Academic Journal of Science and Technology 
ISSN: 2771-3032 | Vol. 9, No. 3, 2024 

 

134 

Comparison	of	Methods	Between	the	Multitube	
Fermentation	Method	and	The	Filter	Membrane	
Method	for	Escherichia	Coli	in	Drinking	Water	
Yuanjing Qiu, Chao Han, Tao Dong 

Kaifeng Product Quality Inspection and Testing Center, Kaifeng 475000, Henan, China 

 

Abstract: Escherichia coli is a normal parasitic bacterium in the intestinal tract and is an important indicator of the degree of 
contamination and hygienic quality of water bodies. In this study, 78 samples of domestic drinking water were tested for 
Escherichia coli by filter membrane method and multi-tube fermentation method. The results showed that the passing rate of 
water samples tested by the multi-tube fermentation method was higher than that of the filter membrane method, which was 
statistically significant (P < 0.05). In the untreated water samples test, the detection rate of Escherichia coli was 92.31% in the 
multi-tube fermentation method compared to 71.79% in the filter membrane method, and the difference was statistically 
significant. The results of the study showed that the detection rate and sensitivity of the multi-tube fermentation method in the 
microbiological testing of domestic drinking water were higher and better than that of the filter membrane method. The selection 
of testing methods should consider its applicability and the accuracy of the test results. Meanwhile, factors affecting the quality 
of microbiological testing include the quality of laboratory personnel, specimen handling, and the quality of instruments and 
reagents, which need to be carefully managed and trained. This study can provide a practical reference for microbiological testing 
of domestic drinking water. 

Keywords: Escherichia coli, domestic drinking water, filtration membrane method, multi-tube fermentation method, test pass 
rate. 

 

1. Summarize 
Escherichia coli is also known as Escherichia coli or 

common coli it is a normal parasitic bacterium in the intestinal 
tract of humans and mongoloid animals and serves as the best 
indicator of stool-like contamination[1] . Coliforms are the 
most prevalent and abundant group of bacteria in the 
intestinal tract and are highly resistant. Moreover, the number 
of coliforms in water is correlated with enteric pathogens, 
which is an important biological indicator for judging the 
degree of contamination and the quality of hygiene of a body 
of water in the international arena. It is of great significance 
for the timely response to the microbial contamination of 
drinking water and surface water sources, and for the effective 
prevention of the outbreak of waterborne diseases[2] . 

2. Information and Methods 

2.1. General information 
The 78 samples of domestic drinking water sent to our 

center for inspection from May 2020 to November 2022 were 
used as experimental subjects, and were divided into two 
groups of 39 samples each based on the inspection method, 
with the control group adopting the filter membrane method 
and the observation group adopting the multi-tube 
fermentation method. The study was carried out in full 
compliance with the relevant provisions of the Hygienic 
Standard for Drinking Water, the total number of coliforms 
in drinking water was ≤100 CFU/mL, all water samples were 
tested within the time limit, and the temperature and humidity 
of the environment met the relevant requirements before the 
test. 

2.2. Methodology 
2.2.1. Filter membrane method test 

Filtration tests were carried out on water samples using a 
microporous filter membrane (0.45 μm). During the operation, 
the membrane was adhered to lactose and then incubated at a 
constant temperature, during which the membrane produced 
gas and acid, indicating the presence of total coliforms in the 
water sample. ① Equipment: micropore filter membrane 
(0.45μm), toothless tweezers, filters, balances, filtration 
equipment, microscopes, petri dishes, conical flasks, 
graduated pipettes, slides. ②  operation process: the first 
sterilization, sterilization, sterilization for the membrane 
sterilization and filter sterilization, membrane sterilization 
step is to paste the membrane into the beaker, at the same time, 
distilled water into the beaker, continue to boil the water 3 
times, so as to play the sterilization effect, each sterilization 
time for 10 ~ 15 min. add hot water, the first and second time 
to change the boiling water, so as to make the container 
residual reagents to be fully cleared, followed by boiling the 
third time to change the water, the first and second time, so 
that the container can be fully cleared, and then the second 
time to boil the water, the first and third times to change the 
water, so that the container can be fully cleared. Subsequently, 
boil the third change into the water; filter sterilization is 
mainly through the flame high temperature to achieve 
sterilization effect. During the actual operation, the alcohol 
cotton ball will be ignited, and the sterilization operation will 
be carried out, and the sterilization time is usually 20~25 
min.Secondly, the water sample needs to be filtered, in this 
operation, the sterile toothless tweezers should be used to 
sterilize the filter membrane, put the filter membrane on the 
sterile filter bed, open the filter, inject 100mL water sample 
into it, open the filter valve, and carry out the pumping 



 

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operation of the water sample, so as to realize the purpose of 
filtering the water sample. The purpose of filtering water 
samples can be realized in this way. Finally, the membrane 
bacteria need to be cultured. This process requires the 
completion of the filtration of water samples, followed by 
water samples immediately start pumping, pumping is 
completed, close the filter valve, the use of sterile toothless 
tweezers will be sent to the filter membrane magenta sodium 
sulfite medium, followed by the use of thermostat for its 
culture. 

2.2.2. Multi-tube fermentation test 
The equipment used in the multi-tube fermentation method 

was the same as that of the filter membrane method to carry 
out the culture of total Escherichia coli flora. ① The culture 
solution was made as follows: add 25g of peptone, 15g of 
lactose, 15g of sodium chloride and 10g of beef paste to 
1000mL of distilled water, heat and dissolve it, adjust the pH 
value to 7.1~7.5, add 2mL of bromocresol violet ethanol 
solution (concentration of 1.50%) to it, mix it thoroughly and 
then put it into the test tubes, put them into autoclave, and 
then put them into autoclave, and then put them into 
refrigerator for storage and reserve at 115℃. Fully sterilized 
for 20 min, to ensure that the sterilization is sufficiently 
removed and stored in the refrigerator. Add 1 mL of drinking 
water sample to the test tube containing 10 mL of culture 
solution of single lactose peptone, inject 10 mL of drinking 
water sample into the test tube containing 10 mL of culture 
solution of double lactose peptone, and at the same time, put 
1 mL of drinking water sample into the test tube containing 9 
mL of physiological saline, mix well, and then select 1 m L of 
the mixture, and then inject 1 m L of culture solution of single 
lactose peptone into it, if the condition of the drinking water 
sample is more serious, it may be appropriate to put it in a 
refrigerator and store it for spare. If the drinking water sample 
is in serious condition, the dilution can be increased 
appropriately. After finishing the above operations, the 
samples were incubated at 36 ℃ in a thermostat. After 24 

hours of incubation, if there is no gas and acid production in 
the test tube, it indicates that the coliforms are negative; if 
there is gas and acid production in the test tube, it is necessary 
to put the test tube into Erythromycin blue agar plate medium. 
Erythromycin agar plate medium was prepared as follows: 25 
g of agar was placed in 500 m L of distilled water, then 8 g of 
peptone and 1.5 g of dipotassium phosphate were added to it, 
and heated to dissolve them completely. Then, based on a 
certain dose, it was filled into a dispensing bottle, cooled, 
sterilized and solidified. After the transfer, it was again put 
into the constant temperature box for cultivation, and after the 
completion of cultivation, it was necessary to carry out colony 
morphology observation and microscopic verification of the 
central area with darker staining color. 

2.3. Observation indicators   
In this study, the test microbial flora is mainly Escherichia 

coli to drinking water health standards as the basis for the two 
test methods of microbiological indicators of water samples 
detection status, detection rate, pass rate to develop a 
statistical comparison. 

2.4. Statistical methods   
Data were processed by SPSS 24.0 statistical software, 

count data were expressed as relative numbers, and 
comparisons between groups were made using the 
𝑥 test, P < 0.05 suggests that the difference is statistically 

significant. 

3. Results 

3.1. Comparison of the passing rate of water 
samples of the two inspection methods   

The passing rate of water sample inspection of multi-tube 
fermentation method was higher than that of filter membrane 
method, and the difference was statistically significant (P < 
0.05). See Table 1. 

 
Table 1. Comparison of the passing rate of water samples of the two inspection methods 

groups n 
Plant water 

(n=10) 
End-of-network water 

(n=9) 
Secondary water 

supply (n=20) 
Unqualified 

water (portions) 
satisfactory 

rate 
Observation 

Group 
39 9 (23.08) 8 (20.51) 20 (51.28) 2 (5.13) 94.87% 

control 
subjects 

39 7 (17.95) 6 (15.38) 17 (43.59) 9 (23.08) 76.92% 

𝑥     5.186 
P    0.023 

 

3.2. Analysis of microbial detection in domestic 
drinking water   

The effect of different water treatments on test results was 
not considered in this study. During the testing of untreated 
water samples, the detection rate of Escherichia coli was 
92.31% (36/39) in the multi-tube fermentation method and 
71.79% (28/39) in the filter membrane method, and the 
microbial detection rate of the multi-tube fermentation 
method was significantly higher than that of the filter 
membrane method, and the difference was statistically 
significant (P < 0.05). 

4. Conclusion and Discussion 
The results of this study showed that the detection rate of 

Escherichia coli was higher in the multi-tube fermentation 
method than in the filter membrane method (P < 0.05), and 
the difference in the data was significant when compared with 
the passing rate of the water samples in the two testing 
methods. It can be seen that the multi-tube fermentation 
method in the microbiological testing of domestic drinking 
water detection rate, sensitivity is higher than the filter 
membrane method. The reason for this is that multi-tube 
fermentation method is a new type of drinking water testing 
method, which can clearly identify the Escherichia coli and 
other flora in drinking water, and the quality of water quality 
identification of high quality, the effect is ideal. For this test 
method, the accuracy of the results will continue to shrink 
with the degree of reliability of the data, reagent dilution 
times and so on. It can be seen that in the microbiological 



 

136 

testing of domestic drinking water, the accuracy of the test 
results need to be determined by the results of the data. In 
order to minimize the degree of contamination of drinking 
water, relevant workers should start from the source, 
strengthen the supervision of water sources, and do a good job 
of related missionary education. He Min [3] to 186 samples 
of domestic drinking water (March 2017 to March 2018) as 
the object of study, of which 93 in the abundant water period, 
93 in the dry water period, respectively, the implementation 
of the filter membrane method, the multi-tube fermentation 
method detection, the study found that the multi-tube 
fermentation method in the dry water period heat-resistant 
bacterial groups, Escherichia coli detection rate [ heat-
resistant bacterial groups (94.62%); Escherichia coli 
(91.40%)] than the filter membrane method The detection rate 
of heat-resistant coliforms and Escherichia coli was higher 
than that of filter membrane method [ heat-resistant coliforms 
(79.57%); Escherichia coli (58.06%)], and the detection rate 
of heat-resistant coliforms and Escherichia coli during the 
plentiful water period of the multitube fermentation method 
[ heat-resistant coliforms (80.65%); Escherichia coli 
(96.77%)] was higher than that of the filter membrane method 
[ heat-resistant coliforms (79.57%); Escherichia coli 
(93.55%)], which showed that both methods were more 
effective than the filter membrane method in detecting heat-
resistant coliforms, and Escherichia coli. It can be seen that 
both methods are suitable for microbiological testing of 
domestic drinking water, and the multitube fermentation 
method is better than the filter membrane method. The results 
of He Min's study were basically the same as those of the 
present study. 

Water is the source of life, and people's lives are closely 
related, whether it is daily life, or business production 
activities are inseparable from water. For drinking water, its 
quality usually has a direct impact on human health and 
safety[4] . Regular testing of drinking water is the key to 
ensuring the safety of drinking water and reducing the 
incidence of related diseases. The World Health Organization 
survey shows that the number of intestinal infectious diseases 
caused by water contamination worldwide is up to 1.2 billion, 
and 4 million children have died due to water contamination 
diseases. Therefore, it is necessary to strengthen the quality of 
drinking water inspection and control of the attention is very 
necessary. And with the advancement of China's industrial 
process, resulting in more and more serious environmental 
pollution problems, this situation also has a direct impact on 
the quality of existing water resources, so that there is a lack 
of water resources in our country, water quality is poor and 
other issues, if you do not take timely and effective measures 
to cope with the problem, it is very likely to have a negative 
impact on the development of the community[5] . In view of 
this, the relevant departments should strengthen the detection 
of drinking water, analyze its problems, and on this basis, the 
application of targeted strategies, timely control, rectification 
of contaminated water sources, to prevent serious 
consequences. In addition, water quality testing can also 
avoid the spread of epidemics, through the detection of 
microorganisms in the drinking water, to see whether there 
are infectious diseases and other adverse factors, from this 
perspective, the microorganisms in the drinking water testing 
has a very high social value. Analyzed from the level of 
detection methods, the current testing methods used are filter 
membrane method, multi-tube fermentation method, etc. 
Multi-tube fermentation method mainly refers to the dilution 

of water samples to culture colonies, based on the difference 
in dilution concentration, respectively, the colonies were 
observed, the number of statistical comparison of the unit 
volume[6] . Membrane test refers to the filter filtration of 
water samples, put the filter membrane in the culture medium 
culture, and then count the number of E. coli unit volume of 
the filter membrane. These two testing methods have their 
own advantages and disadvantages, compared to the filter 
membrane method of testing, multi-tube fermentation test 
higher pass rate, while the filter membrane test is less difficult 
to operate, higher efficiency, both methods are suitable for 
drinking water microbiological testing work . However, the 
comparison of test results show that in the untreated water 
samples test, multi-tube fermentation bacterial detection rate 
is higher than the filter membrane method, and in the 
detection of drinking water, compared with the filter 
membrane method, multi-tube fermentation detection of 
water quality pass rate is higher, on behalf of the multi-tube 
fermentation test is more sensitive, can be more accurately 
detected bacterial colonies. 

This paper is based on continuous optimization measures 
for drinking water microbiological testing work to carry out a 
more in-depth analysis, found that the factors affecting the 
quality of microbiological testing mainly include the 
following points. ①  laboratory personnel's ability and 
quality factors. As microbiological testing work on the quality 
of testing personnel, skills level requirements, if the testing 
personnel skills are rusty or less experience, it will inevitably 
have a certain impact on the quality of test results. ② 
specimen factors. If the water sampling personnel are difficult 
to send samples to the laboratory in a timely manner, easy to 
make the sample contaminated by external factors, or 
improper storage makes the death of bacteria, etc., will affect 
the quality of the test. So the pre-test quality assurance work 
on the quality of the test there is a critical impact, the CDC or 
the relevant departments need to strengthen the importance of 
this area, targeted training to reduce the impact of factors 
related to the test before the test on the quality of the test. ③ 
instrument factors. Microbiological testing work using more 
instruments, instrument calibration, maintenance, etc. are 
likely to affect the accuracy of the test results. ④ reagent 
factors. Reagent quality has a critical impact on the quality of 
microbiological testing. Therefore, the relevant personnel 
must strengthen the reagent quality control, so as to reduce 
the impact of reagent factors on the quality of testing. In order 
to further improve the quality of microbiological testing, the 
CDC and the relevant parts of the CDC can also be targeted 
to set up a professional inspection team, scientific division of 
labor, the construction of a perfect, scientific rules and 
regulations, to improve the sense of responsibility of the 
inspectors, mobilize their enthusiasm. Regular organization 
of professional training, timely consolidation of the 
knowledge base of the test personnel, update, so as to improve 
the business level of the test personnel, to ensure the quality 
of the test. 

In summary, the multi-tube fermentation method and the 
filter membrane method are both applicable to the testing of 
domestic drinking water, and can effectively test the number 
of microbial colonies in water, but compared with the filter 
membrane method, the multi-tube fermentation method has a 
higher detection rate. However, it should be noted that in this 
study  

The relatively small number of samples of drinking water 
selected and the short time of the study will inevitably have 



 

137 

certain deficiencies; follow-up clinical still need to carry out 
a larger sample size, more in-depth study to further determine 
the test value of the two testing methods of multi-tube 
fermentation and membrane method 

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