28 STAINING TAIL FIN CHROMOSOMES 



a. Anesthetize several larvae In l/5,000 MS 222 or chloretone, and transfer 

 from the finger bowl to a Syracuse dish with lamp-hlack and paraffin bottom. 



b. With sharp scissors or scalpel clip off not more than the distal third of 

 the tall. 



c. Transfer tail tip with minimum fluid to the fixative: (use wide-mouthed 

 pipette)** 



Bouln's - 100 CO. 

 Urea - 1 gr. 

 Glacial 



acetic - 5 cc (extra 

 Fix for a minimum of 5 hours, longer does no harm. 



d. Transfer to 70% alcohol for the removal of the yellow picric stain. This may 

 be facilitated by adding a few drops of NHi^^OH, but the ammonia must be com- 

 pletely removed in pure alcohol before staining. 



e. Transfer to Columbia watchglaases or t'o shell vials where the minimum amount 

 of reagents will be used. Hydrate through 5 minute changes in progressively 

 dilute alcohols to distilled water. 



f. Stain for 15 minutes in I/3 Harris' acid haematoxylln. The exact timing and 

 concentration of the stain will vary with the age of the stain and the fresh- 

 ness of the tail tip. DestaiJiing is not practicable. 



HARR IS' ACID HAEMATOXYLIN 



Grubler's haematoxylln O.5 gms. 



Varm 95^ alcohol 5-0 cc. 



Potassium alum 10.0 gms. 



Warm distilled water 100.0 cc. 



Bed mercuric oxide 0.25 gms. 



As soon as the stain turns a deep purple, remove from the flame and cool 

 rapidly under running water. Just befo^-e using, add exactly 2.5 cc. of 

 glacial acetic acid to 50 cc. of the above mixture. Dilute 1/2 to 1/5 for 

 use. Mayer's acid haemalum may be equally good If freshly made up. 



g. Transfer through two changes of tap water to which 5 or 6 drops of 1^ sodium 

 bicarbonate (or ammonia) has been added to each 10 cc. of volume. This 

 alkali nlzat ion will make the stain an Intense bright blue. 



h. Elnse in distilled water; dehydrate with 5 minute changes in progressively 

 more concentrated alcohols to 95^ where two changes are made, followed by 

 3 or i)- changes In absolute alcohol. Transfer to carbol-xylol for 5 minutes, 

 then into two changes of xylol. (If the atmosphere is not humid and the 

 transfer can be made directly from the absolute alcohol to xylol, the stain 

 is apt to be more permanent. ) 



1. Mount in clarite on cleaned slides under slightly compressed (cleaned) cover 

 slips. Several tall tips may be moxinted oh a single slide which can be marked 

 with diamond point for identification. 



** Note: In transferring the tips from one medium to another (if the decanting method 

 is not used) grasp the thick proximal margin of zhe tail tip to prevent injury to the 

 thinner distal portions of the tail tip. 



See section on "Amphibian Germinal Vesicle" for a list of the diploid chromoaome 

 numbers of commonly used forms. 



