The oxidase test is a common microbiology laboratory procedure used to identify and classify bacteria. It serves as a preliminary step based on their respiratory metabolic pathways. This test is straightforward to perform and provides rapid results, making it a valuable diagnostic tool.
Purpose and Underlying Principle
The oxidase test determines if an organism produces the enzyme cytochrome c oxidase, which is a component of the bacterial electron transport chain. This enzyme catalyzes the transfer of electrons from a donor molecule to molecular oxygen, forming water. Bacteria possessing this enzyme are considered “oxidase-positive,” indicating they utilize oxygen as a terminal electron acceptor in aerobic respiration.
The test relies on a chromogenic reagent, typically N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), which acts as an artificial electron donor for the cytochrome c oxidase enzyme. When the enzyme is present, it oxidizes this colorless reagent, causing it to change into a colored compound called indophenol blue, which appears purple or dark blue. If the enzyme is absent, the reagent remains colorless. This biochemical reaction helps differentiate bacterial species, such as distinguishing members of the Enterobacteriaceae family (typically oxidase-negative) from other Gram-negative organisms like Pseudomonas and Neisseria (typically oxidase-positive).
Necessary Materials
Performing the oxidase test requires several specific materials. The key component is the oxidase reagent, most commonly Kovács oxidase reagent, which is a 1% solution of N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride. This reagent should be stored in a dark bottle and refrigerated to maintain stability, as it is light-sensitive and can degrade.
Other essential items include small pieces of filter paper, which serve as the reaction surface. A sterile inoculating loop or wooden applicator stick is needed to transfer bacterial colonies without contamination. Avoid using nichrome wire loops, as their iron can cause false-positive results. Finally, a fresh, well-isolated bacterial culture, typically 18 to 24 hours old, is necessary for accurate enzyme activity.
Step-by-Step Procedure
Begin by placing a small piece of filter paper onto a clean, sterile petri dish or a glass slide. Moisten the filter paper with one or two drops of the oxidase reagent. Apply just enough reagent to moisten the paper without flooding it.
Using a sterile wooden stick, plastic loop, or platinum inoculating loop, carefully pick a well-isolated bacterial colony from a fresh agar plate. Gently rub the collected bacterial mass onto the moistened area of the filter paper. Avoid excessive rubbing, which could damage bacterial cells.
Once the bacterial colony is applied, observe the inoculated area for a color change. A timer should be started immediately upon contact between the bacteria and the reagent. The reaction should be read within a specific timeframe to prevent misinterpretation of results. After use, dispose of the wooden stick or plastic loop in a sharps container, and sterilize any reusable platinum loops according to laboratory protocols.
Interpreting Test Outcomes
The interpretation of the oxidase test results relies on the color change observed on the filter paper and the time it takes for this change to occur. A positive oxidase result is indicated by the development of a dark purple or blue color within approximately 5 to 10 seconds of applying the bacterial colony to the reagent. This rapid color change signifies the presence of cytochrome c oxidase enzyme in the bacterial cells.
Some organisms may exhibit a delayed positive reaction, with the color change appearing within 60 to 90 seconds. A negative oxidase result is recorded if no color change occurs on the filter paper, or if a color change appears only after two minutes or more. This indicates the absence of cytochrome c oxidase, or that the organism uses a different terminal oxidase in its respiratory chain. Disregard any color changes that develop beyond the recommended observation time, as these can lead to false-positive interpretations due to the reagent’s instability and auto-oxidation.
Important Safety Measures
Performing the oxidase test requires adherence to standard laboratory safety practices to protect personnel and prevent contamination. Always wear personal protective equipment, including gloves, a laboratory coat, and eye protection, such as safety goggles, to shield against splashes and contact with bacterial cultures or chemical reagents. Ensure long hair is tied back and loose clothing or jewelry is secured.
Proper handling and disposal of bacterial cultures are essential to prevent the spread of microorganisms. All materials that come into contact with bacteria, including filter paper, applicator sticks, and contaminated gloves, should be placed in designated biohazard waste containers for proper sterilization and disposal. The oxidase reagent itself, N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride, should be handled with care as it is a chemical reagent. Work in a clean, disinfected area, and always wash hands thoroughly after completing the test and before leaving the laboratory.