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Oxidase test

Oxidase test

Oxidase test
A. Two drops of oxidase reagent is applied onto a piece of filter paper.
B and C. Colony material is transferred by a plastic loop to the spot of oxidase on the filter paper.
C. The left spot includes oxidase positive bacteria and the spot to the right oxidase negative bacteria.
D. The final results can be observed after 30 s.
- Click on the image to enlarge it.

General information

Bacteria, which have aerobic respiration, often have cytochrome c and a cytochrome c oxidase. The presence of these components can in combination with other methods be used for typing. A commersial test, which contains an artificial electron acceptor (N, N, N', N'-tetramethyl-p-phenylenediamine, see Fig. 1), is often used. This artificial electron acceptor change colour depending upon redox state. The substance is also referred to as a redox indicator and it can be oxidized by the oxidized form of cytochrome c. Cytochrome c oxidase is the last enzyme of the electron transport chain, where it normally reduces oxygen to water and pump protons to the outside according to the following net reaction:

4 Fe2+-cytochrome cred + 8H+in + O24 Fe3+-cytochrome cox + 2H2O + 4H+out

Cytochrome c oxidase is a transmembrane protein complex (Complex IV), which is also present in the  cytoplasmic (inner) membrane of mitochondria.


  1. Keep the plastic ampoule (which contains a glass ampoule) between your fingers and with the opening upwards and away from your face.  Press the ampouls with your fingers until the glass ampoule breaks.
  2. Apply two drops of the oxidase reagent onto a piece of filter paper.
  3. Transfer bacteria from one colony with a plastic or platinum loop onto the spot with the oxidase reagent. The colonies should have been incubated at the appropriate temperature for 18-24 h .
  • Positive test resultat: Dark blue-purple colour change within 10-30 sec.
  • Negative test resultat: No colour change or colour change after more than 30 sec.

Note that the oxidase reagent is not stable after that the ampoule has been opened. It may be used for a couple of hours, but eventually it will be oxidized by the oxygen in the air.


The oxidase test is used for identification of gram negative bacteria. For instance to identify members of the family Enterobacteriaceae, which are oxidase negative, except members of the genus Plesiomonas (oxidase positive). Members of the family Pseudomonadaceae, and the genera Aeromonas and Campylobacter are oxidase positive.

Updated: 2017-12-20.


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Swedish University of Agricultural Sciences