Oxidase test: Principle Procedure and oxidase positive organisms

Test requirements for Oxidase test: Moisten 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
Positive: Development of dark purple color (indophenols) within 10 seconds
Negative: Absence of color
Principle:
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.


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

Note:

The oxidase test must be performed from 5% Sheep blood agar or another medium without a fermentable sugar (e.g., lactose in MacConkey Agar or Sucrose in TCBS).
Because fermentation of a carbohydrate results in acidification of the medium, and a false negative oxidase test may result if the surrounding pH is below 5.1.

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:

  1. Campylobacter spp
  2. Helicobacter spp
  3. Aeromonas spp
  4. Vibrio cholera
  5. Neisseria spp
  6. Pseudomonas spp
  7. Alcaligens