THE AMPHIBIAN GERMINAL VESICLE 17S 



2. 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 artifacts are most readily- 

 apparent in germinal vesicle fixation, hence this section should be treated as a fur- 

 ther study of the germinal vesicle and its reactions to environmental factors. This 

 in addition to acquaintance with methods of providing permanent preparations. 



A rather new fixative -stain is recommended (see La Cour, 1941) in which aceto- 

 orcein is applied directly to the isolated vesicle. This stain consists of 45% acetic 

 (glacial) acid and 0. 5% orcein into which the vesicle is placed for 30-60 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 (acid- 

 ified) acts in much the same manner. Other permanent mounts may be made with 

 Bouin's fixation followed by Mayer's fixation followed by Mayer's haemalum stain. 



A study of the action of fixatives on the vesicle is very profitable. Isolate 20 to 30 

 full-sized vesicles in N-medium and, while observing them under low-nnagnification 

 of the microscope (under elevated cover slips) add singly such fixatives as Bouin's, 

 4% formalin, saturated HgCl2 in water, etc. Immediate changes in size and con- 

 sistency of the vesicle should be noted. Since Bouin's contains an acid, follow the 

 Bouin-fixation with some alkaline treatment to attempt to counteract the acid factor 

 in this fixation. 



A study of the fixed germinal vesicle raises the legitimate question as to how far we 

 may assume that fixed material accurately represents the structures of the living 

 germinal vesicle. Probably the more accurate picture is a composite one, arrived 

 at by the study of both fresh and fixed material. 



3. IONIC EFFECTS 



A visible iso-electric point can be demonstrated passing through the germinal vesicle 

 by the addition, to one side of the coverslip, of a small drop of dilute HCl. If a slow 

 reaction is desired, use 0. OOlN-HCl; if a fast reaction is desired, use 0. 003N-HC1. 

 The germinal vesicle is negatively charged. Note the Brownian movement of gran- 

 ules. The diffusion of weak acids through the germinal vesicle gives an effect known 

 as the "Ring Phenomenon". As proteins within the vesicle reach the iso-electric 

 point (I. E. P. , hereafter) they become insoluble and appear as floccules. The size 

 of the floccule is relative to the speed of the acid penetration. If penetration is fast, 

 the floccules will be small; if penetration is slow, they will aggregate and be large. 

 Observe the fusion of micelles to build up the so-called "linin reticulum". 



The chromosomes will appear and become distinct only when the solution immediately 

 around reaches the I. E. P. of the chromosomes. Watch the I. E. P. passing to the 

 center of the germinal vesicle and the subsequent swelling of the outer colloidal area, 

 which becomes positively charged and reverses its reaction. 



When the germinal vesicle in weak acid clears, add a similar amount of weak alkali 

 (NaOH) and the reactions will be reversed. In NaOH alone the germinal vesicle will 

 burst. If a very dilute base is used, this swelling can be compensated by acid shrink- 

 ing. The reactions can be made to go back and forth under experimental control. 



4. ELECTRICAL CHARGE AND THE REACTION OF THE GERMINAL VESICLE 



An electrical set-up has been devised by means of which a known current may be sent 

 through the germinal vesicle and then reversed. A brief description of the apparatus 

 is given below. The equipment consists of a 45-volt dry cell, a reversing switch, 

 key, platinum electrodes mounted in glass, with voltmeter and milli-ammeter in the 

 circuit as shown in the accompanying diagram on the following page. 



