Oxidase test is used to identify microorganisms that produce the enzyme, cytochrome-C oxidase. Cytochrome-C oxidase, a respiratory enzyme is an important enzyme of the electron transport chain (ETC), where it catalyzes the transport of electrons from a donor compound (e.g. NADH) to oxygen, the final electron acceptor. If cytochrome-C oxidase is present, it will oxidize tetramethyl-p-phenylenediamine hydrochloride (the redox dye), turning it into a blue-purple colour. The redox dye is usually clear in its reduced form.  Oxidase test is used to differentiate oxidase producing bacteria (e.g. Pseudomonas) from non-oxidase producing ones (e.g. Enterobacteriaceae). Oxidase test which is very useful in differentiating between Pseudomonads and Gram-negative rods is carried out using a redox reagent/dye called tetramethyl-p-phenylenediamine hydrochloride (TMPPEH) which acts as the electron donor. TMPPEH (oxidase reagent) is usually stored in dark (amber) bottles kept in the refrigerator and away from light in order to avoid auto-oxidation which may reduce its potency. The addition of 0.1 % ascorbic acid to oxidase reagent can also help to protect it against auto-oxidation. Pseudomonas, Vibrio, Pasteurella, and Brucella species are oxidase positive because they produce oxidase enzyme. This test can also be performed using an oxidase reagent strip.


  1. Perform this test with a pure culture of the test organism.
  2. Place a piece of filter paper on a clean glass slide or Petri dish.
  3. Add about 2 drops of oxidase reagent onto the filter paper
  4. Pick a speck or colony of the test bacterium using a glass rod or the edge of a clean glass slide. Note: An oxidized or flamed inoculating loop must not be used to pick the test bacterium from the culture plate. 
  5. Smear the inoculum of the bacterium onto the filter paper on the Petri dish plate.
  6. Observe the filter paper for the presence of a blue-purple colour which starts to develop within 5-10 seconds. This is indicative of a positive oxidase test (Figure 1). Absence of a blue-purple colour indicates a negative test result.
Figure 1. Oxidase test. The filter paper on the left-hand is negative while the filter paper on the right-hand side is positive as indicated by the blue colour. Photo courtesy: https://www.microbiologyclass.com

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.

Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

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