CYTOCHEMICAL TESTS ON EMBRYOS 5I15 



THE GLYCOGEN TESTS 



The plaamal reaction is generally used for plasmalogen (a phoaphollpln) in the cyto- 

 plasm and consists of the Feulgen reaction. But this can also he 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 k-'f> chromic acid. 



2. Wash 5 minutes in running water. 



5. Immerse for 15 minutes in Feulgen reagent. 

 h. Wash 3 times in water saturated with SC^. 

 5. Einse in water and mount in glycerine. 



Glycogen makes its first appearance soon after the first fat glohules of the vitellus 

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

 vesicle. 



Bevelander and Johnson (19^+6) give a simple method of hletochemlcal localization of 

 glycogen, as follows : 



1. Fix tissue in Carnoys for 2k hours. 



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

 5. Eemove the paraffin with xylol. 



h. Flood the sections with a saturated solution of Iodine In 100^ alcohol. 

 5. Mount in clarite to study. 



Bensley (1959) describes a stain for glycogen as follows: 



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



Carmine 2 gma. 



Potassium carbonate 1 gm. 



Potassium chloride 5 gms. 



Distilled water 60.O cc. 



2. Add 20 cc. of concentrated ammonia. 



3. Allow to ripen for 2h hours. This becomes the stock solution. 



THE LIPID TESTS 



Identification of the various lipid subatances in the cell ia very difficult (Liaon, 

 1936). Solubility tests are unreliable, and formalin fixation alters normal solubility of 

 some fatty subatances. Cytochemlcal and macrochemical teats may vary, even with the iden- 

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

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

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

 (physical) tests will give substeintially reliable analytical results. 



Serra and Lopes (19'*-5) in studying the cytophyslology of the nucleolus give the 

 following procedure: 



1. Fix for 16 hours in 10^ formol. 



2. Wash tissues well in running water. 

 5. Stain with Sudan III in alcohol: 



At 70° stain for 25 to 75 minutes. 

 At kO° stain for 22 hours. 

 The tests for lipids is not quantitatively reliable. 



ENZYMES* 



1. Hlatochemical Teat for Peroxidase : This test should be applied to immature or 

 post-ovulatlon amphibian ovaries containing oocytes of various alzea. 



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



b. Wash thoroughly in distilled water. 



c. Immerae the tiaaue in saturated aqueous solution of benzidine containing 

 a few drops of acetic acid per 10 cc. 



d. Immerse in 1^ hydrogen peroxide (perhydrol diluted to 1^). Note the oxygen 

 bubbles and the blue followed by brown coloration. The reaction will appear 

 most intense in the ovarian capillaries. 



*See Sumner and Somers 19^7. 



