C. Experiments on Early Development - Operative 



18. EXPERIMENTS WITH THE 

 AMPHIBIAN GERMINAL VESICLE 



PURPOSE: To study the living but isolated nucleus of the amphibian egg, with special 

 emphasis on the chromosome structure and the effects on the chromosomes of var- 

 ious environmental variables. 



MATERIALS: 



Biological: 



Technical: 



Ovaries of any Anuran or Urodele. 



Stenders, Syracuse dishes, depression slides, medicine droppers, 

 electrical apparatus (diagram on p. 175), and various solutions: 

 Phenol red as pH indicator. 



Nuclear medium (N-medium) which is Ca-free Ringers solution 

 made up with glass distilled water as follows: 



NaCl 0. 66 gms. 



KCl 0. 014 gms. 



Water 100 cc (pyrex distilled) 



Buffer: Unnecessary, but with small nuclei can 

 reduce pH to 5. 9 

 Fixatives : Bouin, HgCl^ sat. aq. , 4% formalin, aceto-orcein. 

 Stains: Mayer's Haemalum, 1% crystal violet in N-medium; aceto- 

 orcein (45% glacial acetic acid and 0. 5% orcein), I'y^methyl green 

 in 1% acetic acid. 

 General : 0. 1 N-NaHP04 



0. 001 N, 0. 003 N, and M/lOO HCl 

 0. OOIN, 0. 003 N, andM/lOONaOH 



METHOD: 



Precautions : 



1. All glassware is to be thoroughly washed and rinsed at least once with glass 

 distilled water to remove all traces of metallic ions. 



2. Glass distilled water must be used to make up all solutions, particularly N- 

 medium. Ordinary distilled water often contains traces of copper which are 

 deleterious. 



3. Calcium and all heavy metals must be avoided. (N-medium is calcium-free). 



4. Reduce to a mininnum the amount of light, carbon dioxide, and bacteria. 



5. Keep the isolated germinal vesicle beneath the surface of the solution at all 

 times to avoid contact with air, air bubbles, surface films, and dust par- 

 ticles. 



Controls : 



1. The standard control is the fixed and stained germinal vesicle. 



2. Where environmental variables are used the environment of the nuclear (N) 

 medium is to be considered as the control environment. 



3. Where the "OH ions are used, the +H ions may be used for contrast; where 

 an electric current is used, it may be reversed. 



* This exercise has been organized with the generous aid of Dr. W. R. Duryee. 



- 170 - 



