CYTOCHEMICAL TESTS FOR GAMETES 

 AND EMBRYOS OF AMPHIBIA 



The following tests have all teen tried and proven successful, and should he used on 

 small and large ovarian eggs, Isolated germinal vesicles, testes, the gastrula and neurula 

 stages. The tests are largely for proteins, with emphasis on the amino acids and nucleo- 

 proteins. Some testa for carbohydrates, lipids, and enzymes are Included. 



Specific suggestions are as follows : 



1. Stain immature or post-ovulatlon ovary of Rana pipiens with 



a. Safranin and light green (note yolk nuclei). 



h. Iron haematoxylin (note chromosome and nucleolar structure) 



c. Toluldine hlue - stains both nucleic acids. 



d. Unna's methyl green-pyronine (combine with enzymes). Thymonucleic acid 



green, ribonucleic acid red. 



e. Feulgen - specific for thymonucleic acid. Plasmal reaction. 



2. Glycogen and lipid distribution in oogonia and early oocytes. 



3. Protein tests : 



a. Nucleoprotein extraction - Mlrsky & Pollister (Frog testes). 



b. Biuret - Xanthoproteic - Ninhydrin tests (Frog testes). 



c. Arginine - lyrosine - Tryptophane tests (Frog testes smears; frog ovary). 



d. Test for - SH proteins, nitroprusside -test (Frog testes smears, embryos). 

 h. Tests for enzymes: peroxidases - Indolphenoloxidases (Frog testes smears). 



5. Miscellaneous: Test for Phosphorus and Oxygen. 



PROTEIN TESTS 



NUCLEOPBOTEINS : (Mlrsky & Pollister, I9I+2 ) 



Grind up a large number of whole frog testes in neutral sodium chloride solutions of 

 three concentrations, in two of which the nucleoproteins are soluble and in the third they 

 are Insoluble. 



1. Extract with 1 M NaCl-volume about 10 X that of testes mash. Becomes viscous. 



2. Centrifuge at 10,000 r.p.m. , providing a viscous, slightly opalescent super- 



natant fluid. Viscosity due to nucleoprotein dissolved therein. 

 5. Add 6 volumes of distilled water - nucleoprotein percipitatea in a fibrous 



mass, settling rapidly so that the supernatant fluid can be siphoned off. 

 k. Wash percipitate in 0.1^*- M NaCl (in which it la insoluble). 



5. Redisaolve in 1 M NaCl. 



6. Centrifuge again at high apeed to remove the suspended material. 



7. Be-percipltate by adding 6 volumes of distilled water. 



8. Stir the mixture with a glass rod with a crook at its end, collecting a 



fibrous nucleoprotein which will wind around the glass rod. 



(Further purification can be achieved by repeating the above process) 



************* 



NUCLEIC ACIDS:* 



TOLUIDINE BLUE : 



1. Stain sections (ovary) for 20 minutes in saturated aqueous solution of 

 Toluldine Blue. 



2. Differentiate twice for 10 minutes in 95']^ alcohol. 

 5 . Mount as usual . 



Toluldine blue is taken by both nucleic acids, an orthochromatic 

 (blue) color la given only by nucleic acids, found in both the cytoplasm 

 and nucleus. 



♦The cytoplasm of cells contains principally ribonucleic acid (also known aa yeast or 

 phytonuclelc acid) while the nucleus contains largely desoxyribonuclelc acid (alao known 

 as thymonucleic acid). The ribonucleic acid la transformed Into thymonucleic acid during 

 development. 



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