Introduction
The oxidase test is a biochemical test used to determine the presence of the oxidase enzyme in bacterial cells. The test involves adding a reagent containing an artificial electron acceptor to a bacterial sample and observing the color change that occurs. The test is used to identify certain groups of bacteria, such as Pseudomonas, Neisseria, and Vibrio, that produce the oxidase enzyme.
Purpose of the oxidase test
The purpose of the oxidase test is to determine the presence of the oxidase enzyme in bacterial cells. These bacteria are often associated with infections in humans and animals, and an oxidase test is a useful tool in identifying and diagnosing these infections.
Principle of the oxidase test
The principle of the oxidase test is based on the detection of the electron transport chain component, cytochrome c oxidase, which is involved in aerobic respiration and catalyzes the transfer of electrons from cytochrome c to oxygen.
The oxidase enzyme catalyzes the transfer of electrons from the donor molecule to the acceptor molecule, producing a color change in the reagent.
NADH is an electron donor, and the acceptor molecule is oxygen. But In the case of the oxidase test, N, N, N’, N’-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) acts as an artificial electron acceptor
The reagent used in the oxidase test contains TMPD, which is a colorless compound. When the reagent is added to a bacterial sample, the oxidase enzyme in the bacterial cells transfers electrons from the donor molecule (NADH) to the acceptor molecule (oxygen), producing a blue-purple color in the reagent. The intensity of the color change is proportional to the amount of oxidase enzyme present in the bacterial sample.
Materials required for the oxidase test
- Oxidase reagent (containing N,N,N’,N’-tetramethyl-p-phenylenediamine or TMPD)
- Sterile inoculating loop or swab
- Bacterial sample (pure culture or colony)
- Sterile distilled water
- Sterile test tube or Petri dish
- Gloves
- Safety goggles
Procedure for performing the oxidase test
Filter paper method
- Soak a small piece of filter paper in the oxidase reagent
- Transfer a small amount of bacterial sample onto the filter paper using a sterile inoculating loop or swab
- Observe the filter paper for a color change within 10-30 seconds
Test tube method
- Add a small amount of bacterial sample to a test tube containing oxidase reagent
- Mix the sample and the reagent by gently shaking the test tube
- Observe the mixture for a color change within 10-30 seconds
Interpretation of results:
Positive result: A positive result is indicated by the appearance of a blue-purple color in the oxidase reagent within 10-30 seconds of adding the bacterial sample. This color change indicates the presence of the oxidase enzyme in the bacterial cells.
Negative result: A negative result is indicated by the absence of a color change in the oxidase reagent within 10-30 seconds of adding the bacterial sample. This indicates that the bacterial sample does not produce the oxidase enzyme.
It is important to note that a false-negative result may occur if the bacterial sample is not actively growing or producing the oxidase enzyme at the time of the test. Additionally, a false-positive result may occur if the oxidase reagent is contaminated or if the bacterial sample contains other enzymes that can react with the reagent.
Examples of Positive results
- Pseudomonas aeruginosa
- Neisseria gonorrhoeae
- Vibrio cholerae
- Campylobacter jejuni
- Helicobacter pylori
- Aeromonas hydrophila
- Alcaligenes faecalis
- Moraxella catarrhalis
- Legionella pneumophila
- Pasteurella multocida
Examples of Negative results
- Escherichia coli
- Salmonella enterica
- Staphylococcus aureus
- Streptococcus pyogenes
- Bacillus subtilis
- Listeria monocytogenes
- Clostridium botulinum
- Mycobacterium tuberculosis
- Enterococcus faecalis
- Lactobacillus acidophilus
Applications of Oxidase test
Clinical applications of the oxidase test
Identification of bacterial infections: The oxidase test is used to identify bacterial infections caused by Pseudomonas, Neisseria, and Vibrio species. These infections can range from mild infections, such as otitis externa and conjunctivitis, to severe infections, such as sepsis and meningitis.
Diagnosis of genetic disorders: The oxidase test is also used in the diagnosis of genetic disorders, such as X-linked chronic granulomatous disease (CGD). CGD is caused by a defect in the oxidative burst pathway in phagocytic cells, which leads to an inability to kill bacteria. The oxidase test is used to determine the presence of the NADPH oxidase enzyme, which is required for the oxidative burst pathway.
Industrial applications of the oxidase test
Quality control in food production: The oxidase test is used in the food industry to monitor the quality of food products, such as dairy products and meat. The test is used to detect the presence of spoilage bacteria, such as Pseudomonas, which can cause off-flavors and off-odors in food products.
Environmental monitoring: The oxidase test is also used in environmental monitoring to detect the presence of bacteria in water, soil, and air samples. The test is particularly useful in monitoring the quality of water in swimming pools, spas, and other recreational areas.
Other applications of the oxidase test
Taxonomic identification of bacteria: The oxidase test is used in the taxonomic identification of bacteria. The test is based on the fact that different bacterial species produce different enzymes, and the oxidase test can be used to differentiate between oxidase-positive and oxidase-negative bacteria.
Biochemical research: The oxidase test is also used in biochemical research to study the metabolism of bacteria. The test can be used to determine the role of the oxidase enzyme in bacterial metabolism and to study the effects of inhibitors on the enzyme.
Limitations of Oxidase Test
- Fresh reagents should be used: It is important to use fresh reagents that are no older than 1 week, as the reagents used in the oxidase test are known to auto-oxidize.
- Inhibited oxidase activity: Bacteria and yeast grown on media containing high concentrations of glucose may show inhibited oxidase activity, hence it is recommended to test colonies grown on media without excess sugar such as nutrient agar or tryptic soy agar.
- Aberrant results on media containing dyes: Bacteria grown on media containing dyes may give aberrant results, hence should be avoided.
- Effectiveness of reagents: The test reagents can effectively kill the microorganisms, hence sub-culturing should be done prior to adding any reagent to an active culture.
- Presumptive identification of Neisseria: The oxidase test can be used in the presumptive identification of Neisseria and in the differentiation and identification of gram-negative bacilli. However, it is recommended to confirm the identification with additional biochemical tests.
- False-positive reactions: The use of a nichrome or other iron-containing loop may yield false-positive reactions, therefore platinum loops are recommended.
- False-negative results: Less sensitive strips or reagents may yield false-negative results, especially for Haemophilus organisms. Hence, it is recommended to use highly sensitive strips or reagents.
- Valid results: Oxidase reactions of gram-negative bacilli should be determined on non-selective and non-differential media to ensure valid results. Colonies taken from media containing high levels of glucose may give false-negative reactions.
- Age of colonies: It is recommended to use colonies that are 18-24 hours old as older colonies may produce weaker reactions.
- Disregard of late color changes: Any color changes appearing after 20 seconds should be disregarded as they may not be reliable indicators of oxidase activity.
References
https://asm.org/getattachment/00ce8639-8e76-4acb-8591-0f7b22a347c6/oxidase-test-protocol-3229.pdf
