Oxidase Test

Principle:
Cytochrome containing organisms produce an intracellular oxidase enzyme. This
oxidase enzyme catalyzes the oxidation of cytochrome c. Organisms which contain
cytochrome c as part of their respiratory chain are oxidase-positive and turn the
reagent blue/purple. Organisms lacking cytochrome c as part of their respiratory
chain do not oxidize the reagent, leaving it colorless within the limits of the test,
and are oxidase-negative.
Oxidase positive bacteria possess cytochrome oxidase or indophenol oxidase (an
iron containing haemoprotein). Both of these catalyse the transport of electrons
from donor compounds (NADH) to electron acceptors (usually oxygen). The test
reagent, N, N, N, N-tetramethyl-p-phenylenediamine dihydrochloride acts as an
artificial electron acceptor for the enzyme oxidase. The oxidised reagent forms the
coloured compound indophenol blue.
The cytochrome system is usually only present in aerobic organisms which are
capable of utilising oxygen as the final hydrogen receptor. The end product of this
metabolism is either water or hydrogen peroxide (broken down by catalase).
Reactants:

The oxidase test is used to determine if an organism possesses the

cytochrome oxidase enzyme.

The test is used as an aid for the differentiation of Neisseria, Moraxella,

Campylobacter andPasteurella species (oxidase positive).

It is also used to differentiate pseudomonads from related species.

Examples:
Oxidase Positive Organisms: Pseudomonas, Neisseria, Alcaligens, Aeromonas,
Campylobacter,

Vibrio,

Brucella,

Pasteurella, Moraxella,

pylori, Legionella pneumophila, etc.

Oxidase Negative Organisms: Enterobacteriaceae (e.g. E. coli)
Process:

Helicobacter

There are many method variations to the oxidase test. These include, but are not
limited to, the filter paper test, direct plate method, swab method, impregnated
oxidase test strip method and test tube method. All times and concentrations are
based upon the original recommendations.

Direct Plate Method

Add 2 -3 drops of reagent directly

to suspect colonies on an agar
plate. Do not flood the plate with
the reagent.

Observe for colour change within

10- 30 seconds.

Note: The Direct Plate method should be

carried out on a non-selective agar plate
like NA, BHIA plate.

Reagents:

Kovacs Oxidase Reagent:

1% tetra-methyl-p-phenylenediamine dihydrochloride, in water

Gordon and McLeods Reagent:

1% dimethyl-p-phenylenediamine dihydrochloride, in water

Gaby and Hadley (indophenol oxidase) Reagent:

1% -naphthol in 95% ethanol
1% p-aminodimethylaniline oxalate (use in lab)
Gaby and Hadley developed a modified oxidase test using p-aminodimethylaniline
oxalate with -naphthol to detect oxidase in cultures.
Result:
Positive Result: Development of a deep purple-blue/blue colour indicates oxidase
production within 5-10 seconds.
Negative Result: No purple-blue colour/No colour change.

Limitations:
1.

The reagents used in the oxidase test have been shown to auto-oxidize, so it
is very important to use fresh reagents, no older than 1 week.

2.

Bacteria grown on media containing dyes may give aberrant results.

3.

The test reagents will effectively kill the microorganisms, so sub-culturing

should be done prior to adding any reagent to an active culture.

4.

The

oxidase

test

can

be

used

in

the

presumptive

identification

of Neisseria and in the differentiation and identification of gram-negative bacilli.

Oxidase-positive organisms should be examined by gram stain to determine
morphology and gram reaction. Additional biochemical tests are recommended for
complete identification.
5.

Use of a nichrome or other iron containing loop may yield false-positive

reactions. Platinum loops are recommended.

6.

Oxidase reactions of gram-negative bacilli should be determined on nonselective and non-differential media to ensure valid results. Also, colonies taken
from media containing high levels of glucose may give false-negative reactions.

7.

It is recommended to use colonies that are 18-24 hours old. Older colonies
will produce weaker reactions.