Oxidase Test

introduction image

Oxidase test checks the production of cytochrome-c oxidases, whose presence in the test organism becomes evident by the formation of the blue-purple coloured complex (indophenols) by the oxidation of TMPD (tetramethyl phenylenediamine dihydrochloride) reagent. In the reduced form, the TMPD oxidase reagent remains colourless. Therefore, we can say that the oxidase reagent functions as both “redox indicator and mediator”.

The enzyme cytochrome oxidases carry an iron-containing haemoprotein, generally exists within the mitochondrial cell of aerobic organisms. The enzyme cytochrome oxidases are a part of the respiratory chain, which uses oxygen as a final electron acceptor.

Content: Oxidase Test

  1. Definition
  2. Overview
  3. Principle
  4. Test Reagent
  5. Procedure and Methods
  6. Test Results
  7. Uses
  8. Limitations

Definition of Oxidase Test

Oxidase test can define as a biochemical test that differentiates organism into oxidase-positive and oxidase-negative microorganisms based on the existence of cytochrome oxidase enzyme in their electron transport chain system. The living-organisms which are having cytochrome oxidases can oxidize the TMPD reagent into a blue coloured complex refers as “Indophenol”. Those organisms which lack such enzyme cannot oxidize the reagent and remains in a reduced form (appears colourless).

Oxidase positive organisms: These contains cytochrome oxidases and uses oxygen for energy production in the electron transport chain system.
Example: Neisseria sp, Moraxella sp, Vibrio sp, Helicobacter sp etc.

Oxidase negative organisms: These lacks cytochrome oxidases and cannot use oxygen for energy production in the electron transport chain system.
Example: Enterobacteriaceae sp.

Overview of Oxidase Test

Before moving onto the principle of the oxidase test, we should have some idea of the following terms like:


It can define as a haemoprotein, which belongs to the family of cytochrome-c proteins. Cytochrome-c acts as an electron transport carrier in the electron transport system. It can oxidize as well as reduce in the ferrous and ferric state. It mediates the transfer of electrons within the cytochrome-c reductase (Cyt-Q) and cytochrome-c oxidase (Cyt-IV). In humans, cytochrome-c proteins are coded particularly by the CYCS gene.

Cytochrome-c oxidase

It is the fourth complex of the electron transport system, which commonly occurs in bacteria, archaea and eukaryotes. It is generally a transmembrane protein and the last enzyme of the respiratory chain. This enzyme performs a significant role by receiving an electron from the cytochrome-c complexes and transferring it to the dioxygen electron acceptor. The final electron acceptor releases two water molecules by binding with the four proton ions.

TMPD oxidase reagent

It is also popular by the name of “Wruster’s blue” reagent, which is incompatible with the active oxidizing agent. TMPD reagent works as a redox reagent that detects the presence of cytochrome-c oxidase in the electron transport chain. It also acts as an artificial donor, passes electron to the cytochrome-c complex.


The principle of oxidase test depends upon the mechanism of “Oxidation reaction”, where the organisms possessing cytochrome oxidase enzyme can oxidize the TMPD reagent into end products like blue-coloured complex (Indophenol) and water. Generally, this test differentiates microorganisms into two specific groups, namely oxidase-positive and oxidase negative.

principle of oxidase test

Oxidase positive organisms undergo aerobic respiration, where it makes the use of TMPD as an electron donor. TMPD oxidase reagent will pass electron to the cytochrome-c oxidases, which will oxidize the TMPD reagent into TMPD radicle (stains blue-purple colour). Finally, the terminal oxygen carrier will accept the electron from cytochrome oxidase and binds with a proton to release water. In contrast to this, the organisms deficit of cytochrome oxidases will not oxidize the reagent.

Test Reagent

To perform the oxidase test, we need:
Oxidase reagent: To prepare this reagent, dissolve 0.60 gram of  TMPD (N, N, N’, N’- tetramethyl-p- phenylenediamine dihydrochloride), 0.02 grams of the stabilizing agent in 100ml of dimethyl sulfoxide.

Oxidase discs: These are the filter paper discs that are not only already saturated with DMPD oxalate (N, N – dimethyl-p- phenylenediamine oxalate), α-naphthol, and ascorbic acid but also reduces the time of preparing the oxidase reagent.

Procedure and Methods

Oxidase test includes the following methods:
Methods of oxidase test

Dry filter paper method

In this method, saturate the Whatman’s no.1 filter paper in the 1% N, N, N-tetramethyl p-phenylenediamine dihydrochloride reagent. After that, subject the filter paper to the process of freeze-drying, and later keep it in a screw cap bottle under dark place. While experimenting, take out the filter paper and moisten it with some distilled water. Place the filter paper in a Petri dish and prepare bacterial smear over it. At last, note down the observation result, for any changes in the colour of filter paper.
dry filter paper oxidase test

Wet filter paper method

In this method, take the filter paper discs infused with the oxidase reagent, and directly rub the culture of test organism via a wooden applicator. At last, observe the filter paper for the appearance of a blue-purplish colour.
wet filter paper oxidase test

Direct plate method

It is a very convenient method and a single step procedure, which involves the addition of TMPD oxidase reagent over the suspected colonies.
direct plate oxidase test

Swab method

It is a simple procedure, which involves dipping of the sterile cotton swab in the solution of 1% TMPD reagent. Then gently touch the cotton swab over the suspected colony and look for any colour changes in the swab’s bud.
swab oxidase method

Test tube method

In this method, prepare nutrient broth media and pour up to 4.5ml into the test tube. Then inoculate the test organism and incubate for at least 24-48 hours at 35 degrees Celsius. After incubation, add oxidase reagent (0.2ml) into the culture tube and shake vigorously. At last, look at the culture tubes for the appearance of a bluish-purple colour.
test tube oxidase method

Test Results

There can be three possible outcomes:

  • Positive: Confirms by the appearance of intense deep bluish-purple colour within 10 sec.
  • Delayed positive: Confirms by the appearance of intense deep bluish-purple colour after 10 sec.
  • Negative: Confirms by no colour change by the addition of oxidase reagent.


Oxidase test is used to determine the existence of cytochrome oxidase enzyme within the electron transport chain of mitochondria. It also differentiates organisms into oxidase-positive and oxidase negative, based on the presence of cytochrome oxidase in the electron transport system.

As, cytochrome oxidase is found in the aerobic bacteria, and is a part of the aerobic respiratory chain. So, it also distinguishes aerobic organisms from the facultative aerobes, strict and facultative anaerobes. Oxidase test is useful in the speciation, as it differentiates Pseudomonas from other related species.


The reagents should be freshly prepared; otherwise, it may give diversifying results. A test organism must be 18-24 hours old. The test organism isolated from non-selective and non-differential media is preferred for the identification and classification. Growth medium supplemented with dye can give erroneous results. It makes the use of a platinum loop instead of iron or nichrome to avoid false-positive results due to surface oxidation during flaming.

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