The oxidase test is a common microbiology technique. It identifies bacterial species by detecting a specific enzyme, making it a valuable tool for initial classification.
Understanding the Oxidase Test
The oxidase test identifies bacteria producing the enzyme cytochrome c oxidase. This enzyme is part of the electron transport chain, transferring electrons to oxygen in its final step.
Bacteria with this enzyme use oxygen as a terminal electron acceptor. The test uses a chemical reagent that changes color when oxidized by the enzyme, visually indicating its presence.
Preparation for Testing
Performing the oxidase test requires specific materials. You will need an oxidase reagent, most commonly N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride, available as liquid or impregnated discs. Sterile cotton swabs or wooden applicators are needed for transferring bacterial colonies. Fresh bacterial cultures, typically 18-24 hours old, are also required for reliable testing.
Safety precautions are essential. Always wear disposable gloves and eye protection to prevent contact with bacterial cultures and chemical reagents. Handle bacterial cultures with care to avoid contamination, and dispose of all contaminated materials properly after use. Use fresh oxidase reagent, as older reagents degrade and lead to unreliable results.
Performing the Oxidase Test
The oxidase test can be performed using a direct plate method or a filter paper method. For the direct plate method, carefully pick a well-isolated bacterial colony from an agar plate using a sterile cotton swab or a wooden applicator. Avoid using nichrome or other iron-containing loops, as these can cause false-positive reactions.
Gently rub the bacterial inoculum onto a filter paper strip or pre-wetted oxidase disk. If using liquid reagent, apply one or two drops directly onto the inoculated area. Observe for a color change within 10-30 seconds; a distinct color change indicates a positive result.
The filter paper method involves placing a drop of oxidase reagent onto filter paper. Then, use a sterile swab to pick a bacterial colony and smear it onto the moistened area. Observe for a color change within 10-30 seconds. A deep purple or blue color confirms a positive reaction.
Interpreting Test Results
Interpreting oxidase test results focuses on the presence or absence of a color change. A positive reaction is indicated by a distinct dark purple or blue color within 10-30 seconds of applying the reagent. For example, Pseudomonas aeruginosa yields a positive result.
A negative reaction shows no color change, or only a very faint, delayed discoloration, even after 30 seconds. This indicates the absence of cytochrome c oxidase. Many Enterobacteriaceae family members, such as Escherichia coli, are oxidase-negative.
Interpret results within the specified timeframe to avoid misinterpretations. Observing beyond 60 seconds can lead to false-positives due to spontaneous reagent oxidation. Too much reagent can also cause an artificial color change. Conversely, very old bacterial cultures might lead to false-negatives as enzyme activity diminishes.