Catalase test: Principle, Procedure, Results, Uses

Last updated on June 21st, 2021

Catalase is an enzyme, which is produced by microorganisms that live in oxygenated environments to neutralize toxic forms of oxygen metabolites; H2O2. The catalase enzyme neutralizes the bactericidal effects of hydrogen peroxide and protects them. Anaerobes generally lack the catalase enzyme.

Slide and Tube Catalase Test Methods
Slide and Tube Catalase Test Methods

Principle of Catalase Test

2H2O2 → 2H2O+ O2 (gas bubbles)

Catalase mediates the breakdown of hydrogen peroxide H2O2 into oxygen and water. To find out if a particular bacterial isolate is able to produce catalase enzyme, a small inoculum of a bacterial isolate is mixed into hydrogen peroxide solution (3%) and is observed for the rapid elaboration of oxygen bubbles. The lack of catalase is evident by a lack of or weak bubble production.

Catalase-positive bacteria include strict aerobes as well as facultative anaerobes. They all have the ability to respire using oxygen as a terminal electron acceptor.

Catalase-negative bacteria may be anaerobes, or they may be facultative anaerobes that only ferment and do not respire using oxygen as a terminal electron acceptor (ie. Streptococci).

Percentage of H2O2 used in catalase test

PercentagePurpose
3% H2O2Routine testing of aerobes
15% H2O2Identification of anaerobic bacteria.
30% H2O2In the superoxol catalase (used for the presumptive speciation of certain Neisseria sps)

Procedure of Catalase test

Slide Test

catase test
Catalase Test: Positive and Negative results
  1. Transfer a small amount of bacterial colony to a surface of a clean, dry glass slide using a loop or sterile wooden stick (be sure colony is visible to the naked eye on slide).
  2. Place a drop of 3% H2O2 on to the slide and mix.
  3. A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced by bubbling.
  4. A negative result is no bubbles or only a few scattered bubbles.*
  5. Dispose of your slide in the biohazard glass disposal container.

Tube Catalase Test

  1. Add 4 to 5 drops of 3% H2O2 to in a test tube
  2. Using a wooden applicator stick, collect a small amount of organism from a well-isolated 18- to 24-hour colony and place into the test tube (Note: Be careful not to pick up any agar (especially if using Blood Agar).- Explanation in precaution below)
  3. Place the tube against a dark background and observe for immediate bubble formation (O2 + water = bubbles) at the end of the wooden applicator stick.
Catalase Positive and Catalase Negative Reactions
Catalase Positive and Catalase Negative Reactions

Catalase Test Results:

  • Catalase positive reaction: Evident by immediate effervescence (bubble formation)
  • Catalase negative reaction: No bubble formation (no catalase enzyme to hydrolyze the hydrogen peroxide) or a few bubbles after 20 seconds.

You can perform Catalase Test online here

*Note: Some bacteria possess enzymes other than catalase that can decompose peroxide, a few tiny bubbles forming after 20-30 seconds is not considered a positive test.

Quality control

Each new lot or shipment of the reagent should be tested with positive and negative control prior to using them.
A. Staphylococcus aureus ATCC 25923- catalase-positive
B. Streptococcus pyogenes ATCC 19615- catalase-negative

Precautions/Limitations

  1. Do not use a metal loop or needle with H2O2; it will give a false positive and degrade the metal. Instead, use a platinum loop or wooden stick to perform this test.
  2. If using colonies from a blood agar plate, be very careful not to scrape up any of the blood agar as blood cells are catalase-positive and any contaminating agar (carryover of red blood cells) could give a false positive.
  3. Catalase enzyme is present in viable cultures only, do not test colonies that are older than 24 h. Older cultures may give false-negative results.
  4. Do not test from Mueller-Hinton agar.

Uses of Catalase Test

  1. The catalase test is primarily used to distinguish among Gram-positive cocci: members of the genus Staphylococcus are catalase-positive, and members of the genera Streptococcus and Enterococcus are catalase-negative.
  2. Catalase test is used to differentiate aerotolerant strains of Clostridium (catalase-negative) from Bacillus species (catalase-positive).
  3. A semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis. 
  4. Catalase test is useful to separate among the fastidious Gram-negative rods.
  5. Catalase test can be used as an aid to the identification of Enterobacteriaceae. Members of the Enterobacteriaceae family are catalase positive.
  6. Neisseria gonorrhoeae produces an enhanced elaboration of bubbles not seen with other members of the genus due to superoxol.

References and further readings

About Acharya Tankeshwar 476 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.

33 Comments

  1. how to differentiate between staphylococcus and streptococcus bacteria with catalase test? because almost streptococcus spp. of organisms are aerobic or microaerophilic (beta haemolytic strains).

  2. Ravi Prasad ji
    To differentiate the genus (Streptococcus or Staphylococcus), you can perform catalase test. Streptoccus spp is catalase Negative and Staphylococcus is catalase positive.

  3. Sir i just need to know is it a journal or newspaper article because i have to write a report on it and i need a reference

  4. Something to be careful of when doing Catalse is aerosolization of the organism…….ergo the bubbling.

  5. i tried the tube test and i got absolutely no results. but when i tried the slide test the effervescence came. why do you think this happened.

    • Very hard to say you exact reason on the basis of information you have provided. Please try again with fresh culture with both tube and slide test; adhering with exact protocol and share your results.

  6. Tamara, sounds like you did not have a lot of growth in the tube and the hydrogen peroxide could have been diluted in the liquid media. Culture should be at least 24 hrs old with good growth try this protocol: http://www.microbelibrary.org/library/2-associated-figure-resource/3237-positive-tube-catalase-test
    You can also drop a drop of hydrogen peroxide directly on the media if grown on TSA: http://www.microbelibrary.org/library/2-associated-figure-resource/3234-catalase-test-on-staphlococcus-aureus-streak-plate
    and
    http://www.microbelibrary.org/library/2-associated-figure-resource/3235-catalase-test-on-staphlococcus-aureus-streak-plate-enlarged-view
    Thing to be cautious of 1). use a plastic loop or wood application since hydrogen peroxide can react with a metal loop…ie you could get a false positive., 2). some media reacts with hydrogen peroxide such as blood agar. So you could not drop the hydrogen peroxide directly on the plate. If you do the slide test make sure you do not carry over any agar (blood agar, etc) to avoid a false positive.

  7. i took some useful contents in this article,,help me reference it cause i dont wanna be penalised for stealing others s work

  8. While performing the catalase test on crude protein extract of drosophila species fed on a pomegranate juice supplemented diet and control, the absorbance decreased with time in the case of control but increased in the flies that recieved the polyphenol treatment. Would be great help if you could provide a possible explanation?

  9. thank you for the information, am impressed. kindly help me understand why the other catalases only break down H2O2 in 30s and not as the catalase in staph. could you also write on other culture media such as Todd-Hewitt broth, edward’s agar and tryptone soya broth.

  10. i am really impressed and have also learned a lot from your write up.
    It made me passed excellently my microbiology end of semester exams.

  11. hello my work on ornithobacterium rhinotracheale bacteria. I want any helpful information about its isolation and identification

    • I have the following question for you
      1. Is all salmonella species are catalase positive
      2. What are biochemical test use for the differentiation of salmonella and cetrobacter youngae

  12. I want to check the growth of bacteria in the presence of methylene blue 0.1 %. but I am not confirmed about the formulation to make the media. I need your kind help. Thank You

  13. Hello,Kindly let me know whether it was an error when you wrote that the positive control for catalase test is staphylococcus aureus instead of staphylococcus species.Kindly clarify, because I know coagulase test is done to identify staphylococcus aureus

    • The best practice is to use known strain during Quality Control procedure and most researchers prefer/use ATCC strains. If you have Staphylococcus aureus, it can also be used as a positive control for Catalase test but it’s not mandatory, you can use any species of Staphylococci for this purpose.

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