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.

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

Procedure of Catalase test

Slide Test

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 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

• 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