Fish Microbiology and Quality Assurance

7. Oxidase test

This test helps to determine the ability of bacteria to produce cytochrome oxidase enzyme involved in electron transport chain in aerobic respiration. Aerobic bacteria including some microaerophilic and facultative anaerobesia exhibit oxidase activity by catalyzing the oxidation of a reduced cytochrome by molecular oxygen, resulting in the formation of water or hydrogen peroxide.

Procedure:
A small portion of the test cultures grown on nutrient agar plates are picked using sterile platinum loops or sterile tooth picks and placed on pre-moistened filter paper strips containing test reagent (1% solution of dimethyl- para –phenylenediamine hydrochloride) and observed for 10-15 seconds. The test reagent serving as artificial substrate donates electrons and gets oxidized to a blackish compound in the presence of oxidase and free oxygen. Appearance of black clour is indicative of positive test.

Oxidase reagent
Oxidase reagent is prepared by dissolving 1.0 g of N, N, N’, N’-tetramethyl-p-phenylene diamine dihydrochloride in 100 ml distilled water.

Preparation of oxdase filter paper strips: Whatman filter paper No.1 strips cut to size of 2-5 x 1.0 cm, sterilized in hot air oven at 140⁰c for 1hour, then dipped in oxidase reagent, dried and stored in dark bottle at 4⁰C.