Oxidase test | Principle | Protocol | Results

Ⅰ. Principle of oxidase test

☰ The oxidase test is a test to detect the enzyme cytochrome oxidase in Gram-negative bacteria which produce this enzyme, such as Neisseria or Pseudomonas.

☰ Oxidase Discs (OX) are absorbent paper discs impregnated with N, N, N ’, N’-Tetramethyl-p-phenylenediamine dihydrochloride. In the presence of cytochrome oxidase, N, N, N ’, N’-Tetramethyl-p-phenylenediamine dihydrochloride (colorless) forms a compound.

Ⅱ. Oxidase test protocol

1. Using tweezers, place an oxidase disk on a slide.
2. Choose a colony that is well isolated and representative of the fresh culture to be tested.
3. Collect the chosen colony using a stick or an ose.
4. DO NOT USE a metal loop (except platinum) it may cause false positive reactions.
5. Gently rub the colony on the disc and observe the appearance of a purple / blue color within 30 seconds.

Oxidase test positive and negative (source : ASM MicrobiologyLibrary)

Ⅲ. Oxidase test results

☰ Positive reaction : dark blue to purple coloration appearing within 30 seconds.

☰ Negative reaction : absence of coloration or coloration beyond 30 seconds.

Ⅳ. Bacteria with Oxidase Positive / Negative

☰ Variable oxidase : (positive or negative depending on the strain): Actinobacillus, Brucella, Haemophilus, Pasteurella.

Usage limits:

• Candida albicans will occasionally give positive result with oxidase test when grown on chocolate agar but give negative reactions when grown on Sabouraud dextrose agar.
• Kovac’s oxidase reagent, 1% aqueous solution of N, N, N′, N′ -tetramethyl-p-phenylenediamine dihydrochloride is less toxic and more sensitive than the 6% solution of N, N, N′, N′-tetramethyl-p-phenylenediamine in dimethyl sulphoxide (DMSO) but more expensive and relatively unstable.
• It is necessary to carry out additional biochemical and / or serological tests for a precise identification of the species.
• Insufficient inoculum may cause weak or late staining.
• Colonies taken from media containing nitrates may produce an inconsistent result.
• Media containing a high proportion of blood may generate a false positive result.
• It is essential to proceed from pure and fresh cultures so that the results can be interpreted.

Ⅴ. Frequently asked questions

Q : What does the oxidase test determine?
A : The oxidase test is a biochemical reaction that determines the presence or absence of cytochrome oxidase, it is one of the main bacterial identification tests.

Q : What color is a positive oxidase test?
A : Dark purple

Q : Does E coli test positive for oxidase?
A : Non, E coli test negative for oxidase, the entire family of Enterobacteriaceae are oxidase negative

Q : Why is it important to read oxidase results within 30 seconds?
A : In this test, the timing is important. The reagents of oxidase test is unstable, If it is exposed to air for too long , the oxygen will change the reagent color as time passes (spontaneously become oxidized