Enzymes 165 



immediately, in the shade of the original dye. Counterstain 

 as desired. Mount in balsam. 



B ) The benzidine method will stain even somewhat stale, 

 degraded hemoglobin ( nonenzymatic heme catalysis?). Of 

 the numerous modifications of this method, the technique of 

 Osgood^^ and Washburn^^ is recommended. 



Prepare a 0.2-0.3 per cent solution of benzidine in 95 per 

 cent alcohol. To each 100 ml. of the solution add about 

 0.5 g. Na nitroprusside dissolved in a few ml. of water (nitro- 

 prusside appears to enhance blue shades in preference to 

 brown ones). This stock solution will keep for months if 

 refrigerated. 



When staining smears, pour the solution over the slide for 

 3 minutes. The alcoholic solution will serve as a fixative. This 

 step may be omitted in the case of tissue sections. Decant. 

 Mix equal volumes of the solution and a 1:5 dilution (for 

 hemoglobin) or a 1:50 dilution (for myeloid granules) of 

 commercial (3 per cent) hydrogen peroxide; pour it over 

 the slide and leave it on for about 5 minutes. Decant, wash 

 slide briefly in water, counterstain with a red nuclear stain, 

 dehydrate, and mount in balsam. Myeloid granules appear 

 intensely dark blue; hemoglobin appears in shades of dark 

 brown to blue. The addition of a few drops of an acetate 

 buffer of about pH 4.5 to the benzidine-H202 mixture will 

 make hemoglobin stain in a clearer blue shade. The naphthol 

 technique of Loele"^^ is not recommended. 



APPENDIX 



Unna's "Reduktionsorte" and "Sauerstofforte'* 



The terms Reduktionsorte ("sites of reduction") and Sauerstofforte 

 ("sites of oxygen") were coined by the German dermatologist Unna*^^ 

 to denote structures which reduce oxidizing substances or show the 

 presence of "free" oxygen, respectively. The first are demonstrated by 



74. Osgood, E. E.: Atlas of hematology (San Francisco: Stacey, 1937). 



75. Washburn, A. H.: J. Lab. & Clin. Med., 14:246, 1928. 



76. Unna, P. G.: Arch. f. mikr. Anat, 78:1, 1911; Unna, P. G.: Med. 

 KHn., 8:951, 1912; Unna, P. G.: Berl. klin. Wchnschr., 50:809, 1913; Unna, 

 P. G.: Abderhalden's Handb. d. biol. Arbeitsmeth., V-22:62, 1928. 



