CYTOCHEMICAL TESTS ON EMBRYOS 339 



THE GLYCOGEN TESTS 



The plasmal reaction is generally used for plasmalogen (a phospholipin) in the cytoplasm 

 and consists of the Feulgen reaction. But this can also be used for glycogen if the proper 

 preliminary treatment is given to the tissues, as follows: 



1. Fix pieces (small) of ovary (amphibian) for 1 hour in 4% chromic acid. 



2. Wash 5 minutes in running water. 



3. Immerse for 15 minutes in Feulgen reagent. 



4. Wash 3 times in water saturated with SO2. 



5. Rinse in water and mount in glycerine. 



Glycogen makes its first appearance soon after the first fat globules of the vitellus appear, 

 with a concentration in a ring about the nucleus. There is none in the germinal vesicle. 



Bevelander and Johnson (1946) give a simple method of histochemical localization of 

 glycogen, as follows: 



1. Fix tissue in Carnoys for 24 hours. 



2. Mount sections on albumen smeared slides, flooded with Lugol's solution at 40 C. 



3. Remove the paraffin with xylol. 



4. Flood the sections with a saturated solution of Iodine in 100% alcohol. 



5. Mount in clarite to study. 



Bensley (1939) describes a stain for glycogen as follows: 



1. Boil the following gently until the color darkens, then cool. 



Carmine 2 gms. 



Potassium carbonate 1 gm. 



Potassium chloride 5 gms. 



Distilled water 60. cc. 



2. Add 20 cc. of concentrated ammonia. 



3. Allow to ripen for 24 hours. This becomes the stock solution. 



THE LIPID TESTS 



Identification of the various lipid substances in the cell is very difficult (Lison, 1936). 

 Solubility tests are unreliable, and formalin fixation alters normal solubility of some 

 fatty substances. Cytochemical and macrochemical tests may vary, even with the iden- 

 tical lipids. Glycerides and fatty acids are never birefringent in the dissolved condition 

 when examined in vivo, but after treating with formalin or freezing they may become 

 crystalline and birefringent. Tests with Osmic Acid, Sudan III, when coupled with other 

 (physical) tests will give substantially reliable analytical results. 



Serra and Lopes (1945) in studying the cytophy siology of the nucleolus give the following 

 procedure: 



1. Fix for 16 hours in 10% formol. 



2. Wash tissues well in running water. 



3. Stain with Sudan III in alcohol: 



At 70° stain for 25 to 75 minutes. 

 At 40° stain for 22 hours. 

 The tests for lipids is not quantitatively reliable. 



ENZYMES* 



1. Histochemical Test for Peroxidase : This test should be applied to immature or 

 post-ovulation amphibian ovaries containing oocytes of various sizes. 

 a. Fix the ovary in 10% formol for 10 minutes. This destroys the catalase. 



* See Sumner and Somers 1947. 



