Catalase test: Principle, Procedure, Results, Uses

By Acharya Tankeshwar • Updated: 06/05/22 • 4 min read

Catalase is an enzyme produced by microorganisms that live in oxygenated environments to neutralize toxic forms of oxygen metabolites such as hydrogen peroxide (H₂O₂). The catalase enzyme neutralizes the bactericidal effects of hydrogen peroxide and protects aerobes or facultative anaerobes. Anaerobes generally lack the catalase enzyme.

Slide and Tube Catalase Test Methods — Slide and tube catalase test methods

Table of Contents

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

Percentage	Purpose
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

Slide Test

catase test — Catalase Test: Positive and Negative results

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 the slide).
Place a drop of 3% H₂O₂ onto 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 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 it 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 Positive and Catalase Negative Reactions

Catalase Test Results

Catalase positive reaction: Evident by immediate effervescence (bubble formation)
Catalase negative reaction: No (effervescence) bubble formation 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

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

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.