Oxidase test: Principle Procedure and oxidase positive organisms

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.

Test requirements for Oxidase test: Moist filter paper with the substrate (1% tetramethyl-p-phenylenediamine dihydrochloride), or commercially prepared paper disk, wooden wire or platinum wire.
 Expected results of Oxidase test
  1. Positive: Development of dark purple color (indophenols) within 10 seconds
  2. Negative: Absence of color

Related posts: Modified Oxidase Test (Microdase): Principle, Procedure and Uses

Procedure of Oxidase test:

  1. Take a filter paper soaked with the substrate tetramethyl-p-phenylenediamine dihydrochloride
  2. Moisten the paper with a sterile distilled water
  3.  Pick the colony to be tested with wooden or platinum loop and smear in the filter paper
  4. 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:  
  1. 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
  2. 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 a 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.

During 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. Genera of the Enterobacteriaceae family are characterized as oxidase negative.

Name of Oxidase positive bacteria are: Mneomoics for Oxidase Positive Organisms- PVNCH ( Its just an acronyms inspired by the famous mneomonic for Urease Positive organisms-PUNCH)

  1. PPseudomonas spp
  2. V: Vibrio cholerae
  3. NNeisseria spp
  4. C: Campylobacter spp
  5. H: Helicobacter spp/ Haemophilus spp. 
  6. Aeromonas spp
  7. Alcaligens