<& 



PHOSPHATE AND NITRATE 3o 



acetic acid to 10 ml. of a mixture of 2 vol. 96% alcohol and 1 vol. 



formalin. 

 Molybdate Solution. Dissolve 0.5 g. ammonium molybdate in 20 



ml. distilled water, add 10 ml. cone. (30%) hydrochloric acid, and 



dilute to 50 ml. with distilled water. 

 Acetic Benzidine Solution. Dissolve 25 mg. benzidine in 5 ml, 



glacial acetic acid and dilute to 50 ml. with distilled water. 

 Saturated Sodium Acetate Solution. 



PROCEDURE 



1. Fix the tissue in the acetic alcohol-formalin mixture and wash 

 well in water. 



2. In order to hydrolyze organic phosphates and precipitate the 

 free phosphate, treat small pieces of the tissue or frozen sections with 

 the molybdate reagent at 10-12° for 2-3 weeks, followed by 2-3 

 days at 20-25°. The temperature is kept low to prevent alteration of 

 the tissue and the rather long time is required to effect hydrolysis 

 with the relatively weak acid concentration of the reagent. 



3. Cover the tissue with a drop of acetic benzidine soln. for 3 

 min. and then add 2 drops of the sodium acetate soln. 



4. Mount in glycerol which has been stoi-ed with crystals of 

 sodium acetate in the bottle. 



Result. An intense blue coloration characterizes phosphate. 



note: Sena and Queiroz Lopez employ a digestion with nuclease to liberate 

 phosphate from nucleic acid. The visualization of this phosphate then serves 

 to indicate the nucleic acid. 



NITRATE 



Cramer ( 1940) developed a method for the histological demon- 

 stration of nitrate which is based on the doubly refractive property 

 in polarized light of the insoluble salt formed by the interaction of 

 nitrate with Nitron (4,5-dihydro-l,4-diphenyl-3,5-phenylimino-l,2,4- 

 triazole). Busch (1905) originally employed this reaction for the 

 gravimetric determination of nitric acid. 



Cramer Method for Nitrate 



SPECIAL REAGENTS 



Nitron Reagent. 10% Nitron in 5% acetic acid. 



