Oxidase test - Purpose, Principle, Biochemistry, Reagent, Procedure, Result, Interpretation

Introduction to Oxidase test

The oxidase test is a biochemical test used to determine the presence of oxidase enzyme in microorganisms as this enzyme is capable of catalyzing the transport of electrons between a redox dye tetramethyl-p-phenylene-diamine and electron donors.

Purpose of Oxidase test

The purpose of the oxidation reaction is able to take place due to the presence of a cytochrome oxidase system. This system with the help of molecular oxygen is responsible for the activation of reduced cytochrome and in the terminal stage of the electron transfer system, the reduced cytochrome acts as an electron acceptor.

Principle of Oxidase test

During the process of respiration, a substrate is oxidized by molecular oxygen by going through the electron transport system. In aerobic organisms, the presence of a cytochrome system makes it possible to utilize oxygen as a final hydrogen acceptor to reduce molecular oxygen to hydrogen peroxide.

In microorganisms, all oxidase-positive organisms are aerobic or facultatively anaerobic. One noticeable exception is the Vibrio fetus which is microaerophilic. Obligate anaerobes on the other hand lack oxidase activity as well as a cytochrome oxidase system due to their inability to live in the presence of atmospheric oxygen.

Oxidase-positive organisms can also be catalase test positive.

Biochemistry of Oxidase test

Microorganisms producing oxidase enzymes in the presence of cytochrome C, atmospheric oxygen, and an oxidase reagent are able to oxidize the reagent to form indophenol, a colored compound.

The oxidizing agent p-phenylenediamine can be used in the oxidase test. The p-phenylenediamine is the primary aromatic amines diamino which is a derivative of benzene. These oxidizing agents, in presence of cytochrome oxidase and atmospheric oxygen, are oxidized to produce a colored compound - indophenol.

If α-naphthol is used as an oxidizing agent, indophenol blue is formed.

Reagent of Oxidase test

The reagent of the oxidase test is as follows:

• Kovac's reagent:

  1% tetramethyl-p-phenylenediamine dihydrochloride

• Gordon and McLeod's reagent:

  1 to 1.5% dimethyl-p-phenylenediamine dihydrochloride

Procedure of Oxidase test

The procedure of the Oxidase test can be done by the wet filter paper method and the dry filter paper method.

Wet filter paper method

1. Take a piece of Whatman's filter paper and place it on a clean, dry petri dish/glass slide

2. Add 2-3 drops of fresh oxidizing agent solution (1% tetramethyl-p-phenylenediamine dihydrochloride)

3. With the help of a wooden stick or glass rod, pick up a colony from fresh bacterial culture and smear it on the filter paper

   (do not use a metal wire loop to pick up a colony and take careful measures not to pick up media)

4. Observe for color change within 5-10 seconds on the filter paper smeared with the test organism

Dry filter paper method

1. Take an oxidase-impregnated disc and place it on a dry petri dish/glass slide

2. Pour 1-2 drops of distilled water to moist the paper

3. With the help of a wooden stick or glass rod, pick up a colony from fresh bacterial culture and smear it on the wet area of the disc

   (do not use a metal wire loop to pick up a colony and take careful measures not to pick up media)

4. Observe for color change within 5-10 seconds on the filter paper smeared with the test organism

Result, Interpretation of Oxidase test

The result of the oxidase test can be interpreted as follows:

Positive

Within 5-10 seconds, a blue-purple color develops where the test organism was smeared.

Negative

Absence of blue-purple color where the test organism was smeared. If the paper is left out for a while, coloration may develop on the paper.

Oxidase positive organism

A list of oxidase-positive organisms are:

• Neisseria

• Pseudomonas

• Moraxella

• Helicobacter pylori

• Vibrio cholerae

• Campylobacter jejuni

• Alcaligenes

• Aeromonas

Oxidase negative organism

A list of oxidase-negative organisms are:

• Enterobacteriaceae family

• Acetobacter

• most gram-positive bacteria

Oxidase variable organism

A list of oxidase-variable organisms are:

• Legionella pneumophila