346 description of plate culture, 261 descriptive terms, 261 desiccation, effects of, 306 table, 501 desiccator, mueller's, 307 dextrose solution, preparation of, 178 diaphragm, iris, 53 diastatic enzymes, tests for, 278 differential atmosphere cultivation, 257 incubation, 255 media, 255 staining, 108 sterilisation, 256 diluting chamber, 248 dilution by teat pipette, 383 of serum, 382 tables, 498 dilutions, preparations of, 496 diphtheria, bacillus of, in milk, 452 diplobacilli, morphology of, 133 diplococci, morphology of, 133 diplococcus pneumoniæ, immunisation against, 322 discontinuous sterilisation, 36 discs of plaster-of-paris, 192 disinfectants, action of, 310 chemical, 27 testing of, 480 dissociating fluid, price jones; 400 dosage of inoculum, 316 double nosepiece, 58 stains for spores, 106 sugar agar (russell), 207 drop-bottle, 73 dry heat, 28 dunham's solution, 177 dyes, aniline, 83 earthenware box for dirty slides, 70 earthy salts agar (lipman and brown), 197 edge of individual colonies, characters of, 267 egg albumin agar, 213 broth, (lipschuetz), 213 media (dorset), preparation of, 174 inspissated, 212 (lubenau), 209 (tarchanoff and kolesnikoff), 212 to clear nutrient media with, 166 ehrlich's eyepiece, 55 eikonometer, 65 eisenberg's milk-rice medium, 189 electric dental engine, 360 signal clock, 38 warm stage, 59 elevation of colonies, 263 eisner's gelatine, 204 method of isolating coli: typhoid group, 438 endogenous spores, 138 varieties of, 139 endo-germination, 139 english proof agar, blaxall, 193 enumerating colonies on plates, 423 discs, jeffer's, 424 pakes', 424 enrichment method in water analysis, 427 enumeration of micro-organisms, 423 environmental conditions, 142 enzyme production, investigation of, 277 eosin, 93 equatorial germination, 140 erlenmeyer flask, 4 ernstschen koerner, 136 esmarch's roll culture, 226 water collecting bottle, 417 estimation of reaction of media, 280 ether flame, 28 soluble acids, 284 eucaine, 345 exalting virulence of organisms, 320 examination of milk, 441 experimental infections, study of, during life, 370 inoculation of animals, 332 extracellular toxins, 144 eyepiece, ehrlich, 55 _micrometer_, 63 eyepieces, 55 eye-shade, 57 fahrenheit degrees, conversion of, 495 feeding experiments, 369 fermentation reactions, 279 tubes, 17 field of objective, 56 filar micrometer, 66 filling tubes, etc., with medium, 160 film preparations, 81 fixing, 81 making, 81 mounting, 85 staining, 83 filter candle, closed, 47 open, 43 testing efficiency of, 478 to disinfect, 28 to sterilise, 29 flask, 6 papers, to fold, 156 filters, cotton-wool, 40 porcelain, 42 testing of, 478 filtration, 40 by aspiration, 42 of media, 156 under pressure, 45 fine adjustment, 51 spindle head, 52 fish, analysis of, 460 bouillon, 190 fish gelatine, 190 gelatine-agar, 190 fishing colonies, 253 fission, reproduction by, 136 fixation, 81 by heat, 81 of tissues, 114 fixing fluids, for films, 82 flagella, classification of bacilli by, 136 to stain, 101 flask bohemian, 4 erlenmeyer, 4 filter, 6 kitasato'a serum, 6 kolle's culture, 4 flasks and test tubes, to plug, 24 to clean dirty, 20 new, 18 to sterilise, 31 fleischwasser, 148 fluid cultures, description of, 271 media, 146 foot of microscope, 50 formaldehyde in milk, hehner's test for, 442 formalin method of preserving cultures, 407 tissues, 404 fractional sterilisation, 33 fraenkel and voge's solution, 183 fraenkel's earth borer, 472 freezing method for sections, 115 french mannite agar (sabouraud), 193 proof agar (sabouraud), 193 fresh preparations of bacteria, 74 friedländer's capsule stain for sections, 123 frost's mounting fluid, 406 frozen sections, rapid method, 116 fuchsin, 92 agar (braun), 205 sulphite agar (endo), 206 gas analysis, qualitative, 290 quantitative, 290 collecting apparatus, 291 generators, 242 production by bacteria, 289 tubes for media, 161 gasperini's solution, 193 gelatin agar, 193 preparation of, 164 surface plates, 231 general anæsthetics, 345 gentian violet, 91 german lined paper, 69 germicides, 27 testing power of, 480 germination, 140 geryk air-pump, 43 glass apparatus in common use, 3 to clean, 18 glass-cutting knife, 8 glucose formate agar (kitasato), 180 bouillon (kitasato), 180 gelatine (kitasato), 180 glycerinated potato, 209 glycerine agar, 209 blood-serum, 208 bouillon, 209 potato bouillon, 203 broth, 203 goadby's gelatine, 214 gonidium, 128 goniodophore, 128 graduated capillary pipettes, 13 pipettes, 6 gram-claudius' differential stain, 109 gram's differential stain, 108 gram-weigert for sections, 121, 122 gram-weigert's differential stain, 109 modified, 110 grease pencils, 72 grouping of bacteria for study, 410 guarded trepine, 360 guarniari's agar gelatine, 194 guinea-pig cages, 343 holder, 350 gulland's solution, 82 gum solution, preparation of, 116 guy's culture bottle, 5 gypsum blocks (engel and hansen), 192 hæmatin, 95 hæmatocytometer, 248 hæmatoxilin, 95 hæmolysin, definition of, 326 preparation of, 327 storage of, 331 hæmolytic serum, titration of, 328 hanging-block culture (hill), 235 hanging-drop cultures, 233 examination of, 86, 79 preparation of, 78 permanent staining of, 80 slides, 70 hardening tissues, 114 haricot agar, 200 bouillon, 200 hay infusion, 200 hearson's water bath, 299 heat effect of, 299 hehner's test, 442 heiman's serum agar, 210 hesse's anaerobic culture method, 237 histological examination of blood, 373 holder for guinea-pigs, 350 hot air, 29 steriliser, 30 to use, 31 incubator, 217 hot-water funnel, 158 human blood agar plates, 250 huyghenian eyepiece, 55 hydrogen, generating apparatus, 242 in culture, test for, 289 peroxide in milk, test for, 442 hyphomycetes, morphology of, 126 reproduction of, 126 ice-box, for water samples, 419 ice cream, analysis of, 457 illuminant for microscope, 67 immune body, 393 immunisation, methods of, 321 imperial system, 492 factors for converting, 493 impression films, 85 incubators, 216 index cards, 336, 403 indol, test for, 286 infection, definition of, 370 general observations during life, 371 results of, 404 influence of environment on bacterial growth, 142 inhalation, fluid inoculum, 365 powdered inoculum, 366 inhibition coefficient, 310, 311 inoculation card index, 336 cutaneous, 352 intracranial, 360 intramuscular, 355 intraocular, 362 intraperitoneal, 355 intrapulmonary, 363 intravenous, 363 of collodion capsules, 357 subcutaneous, 353 syringe, 344 inoculum, character of, 346 preparation of, 346 inosite-free media--bouillon (durham), 183 inseparate toxins, 144 intermittent sterilisation, 36 intracellular toxins, 144 intracerebral inoculation, 362 intracranial inoculation, 360 intragastric inoculation, large animals, 367 marks method, 367 intramuscular inoculation, 355 intraocular inoculation, 362 intraperitoneal inoculation, 355 intrapulmonary inoculation, 363 intravenous inoculation, 363 in vacuo anaerobia cultures, 289 invertin enzymes, tests for, 279 involution forms, 137 iodine solution, 108 iron bouillon, 185 peptone solution (pakes), 185 isolation by animal experiments, 258 by differential atmosphere, 257 incubation, 255 media, 255 sterilisation, 256 by dilution, 248 by plate cultures, 250 subcultures, preparation of, 254 jeffer's counting disc, 424 jenner's stain, 97 jores' mounting fluid, 405 kaiserling fixing solution, 405 kanthack's serum agar, 211 killed cultivations, 318 kipp's hydrogen apparatus, 242 kitasato's serum flask, 6 klebs-loeffler bacillus in milk, 452 koch's steam steriliser, 34 kohle's culture flask, 4 lab enzymes, test for, 279 laboratory animals, 335 comparative hæmatocytology of, 374 normal temperature, 372 regulations, 1 lactose litmus agar (wurtz), 203 bouillon, 203 gelatine (wurtz), 203 lakmus molke, 203 lang's solution, 82 lead bouillon, 185 peptone solution, 186 leishman's stain, 98 for sections, 125 lemco broth, 163 leptothrix, morphology, 133 lethal dose, minimal, 316 leviditi's staining method, 124 light, action of, 308 liquefiable media, 147 liquid soap, 346 lithium carmine, 96 litmus bouillon, 186 gelatine, 202 milk cultures, description of, 272 preparation of, 172 nutrose agar (drigalski-conradi), 205 whey, 195 agar, 196 gelatine, 196 (petruschky), 195 local anæsthetics, 345 reaction to infection, 372 locomotive movement, 80 loeffler's capsule stain, 103 serum, 208 lophotrichous bacilli, 136 lorrain smith electric warm stage, 59 serum, 208 lugol's solution, to prepare, 108 lysol, 27 macconkey's capsule stain, 99 media, 180, 199, 205 maccrorrie's capsule stain, 103 macroscopical examination of cultures, 261 malachite green agar (loeffler), 207 malt extract solution (herschell), 196 margin of individual colonies, 267 martin's filtering apparatus, 320 material for inoculation, 346 mayer's albumin, 120 mean phenol coefficient, 490 measuring bacteria, 61 meat, bacteriological analysis of, 460 extract preparation of, 148 reaction of, 149 mechanical separation of bacteria, 249 stage, 52 media, filtration of, 156 preparation of, 163 aerobic culture, 222 aesculin agar, 204 agar-agar, 167 agar gelatine (guarniari), 194 media, preparation of anaerobic culture, 180 animal tissue (frugoni), 210 ascitic bouillon, 210 fluid agar (wassermann), 213 asparagin (fraenkel and voge's), 183 (uschinsky), 183 beer wort, 175 beetroot, 200 beyrinck's solution i, 197 ii, 198 bile salt agar (macconkey), 205 broth (macconkey), 180 double strength, 199 blood agar (washbourn), 214 blood-serum, 168 (councilman and mallory), 208 (loeffler), 208 (lorrain smith), 208 bouillon, 163 bread paste, 193 brilliant green agar (conradi), 206 bile salt agar (fawcus), 206 capaldi-proskauer, no. i, 186 no. ii, 187 carbohydrate, 177 carbolised agar, 202 bouillon, 202 gelatine, 202 carrot, 200 china green agar (werbitski), 207 citrated blood agar, 171 cohn's solution, 191 dextrose solution, 178 double sugar agar (russell), 207 earthy salt agar (lipman and brown), 197 egg dorset, 174 lubenau, 209 egg-albumen, inspissated, 212 (tarchanoff and kolesnikoff), 212 egg-albumin agar, 213 broth (lipschuetz), 213 english proof agar (blaxall), 193 fish bouillon, 190 gelatine, 190 agar, 190 fluid, 146 french mannite agar (sabouraud), 193 media, preparation of french proof agar (sabouraud), 193 fuchsin agar (braun), 205 sulphite agar (endo), 206 gelatine, 193 agar, 193 glucose formate agar (kitasato), 180 bouillon (kitasato), 180 gelatine (kitasato), 180 glycerinated broth, 209 potato, 209 glycerine agar, 209 blood-serum, 208, 209 bouillon, 209 potato bouillon, 203 gypsum blocks (engel and hansen), 192 haricot agar, 200 bouillon, 200 hay infusion, 200 inosite free-bouillon (durham), 183 iron bouillon, 185 peptone solution (pakes), 185 lactose litmus agar (wurtz), 203 bouillon, 203 gelatine (wurtz), 203 lakmus molke, 203 lead bouillon, 185 peptone solution, 186 lemco broth, 163 liquefiable, 147 litmus bouillon, 186 gelatine, 202 milk, 172 nutrose agar (drigalski-conradi), 205 whey, 195 agar, 196 gelatine, 196 (petruschky), 195 malachite green agar (loeffler), 207 malt extract solution (herschell), 196 milk, 172 rice (eisenberg), 189 (soyka), 189 naegeli's solution, 191 naehrstoff agar (hesse and niedner), 199 neutral litmus solution, 179 nitrate bouillon, 185 peptone solution (pakes), 186 nutrient, 146 agar-agar, 167 media, preparation of nutrient bouillon, 163 gelatine, 164 nutrose agar (eyre), 172 oleic acid agar (fleming), 201 omeliansky's nutrient fluid, 189 parietti's bouillon, 202 parsnip, 200 pasteur's solution, 191 peptone rosolic acid water, 186 water (dunham), 177 plaster-of-paris discs, 192 potato, 174 gelatine (elsner), 204 (goadby), 214 proteid free broth (uschinsky), 183 rosolic acid peptone solutions, 186 serum, bouillon, 210 dextrose water, (hiss), 188 sugar, (hiss), 188 water, 170 serum-agar (heiman), 210 (kanthack and stevens), 211 (libman), 212 (wertheimer), 211 silicate jelly (winogradsky), 198 solid, 147 special, 182 stock nutrient, 163 sugar, 177 agar, 185 (dextrose) bouillon, 184 gelatine, 184 sulphindigotate agar, 181 bouillon (weyl), 181 gelatine (weyl), 181 tissue (noguchi), 214 turnip, 200 urine agar, 188 bouillon, 187 gelatine, 187 (heller), 188 wheat bouillon (gasperini), 193 whey agar, 195 gelatine, 195 wine must, 192 winogradsky's solution (for nitric organisms), 198 (for nitrous organisms), 198 wood ash agar, 201 wort agar, 176 gelatine, 176 media, preparation of yeast water (pasteur), 191 standardisation of, 154 storage of, in bulk, 159 storing tubes of, 161 sore boxes, 162 titration of, 150 tubing of nutrient, 160 merismopedia, morphology of, 132 mesophilic bacteria, 143 pathogenic effects, 315 metabolic end-products, 145 metachromatic granules, 136 metal instruments, to sterilise, 28 metatrophic bacteria, 131 methods of cultivation, 221 of identification of bacteria, 259 of inoculation, 352 of isolation, 248 of sterilisation, 26 methylene-blue, 90 metric system, 492 factors for converting, 493 meyer's carmine, 96 microbes of indication, 426 micrococci, morphology, 132 micrococcus, melitensis in milk, 456 micrometer, filar, 66 net, 63 ocular, 63 stage, 62 micrometry, methods of, 61 micron, 61 microscope, 49 microscopical examination of bacteria, 86 stained, 88 unstained, 86 observations of cultures, 272 milk, analysis of, qualitative, 446 quantitative, 444 condensed, analysis of, 444 media, 193 preparation of, 172 rice (eisenberg), 193 (soyka), 189 samples, collection of, 443 sedimenting tubes, 449 minimal lethal dose, 316 mirror for microscope, 55 moeller's stain for spores, 107 moist heat, 32 molecular movement, 79 monotrichous bacilli, 136 motility, examination for, 79 true, 80 moulds, examination of, 126 for paraffin imbedding, 117, 119 mounting film preparations, 85 paraffin sections, 119 mouse cages, 342 holder, 351 scales, 341 mucor mucedo, 126 mucorinæ, 126 mueller's desiccator, 307 muffle furnace, 28 muirs's capsule stain, 100 flagella stain, 101 museum preparations of bacteria, 407 of tissues, 404 sealing of, 406 mycelium, 126 mycoprotein, 135 naegeli's solution, 191 naehrstoff agar (hesse and niedner), 199 naked flame, 28 neisser's stain modified, 111 net micrometer, 63 neutral litmus solution, preparation of, 179 red, 94 nitrate bouillon, 185 peptone solution (pakes), 186 nitric organisms in soil, 478 nitrosoindol reaction, 287 nitrous organisms in soil, 477 normal averages (_t.p.r._), 372 serum, 375 nosepiece, 57 double, 58 triple, 58 navy's anaerobic method, 244 jars, 245 nuclei, to stain, 105 nucleus of bacteria, 135 numerical aperture, 56 nutrient media, 146 nutrose agar (eyre), preparation of, 172 object marker, 61 objectives, 55 oblique tube cultures, 223 ocular micrometer, 63 oculars, 55 oese, platinum, 71 oïdium, 128 oil of garlic, 27 of mustard, 27 oleic acid agar (fleming), 201 omeliansky's nutrient fluid, 189 operation tables (eyre's), 352 (tatin's), 351 opsonic index, 393 opsonic index, determination of, 390 opsonin, 387 optical characters of colonies, 267 optimum reaction of medium, determination of, 305 temperature, determination of, 298 organisms of suppuration, 409 orsat-lunge gas apparatus, 292 orth's carmine, 96 oxford stain for actinomyces, 112 oysters, analysis of, 463 pakes' counting disc, 424 filter reservoir, 45 papier chardin, 158 pappenheim's stain, 111 paraboloid condenser, 60 parachromophorous bacteria, 144 paraffin method for sections, 117 sections, mounting of, 119 to stain, 121 paratrophic bacteria, 131 parietti's bouillon, 202 method of isolating coli-typhoid group, 437 parsnip medium, 200 passages of virus, 320 pasteur-chamberland filter, 42 pasteur's pipettes, 10 solution, 191 pathogenesis, investigation of, 315 pathogenic bacteria, 131 study of, 408 pediococci, morphology of, 132 penicillium, 128 peptone rosolic acid water, 186 water (dunham), preparation of, 177 percentage formula, 496 perchloride of mercury, 27 perisporaceæ, 127 peritrichous bacilli, 136 permanent preparations of bacteria, 407 of tissues, 404 petri's dishes, 6 phagocytic index, 392 phenol coefficient, 489 production, test for, 287 photogenic bacteria, 131, 144 physiological filter, 156 picric acid solution, 121 (spengler's), 112 picrocarmine, 97 pigment production, observations on, 288 pipettes, automatic, 13 blood, 11 capillary, 10 cases for, 7 graduated, 6 capillary, 13 pasteur's, 10 sedimentation, 16 standard graduated, 7 teat, 10 throttle, 13 to clean infected, 20 new, 18 to sterilise, 31 piridin method of staining spirochætes, 124 pitfield's flagella stain, 103 plasmolysis, 135 plaster-of-paris discs, 192 plate box, 7 cultures, description of, 261 preparation of, 226 levelling stand, 228 plates, petri's, 6 to clean infected, 20 new, 18 to sterilise, 31 platinum needles, 71 method of mounting, 71 pleomorphism, 133 polar germination, 140 granules, 136 polkoerner, 136 polychrome blood stains, 97 pooled serum, 379 porcelain filter, 42 berkefeld, 42 chamberland, 42 doulton, 42 post-mortem examination of experimental animals, 396 potato gelatine (eisner), 204 (goadby), 214 medium, preparation of, 174 potted meat, analysis of, 460 pouring plates, 227 preparation of experimental animals, 335 preservatives in milk, 442 pressure temperature table, 500 primary colours, action of, 309 proteid free broth (uschinsky), 183 proteolytic enzymes, tests for, 277 prototrophic bacteria, 131 psychrophilic bacteria, 143 pathogenic effects, 315 pus, collection of, 373 pyrogallic acid solution, 293 qualitative analysis of air, 470 of milk, 446 of sewage, 467 of soil, 476 of unsound meat, 462 of water, 426 quantitative analysis of air, 468 of milk, 444 of sewage, 466 of soil, 473 of unsound meat, 460 rabbit cages, 343 scabies, treatment of, 338 scales, 340 raising virulence of organisms, 320 ramsden's micrometer, 66 range of medium reaction, measurement of, 305 of temperature, measurement of, 298 rat cages, 342 raw milk, saul's test for, 442 reaction of medium, 305 optimum, 305 range of, 305 scale, 153 reduced pressure and temperature table, 501 reducing agents, production, 389 tests for, 289 reduction of nitrates, 389 reichert's thermo-regulator, 218 relation of bacteria to environment, 142 removal of material from culture tubes, 74 rennin enzymes, tests for, 279 reproduction of bacteria, 136 resistance glass, 6 to lethal agents, 306 resting stage of bacteria, 137 restrictions upon experimental inoculations, 334 ribbert's capsule stain, 101 roll cultures, 226 rosolic acid peptone solution, 186 rosindol reaction, 286 roux's anaerobic culture method, 237 culture bottle, 5 sabouraud's medium, 193 saccharomyces, morphology of, 129 safranine, 94 salicylic acid in milk, test for, 443 saprogenic bacteria, 131 sarcinæ, morphology of, 132 saul's test, 442 scales, decimal, 340 trip, 164 scalpels, to sterilise, 32, 33 schallibaum's solution, 121 scheme for study of bacteria, 259 schizomycetes, classification of, 131 morphology of, 131 scissors, to sterilise, 32 sealing museum jars, 406 searing iron, 397 sections, special staining methods for, 121 sedimentation pipettes, 16 tubes, 9 selecting objectives, 57 sensitising red blood cells, 395 serial cultivations, 251 serological examination of blood, 378 serum agar (heiman), 210 (kanthack and stevens), 211 (libman), 212 plates, 250 (wertheimer), 211 bouillon, 210 collection of, 379 dextrose water (hiss), 188 inspissator, 169 sugar media (hiss), 188 water, preparation of, 170 sewage, analysis of, qualitative, 467 quantitative, 466 shake cultivations, 225 description of, 271 shape of colonies, 262 shaving experimental animals, 349 shellfish, analysis of, 463 silicate jelly (winogradsky), 198 single stain for spores, 106 size of colonies, 262 slanted tube cultures, 223 slides, to clean new, 22 used, 23 smear culture, 224 description of, 268 soap liquid, 346 soda solution, storage of stock, 154 sodium bicarbonate in milk, test for, 443 soil, analysis of, qualitative, 476 quantitative, 473 collection of samples, 471 solid media, 147 soluble toxins, 144 soyka's milk rice, 189 spear-headed spatula, 402 special media, 182 specific serum, 379 dilution of, 382 spherical aberration, 55 spirillum, morphology of, 133 spirochæta, morphology of, 133 spirochætes in tissues, to stain, 124 spleen extract, 149 sporangium, 127 spore formation, arthrogenous, 138 endogenous, 138 method of, 138, 273 germination, method of, 140, 274 observation of, 140, 273 spores, characters of, 139 classification of, 139 double stain for, 106 to stain, 106 stab culture, 224 description of, 265 stage micrometer, 62 of microscope, 52 staining methods, 90 paraffin sections, 121 reactions of bacteria, 274 stains intra-vitam, 77 negative (burri), 77 rack for, 72 standard graduated pipettes, 7 soda solution, 154 standardisation of media, 154 standardising bouillon, 155 staphylococci, morphology, 132 staphylococcus in milk, 456 steam steriliser, arnold, 35 koch, 35 to use, 35 streaming, 35 sterigma, 127 sterilisation by chemicals, 27 by dry heat, 28 by filters, 40 by moist heat, 32 by streaming steam, 35 by superheated steam, 36 of albuminous liquids, 32 of gases, 40 sterilising agents, 26 stichcultur, 224 stock dilutions, 497 nutrient media, 163 plate for isolation work, 253 storage of media in bulk, 159 of tubed media, 161 store boxes for media, 161 streak culture, 224 description of, 268 streaming movement, 80 steam, 35 streptobacilli, morphology, 133 streptococci in soil, 477 in water, detection of, 432 morphology of, 132 streptococcus pyogenes longus in milk, 455 streptothrix, morphology of, 133 strichcultur, 223 structure, internal, of colonies, 265 study of pathogenic bacteria, 408 subcutaneous inoculation, 353 subdural inoculation, 361 substage condenser, 54 sugar agar, 185 dextrose bouillon, 184 gelatine, 184 media, preparation of, 177 sulphindigotate agar, 181 bouillon (weyl), 181 gelatine (weyl), 181 sulphuretted hydrogen in cultures, test for, 290 sunlight, action of, 309 superheated steam, 36 superior lethal coefficient, 310, 313 suppuration, organisms of, 409 surface characters of colonies, 264 plates, 230 surgical motor, electric, 360 swarm spores, 127 syringe for subcutaneous inoculation of solid material, 354 hypodermic, 344 tatin's operating table, 351 taxonomy, 262 teat-pipettes, 10 temperature, action of, 299 optimum, 298 pressure table, 500 range, 298 taking, 340 test objects for objectives, 57 testing filters, 478 test-tubes, 3 to clean infected, 19 new, 18 to plug, 24 to sterilise, 31 tetracocci, morphology of, 132 thermal death-point, 143 determination of, 298 of spores, 301, 304 of vegetative forms, 298, 303 thermophilic bacteria, 143 thermo-regulators, hearson's capsule, 218 reichert's, 218 thionine blue, 92 thiothrix, morphology of, 133 thresh's water collecting bottle, 418 throttle pipettes, 13 tinned meat, analysis of, 460 tissue medium (noguchi), 214 stains, 95 tissues for sectioning, fixing, 114 freezing, 116 hardening, 114 imbedding, 118 preparation of, 114 washing, 115 titration of media, 150 torulæ, differentiation from saccharomyces, 130 total acidity, 280 toxins, testing of, 318 trephines, 360 triple nosepiece, 58 true motility, 80 tube cultures, preparation of, 222 length, 50 tubercle bacillus in milk, 453 to stain, 110, 124 tuberculous guinea-pig, cadaver of, 454 tubing nutrient media, 160 turnip media, 200 unna-pappenheim's stain for sections, 123 unsound meat, analysis of, 460 urine agar, 188 gelatine, 187 (heller), 188 media bouillon, 187 uschinsky's solution, 183 valency of specific sera, 386 van ermengem's flagella stain, 104 vegetative stage of bacteria, 136 vesuvin, 94 vibrio choleræ in milk, 452 in water, 439 morphology of, 133 virulence, attenuating, 321 of organisms, 320 raising, 320 vivisection license, 334 voges holder, 350 volatile oils as disinfectants, 27 warm stage, 58 washing red blood cells, 388 tissues, 115 water, analysis of, qualitative, 426 quantitative, 416 steriliser, 33 weighing animals, 340 welch's capsule stain, 101 wertheimer's serum agar, 211 wheat bouillon (gasperini), 193 whey agar, 195 gelatine, 195 wine must, 192 winogradsky's solution i, 198 ii, 198 wire crates for test-tubes, 31 wood ash agar, 201 working up plates, 252 wort agar, 176 gelatine, 176 wright's anaerobic method, 239 yeast water (pasteur), 191 ziehl-neelsen's stain, 110 zoogloea, 134 zymogenic bacteria, 131 saunders' books on pathology, physiology histology, embryology bacteriology, biology * * * * * w. b. saunders company west washington square philadelphia 9, henrietta street covent garden, london * * * * * literary superiority the excellent judgment displayed in the publications of the house at the very beginning of its career, and the success of the modern business methods employed by it, at once attracted the attention of leading men in the profession, and many of the most prominent writers of america offered their books for publication. thus, there were produced in rapid succession a number of works that immediately placed the house in the front rank of medical publishers. one need only cite such instances as musser and kelly's treatment, keen's surgery, kelly and noble's gynecology and abdominal surgery, cabot's differential diagnosis, de lee's obstetrics, mumford's surgery, cotton's dislocations and joint fractures, crandon and ehrenfried's surgical after-treatment, sisson's veterinary anatomy, anders and boston's medical diagnosis, gant's constipation and obstruction, jordan's bacteriology, and kemp on stomach, intestines, and pancreas. these books have made for themselves places among the best works on their respective subjects. ~a complete catalogue of our publications will be sent upon request~ mallory's pathologic histology ~pathologic histology.~ by frank b. mallory, m. d., associate professor of pathology, harvard university medical school. octavo of 677 pages, with 497 figures containing 683 original illustrations, 124 in colors. cloth, $5.50 net; half morocco, $7.00 net. ~reprinted in three months~ dr. mallory here presents _pathology_ from the morphologic point of view. he presents his subject biologically, first by ascertaining the cellular elements out of which the various lesions are built up; then he traces the development of the lesions from the simplest to the most complex. he so presents pathology that you are able to trace backward from any given end-result, such as sclerosis of an organ (cirrhosis of the liver, for example), through all the various acute lesions that may terminate in that particular end-result to the primal _cause_ of the lesion. the _illustrations_ are most beautiful. ~dr. w. g. maccallum~, _columbia university_ "i have looked over the book and think the plan is admirably carried out and that the book supplies a need we have felt very much. i shall be very glad to recommend it." * * * * * howell's physiology ~a text-book of physiology.~ by william h. howell, ph.d., m. d., professor of physiology in the johns hopkins university, baltimore, md. octavo of 1020 pages, 306 illustrations. cloth, $4.00 net. ~the new (5th) edition~ dr. howell has had many years of experience as a teacher of physiology in several of the leading medical schools, and is therefore exceedingly well fitted to write a text-book on this subject. main emphasis has been laid upon those facts and views which will be directly helpful in the practical branches of medicine. at the same time, however, sufficient consideration has been given to the experimental side of the science. the entire literature of physiology has been thoroughly digested by dr. howell, and the important views and conclusions introduced into his work. illustrations have been most freely used. ~the lancet, london~ "this is one of the best recent text-books on physiology, and we warmly commend it to the attention of students who desire to obtain by reading a general, all-round, yet concise survey of the scope, facts, theories, and speculations that make up its subject matter." mallory _and_ wright's pathologic technique ~fifth edition~ ~pathologic technique.~ a practical manual for workers in pathologic histology, including directions for the performance of autopsies and for clinical diagnosis by laboratory methods. by frank b. mallory, m. d., associate professor of pathology, harvard university; and james h. wright, m. d., director of the pathologic laboratory, massachusetts general hospital. octavo of 500 pages, with 152 illustrations. cloth, $3.00 net. in revising the book for the new edition the authors have kept in view the needs of the laboratory worker, whether student, practitioner, or pathologist, for a practical manual of histologic and bacteriologic methods in the study of pathologic material. many parts have been rewritten, many new methods have been added, and the number of illustrations has been considerably increased. ~boston medical and surgical journal~ "this manual, since its first appearance, has been recognized as the standard guide in pathological technique, and has become well-nigh indispensable to the laboratory worker." * * * * * eyre's bacteriologic technic ~bacteriologic technic.~ a laboratory guide for the medical, dental, and technical student. by j. w. h. eyre, m. d., f. r. s. edin., director of the bacteriologic department of guy's hospital, london. octavo of 520 pages, 219 illustrations. cloth, $3.00 net. ~just ready--new (2d) edition, rewritten~ dr. eyre has subjected his work to a most searching revision. indeed, so thorough was his revision that the entire book, enlarged by some 150 pages and 50 illustrations, had to be reset from cover to cover. he has included all the latest technic in every division of the subject. his thoroughness, his accuracy, his attention to detail make his work an important one. he gives clearly the technic for the bacteriologic examination of water, sewage, air, soil, milk and its products, meats, etc. and he gives you good technic--methods attested by his own large experience. to any one interested in this line of endeavor the new edition of dr. eyre's work is indispensable. the illustrations are as practical as the text. mcfarland's pathology ~a text-book of pathology.~ by joseph mcfarland, m. d., professor of pathology and bacteriology in the medico-chirurgical college of philadelphia. octavo of 856 pages, with 437 illustrations, many in colors. cloth, $5.00 net; half morocco, $6.50 net. ~the new (2d) edition~ you cannot successfully treat disease unless you have a practical, _clinical_ knowledge of the pathologic changes produced by disease. for this purpose dr. mcfarland's work is well fitted. it was written with just such an end in view--to furnish a ready means of acquiring a thorough training in the subject, a training such as would be of daily help in your practice. for this edition every page has been gone over most carefully, correcting, omitting the obsolete, and adding the new. some sections have been entirely rewritten. you will find it a book well worth consulting, for it is the work of an authority. ~st. paul medical journal~ "it is safe to say that there are few who are better qualified to give a résumé of the modern views on this subject than mcfarland. the subject-matter is thoroughly up to date." ~boston medical and surgical journal~ "it contains a great mass of well-classified facts. one of the best sections is that on the special pathology of the blood." * * * * * mcfarland's biology: medical and general ~biology: medical and general~--by joseph mcfarland, m. d., professor of pathology and bacteriology in the medico-chirurgical college of phila. 12mo, 457 pages, 160 illustrations. cloth, $1.75 net. ~just ready--new (2d) edition~ this work is both a _general_ and _medical_ biology. the former because it discusses the peculiar nature and reactions of living substance generally; the latter because particular emphasis is laid on those subjects of special interest and value in the study and practice of medicine. the illustrations will be found of great assistance. ~frederic p. gorham, a. m.~, _brown university_. "i am greatly pleased with it. perhaps the highest praise which i can give the book is to say that it more nearly approaches the course i am now giving in general biology than any other work." mcfarland's pathogenic bacteria and protozoa ~pathogenic bacteria and protozoa.~ by joseph mcfarland, m. d., professor of pathology and bacteriology in the medico-chirurgical college of philadelphia. octavo of 878 pages, finely illustrated. cloth, $3.50 net. ~new (7th) edition, enlarged~ dr. mcfarland has subjected his book to a most vigorous revision, bringing this edition right down to the minute. important new additions have increased it in size some 180 pages. by far the most important addition is the inclusion of an entirely new section on _pathogenic protozoa_. this section considers every protozoan pathogenic to man; and in that same clean-cut, definite way that won for mcfarland's work a place in the very front of medical bacteriologies. the illustrations are the best the world affords, and are beautifully executed. ~h. b. anderson, m. d.~, _professor of pathology and bacteriology, trinity medical college, toronto._ "the book is a satisfactory one, and i shall take pleasure in recommending it to the students of trinity college." ~the lancet, london~ "it is excellently adapted for the medical students and practitioners for whom it is avowedly written.... the descriptions given are accurate and readable." * * * * * hill's histology and organography ~a manual of histology and organography.~ by charles hill, m. d., formerly assistant professor of histology and embryology, northwestern university, chicago. 12mo of 468 pages, 337 illustrations. flexible leather, $2.00 net. ~the new (2d) edition~ dr. hill's work is characterized by a completeness of discussion rarely met in a book of this size. particular consideration is given the mouth and teeth. ~pennsylvania medical journal~ "it is arranged in such a manner as to be easy of access and comprehension. to any contemplating the study of histology and organography we would commend this work." * * * * * get the new the best standard american illustrated dictionary ~new (7th) edition--5000 sold in two months~ ~the american illustrated medical dictionary.~ a new and complete dictionary of the terms used in medicine, surgery, dentistry, pharmacy, chemistry, veterinary science, nursing, and kindred branches; with over 100 new and elaborate tables and many handsome illustrations. by w. a. newman dorland, m.d., editor of "the american pocket medical dictionary." large octavo, 1107 pages, bound in full flexible leather. price, $4.50 net; with thumb index, $5.00 net. ~it defines all the new words--it is up to date~ the american illustrated medical dictionary defines hundreds of the newest terms not defined in any other dictionary--bar none. these new terms are live, active words, taken right from modern medical literature. it gives the capitalization and pronunciation of all words. it makes a feature of the derivation or etymology of the words. in some dictionaries the etymology occupies only a secondary place, in many cases no derivation being given at all. in the american illustrated practically every word is given its derivation. every word has a separate paragraph, thus making it easy to find a word quickly. the tables of arteries, muscles, nerves, veins, etc., are of the greatest help in assembling anatomic facts. in them are classified for quick study all the necessary information about the various structures. every word is given its definition--a definition that _defines_ in the fewest possible words. in some dictionaries hundreds of words are not defined at all, referring the reader to some other source for the information he wants at once. ~howard a, kelly, m. d.~, _johns hopkins university, baltimore._ "the american illustrated dictionary is admirable. it is so well gotten up and of such convenient size. no errors have been found in my use of it." ~j. collins warren, m. d., ll.d., f.r.c.s. (hon.)~, _harvard medical school_ "i regard it as a valuable aid to my medical literary work. it is very complete and of convenient size to handle comfortably. i use it in preference to any other." * * * * * stengel's text-book of pathology ~fifth edition~ ~a text-book of pathology.~ by alfred stengel, m. d., professor of medicine in the university of pennsylvania. octavo volume of 979 pages, with 400 text-illustrations, many in colors, and 7 full-page colored plates. cloth, $5.00 net; sheep or half morocco, $6.50 net. ~with 400 text-cuts, many in colors, and 7 colored plates~ in this work the practical application of pathologic facts to clinical medicine is considered more fully than is customary in works on pathology. while the subject of pathology is treated in the broadest way consistent with the size of the book, an effort has been made to present the subject from the point of view of the clinician. in the second part of the work the pathology of individual organs and tissues is treated systematically and quite fully under subheadings that clearly indicate the subject-matter to be found on each page. in this edition the section dealing with general pathology has been most extensively revised, several of the important chapters having been practically rewritten. ~the lancet, london~ "this volume is intended to present the subject of pathology in as practical a form as possible, and more especially from the point of view of the 'clinical pathologist.' these objects have been faithfully carried out, and a valuable text-book is the result. we can most favorably recommend it to our readers as a thoroughly practical work on clinical pathology." * * * * * stiles' nutritional physiology ~nutritional physiology.~ by percy goldthwait stiles, assistant professor of physiology at simmons college, boston. 12mo of 295 pages, illustrated. cloth, $1.25 net. ~illustrated~ this new work expresses the most advanced views on this important subject. it discusses in a concise way the processes of digestion and metabolism. the key-word of the book throughout is "energy"--its source and its conservation. "it is remarkable for the fineness of its diction and for its clear presentation of the subject, relieved here and there by a quaintly humorous turn of phrase that is altogether delightful."--_colin c. stewart, ph. d., dartmouth college._ * * * * * jordan's general bacteriology ~a text-book of general bacteriology.~ by edwin o. jordan, ph.d., professor of bacteriology in the university of chicago and in rush medical college. octavo of 623 pages, illustrated. cloth, $3.00 net. ~new (3d) edition~ professor jordan's work embraces the entire field of bacteriology, the non-pathogenic as well as the pathogenic bacteria being considered, giving greater emphasis, of course, to the latter. there are extensive chapters on methods of studying bacteria, including staining, biochemical tests, cultures, etc.; on the development and composition of bacteria; on enzymes and fermentation-products; on the bacterial production of pigment, acid and alkali; and on ptomaines and toxins. especially complete is the presentation of the serum treatment of gonorrhea, diphtheria, dysentery, and tetanus. the relation of bovine to human tuberculosis and the ocular tuberculin reaction receive extensive consideration. this work will also appeal to academic and scientific students. it contains chapters on the bacteriology of plants, milk and milk-products, air, agriculture, water, food preservatives, the processes of leather tanning, tobacco curing, and vinegar making; the relation of bacteriology to household administration and to sanitary engineering, etc. ~prof. severance burrage~, _associate professor of sanitary science, purdue university._ "i am much impressed with the completeness and accuracy of the book. it certainly covers the ground more completely than any other american book that i have seen." * * * * * buchanan's veterinary bacteriology ~veterinary bacteriology.~ by robert e. buchanan, ph.d., professor of bacteriology in the iowa state college of agriculture and mechanic arts. octavo, 516 pages, 214 illustrations. cloth, $3.00 net. ~the best published~ professor buchanan discusses thoroughly all bacteria causing diseases of the domestic animals. he goes minutely into the consideration of immunity, opsonic index, reproduction, sterilization, antiseptics, biochemic tests, culture-media, isolation of cultures, the manufacture of the various toxins, antitoxins, tuberculins, and vaccines that have proved of diagnostic or therapeutic value. then, in addition to bacteria and protozoa proper, he considers molds, mildews, smuts, rusts, toadstools, puff-balls, and the other fungi pathogenic for animals. ~b. f. kaupp, d. v. s.~, _state agricultural college, fort collins._ "it is the best in print on the subject. what pleases me most is that it contains all the late results of research. it fills a long felt want." heisler's embryology ~a text-book of embryology.~ by john c. heisler, m.d., professor of anatomy in the medico-chirurgical college, philadelphia. octavo volume of 435 pages, with 212 illustrations, 32 of them in colors. cloth, $3.00 net. ~third edition--with 212 illustrations, 32 in colors~ this edition represents all the advances recently made in the science of embryology. many portions have been entirely rewritten, and a great deal of new and important matter added. a number of new illustrations have also been introduced and these will prove very valuable. heisler's embryology has become a standard work. ~g. carl huber, m.d.~, _professor of embryology at the wistar institute, university of pennsylania._ "i find this edition of 'a text-book of embryology,' by dr. heisler, an improvement on the former one. the figures added increase greatly the value of the work. i am again recommending it to our students." * * * * * böhm, davidoff, _and_ huber's histology ~a text-book of human histology.~ including microscopic technic. by dr. a. a. böhm and dr. m. von davidoff, of munich, and g. carl huber, m.d., professor of embryology at the wistar institute, university of pennsylvania. handsome octavo of 528 pages, with 361 beautiful original illustrations. flexible cloth, $3.50 net. ~second edition, enlarged~ the work of drs. böhm and davidoff is well known in the german edition, and has been considered one of the most practically useful books on the subject of human histology. this second edition has been in great part rewritten and very much enlarged by dr. huber, who has also added over one hundred original illustrations. dr. huber's extensive additions have rendered the work the most complete students' text-book on histology in existence. ~boston medical and surgical journal~ "is unquestionably a text-book of the first rank, having been carefully written by thorough masters of the subject, and in certain directions it is much superior to any other histological manual." * * * * * wells' chemical pathology ~chemical pathology.~--being a discussion of general pathology from the standpoint of the chemical processes involved. by h. gideon wells, ph.d., m.d., assistant professor of pathology in the university of chicago. octavo of 616 pages. cloth, $3.25 net. ~just ready--new (2d) edition~ dr. wells' work is written for the physician, for those engaged in research in pathology and physiologic chemistry, and for the medical student. in the introductory chapter are discussed the chemistry and physics of the animal cell, giving the essential facts of ionization, diffusion, osmotic pressure, etc., and the relation of these facts to cellular activities. special chapters are devoted to _diabetes_ and to _uric-acid metabolism and gout_. ~wm. h. welch, m.d.~ _professor of pathology, johns hopkins university._ "the work fills a real need in the english literature of a very important subject, and i shall be glad to recommend it to my students." * * * * * lusk's elements of nutrition ~elements of the science of nutrition.~ by graham lusk, ph.d., professor of physiology at cornell medical school. octavo volume of 302 pages. cloth, $3.00 net. ~the new (2d) edition--translated into german~ prof. lusk presents the scientific foundations upon which rests our knowledge of nutrition and metabolism, both in health and in disease. there are special chapters on the metabolism of diabetes and fever, and on purin metabolism. the work will also prove valuable to students of _animal dietetics_ at agricultural stations. ~lewellys f. barker, m. d.~ _professor of the principles and practice of medicine, johns hopkins university._ "i shall recommend it highly to my students. it is a comfort to have such a discussion of the subject in english." daugherty's economic zoölogy ~economic zoölogy.~ by l. s. daugherty, m. s., ph. d., professor of zoölogy, state normal school, kirksville, mo., and m. c. daugherty, author with jackson of "agriculture through the laboratory and school garden." part i: _field and laboratory guide_. 12mo of 237 pages, interleaved. cloth, $1.25 net. part ii: _principles._ 12mo of 406 pages, illustrated. cloth, $2.00 net. ~illustrated~ there is no other book just like this. not only does it give the salient facts of structural zoölogy and the development of the various branches of animals, but also the natural history--the _life and habits_--thus showing the interrelations of structure, habit, and environment. in a word, it gives the principles of zoölogy and _their actual application_. the economic phase is emphasized. part i--the _field and laboratory guide_--is designed for practical instruction in the field and laboratory. to enhance its value for this purpose blank pages are inserted for notes. * * * * * drew's invertebrate zoölogy ~a laboratory manual of invertebrate zoölogy.~ by gilman a. drew, ph. d., assistant director at marine biological laboratory, woods hole, mass. with the aid of former and present members of the zoölogical staff of instructors. 12mo of 213 pages. cloth, $1.25 net. ~just ready--new (2d) edition~ the subject is presented in a logical way, and the type method of study has been followed, as this method has been the prevailing one for many years. ~prof. allison a. smyth, jr., virginia polytechnic institute~ "i think it is the best laboratory manual of zoölogy i have yet seen. the large number of forms dealt with makes the work applicable to almost any locality." * * * * * norris' cardiac pathology ~studies in cardiac pathology.~ by george w. norris, m.d., associate in medicine at the university of pennsylvania. large octavo of 235 pages, with 85 superb illustrations. cloth, $5.00 net. ~superb illustrations~ the wide interest being manifested in heart lesions makes this book particularly opportune. the illustrations are superb and are faithful reproductions of the specimens photographed. each illustration is accompanied by a detailed description; besides, there is ample letter press supplementing the pictures. considerable matter of a diagnostic and therapeutic nature has been interwoven. ~boston medical and surgical journal~ "the illustrations are arranged in such a way as to illustrate all the common and many of the rare cardiac lesions, and the accompanying descriptive text constitutes a fairly continuous didactic treatise." * * * * * mcconnell's pathology ~a manual of pathology.~ by guthrie mcconnell, m.d., professor of bacteriology and pathology at temple university, philadelphia. 12mo of 523 pages, with 170 illustrations. flexible leather, $2.50 net. ~new (2d) edition~ dr. mcconnell has discussed his subject with a clearness and precision of style that make the work of great assistance to both student and practitioner. the illustrations have been introduced for their practical value. ~new york state journal of medicine~ "the book treats the subject of pathology with a thoroughness lacking in many works of greater pretension. the illustrations--many of them original--are profuse and of exceptional excellence." * * * * * hektoen and riesman's pathology american text-book of pathology. edited by ludvig hektoen, m.d., professor of pathology, rush medical college, chicago; and david riesman, m.d., professor of clinical medicine, philadelphia polyclinic. octavo of 1245 pages, 443 illustrations, 66 in colors. cloth, $7.50 net; half morocco, $9.00 net. dürck _and_ hektoen's special pathologic histology ~atlas and epitome of special pathologic histology.~ by dr. h. dürck, of munich. edited, with additions, by ludvig hektoen, m. d., professor of pathology, rush medical college, chicago. in two parts. part i.--circulatory, respiratory, and gastro-intestinal tracts. 120 colored figures on 62 plates, and 158 pages of text. part ii.--liver, urinary and sexual organs, nervous system, skin, muscles, and bones. 123 colored figures on 60 plates, and 192 pages of text. per part: cloth, $3.00 net. _in saunders' hand-atlas series._ the great value of these plates is that they represent in the exact colors the effect of the stains, which is of such great importance for the differentiation of tissue. the text portion of the book is admirable, and, while brief, it is entirely satisfactory in that the leading facts are stated, and so stated that the reader feels he has grasped the subject extensively. ~william h. welch, m.d.,~ _professor of pathology, johns hopkins university, baltimore._ "i consider dürck's 'atlas of special pathologic histology,' edited by hektoen, a very useful book for students and others. the plates are admirable." * * * * * sobotta _and_ huber's human histology ~atlas and epitome of human histology.~ by privatdocent dr. j. sobotta, of würzburg. edited, with additions, by g. carl huber, m. d., professor of histology and embryology in the university of michigan, ann arbor. with 214 colored figures on 80 plates, 68 text-illustrations, and 248 pages of text. cloth, $4.50 net. _in saunders' hand-atlas series._ ~including microscopic anatomy~ the work combines an abundance of well-chosen and most accurate illustrations, with a concise text, and in such a manner as to make it both atlas and text-book. the great majority of the illustrations were made from sections prepared from human tissues, and always from fresh and in every respect normal specimens. the colored lithographic plates have been produced with the aid of over thirty colors. ~boston medical and surgical journal~ "in color and proportion they are characterized by gratifying accuracy and lithographic beauty." bosanquet on spirochætes ~spirochætes~: a review of recent work, with some original observations. by w. cecil bosanquet, m.d., fellow of the royal college of physicians, london. octavo of 152 pages, illustrated. $2.50 net. ~illustrated~ this is a complete and authoritative monograph on the spirochætes, giving morphology, pathogenesis, classification, staining, etc. pseudospirochætes are also considered, and the entire text well illustrated. the high standing of dr. bosanquet in this field of study makes this new work particularly valuable. * * * * * levy _and_ klemperer's clinical bacteriology ~the elements of clinical bacteriology.~ by drs. ernst levy and felix klemperer, of the university of strasburg. translated and edited by augustus a. eshner, m. d., professor of clinical medicine, philadelphia polyclinic. octavo volume of 440 pages, fully illustrated. cloth, $2.50 net. ~s. solis-cohen, m.d.~, _professor of clinical medicine, jefferson medical college_, philadelphia. "i consider it an excellent book. i have recommended it in speaking to my students." * * * * * lehmann, neumann, _and_ weaver's bacteriology ~atlas and epitome of bacteriology~: including a text-book of special bacteriologic diagnosis. by prof. dr. k. b. lehmann and dr. r. o. neumann, of würzburg. _from the second revised and enlarged german edition._ edited, with additions, by g. h. weaver, m. d., assistant professor of pathology and bacteriology, rush medical college, chicago. in two parts. part i.--632 colored figures on 69 lithographic plates. part ii.--511 pages of text, illustrated. per part: cloth, $2.50 net. _in saunders' hand-atlas series._ * * * * * dürck and hektoen's general pathologic histology atlas and epitome of general pathologic histology. by pr. dr. h. dürck, of munich. edited, with additions, by ludvig hektoen, m. d., professor of pathology in rush medical college, chicago. 172 colored figures on 77 lithographic plates, 36 text-cuts, many in colors, and 353 pages. cloth, $5.00 net. _in saunders' hand atlas series._ american text-book of physiology second edition american text-book of physiology. in two volumes. edited by william h. howell, ph. d., m.d., professor of physiology in the johns hopkins university, baltimore, md. two royal octavos of about 600 pages each, illustrated. per volume: cloth, $3.00 net; half morocco, $4.25 net. "the work will stand as a work of reference on physiology. to him who desires to know the status of modern physiology, who expects to obtain suggestions as to further physiologic inquiry, we know of none in english which so eminently meets such a demand."--_the medical news._ warren's pathology and therapeutics second edition surgical pathology and therapeutics. by john collins warren, m. d., ll. d., f. r. c. s. (hon.), professor of surgery, harvard medical school. octavo, 873 pages, 136 relief and lithographic illustrations, 33 in colors. with an appendix on scientific aids to surgical diagnosis and a series of articles on regional bacteriology. cloth, $5.00 net; half morocco, $6.50 net. gorham's bacteriology a laboratory course in bacteriology. for the use of medical, agricultural, and industrial students. by frederic p. gorham, a. m., associate professor of biology in brown university, providence, r. i., etc. 12mo of 192 pages, with 97 illustrations. cloth, $1.25 net. "one of the best students' laboratory guides to the study of bacteriology on the market.... the technic is thoroughly modern and amply sufficient for all practical purposes."--_american journal of the medical sciences._ raymond's physiology new (3d) edition human physiology. by joseph h. raymond, a. m., m. d., professor of physiology and hygiene, long island college hospital, new york. octavo of 685 pages, with 444 illustrations. cloth, $3.50 net. "the book is well gotten up and well printed, and may be regarded as a trustworthy guide for the student and a useful work of reference for the general practitioner. the illustrations are numerous and are well executed."--_the lancet_, london. ball's bacteriology seventh edition, revised essentials of bacteriology: being a concise and systematic introduction to the study of micro-organisms. by m. v. ball, m. d., late bacteriologist to st. agnes' hospital, philadelphia. 12mo of 289 pages, with 135 illustrations, some in colors. cloth, $1.00 net. _in saunders' question-compend series._ "the technic with regard to media, staining, mounting, and the like is culled from the latest authoritative works."--_the medical times_, new york. budgett's physiology new (3d) edition essentials of physiology. prepared especially for students of medicine, and arranged with questions following each chapter. by sidney p. budgett, m. d., formerly professor of physiology, washington university, st. louis. revised by havan emerson, m. d., demonstrator of physiology, columbia university. 12mo volume of 250 pages, illustrated. cloth, $1.00 net. _saunders' question-compend series._ "he has an excellent conception of his subject.... it is one of the most satisfactory books of this class"--_university of pennsylvania medical bulletin._ leroy's histology new (4th) edition essentials of histology. by louis leroy, m. d., professor of histology and pathology, vanderbilt university, nashville, tennessee. 12mo, 263 pages, with 92 original illustrations. cloth, $1.00 net. _in saunders' question-compend series._ "the work in its present form stands as a model of what a student's aid should be; and we unhesitatingly say that the practitioner as well would find a glance through the book of lasting benefit."--_the medical world_, philadelphia. barton and wells' medical thesaurus a thesaurus of medical words and phrases. by wilfred m. barton, m. d., assistant professor of materia medica and therapeutics, and walter a. wells, m. d., demonstrator of laryngology, georgetown university, washington, d.c. 12mo, 534 pages. flexible leather, $2.50 net; thumb indexed, $3.00 net. american pocket dictionary new (8th) edition dorland's pocket medical dictionary. edited by w. a. newman dorland, m. d., editor "american illustrated medical dictionary." containing the pronunciation and definition of the principal words used in medicine and kindred sciences, with 64 extensive tables. 677 pages. flexible leather, with gold edges, $1.00 net; with patent thumb index, $1.25 net. "i can recommend it to our students without reserve."--j. h. holland, m.d., _of the jefferson medical college_, philadelphia. 