Oxidase test reagent manually prepared: Introduction, principle, procedure

Oxidase Test Reagent Manually Prepared: Introduction, Principle, Procedure and Result Interpretation

Introduction of Oxidase Test

Oxidase test manually prepared is as shown above image. It was then tested with Pseudomonas aeruginosa and result found positive i.e. purple color.

Oxidase positive bacteria are-

Pseudomonas
Aeromonas
Vibrio
Brucella
Haemophilus
Alkaligenes
Neisseria
Moraxella
Kingella

Note: Tetramethyl p-phenylene diamine dihydro chloride i.e. substrate used in oxidase test is more expensive.

Kovac’s Oxidase Test

Kovac’s oxidase test determines the presence of cytochrome oxidase. Kovac’s oxidase reagent, tetramethyl-p-phenylenediamine dihydrochloride, is turned into a purple compound by organisms containing cytochrome c as part of their respiratory chain. This test helps in the recognition of Neisseria meningitidis, but other members of the genus Neisseria, as well as unrelated bacterial species, may also give a positive reaction. Positive and negative quality control (QC) strains should be tested along with the unknown isolates to ensure that the oxidase reagent is working properly.

Preparation of 1% Oxidase Reagent from Oxidase Powder

To prevent deterioration of stock oxidase powder, the powder should be stored in a tightly sealed desiccator and kept in a cool, dark area. Kovac’s oxidase reagent is intended only for in vitro diagnostic use. Avoid contact with the eyes and skin as it can cause irritation. In case of accidental contact, immediately flush eyes or skin with water for at least 15 minutes.

Prepare a 1.0% Kovac’s oxidase reagent by dissolving 0.1 g of tetramethyl-pphenylenediamine dihydrochloride into 10 ml of sterile distilled water.
Mix well and then let stand for 15 minutes.
The solution should be made fresh daily and the unused portion should be discarded.
Alternatively, the reagent could be dispensed into 1 ml aliquots and stored frozen at -20°C.
The aliquots should be removed from the freezer and thawed before use. Discard the unused portion each day the reagent is thawed.

Filter Paper Method

On a nonporous surface like Petri dish or glass plate and wet a strip of filter paper with a few drops of Kovac’s oxidase reagent.
Let the filter paper strip air dry before use.
Use a disposable plastic loop, a platinum inoculating loop, or a wooden applicator stick to pick a portion of a colony from overnight growth on the blood agar and rub it onto the treated filter paper
Do not use a nichrome loop, as it may produce a false-positive reaction.
Observe the filter paper for color change to purple.

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