172 THE AMPHIBIAN GERMINAL VESICLE 



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 

 dropper 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 adher- 

 ent yolk. 



B. EXAMINATION OF THE GERMINAL VESICLE 



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

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

 of the corners 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 

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

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

 readily from the side. 



OBSERVATIONS AND EXPERIMENTS: 



1. NORMAL APPEARANCE OF THE GERMINAL VESICLE 



a. The isolated germinal vesicle should appear exactly as it does in the egg except 

 for a very slight swelling. 



b. The hyaline condition of the ground substance should persist for several hours. 



c. The nucleoli and the hyaline chromosomes should maintain their relative centred 

 positions in respect to each other. 



d. Fixation pictures are distinctly different. 



To see the chromosomes within the central chromosome core it will We necessary to 

 add a drop of 0. 1 -N NaHPO^ or a small amount of calcium (as in normal Ringer's 

 solution). The chromosomes may then be stained with 1% Crystal Violet or with 

 Aceto-orcein and studied under high power magnification. With Aceto-orcein, which 

 is a combination fixative and stain, the vesicle is permanently fixed. 



The germinal vesicles of large, medium, and small ovarian eggs are structurally 

 different and should be studied in detail before applying any of the experimental pro- 

 cedures. The smaller eggs are relatively more transparent, due to the lack of yolk 

 or pigment. The general characteristics of the three types are as follows: 



Large egg vesicle (nucleus) 



1. Nuclear membrane has outside sac-like bulges (see photographs). 



2. Nucleoli clusters appear in the center. 



3. The chromosome frame appears in the center of the ring of nucleoli. This 

 is a gel structure which gives rise to the first maturation spindle. 



4. Very small contracted chromosomes appear on the framework. The dip - 

 loid numbers of a few of the common forms are as follows: 



Bufo (various species) - 22 chromosomes 



Rana esculanta - 24 



Rana pipiens, R. fusca - 26 



Rana catesbiana - 28 (26?) 



Hyla arborea - 24 



Triturus (various species) - 24 



T. viridescens - 22 



Desmognathus - 24 



Salamandra - 24 



Amblystoma tigrinum - 28 (axolotl 16, 24, 28, 30) 



Plethodon - 24 



