31.14: Oxidase
- Page ID
- 165425
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The oxidase test is used to determine whether a bacterial organism produces the enzyme cytochrome c oxidase, which plays a key role in the electron transport chain during aerobic respiration. This enzyme facilitates the transfer of electrons from cytochrome c to molecular oxygen, serving as the final electron acceptor in the process. The presence or absence of cytochrome c oxidase helps differentiate bacterial groups, particularly among Gram-negative species.
For example, genera such as Pseudomonas, Vibrio, and Neisseria are typically oxidase positive, while members of the Enterobacteriaceae family are usually oxidase negative. This distinction makes the oxidase test an important tool in microbial identification.
The test is based on a redox reaction involving the reagent p-phenylenediamine dihydrochloride. When this reagent is oxidized by cytochrome c oxidase, it turns from colorless to dark purple or blue. A color change within one minute indicates a positive result. If the organism does not produce the enzyme, the reagent remains colorless or shows only the natural pigmentation of the cells.
It is essential to perform the test under controlled conditions. A color change that appears after one minute should not be interpreted as a positive result, as it may be due to atmospheric oxidation rather than enzymatic activity. Additionally, using a fresh, actively growing culture is important because older or stressed cells may not express the enzyme, leading to a false-negative result.
*Do not perform this test on cultures you need to preserve, If you have more tests to do prepare a fresh nutrient agar slant first.
Procedure
- Using a sterile wooden stick (not a metal loop or needle), collect a small amount of the organism
- Smear the colony onto a piece of clean filter paper, forming a visible spot. Avoid picking up any agar or growth medium
- Add one to two drops of oxidase reagent directly onto the smear
- Start a timer immediately. Results must be read within 15-30 seconds
Interpretation of Results
- Purple or dark blue color within one minute: positive result, cytochrome c oxidase is present
- No color change or a color that appears after one minute: negative result, or not producing detectable enzyme under current conditions
- Purple color developing after one minute should be ignored, as this is likely due to oxidation by air, not enzyme activity