Oxidase test

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The oxidase test is a test used in microbiology to determine if a bacterium produces certain cytochrome c oxidases. It uses disks impregnated with a reagent such as N,N,N′,N′-Tetramethyl-p-phenylenediamine (TMPD) or N,N-Dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator. The reagent is a dark blue to maroon color when oxidized, and colorless when reduced.

Strains may either be oxidase positive (OX+) or negative (OX-). OX+ normally means that the bacterium contains cytochrome c oxidase and can therefore utilize oxygen for energy production with an electron transfer chain.

Typically the Pseudomonadaceae are OX+ and Enterobacteriaceae are OX-. Another example is the preliminary identification of Neisseria and Moraxella genera, which are both oxidase positive, Gram-negative diplococci.

Procedures

Wet each disk with about 4 inoculating loops of de-ionized water.
Use a loop to aseptically transfer a large mass of pure bacteria to the disk.
Observe the disk for up to 3 minutes. If the area of inoculation turns dark blue to maroon to almost black, then the result is positive. If a color change does not occur within three minutes, the result is negative.

Alternatively, live bacteria cultivated on trypticase soy agar plates may be prepared using sterile technique with a single-line streak inoculation. The inoculated plates are incubated at 37°C for 24-48 hours to establish colonies. Fresh bacterial preparations should be used. After colonies have grown on the media, two-to-three drops of the reagent DMPD is added to the surface of each organism to be tested. A positive test (OX+) will result in a color change to pink, through maroon and into black, within 10-30 seconds. A negative test (OX-) will result in a light pink coloration or absence of coloration.

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