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.
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
|3% H2O2||Routine testing of aerobes|
|15% H2O2||Identification of anaerobic bacteria.|
|30% H2O2||In the superoxol catalase (used for the presumptive speciation of certain Neisseria sps)|
Procedure of Catalase test
- 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).
- Place a drop of 3% H2O2 on to the slide and mix.
- A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced by bubbling.
- A negative result is no bubbles or only a few scattered bubbles.*
- Dispose of your slide in the biohazard glass disposal container.
Tube Catalase Test
- Add 4 to 5 drops of 3% H2O2 to in a test tube
- 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)
- 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 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.
*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.
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
- 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.
- 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.
- 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.
- Do not test from Mueller-Hinton agar.
Uses of Catalase Test
- 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.
- Catalase test is used to differentiate aerotolerant strains of Clostridium (catalase-negative) from Bacillus species (catalase-positive).
- A semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis.
- Catalase test is useful to separate among the fastidious Gram-negative rods.
- Catalase test can be used as an aid to the identification of Enterobacteriaceae. Members of the Enterobacteriaceae family are catalase positive.
- Neisseria gonorrhoeae produces an enhanced elaboration of bubbles not seen with other members of the genus due to superoxol.
References and further readings
- Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology (14 edition). Mosby.
- Procop, G. W., & Koneman, E. W. (2016). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Seventh, International edition). Lippincott Williams and Wilkins.
- Karen Reiner. 2010. Catalase test protocol.
- Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology. https://doi.org/10.1128/9781555818814