130 



TECHNIQUES FOR STAINING CHROMOSOMES 



3. Transfer tail tip with minimum fluid to the fixative: (use wide -mouthed pipette)* 



Bouin's - 100 cc. 



Urea - 1 gr. 



Glacial acetic - 5 cc. (extra) 



Fix for a minimum of 3 hours, longer does no harm. 



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

 facilitated by adding a few drops of NH^OH, but the ammonia must be completely 

 removed in pure alcohol before staining. 



5. Transfer to Columbia watchglasses or to shell vials where the minimum amount 

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

 dilute alcohols to distilled water. 



6. Stain for 15 minutes in 1/3 Harris' acid haematoxylin. The exact timing and con- 

 centration of the stain will vary with the age of the stain and the freshness of the 

 tail tip. Destaining is not practicable. (This procedure is from Henley-Costello 

 modified after Fankhauser) 



HARRIS' ACID HAEMATOXYLIN 



Grubler's haematoxylin 0. 5 gms. 



Warm 95% alcohol 5. cc. 



Potassium alum 10. gins. 



Warm distilled water 100. cc. 



Red mercuric oxide 0. 25 grns. 



SALAMANDER TAIL-TIP PREPARATIONS FROM G. FANKHAUSER 



1 - Haploid chromosome complex 3 - Triploid 



2 - Diploid 4 - Pentaploid 



(Courtesy of G. Fankhauser from Sharp's "Fundamentals of Cytology" 1943) 



♦ Note: In transferring the tips from one medium to another (if the decanting method is not used) grasp the thick proximal margin 

 of the 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 chromosome numbers of commonly used forms. 



