Catalase test: principle, uses, procedure and results 4.67/5 (45)

in Biochemical tests in Microbiology, Microbiology for Beginners · 29 Comments
catase test
Catalase Test: Positive and Negative results

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.

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, small inoculum of bacterial isolate is mixed into hydrogen peroxide solution (3%) and is observed for the rapid elaboration of oxygen bubbles occurs.  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 on catalase test:

  1. For routine testing of aerobes, 3% hydrogen peroxide is used.
  2. 15% H2O2 solution: for the identification of anaerobic bacteria
    Catalase test is used to differentiate aerotolerant strains of Clostridium (catalase negative) from Bacillus species (catalase positive).
  3. The superoxol catalase test used for the presumptive speciation of certain Neisseria organisms requires a different concentration of H2O2.

Uses Catalase Test Results 

  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, which are catalase negative, from Bacillus species, which are positive.
  3. Semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis. 
  4. Catalase test can be used as an aid to the identification of Enterobacteriaceae. Members of Enterobacteriaceae family are catalase positive.

Procedure of Catalase test (Slide Test)

  1. Transfer a small amount of bacterial colony to a surface of clean, dry glass slide using a loop or sterile wooden stick
  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-Procedure and Results 

  1. Add 4 to 5 drops of 3% H2O2 (Hydrogen peroxide) 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 (esp 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

Results:

  • Catalase Positive reactions: Evident by immediate effervescence (bubble formation)
  • Catalase Negative reaction: No bubble formation (no catalase enzyme to hydrolyze the hydrogen peroxide)

You can perform Catalase Test online here

Precautions while performing catalase test

  1. Do not use a metal loop or needle with H2O2; it will give a false positive and degrade the metal.
  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. Because some bacteria possess enzymes other than catalase that can decompose hydrogen peroxide, a few tiny bubbles forming after 20 to 30 seconds is not considered as positive test.

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29 thoughts on “Catalase test: principle, uses, procedure and results”

  2. raviprasad k v

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

  3. Tankeshwar Acharya

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    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.

  5. Salman Khalid

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    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

  9. Tamara G.

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    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.

    1. Tankeshwar Acharya

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      Reply

      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.

  10. Tasha

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    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.

  13. ntshuxeko Confidence

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

  15. Anonymous

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    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?

  23. jesca

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    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.

  27. Bawade Raymond

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    Reply

    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.

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