The oxidase biochemical test was originally devised to identify Neisseria species, but later was used to separate the Pseudomonadaceae from the oxidase-negative Enterobacteriaceae.This test identifies organisms that produce the enzyme cytochrome oxidase that participates in the electron transport chain by transferring electrons from a donor molecule to oxygen.
The Kovac oxidase reagent contains a chromogenic reducing agent, which is a compound that changes color when it becomes oxidized within 15 seconds.
Majority of most gram-positive bacteria are oxidase-negative and many of the gram-negative bacilli, other than the Enterobacteriaceae, are variable. This formulation is based on the oxidase reagent formula of Kovacs.
Principle of Oxidase test
- Based on the bacterial production of an intracellular oxidase enzyme.
- The oxidase reaction is due to the presence of a cytochrome oxidase system, which activates the oxidation of reduced cytochrome by molecular oxygen, which in turn acts as an electron acceptor in the terminal stage of the electron transfer system.
- Those microorganisms that produces oxidase enzyme, when in the presence of atmospheric oxygen, cytochrome c, and phenylenediamine oxidase reagent, oxidize to form a purple colored compound, indophenol.
- Ascorbic acid is incorporated in the reagent as a reducing agent to reduce auto-oxidation and to improve stability.
Materials required for Oxidase test
- Inoculating loop
- collection containers
- Supplemental media
- Whatman (No.1) filter paper or oxidase test strip
- Platinum loop or needle, or wooden applicator stick.
Reagents required for Oxidase Test
|Ascorbic Acid||2.0 g|
|Demineralized Water (Distilled water)||1000.0 ml|
Oxidase test procedure (Fresh reagent method)
- Dispense 1-2 drops of reagent onto a strip of Whatman (No. 1) filter paper (or an equivalent)
- Using a platinum loop or needle, or wooden applicator stick, smear the growth of an 18-24-hour old isolate onto the saturated filter paper.
- Observe for color development within 10-30 seconds.
Oxidase test procedure (Reagent strip method)
- Moisten the strip with a drop of sterile water or distilled water
- Using a piece of an applicator stick or glass rod (Avoid using an oxidized wire loop) remove a colony of the test organism and rub it on the oxidase test strip
Interpretation of oxidase test result
|Positive test||Development of a blue,red or violet to purple color within 10-30 seconds|
|Negative Test||Delayed reaction or no color development within 10-30 seconds|
NB : Ignore any violet to purple color that develops after 10 – 30 seconds
Possible Oxidase positive and Negative Organisms (Controls)
Possible limitation of Oxidase test
- Select colonies for testing which have not been cultured on selective media or media containing glucose, as selective agents and fermentation may inhibit oxidase enzyme activity resulting in possible false-negative results
- Weak oxidase producers, such as Pasteurella, may appear negative within the time limits of the test. Results inconsistent with other biochemical reactions or with the organism should be repeated
- A platinum inoculating loop or needle, or a wooden applicator stick is recommended for use in oxidase testing.The presence of any trace of iron (nichrome) can catalyze an oxidase reaction, resulting in a false-positive reaction.
- False negative results may occur if mixed cultures contain the two genera Pseudomonas and Neisseria. An inhibitory substance is produced by Pseudomonas species, which interferes with the production of oxidase by Neisseria species
- The oxidase reagent quickly auto-oxidizes and loses its sensitivity. Unused reagent should be discarded at the end of each workday
- The oxidase test is an oxidizing reaction; oxygen must reach the colonies for optimal results.
- Kovacs, N. 1956. Nature. 178:703.
- Cowan, S.T. and K.J. Steel. 1966. Manual for the Identification of Medical Bacteria. Cambridge University Press, Cambridge, UK.
- Steel, K.J. 1962. J. Appl. Bacteriol. 25:445-455.
- Murray, P.R., E.J. Baron, J.H. Jorgensen, M.A. Pfaller, and R.H. Yolken. 2003. Manual of Clinical Microbiology. 8th ed., Vol. 1. ASM, Washington, D.C.
- MacFaddin, J.F. 2000. Biochemical Tests for Identification of Medical Bacteria. 3rd ed. Lippincott Williams & Wilkins, Philadelphia, PA.
- Steel, K.J. 1961. J. Gen. Microbiol. 25:297-306.