PEROXIDASE AND DOPA OXIDASE 91 



6. Dehydrate in 95% alcohol for 30 sec, and absolute alcohol 

 for 5 sec. 



7. Clear in xylol and mount in balsam. 



8. Run controls in which the benzidine is omitted. 



Result. Peroxidase manifests itself by an initial blue color which 

 changes to brown. Diffusibility, particularly of the color produced, 

 can be expected to interfere with proper localization of the enzyme. 



Armitage Method for Peroxidase in Blood or Bone 

 Marrow Smears 



SPECIAL REAGENTS 



Fixing Solution. 10% Formalin in 96% alcohol. Prepare the soln. 



just before using. 

 Benzidine Reagent. Dissolve 750 mg. benzidine in 500 ml. 40% 



alcohol, filter, add 0.7 ml. 3% hydrogen peroxide, and shake 



before using. If stored in the dark, the reagent will be good for 



months. 



PROCEDURE 



1. Fix the smear in the alcoholic formalin. 



2. Cover the material with the benzidine reagent for about 2 min. 

 if the smear is fresh, and up to 20 min. if it is old. 



3. Wash in 40% alcohol until yellow granules appear in the 

 leucocytes. 



4. Dehydrate in absolute alcohol and dry at about 37°. 



5. A counterstain of dilute Giemsa or dilute Leishman stain may 

 be applied for 30 min. followed by washing in water, blotting, and 

 drying. 



Result. The appearance of yellow granules is a positive test for 

 peroxidase. 



DOPA OXIDASE 



The enzymatic oxidation of 3,4-dihydroxyphenylalanine — or dopa 

 — has been applied, histologically, to the identification of melano- 

 blasts, since these appear to be the seat of the oxidase activity and 

 the conversion of dopa to melanin results in their becoming black- 

 ened. Bloch's earlier work has been adapted by Laidlaw (1932) and 

 Laidlaw and Blackberg ( 1932 ) to the demonstration of dopa oxidase 



