70 MICROSCOPIC TECHNIQUES 



Bennett ( 1940) first removed ascorbic acid from the tissue to pre- 

 vent its interference with the tests. Albert and Leblond (1946) 

 substituted 2,4-dinitrophenylhydrazine for the phenylhydrazine of 

 Bennett. This enabled a more intense staining in thinner sections. 



Bennett Use of Phenylhydrazine Reaction for Water-Insoluble 

 Aldehydes and Ketones 



SPECIAL REAGENTS 



M/10 Acetate Buffer, pH 6.0 to 6.5. 



1 % Iodine in Alcohol. 



1% Sodium Thiosulfate Solution. 



1% Buffered Phenylhydrazine. Prepare just before use by mixing 

 equal vol. of 2% phenylhydrazine hydrochloride and the acetate 

 buffer. Gently bubble carbon dioxide through the solution for 15 

 min. to remove oxygen. 



Control Reagent. Same as the 1 % buffered phenylhydrazine with- 

 out the phenylhydrazine. 



PROCEDURE 



1. Transfer frozen sections of fresh tissue from the microtome 

 directly into acetate buffer. If fixed tissue is employed, transfer to 

 water. 



2. Add the iodine solution dropwise until a faint straw color 

 persists, and let stand 15 min. 



3. Add sodium thiosulfate solution dropwise until the color is 

 discharged and a little more has been added ; let stand 5 min. 



4. Wash the sections several times in distilled water. 



5. Place the sections in glass-stoppered bottles containing 

 buffered phenylhydrazine solution. Fill the bottles to the top so that 

 no air bubbles are present under the stopper. 



6. Run control sections as in previous steps, only use the control 

 reagent in place of phenylhydrazine. 



7. After standing several hr. or overnight, wash all sections 

 with distilled water a few times. 



8. Mount in glycerol or glycerol-gelatin and examine by means 

 of incident illumination from above. 



Result. A yellow color appears in areas giving the positive test. 

 It is essential that care be taken in conducting this test since the 

 appearance of a yellow deposit on the walls of the bottle or on top 

 of the liquid indicates decomposition of the reagent, and when this 



