340 CYTOCHEMICAL TESTS ON EMBRYOS 



b. Wash thoroughly in distilled water. 



c. Immerse the tissue 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. 



2. Histochemical Test for Indophenoloxidase: This test should be applied to the im- 

 mature or post-ovulation ovary of any amphibian. The method is essentially that 

 of Voss (1924). 



a. Make up Solution A by dissolving 0. 5 gm. of alpha-naphthol in 100 cc. of 

 boiling distilled water; boil for 5 minutes; cool and filter. 



b. Make up Solution B by dissolving 0. 5 gm. dimethylparaphenylenediamine* in 

 100 cc. of cold distilled water and allow it to stand for 24 hours. Filter. 



c. When ready to make the test, mix equal parts of "A" and "B"; add an equal 

 volume of 0. 64% NaCl and mix thoroughly. 



d. Pour some of the mixture in a Stender and add the tissue to be tested. The 

 mixture can be further diluted with physiological saline solution to prevent 

 too intense a reaction. 



This procedure has been modified by Child by diluting the reagent as follows: 



a. To 10 cc. of distilled water add 1 drop of dimethylparaphenylenediamine. * 



b. To 10 cc. of distilled water add 1 mgm. of alpha-naphthol. 



c. Place the fresh tissue to be tested in physiological saline and to each 1 cc. of 

 this solution add 1 drop of each of the solutions "a" and "b". 



Note which of these methods stains the yolk nucleus of the sniall oocytes. 



When tissue is placed in an alkaline solution of sodium glycerophosphate, en- 

 zymatic activity liberates phosphate which, as it is formed, is precipitated by 

 the calcium ions. This calcium phosphate is then made visible by conversion to 

 cobalt sulfide which gives the black coloration, indicating the presence of the 

 enzyme. 



THE TEST FOR PHOSPHORUS 



Phosphorus is found in the nucleolus, the chromosomes, and the protoplasm generally. 

 It is in the thymo- and ribonucleic acids, in conjugated phosphoproteins and in the nucleo- 

 proteins. Serra and Lopes (1945) say: "It seems that we can safely conclude that the 

 phosphorus reaction, the coloration with basic and acidic stains and the nuclease reac- 

 tion, show the existence of nucleotides of the ribose type in the nucleolus. " The nucleo- 

 lar inclusions probably have a greater concentration of these nucleotides than does the 

 remaining part of the nucleolus. The nucleoli are richer in nucleotides, as determined 

 by this phosphorus test, when they are young. 



The method of Angeli, A . , (1933 Riv. di Biol. 10:702) 



1. Sections treated for 20 minutes with solution made up of 



3 grns. ammonium molybdate 

 20 cc. distilled water 

 20 cc. of 30% aqueous hydrochloric acid 



2. Reduced in N/50 stannous chloride. 



3. Rinsed quickly in distilled water. 



4. Washed in 2. 5% aqueous ammonia. If phosphorus is present in any form there 

 will develop a blue-green color 



* If the reagent is in solid form, it should be heoted on a water bath until it melts. 



