The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain. When present, the cytochrome c oxidase oxidizes the reagent (tetramethyl-p-phenylenediamine) to (indophenols) purple color end product. When the enzyme is not present, the reagent remains reduced and is colorless.
Note: All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron acceptor in respiration. This does NOT mean that they are strict aerobes. Bacteria that are oxidase-negative may be anaerobic, aerobic, or facultative; the oxidase negative result just means that these organisms do not have the cytochrome c oxidase that oxidizes the test reagent. They may respire using other oxidases in electron transport.
Purpose of Oxidase test
Oxidase test is most helpful in screening colonies suspected of being one of the Enterobacteriaceae (all negative) and in identifying colonies suspected of belonging to other genera such as Aeromonas, Pseudomonas, Neisseria, Campylobacter, and Pasteurella (positive).
Expected results of Oxidase test
- Positive: Development of dark purple color (indophenols) within 10 seconds
- Negative: Absence of color
Quality Control of Oxidase Test
Bacterial species showing positive and negative reactions should be run as controls at frequent intervals. The following are suggested:
A. Positive control: Pseudomonas aeruginosa
B. Negative control: Escherichia coli
Procedure of Oxidase test:
- Take a filter paper soaked with the substrate tetramethyl-p-phenylenediamine dihydrochloride
- Moisten the paper with a sterile distilled water
- Pick the colony to be tested with wooden or platinum loop and smear in the filter paper
- Observe inoculated area of paper for a color change to deep blue or purple within 10-30 seconds
Precaution to be taken while performing oxidase test:
- Do not use Nickel-base alloy wires containing chromium and iron (nichrome) to pick the colony and make smear as this may give false positive results
- Interpret the results within 10 seconds, timing is critical
The oxidase test must be performed from 5% sheep blood agar or another medium without a fermentable sugar. Fermentation of carbohydrate results in acidification of the medium (e.g., lactose in MacConkey Agar or Sucrose in TCBS), and a false negative oxidase test may result if the surrounding pH is below 5.1. Subinoculation on Nutrient Agar is required before the oxidase test can be performed.
During the identification of suspected Vibrio cholerae isolate, it is not possible to perform an oxidase test directly from a TCBS culture because the acid produced by the sucrose fermenting colonies will inhibit the oxidase reaction.
Oxidase test results
Bacterial genera characterized as oxidase positive include Neisseria and Pseudomonas etc. Genera of the Enterobacteriaceae family are characterized as oxidase negative.
Name of Oxidase positive bacteria are: Mneomoics for Oxidase Positive Organisms- PVNCH (It’s just an acronym inspired by the famous mneomonic for Urease Positive organisms-PUNCH)
- P: Pseudomonas spp
- V: Vibrio cholerae
- N: Neisseria spp
- C: Campylobacter spp
- H: Helicobacter spp/ Haemophilus spp.
- Aeromonas spp