Oxidase test: Principle, Procedure, Results

By Acharya Tankeshwar •  Updated: 05/04/22 •  6 min read

Oxidase Test 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 dihydrochloride) to indophenols, a purple or dark blue color end product. When the enzyme is not present, the reagent remains reduced and is colorless.

Mechanism of the Cytochrome Oxidase Reaction
Oxidase test result

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

Various types of Oxidase test
Various types of oxidase test procedure

Procedure of Oxidase test

Oxidase test can be performed in various ways. These include, but are not limited to, the filter paper test, filter paper spot test, direct plate method, and test tube method.

Filter Paper Test Method

  1. Soak a small piece of filter paper in 1% Kovács oxidase reagent and let dry.
  2. Use a loop and pick a well-isolated colony from a fresh (18- to 24- hour culture) bacterial plate and rub onto treated filter paper
  3. Observe for color changes.
Oxidase test filter paper test method
Oxidase-positive Pseudomonas aeruginosa (left) and
oxidase-negative Escherichia coli (right).

Results

Filter Paper Spot Method

  1. Use a loop and pick a well-isolated colony from a fresh bacterial plate and rub it onto a small piece of filter paper.
  2. Place 1 or 2 drops of 1% Kovács oxidase reagent on the organism smear.
  3. Observe for color changes.
Oxidase test on filter paper
Oxidase-positive Pseudomonas aeruginosa (left) and
oxidase-negative Escherichia coli (right).

Results

Direct Plate Method

  1. Grow a fresh culture (18 to 24 hours) of bacteria on nutrient agar or trypticase soy agar using the streak plate method so that well-isolated colonies are present.
  2. Place 1 or 2 drops of 1% Kovács oxidase reagent on the organisms.
  3. Do not invert or flood plate.
  4. Observe for color changes.
Oxidase-positive Vibrio cholerae showing purple colonies, and oxidase-negative Escherichia coli with lack of color change

Results

Test Tube Method

  1. Grow a fresh culture (18 to 24 hours) of bacteria in 4.5 ml of nutrient broth (or standard media that does not contain a high concentration of sugar).
  2. Add 0.2 ml of 1% α-naphthol, then add 0.3 ml of 1% paminodimethylaniline oxalate (Gaby and Hadley reagents).
  3. Observe for color changes.
Test tube method of oxidase test

Oxidase positive Neisseria sicca (left) and oxidase negative Staphylococcus aureus (right)

Results

Uses of oxidase test

Quality Control

Bacterial species showing positive and negative reactions should be run as controls at frequent intervals. The following are suggested:
A. Oxidase positive: Pseudomonas aeruginosa
B. Oxidase negative: Escherichia coli

 Precautions and Limitations

The oxidase test must be performed from 5% sheep blood agar or another medium without fermentable sugar. Fermentation of carbohydrates 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.

List of oxidase-positive organisms

Bacterial genera characterized as oxidase-positive include Neisseria and Pseudomonas etc. Genera of the Enterobacteriaceae family are characterized as oxidase negative. 

Mneomonics for oxidase positive organisms- PVNCH (It’s just an acronym inspired by the famous mnemonic 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

References and further readings

  1. Patricia Shields, Laura Cathcart. 2010. Oxidase test protocol.
  2. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology. https://doi.org/10.1128/9781555818814

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.