94 MICROSCOPIC TECHNIQUES 



CYTOCHROME OXIDASE 



Tests for cytochrome oxidases have been adapted to histochemical 

 work and a discussion of them has been given by Lison ( 1936, pages 

 269-290). The enzyme has been referred to as "nadi oxidase" and 

 "indophenol oxidase/' but Keilin and Hartree ( 1938) have made it 

 clear that it should be called "cytochrome oxidase" since its catalytic 

 effect applies to the oxidation of reduced cytochrome. In the 

 presence of cytochrome c, cytochrome oxidase effects the oxidation of 

 a mixture of p-aminodimethylaniline and a-naphthol (nadi reagent) 

 to indophenol, or of p-phenylenediamine to the diimine. The diffusi- 

 bility of the colored compounds produced must be considered with 

 reference to localizations of the enzyme in tissue. 



In order to check whether a nadi reaction is being given by cyto- 

 chrome oxidase or some other factor, Moog (1943b) exposed fresh 

 tissue (chick embryo) to a 0.005 M azide solution in acidified 

 physiological saline (pH 5.8) for 3 min., and then transferred it to 

 freshly prepared nadi reagent containing 0.005 M azide. Azide 

 specifically inhibits cytochrome oxidase. As a control of the possi- 

 bility that indophenol blue might be reduced to the leuco form as 

 fast as formed, Moog also placed the tissue in 0.003 M phenylurethan 

 in saline for 3 min. to saturate the reducing systems, and then trans- 

 ferred to the nadi reagent containing 0.003 M phenylurethan. The 

 reagent used by Moog was prepared by combining, just before use, 

 equal parts of 0.01 M /^-aminodimethylaniline in 1 % sodium chloride, 

 0.01 M a-naphthol in 1% sodium chloride, and 0.066 M phosphate 

 buffer. The reaction was carried out at 38° for the interval required 

 to attain a standard coloration (5-14 min.). Under the conditions 

 employed, identical results were obtained at pH 5.8 and 7.2. 



Graff Method for Cytochrome Oxidase in Fixed Tissue 

 ("M. Nadi Oxidase") 



SPECIAL REAGENTS 



a-Naphthol Solution. Boil 1 g. a-naphthol in 100 ml. distilled water 

 and add 25% potassium hydroxide dropwise until the melted 

 a-naphthol is dissolved. Store in the dark; keeps for at least 1 

 month. 



1% p-Aminodimethylaniline or Its Hydrochloride. Boil to dissolve 

 solid in the water. Store in the dark; may be used for 2-3 weeks. 



