EXPERIMENTS WITH THE 

 AMPHIBIAN GERMINAL VESICLE* 



PURPOSE : To study the living but Isolated nucleus of the amphihlan egg, with special 



emphasis on the chromosoine structure and the effects on the chromoaomes of various en- 

 vironmental variables. 



MATERIALS : 



Biological : Ovaries of any Anuran or Urodele. 



Technical : 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 Singers solution made up 



with glass distilled water. 

 Fixatives: Bouin, HgClg sat. aq.., h% formalin, aceto-orcein. 

 Stains: Mayer's Eaemalum, 1^ crystal violet in N-medium; aceto-orcein 



(1+5^ glacial acetic acid and 0.55^ orcein), 1^ methyl green in 1^ 



acetic acid. 

 General: 0.1 N-NaHPOi^ 



0.001 N, 0.005 N, and M/iOO HCl 

 0.001 N, 0.003 N, and m/100 NaOH 



METHOD : 



Precautions : 



1. All glassware Is to be thoroughly washed and rinsed at least once with glass dis- 

 tilled water to remove all traces of metallic ions. 



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

 Ordinary distilled water often contains traces of (Jopper which are deleterious. 



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

 h. Reduce to a minimum the amount of light, carbon dioxide, and bacteria. 

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

 times to avoid contact with air, air bubbles, surface films, and dust particles. 



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. 



5. Where the ~0H ions are used, the +H Ions may be iised for contrast; where an elec- 

 tric current is used, it may be reversed. 



Procedure : 



Where possible, use the eggs of some Urodele for in these the germinal vesicles 

 are relatively larger and the chromosomes are the more easily studied than in the 

 eggs of other amphibia. However, since the frog (Anura) is more readily available, 

 the following description will be concerned with the germinal vesicles of Rana 

 pipiens. 



A. REMOVAL OF THE GERMINAL VESICLE 



Remove the ovary of a sexually mature and hibernating female frog and place It in 

 Amphibian Ringer's solution in a finger bowl. Wash free any adherent blood. This ovary 

 will remain healthy for about 2k hours at 20°C. or for about h days at 10°C. 



Cut off a small portion of the ovary (20 to 50 eggs) and transfer to a #2 Stender 

 containing Nuclear Medium. Pour off this solution after a few minutes and replace with 



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



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