TECHNIQUE FOR STAINING 

 CHROMOSOMES IN TAIL-FIN 



It Is often desirable to determine the chromosome count in an androgenetic, gynogene- 

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

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

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

 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 l/j or the tail fin is cut off. 



THE METHOD OF PARMEMTER (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: 



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



b. Transfer to 70^ alcohol to which 2% ammonia has been added. This will 

 shortly remove the yellow coloring of the picric acid. 



c. Transfer through appropriate (alcohol) steps to water; leave for 12-2U 

 hours. This will tend to soften the tissues somewhat. 



d. Place the head of the tadpole in a depression of a shell-depression 

 slide, with the tall flattened on the slide in a few drops of water. 



e. With a sharp scalpel trim off the entire margin of the tail fin. Eemove 

 as little of the tissue as possible, but cut to the Junction with the 

 body. 



f. With sharp scissors make a circular cut around the body of the tadpole 

 just behind the mouth. The cut should not be so deep that it injures 

 internal organs. 



g. 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 lateral tall-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 proper- 

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



be secured, 

 h. If the sheets of epidermis tend to curl, but a few knicks in the edges with 



a sharp scalpel. Transfer with wide-mouthed pipette. 

 1. Stain, dehydrate, and clear tail-fins in shell vials or small Stendere. 



1. Stain: 



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

 destain under binocular observation. This is still the best 

 chromosome stain but it has the disadvantage that the destaining 

 must be exact and any persistent yolk material stains black. 



Harris' Acid Haemalum :* The time and concentration of stain must 

 be determined empirically. It is suggested that 50^ stain be used 

 for 8 to 10 minutes, without destaining. Wash in alkaline tap 

 water. 



# 



Mayers Haemalum or Conkllns Haematoxylin are also satisfactory. 



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