Bull


 

 

73 

Muhammad. I. G. Al-Janabi 
 

Bull. Iraq Nat. Hist. Mus. 

(2014) 13 (1): 73-79 

 

A DESCRIPTION STUDY OF TWO LOCAL FISH  HIMRI 

CARASOBARBUS LUTEUS (Heckel, 1843)(CYPRINIFORMES: 

CYPRINIDAE) AND HISHNI  LIZA ABU (Heckel, 1843)            

(MUGILOIDEI : MUGILIDAE) BY BONES STAINING METHOD 
 

Muhammad. I. G. Al-Janabi 

Iraq Natural History Research Center and Museum, University of Baghdad, 

Bab Al-Muadham, Baghdad, Iraq 

 

ABSTRACT  
    Two local fish Himri Carasobarbus luteus (Heckel, 1843)  and Hishni Liza abu (Heckel, 

1843) were stained with Alizarin Red and featured some anatomical qualities which cleared 

the difference of the muscular and skeletal fabric for each fish. Since clear Histologic 

differences appeared in these two species, it was intended from this study the possibility of 

adopting a diagnosis between local fish species by staining bones and tissues. 

 

Key words : Carasobarbus luteus; Liza abu; Cyprinidae; Mugilidae; Baghdad; Iraq. 

 

INTRODUCTION  
    The method of staining bones is one of the means adopted in the study of tissue and bone, 

and organs too, through which taxonomic studies can be conducted among species of fish as 

stated by  (Potthoff,1984).as well as differences between taxonomic species of fish known and 

conventional. Whole fish or some parts of the body such as staining bones and connective 

tissue could be stain in two colors and clear as pointed out in (Dingerkus and Uhler, 1977). 

 

    This protocol was originally modified from Klymkowski and Hanken (1991) for amphibians. 

Although both cartilage and bone in the same specimen could be stain. It is better to stain bone 

and cartilage in different specimens of the same developmental stage, (Jonathan Knight, 2009). 

 

    Green (1952) stated that  alizarin staining method for the preparation of whole skeletons has 

proved very useful for the study of bones of embryos and small animals. It has certain 

advantages over methods in which the carcass is macerated and the bones are separated and 

dried. Among these advantages are: (a) there is no chance of losing the small bones, (b) all 

bones are retained in their original position, (c) there is no chance of wrongly identifying 

similar bones, (d) in the finished preparations the bones, after identification, may be 

disarticulated and examined from all angles, equally as well as in dried preparations, and (e) 

many animals may be processed together without danger of mixing their bones, a great saving 

in time and effort. 

 

    The main purpose of conducting this study is to identify the possibility of textile and 

structural differences between two local Iraqi fishes using staining with Alizarin Red. 

 

MATERIALS AND METHODS  
Ten local Himri Carasobarbus luteus (Heckel, 1843) Fish brought from the local market at 

Baghdad city at period of collection, Total length of these fish was  (12 cm ± 1.5 cm), and 

 



 

 

74 

A Description Study Of Two Local Fish  Himri 

average weight (65 g ± 5 g). also brought ten local Hishni Liza abu (Heckel, 1843), The 

averaged of total length was (14.5 cm ±1 cm) and average of weight (70 g ± 2 g). 

 

    The fishes were put in ice water with small ice Cubes, preparing a solution of formalin 

concentration (10%), to ease the formalin concentration (37%) to the concentration (10%) with 

adding 3 portion of distilled water to 1 part solution of formalin to get the concentration of 

formalin (10%), The fish flooded with a solution of formalin diluted Group for more than 48 

hours with continuous monitoring, then they washed with water for a full hour to get rid of the 

remnants of the diluted solution of formalin. 

 

   Then potassium hydroxide solution was prepared  by dissolving KOH (60 g) KOH crystalls 

in 1 liter of distilled water, with the preparation of dye alizarin dissolving (0.1 g) in (100 ml) of 

distilled water. The fish was flooded with a solution of KOH and then added alizarin dye the 

fish gradually until the arrival of the amount of dye to 50 ml, which is added to a solution of 

KOH submerging the Fish. 

 

    After that the Fish left in staining solution for two days, The scales were removed full of 

body and quietly and back again to the same KOH solution to stay for Other 5-6 days. a 

solution of pure Glycerine of more than (70%), was prepared to be placed where the fish is. 

Immediately after the end of six days in a solution of KOH staining with red alizarin, leaving 

the fish in Glycerine for 3-4 days. Then bottles were brought to save fish to create an imaging 

process after Shedding and create the appropriate lighting. 

 

RESULTS AND DISCUSSION 
    The two studied fishes were identified according to (Khalaf, 1961; Mahdi, 1962 and Forese 

and Pauly, 2004). The results showed differences in the distribution of the staining on the 

bones of both fishes. Figure (1) shows the distribution of the staining on the skeleton of C. 

luteus. Figure (2), shows the distribution of the stain on the skeleton of L.abu. 

 

    Figures (1) and (2) Shows differences textile and distribution of the stain on the skeleton of 

each species. L.abu shoed with pink to red, In addition to the brightness of the color dark 

purple on the bones of it, while a variation was observed  in the distribution of color of bone 

between the different species, as seen in (1), just like  what it was in (Potthoff,1984). The 

author can distinguish the species of fish from the stain of bone only, as seen from Figure (3). 

Severity of staining in the bone area  near the tail in C.luteus which shows the different bone 

tissue of these fish from L.abu as in Figure (4). It was also noticed that the difference in 

coloration of the bones of the head of each studied fish  as it was more pronounced in the first 

of the second, as in Figure (5).  

 

    This shows that the discrepancy in the distribution of the stain between the species probably 

may be due to the differences of bone tissue between fish species, as show in (Potthoff,1984). 

While (Klymkowsky and Hanken, 1991) pointed to the raise of the level of clarity when 

staining, for we can keep the fish in a solution of KOH concentration (1 %), after being placed 

in formalin and the duration depends on the size of the model, Then the possibility of 

removing the scales and even the skin gently, to raise the level of staining bones and increase 

the clarity which was enhanced as stated by (Potthoff,1984) knowing that the second pointed 

to the possibility stain even fish larvae without resorting to remove scales or remove the skin 

due to their small size. As for the rest of the small vertebrates such as fish, it was placed in a 

solution (20 ml acetic acid plus a 80 ml alcohol and 15 mg Alcian blue) then used immediately 

after immersion to dilute formalin (10%). 



 

 

75 

Muhammad. I. G. Al-Janabi 
 

Lewis and Witten (2004) referred to the possibility of stain connective tissue and cartilage 

without muscle tissue after the change in the concentration of formalin and potassium 

hydroxide and adoption Glycerine purity of up to (100%), and the different periods of 

immersion fish with the removal of muscle tissue gently to one aspect of the fish intended for 

study, and came this way also identical with source (Klymkowsky and Hanken, 1991) as in 

Figure (6), which shows the degree of clarity and form in this way. (Lewis and Witten, 2004) 

with the length of the fish immersion in staining solution, if the length of the fish is between 

(1cm - 8cm) dive for a one-day staining  solution, but if the length of the fish is between (8cm 

- 20cm), two days, but if the length of the fish above (20cm) the period should be  4 days of 

immersion and above depending on the size and the length of the form, in this study the fish 

dive in staining solution for two days and after removing the scales for 5 more days. Each of 

the sources (Klymkowsky and Hanken, 1991) and (Jonathan, 2009), point out to the different 

ways of staining for the previous method,  and at the same time for a way to hold staining in 

this study.  

 

    Gavaia and Cancela (2000) noted that  the accuracy and clarity of the models are due to 

differences in the concentrations of the chemicals used, and when additives are used, Then 

tend to promote the work and raise the level of clarity of form for a taxonomic studies on the 

fish species studied. 

 

LITERATURE CITED 
 Dingerkus, G., Uhler, L. D. 1977. Enzyme clearing of Alcian   Blue stained whole small 

vertebrates dor demonstration of cartilage. Stain Technology 52(4): 229-232. 

 

Forese,R. and Pauly,D. 2004. FisheBase.World wid web electronic 

publication.www.fishbase.org,version (02/2014). 

 

Gavaia, P. J., Sarasquete, C., Cancela, M. L.2000. "Detection of mineralized struc tures in 

early stages of development of marine Teleostei using a modified Alcian Blue-

Alizarin Red double staining technique for bone and cartilage. Biotechnic and 

Histochemistry 75(2): 79-84. 

 

Green, Margaret C.1952. A Rapid Method For Clearing and  Staining  Specimens For The 

Demonstration Of Bone. The Ohio Journal of Science. v52 n1 (January, 1952), 

31-33. 

 

Jonathan K. 2009.  A Simple Whole-Mount Staining Protocol for Bone and or Cartilage in 

adults and Larvae. from Zebrafish Book 5th Edition, University, Canada) and after 

discussions with P.E. Witten (The Institute of Aquaculture Research in 

Sunndalsøra, Norway). 

 

Khalaf, K. T. 1961. The marine and freshwater fishes of Iraq. Ar-Rabitta Press, Baghdad. 164 

pp.  

 

Klymkowsky MW, Hanken J. 1991. Whole-mount staining of Xenopus and other vertebrates. 

Methods in Cell Biology 36:419-441. 

 

Lewis, L. M., Lall, S. P. Witten, E. P. 2004. Morphological descriptions of the early stages of 

spine and vertebral development in hatchery-reared larval and juvenile Atlantic 

halibut (Hippoglossus hippoglossus). Aquaculture 241(1-4): 47-59. 

http://zebrafish.org/zirc/orders/buyBookQ.php?item=Book&id=book&detail=The%20Zebrafish%20Book


 

 

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A Description Study Of Two Local Fish  Himri 

 

Mahdi, N. 1962. Fishes of Iraq. Ministry of Education, Baghdad. 82 pp.  

 

Potthoff, T. 1984. Clearing and staining techniques. In: Ontogeny and Systematics of Fishes 

(based on an international symposium dedicated to the memory of Elbert Halvor 

Ahlstrom). 

 

Moser,H.G., Richards,W.J., Cohen,D.M., Fahay,M.P., Kendall,A.W., Jr., and Richardson S.L., 

eds. Lawrence, KS, Special Publication 1, American Society of Ichthyologists and 

Herpetologists, Allen Press, pgs. 35-37. 

 

 

 
 

Fig.1: Carasobarbus luteus (Heckel, 1843). 

 

 

 
 

Fig.2: Liza abu (Heckel, 1843). 

 

 

 

 

 

 

 

 



 

 

77 

Muhammad. I. G. Al-Janabi 
 

 

 
 

Fig.3: The distribution of the stain and coloration in the tail of C. luteus (Heckel, 1843). 

 

 
 

Fig.4: The distribution of the stain and coloration in the tail of L. abu (Heckel, 1843). 

 

 

 

 

 

 

 

 

 

 



 

 

78 

A Description Study Of Two Local Fish  Himri 

 

 
 

Fig.5: The distribution of the stain and the clarity of head bones of A. L. abu and B. C. luteus  
 

 

 

 
 

Fig.6: The degree of staining clarity Association of tissues and bones, (Klymkowsky and 

Hanken, 1991). 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 

79 

Muhammad. I. G. Al-Janabi 
 

Bull. Iraq Nat. Hist. Mus. 

(2014) 13 (1): 73-79 

 

 ,CARASOBARBUS LUTEUS (Heckel دراسة  وصفية لنوعي االسماك الحمري

 : LIZA ABU (Heckel, 1843) (MUGILOIDEIو الخشني  (1843

MUGILIDAE)  المحلية بطريقة تصبيغ العظام 

 

 محمد عناد غزوان الجنابي

 جامعة بغداد-مركز بحوث ومتحف التاريخ الطبيعي

muhammadinad@yahoo.com  

 

 الخالصة 

 ,Carasobarbus luteus (Heckelتم تصبيغ كل من االسماك المحلية الحمري     

االليزارين و ظهرت بعض بصبغة   Liza abu (Heckel, 1843)والخشني   (1843
الصفات التشريحية التي يمكن من خاللها دراسة التنوع و االختالف بالنسيج العضلي و 

الهيكل العظمي لكل من هذين النوعين المحليين ، كان الهدف من هذه الدراسة امكانية اعتماد 

 .التشخيص بين االنواع السمكية المحلية بطريقة تصبيغ العظام و االنسجة