Oxidase test: Principle Procedure and oxidase positive organisms

Last updated on June 4th, 2020

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.

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.

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).

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

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:

  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 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)

  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
About Acharya Tankeshwar 466 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


  1. @Mohammed Yusuf, No. Enterobacteriaceae family is facultative anaerobe and their group characteristics is Oxidase Negative, Catalase positive.

    • There are lots of Gram Negative Bacteria that are oxidase negative. Catalase positive and Oxidase negative is the group characteristics of family Enterobacteriaceae (which includes member such as Salmonella, Shigella, E.coli, Klebsiella etc).

  2. Hello, I would like to ask if all Micrococci are oxidase positive. If so, then what could be the reason for oxidase (not modified oxidase) test yielding negative results?
    Thank you very much for your answer.

  3. Can I use human plasma for coagulase test with the same concentration and volume as we use in rabbit plasma?

  4. Thank you for your help! I am a little confused at this time, my lecture notes indicate the final hydrogen in the ETC oxidises oxygen into hydrogen peroxide. Yet, in my previous class, hydrogen oxidised oxygen into water.

    I’ve read many of your posts and I’m super appreciative of your work and passions! Thank you!

    • Thank you Mahala. I appreciate your feedback. I request you to check the concept of oxidation-reduction again. This resource can help: read here

  5. Hello, I’m not a microbiologist but I need some information for my samples…
    I have 1 sample and sent to a micro lab for EB per 10g as per ISO method test (n=10). 1 out of 10 is detected for EB.
    The lab said 1 of the sample after confirmatory test is oxidase – and glucose fermenting so they confirmed it is EB positive.

    What is the technical way to explain my case why only 1 out of 10 test turned out + for EB?
    Is there any posibility of false positive EB result using ISO method?
    My sample has also some detection of lactic acid bacteria – will this interefere with the EB test and give false positive result?

    Please help. Thanks!

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