Abstract
The indophenol reaction employed depends upon the oxidation of a mixture of a-naphthol and dimethyl-paraphenylene-diaminehydrochloride, in alkaline solution, into indophenol (blue) in accordance with the following equation:
(CH3)2N.C6H4.NH3Cl+CO10H7.OH+O2=(CH3)2N.C6H4.N=C10H6O+2H2O+HCl
The HCl, which prevents the reaction, is neutralized by the alkali present. This oxidation occurs spontaneously at a very slow rate when the reacting substances are exposed to air, but may be accelerated twenty times or more when minced animal tissue is added. The determinations were always carried out in open flat-bottomed Petri dishes.
The substrate in use at the present time contains 0.072 per cent of a-naphthol (M/200), 0.08624 per cent of dimethyl-paraphenylenediamine-hydrochloride (M/200) and 0.0625 per cent sodium carbonate in 19 per cent alcohol. In order to prevent spontaneous oxidation in the substrate prior to the beginning of the oxidase test, the reagents which make up the substrate must not be mixed until needed. By having stock solutions of: (1) 0.18 per cent a-naphthol in equal parts of distilled water and 95 per cent alcohol, (2) 0.216 per cent aqueous solution of dimethyl-paraphenylene-diamine-hydrochloride, and (3) 0.3125 per cent aqueous solution of sodium carbonate, the amount of substrate needed for a test may be obtained by pipetting 2 cc. of each of solutions number one and two, and 1 cc. of the carbonate solution into the Petri dish. The success of the reaction depends upon the freshness of the diamine-hydrochloride solution which is relatively unstable and oxidizes readily in the presence of air. It is stable in the powdered form and, therefore, the solution should be prepared fresh daily.
The tissue to be studied is ground thoroughly with an equal weight of quartz sand in a mortar for exactly 10 minutes. One gram of this minced tissue-sand mixture is now accurately weighed out in a flat-bottomed Petri dish 9.5 cm. in diameter.
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