ASCORBIC ACID 55 



ascorbic acid is absent. Tonutti (1938) washed the tissue in 5.4% 

 levulose solution before staining in order to remove blood. The 

 reliability of the localizations obtained with the silver stain remains 

 to be proved, according to Danielli ( 1946a) . It would be necessary 

 to establish that the ascorbic acid is attached to a nondiffusible 

 body and that the reaction product could not diffuse, or that the 

 ascorbic acid site has a high affinity for the reaction product. 



Bourne Silver Stain for Ascorbic Acid 



/. Reduced Ascorbic Acid 

 SPECIAL REAGENTS 



Acid Silver Nitrate. Add 5 ml. glacial acetic acid to 100 ml. 5% 



silver nitrate. 

 5% Ammonium Hydroxide. 



PROCEDURE 



1. Place frozen sections of fresh tissue in the acid silver nitrate 

 soln. for a few minutes, and then treat with 5% ammonium 

 hydroxide. 



2. Wash with distilled water. If desired, lipids can be then 

 stained with a Sudan dye in 90% alcohol. 



3. After clearing, mount in glycerin. 



Result. Granules containing reduced ascorbic acid appear black. 



11. Reduced and Oxidized Ascorbic Acid 

 PROCEDURE 



1. Expose the fresh tissue to the vapor of glacial acetic acid for 

 several minutes. 



2. Cut into thin pieces and subject to an atmosphere of hydrogen 

 sulfide for 15 min. in order to reduce the oxidized form. 



3. Remove hydrogen sulfide by placing in a vacuum for 10-30 

 min. followed by a good stream of nitrogen gas for 15 min. 



4. Treat with acid silver nitrate soln. followed by ammonium 

 hydroxide as above. 



Should glutathione be present in quantities sufficient to inhibit the 

 test, wash the tissue momentarily after the hydrogen sulfide treat- 

 ment and immerse at once into a mercuric acetate soln. for a few 

 minutes. After washing, apply the acid silver nitrate solution and 

 then the ammonium hydroxide. 



