Biochemical tests

OXIDASE TEST

Written by MicroDok

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 TMPPEH (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.

Procedure

  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. Absence of a blue-purple colour indicates a negative test result.

Illustration of oxidase test

The left-hand side of the filter paper is negative while the right hand side indicate a positive test reasult as observed by the formation of a blue colour. The formation of the colour follows the reaction of oxidase (released by the organism) with the redox dye, TMPPEH.

REFERENCES

Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. 2nd Cambridge University Press, UK. Pp. 178-187.

Willey J.M, Sherwood L.M and Woolverton C.J (2008). Harley and Klein’s Microbiology. 7th ed. McGraw-Hill Higher Education, 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.

World Health Organization (1993). Laboratory Biosafety Manual, 2nd edn. Geneva: WHO.

World Health Organization (2003). Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens.  WHO/EMC/97.3. Geneva: WHO.

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MicroDok

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