What is Oxidase test?
The oxidase test is a straightforward biochemical test used in microbiology to detect the enzyme cytochrome c oxidase in bacteria. This enzyme is crucial for bacterial respiration, specifically in the electron transport chain, and the test helps distinguish between different types of bacteria, especially Gram-negative bacteria, like identifying Pseudomonas from Enterobacteriaceae. Here’s an explanation of the test’s principle, procedure, interpretation of results, and precautions.
Table of Contents
Principle of the Oxidase Test
The oxidase test detects the enzyme cytochrome c oxidase, which is involved in the electron transport chain in aerobic respiration. The test uses a chemical reagent called tetramethyl-p-phenylenediamine (oxidase reagent), which reacts with cytochrome c oxidase. If the enzyme is present, it oxidizes the reagent, causing a color change. This indicates the presence of cytochrome c oxidase and can be used to differentiate bacterial species.
Procedure of the Oxidase Test
Performing the oxidase test is easy and requires minimal materials, including the oxidase reagent, filter paper or oxidase strips, and a fresh bacterial culture.
Gather Materials
You need a sterile swab, oxidase reagent (1% tetramethyl-p-phenylenediamine), filter paper or test strips, and a fresh bacterial culture grown on an appropriate medium like tryptic soy agar.

Sample Collection
Use a sterile swab or toothpick (not a metal loop, to avoid false positives) to pick a small sample from a fresh bacterial colony (no older than 24 hours).
Test Application
Smear the bacterial sample on filter paper or an oxidase strip. Apply a few drops of oxidase reagent or press the sample to an oxidase disc.
Observe for Color Change
If cytochrome c oxidase is present, the reagent will turn blue or purple within 10-30 seconds. If no color change occurs within this time, the result is negative.
Interpretation of Results

Positive Result
A blue or purple color within 10-30 seconds indicates the bacteria produce cytochrome c oxidase. Bacteria like Pseudomonas, Neisseria, and Vibrio are typically oxidase-positive.

Negative Result
If no color change occurs within the timeframe, the bacteria do not produce cytochrome c oxidase. Most bacteria in the Enterobacteriaceae family (like E. coli) are oxidase-negative.
Precautions to Ensure Accuracy
Even though the oxidase test is simple, several precautions are necessary for accurate results:
Fresh Cultures
Always use bacterial cultures that are 18-24 hours old. Older cultures may produce unreliable results due to changes in enzyme activity.
Use Non-Metal Instruments
Avoid using metal loops, as they can cause false-positive results. Instead, use sterile swabs, plastic loops, or toothpicks.
Proper Reagent Handling
The oxidase reagent is sensitive to oxygen and may oxidize if exposed to air for too long, leading to false positives. Ensure it’s fresh or properly stored.
Observe Timely
Check for a color change within 10-30 seconds. Any color change after 30 seconds is considered invalid and should not be interpreted as positive.
Avoid Overuse of Reagent
Apply the reagent sparingly to avoid over-dilution, which could make the results harder to interpret.
Pure Culture
Ensure that the bacterial culture is uncontaminated, as other microorganisms may interfere and skew the results.
Conclusion
The oxidase test is a quick and efficient way to determine if bacteria produce the enzyme cytochrome c oxidase, helping to differentiate between oxidase-positive and oxidase-negative bacteria. With the proper technique and attention to precautions, this test is a reliable tool in microbiology labs for identifying bacterial species such as Pseudomonas or distinguishing them from Enterobacteriaceae.
Frequently Asked Questions (FAQ)
Define sterile swab in short?
A sterile swab is a medical tool designed for single-use applications that has undergone a sterilization process to eliminate microorganisms and prevent contamination. This process is vital in medical settings to ensure patient safety, prevent infections, and maintain the integrity of biological samples.
Define cytochrome c oxidase ?
Cytochrome c oxidase is an enzyme in the electron transport chain of aerobic respiration that facilitates the transfer of electrons from cytochrome c to oxygen, the final electron acceptor, thereby helping produce energy in the form of ATP.
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