10. a. TECHNIQUES FOR STAINING 

 CHROMOSOMES IN THE TAIL-FIN 



It is often desirable to determine the chromosome count in an androgenetic, gynogenetic, 

 or parthenogenetic embryo or tadpole. This can be accomplished the better with the 

 Urodela than with the Anura, due in part to the larger amount of pigment in the epidermis 

 of most Anura. There are two methods of preparing the material. The one involves fix- 

 ing the entire larva and subsequently peeling off the entire tail epidermis. This is better 

 with Anuran material. With such a large sheet of cells one can generally find abundant 

 chromosome figures. There is a technical difficulty of keeping the tail epidermis flat- 

 tened through the staining procedure. The second method is suitable for Urodele larvae 

 and allows them to survive, since only the distal 1/3 or the tail fin is cut off. 



THE METHOD OF PARMENTER (for Anura) 



There is a stage in larval development when the yolk in the tail fin has been reduced to a 

 minimum and yet the cells themselves have not become so small that the chromosomes 

 are difficult to identify. For the frog tadpole this stage is attained in from 15 to 20 days 

 at laboratory temperatures, at the beginning of feeding. The tail fin should be well 

 formed, thin, and transparent. The steps in the process are as follows: 



1. Fix the entire tadpole in Bouin's or Michaelis' fluid for 2 hours. 



2! Transfer to 70% alcohol to which 2% ammonia has been added. This will shortly 

 remove the yellow coloring of the picric acid. 



3. Transfer through appropriate (alcohol) steps to water; leave for 12-24 hours. 

 This will tend to soften the tissues somewhat. 



4. Place the head of the tadpole in a depression of a shell-depression slide, with 

 the tail flattened on the slide in a few drops of water. 



5. With a sharp scalpel trim off the entire margin of the tail fin. Remove as little 

 of the tissue as possible, but cut to the junction with the body. 



6. With sharp scissors make a circular cut around the body of the tadpole just be- 

 hind the mouth. The cut should not be so deep that it injures internal organs. 



7. Along the mid-dorsal line cut through the body flap to the junction with the tail. 

 Cut through the mid-ventral line in a similar manner. These cuts will provide 

 lateral flaps of relatively tough body epidermis which is continuous with the lat- 

 eral tail-fin epidermis. With the tadpole on its side, the tail fin immersed in 

 water, it will now be possible to grasp the tough body epidermis with forceps and 

 gradually peel off the lateral tail-fin epidermis. This can be done with the aid of 

 a hair loop which can be worked beneath the epidermis as it is raised with the 

 forceps. It is not recommended that a needle be used. If the tail-fin has been 

 properly trimmed, two continuous sheets of epidermis from a single tadpole may 

 be secured. 



8. If the sheets of epidermis tend to curl, put a few knicks in the edges with a sharp 

 scalpel. Transfer with wide-mouthed pipette. 



9. Stain, dehydrate, and clear tail-fins in shell vials or small Stenders. 



a. Stain: 



Heidenhain's Iron Haematoxylin : Mordant 12 hours, stain 2 hours, destain 

 under binocular observation. This is still the best chromosome stain but 



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