Oxidase test | Principle | Protocol | Results

☰ Content :

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

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

Oxidase Positive Bacteria Neisseria (toutes espèces) / Actinobacillus lignieresii / Aeromonas spp. / Actinobacillus equuli / Pasteurella spp. / Bordetella pertussis / Vibrio spp. / Bac. anthracis / Cordiobacterium hominis / Bac. subtillis / Pseudomonas spp. / Brucella spp. / Flavobacterium spp. / Chromobacterium spp. / Alcaligenes spp. / Eikenella corrodens / Moraxella spp. / Plesiomonas spp. / Campylobacter spp. / Branhamella catarrhalis / Micrococcus spp.
Negative Oxidase Bacteria Staphylococcus spp. / Pseudomonas mallei / Streptococcus spp. / Pseudomonas maltophilia / Gemella haemolysans / Bordetella parapertussis Peptococcus spp. / Actinobacillus / Peptostreptococcus spp. / actinomycetem-comitans / Leuconostoc spp. / Anaerobier (tous) / Corynebacterium spp. / Listeria spp. / Pasteurella haemolytica / Lactobacillus spp. / Bacillus spp. / Streptobacillus / Enterobacteriaceae / Mycoplasma spp. / Acholeplasma spp.

☰ 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