Some bacteria like Pseudomonas, Pasteurellaceae, Campylobacter, Neisseria, Moraxella, can produce cytochrome
C oxidase located in their membrane which can catalyse the transport of electrons from donor compounds to
electron acceptors (oxygen). This respiratory system is present in aerobic bacteria which are capable to use
oxygen as the final hydrogen receptor.
In the presence of oxygen the cytochrome C enzyme oxidizes the
phenylendiamine reagent, resulting indophenol, a purple-blue compound.
There are several ways to perform oxidase test: using filter paper, using a
swab or adding the reactive directly on plate.
Filter paper method: soak a piece of filter paper in Kovacs reagent
(N, N, N', N'-tetra-methyl-p-phenylenediamine dihydrochloride). Pick a
colony and dispense on filter paper.
Swab method: harvest culture from a solid media then add 1-2 drops of
Direct method: add 1-2 drops of reagent to one colony from an agar plate.
For all methods a blue color appears within 1 minute if reaction is positive.
Bacteria culture should not be older than 48 hours.
Do not use an iron or nichrome inoculating loop; false positive reaction may occur.
Test should not be performed on cultures from media containing carbohydrates or tellurite; false negative
reaction may occur.
Phenylenediamine reagent auto-oxidise quickly. Add 1% ascorbic acid for a longer preservation. Do not use it, if
it's already blue.
(c) Costin Stoica