172 



THE AMPHIBIAN GERMINAL VESICLE 



fresh N-medium. Cut this piece of ovary Into smaller pieces each containing 3 to U eggs 

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



DISPERSED 

 OERMIKAl fOLK 

 VESICLE 



GERHlmi VESICLE 



•NIHAL POLE 



FOLLICLE WALL 

 FOLLICLE CELLS- 



FOLLICLE CELL 

 FOLLICLE WALL. 



FORCEPS 



REMOVAL OF THE AMPHIBIAN GERMINAL VESICLE 



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

 email 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 1/5 the 

 diameter 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-medlum over the Isolated 

 germinal vesicle to wash away the yolk globules which are adherent to it. This will be- 

 come increasingly difficult with time so that the cleaning procedure should Immediately 

 follow the liberation of the vesicle. The clean germinal vesicle should appear 9s a clear 

 spherical 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 

 vesicle, followed by more of the same fluid. The transfer must be made in nuclear fluid 

 to a position beneath the surface of the fluid in the new container. The vesicle must not 

 come into contact with air. Re-examine the vesicle and clean it further if necessary. The 

 yolk actions tend to coagulate the vesicle and must be removed entirely. A medicine drop- 

 per with diameter slightly greater than the diameter of the vesicle can be used to suck 

 the vesicle in and out (gently), the edges of the dropper thus scraping off the adherent 

 yolk. 



B. EXAMINATION OF THE GERMINAL VESICLE 



Place a small amount of Permoplast on each of the four corners of a small, square 

 coverslip (a quarter size coverslip is satisfactory). Add equal amounts of Permoplast to 

 each of the comers but only enough to elevate the coverslip a bit more than the diameter 

 of the vesicle. Now transfer the germinal vesicle, in a drop or two of N-medium, to a 

 cleeua slide and gently cover with the coverslip. Add more N-medium from the side of neces- 

 sary. The advantage of a small coverslip is that various reagents may be added readily 

 from the side. 



OBSERVATIONS AND EXPEBIMENTS: 



NORMAL APPEARANCE OF THE GERMINAL VESICLE 



1. The isolated germinal veaicle should appear exactly as it does in the egg 

 except for a very alight swelling. 



