Identification of Haemoglobin by its Catalase Reaction with Peroxide ando-Dianisidine

Abstract

o-Dianisidine (3,3′-dimethoxybenzidine) when used as an indicator in the peroxidase activity of haemoglobin forms a clear and distinct orange stain. The reaction takes from 5 to 15 rain in the presence of H₂O₂ and completes both fixation and staining. This may be followed by dehydration in dioxane, clearing in xylene and mounting in D.P.X. or Canada balsam. The detergent Tepol can be used to spread or to disintegrate the chick embryo to obtain a monolayer of cells after the staining reaction has been completed. Background staining is negligible, the reaction is very sensitive and the colour developed is permanent Stock solutions are: (1) o-dianisidine, 100 mg per 70 ml of ethanol; (2) acetate buffer, 0.1 M at pH 4.6; and (3) hydrogen peroxide, 30% aqueous solution. The stock solutions should be refrigerated. The staining mixture contains o-diarusidine solution, acetate buffer, distilled water and hydrogen peroxide in the proportion of 4:1:1.5:0.2.