6. Chemical Embryology 



43. CYTOCHEMICAL TESTS FOR GAMETES 

 AND EMBRYOS OF AMPHIBIA 



The following tests have all been tried and proven successful, and should be 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 tests for carbohydrates, lipids, and enzymes are included. 



Specific suggestions are as follows: 



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



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



b. Iron haematoxylin (note chromosome and nucleolar structure) 



c. Toluidine blue - stains both nucleic acids. 



d. Unna's methyl gr een-py ronine (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 - Mirsky & Pollister (Fred testes). 



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



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



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



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



5. Miscellaneous: Test for Phosphorus and Oxygen. 



PROTEIN TESTS 



NUCLEOPROTEINS: (Mirsky & Pollister, 1942) 



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 supernat- 

 ant fluid. Viscosity due to nucleoprotein dissolved therein. 



3. Add 6 volumes of distilled water - nucleoprotein percipitates in a fibrous mass, 

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



4. Wash percipitate in 0. 14 M NaCl (in which it is insoluble). 



5. Redissolve in 1 M NaCl. 



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



7. Re-percipitate 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) 



- 325 



