THE AMPHIBIAN GERMINAL VESICLE 



171 



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 Am- 

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

 will remain healthy for about 24 hours at 20°C. or for about 4 days at 10°C. 



Cut off a small portion of the ovary (20 to 30 eggs) and transfer to a #2 Slender containing 

 Nuclear Medium. Pour off this solution after a few minutes and replace with fresh N- 

 medium. Cut this piece of ovary into smaller pieces each containing 3 to 4 eggs and 

 transfer one such cluster of eggs to a Syracuse dish containing N-medium. 



GEHMIHAL VESICLC 



ANIHiL POLE 



FOLLICLE »LL 

 FOlLltLE CELLS' 



DISPEDSEO 



iCHHiui roil 



»£SICIE 



OLLICLE CEIL 

 FOLLICLE IHLl. 



REMOVAL OF THE AMPHIBIAN GERMINAL VESICLE 



With very sharp forceps and under the low magnification of the microscope, make a 

 small tear at a point in the follicle sac and egg wall in the region of the animal hemi- 

 sphere but close to the margin of the vegetal material. The germinal vesicle is located 

 in the center of the animal hemisphere. The tear should not be larger than 1/3 the di- 

 ameter of the egg. The egg substance will immediately flow out, carrying with it the 

 rather large and spherical germinal vesicle. When working with the smaller, immature 

 oocytes, a needle puncture may suffice to liberate the vesicle. 



Using a clean medicine dropper, force a gentle flow of N-medium over the isolated ger- 

 minal vesicle to wash away the yolk globules which are adherent to it. This will become 

 increasingly difficult with time so that the cleaning procedure should immediately follow 

 the liberation of the vesicle. The clean germinal vesicle should appear as a clear spher- 

 ical sac without visible contents. 



The chromosomes cannot be seen at low magnification. Transfer the germinal vesicle to 

 fresh N-medium in another Syracuse dish for further cleaning. The transfer can be made 

 by sucking up a small amount of fluid into the medicine dropper and then drawing up the 



