nh THE AMPHIBIAN GERMINAL VESICLE 



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



hours . 

 5. The nucleoli and the hyaline chromoaomes should maintain their relative 



central positions In respect to each other. 

 k. Fixation pictures are distinctly different. 



To see the chromoaomes within the central chromosome core it will be neceasary to add 

 a drop of 0.1-N NaHPOj; or a small amount of calcium (as in normal Elnger's solution). The 

 chromosomes may then be stained with 1^ Crystal Violet or with Aceto-orceln and studied 

 under high power magnification. With Aceto-orceln, which is a combination fixative and 

 stain, the vesicle is permanently fixed. 



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

 ferent and should be studied in detail before applying any of the experimental procedures. 

 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 veaicle (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. 



h. Very small contracted chromoaomes appear on the framework. The diploid num - 

 bers of a few of the common forms are as follows : 



Bufo (various species) - 22 chromosomes 



Sana esculanta - 2k 



Sana plpiens, E. fusca - 26 



Eana catesbiana - 28 (26?) 



Hyla arborea - 214- 



Triturus (various species) - 2k 



T. vlrldescena - 22 



Deamognathus - 2k 



Salamandra - 2k 



Amblystoma tigrlnum - 28 (axolotl 16, 2k, 28, 30) 



Plethodon - 2k 



Half or medium- si zed egg veaicle (nucleus) 



1. Nuclear aacs small. 



2. Nucleoli are next to nuclear wall. 



5. Chromosome frame fills the entire nucleus. 



k. Chromosomes are spread out to their maximum extension, and possess large 

 lateral loops . 



Small-sized egg vesicle (nucleus) 



1. Nuclear aacs barely visible. 



2. Nucleoli peripheral. 



3- Chromosome frame fills the entire nucleus. 



'+. Chromosomes are much smaller than in the larger nuclei. 



B. PERMANENT FIXATION AND STAINING OF THE GERMINAL VESICLE 



The student is again cautioned about the use of fixatives in the laboratory where 

 living material is also to be kept. Further, fixation artefacts are most readily apparent 

 in germinal vesicle fixation, hence this section should be treated as a further study of 

 the germinal vesicle and its reactions to environmental factors. This in addition to ac- 

 quaintance with methods of providing permanent preparations. 



A rBther new fixative-stain is recommended ( aee La Cour, 19'+l) In which aceto-orceln 

 Is applied directly to the Isolated vesicle. This stain consists of k'^'fi acetic (glacial) 

 acid and 0.5']^ orcein into which the vesicle la placed for 3O-60 seconds. It is then run 

 up rapidly through the alcohols (in which some of the dye will dissolve out) and into an 

 alcohol-free mounting medium. A stain of 1^ Methyl Green (acidified) acts in much the 

 same manner. Other permanent mounts may be made with Bouln's fixation followed by Mayer's 

 fixation followed by Mayer's haemalum stain. 



