5^1^ CYTOCHEM ICAL TESTS ON EMBRYOS 



2. Hlatochemlcal Test for Indophenoloxldase : This teat should be applied to the im- 

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

 of Voss (1921+). 



a. l^ke up Solution A by dissolving 0.5 gm- of alpha-naphthol in 100 cc. of 

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



b. Make up Solution B by dissolving 0.5 gm. dimethylparaphenylenedi amine* in 

 100 cc. of cold distilled water and allow it to stand for 2U 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 dlmethylparaphenylenediamlne.* 



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 small oocytes. 



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 pliosphoproteins and in the nucleo- 

 proteins. Serra and Lopes ( 19^+5 ) say: "It seems that we can safely conclude that the 

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

 tion, show the existence of nucleotides of the riboae 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 teat, when they are young. 



The method of Angell, A., (1955 Elv. di Biol. 10:702) 



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



5 gms. ammonium molybdate 

 20 cc. distilled water 

 20 cc. of 50^ aqueous hydrochloric acid • 



b. Eeduced in N/50 stannous chloride. 



c. Blnsed quickly in distilled water. 



d. Washed in 2.5^ aqueous ammonia. If phosphorus is present in any form there 

 will develop a blue-green color. 



The method of Serra and Lopes ( 19'*-5 ) 



a. Fix small pieces of tissue in a mixture of 2 volumes of 95^ alcohol, 1 volume 

 of formol, and a few drops of glacial acetic acid per 10 cc. of total volume. 

 Carnoy's fixative is also satisfactory. 



b. Wash In running water, then in distilled water. 



c. Hydrolyze for 5 days or more at 10° to 12°C. and in darkness, if possible. Use 

 5 cc. of the following reagent: to 20 cc. of diatilled water add 0-5 gms. 

 ammonium molybdate and 10 cc. of 50^ HCl, dilute the mixtxire to a total of 



50 cc. with distilled water. 



d. Transfer to temperature of 20° to 25° for several days. 



e. Add 1 drop of acetic benzedine. (Dissolve 25 mgms. of benzedine in 5 cc. of 

 pur^ glacial acetic acid. Dilute to 50 cc. with diatilled water. Stir or 

 agitate for 5 minutea.) 



f. Add 2 drops of pure saturated sodium acetate. The tissue, if phosphorus is 

 present, will rapidly develop an intense blue color. The color is durable. 



g. Mount In glycerine to which has been addec" a few crystals of sodium acetate. 



* If the reagent ie in solid form. It should be heated on a water bath until it melts. 



