Oxidase test by Kovac’s method (Principle, Procedure, Result Interpretation and Precautions)


This test is performed to determine or identify the presence of an enzyme cytochrome oxidase (of the electron transport chain) in bacterial cells. The substrate used is tetramethyl-p-phenylenediamine dihydrochloride, which is oxidized to a purple colored end product called indophenol by the enzyme oxidase. The development of a dark purple color is a positive test that indicates the presence of oxidase, whereas if the enzyme is not present, the reagent remains reduced and is colorless.


  • Take a filter paper and moisten it with the substrate i.e. 1% tetramethyl-p-phenylenediamine dihydrochloride or select a commercially available paper disk that has been impregnated with the same substrate.
  • Remove a small portion of a bacterial colony (preferably not more than 24 hours old) from the agar surface with a sterile platinum wire or wooden stick.
  • Rub the sample on the filter paper or commercial disks.
  • Observe the inoculated area of the paper of disks for the color change to deep blue or purple within 10 seconds because timing is very critical.

Result interpretation:

  • Positive result: Development of a dark purple color within 10 seconds of inoculation. E.g. Neisseria gonorrhoeae, Vibrio cholera, Pseudomonas are oxidase positive.
  • Negative result: No change in color (i.e. no blue color seen) e.g. Members of family Enterobacteriaceae like E.coli are oxidase negative.


  • Avoid using Nickel-base alloy wires containing chromium and iron (nichrome) to rub the colony paste onto the filter paper as this may give false positive results
  • Interpret the results within 10 seconds.
  • Lactose in MacConkey Agar and Sucrose in TCBS are fermentable carbohydrates that results in acidification of medium giving false negative oxidase. Hence, oxidase test must be performed from 5% Sheep blood agar or another medium without a fermentable sugar.

Oxidase test by Kovac’s method (Principle, Procedure, Result Interpretation and Precautions)