Catalase test: Principle, Procedure, Results, Uses

Catalase is an enzyme produced by microorganisms that live in oxygenated environments to neutralize the bactericidal effects of toxic forms of oxygen metabolites such as hydrogen peroxide (H₂O₂). The catalase enzyme protects aerobes and facultative anaerobes from oxidative damage. Anaerobes generally lack the catalase enzyme. Catalase is also a significant staphylococcal virulence factor.

Principle

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 can produce catalase enzyme, a small inoculum of a bacterial isolate is mixed into hydrogen peroxide solution (3%). It 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 can respire using oxygen as a terminal electron acceptor.

Catalase-negative bacteria may be anaerobes or facultative anaerobes that only ferment and do not respire using oxygen as a terminal electron acceptor (i.e. streptococci).

Percentage of H₂O₂ used in catalase test

Procedure

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 the colony is visible to the naked eye on the slide).
2. Place a drop of 3% H₂O₂ onto the slide and mix.
3. A positive result is the rapid evolution of oxygen (within 5-10 seconds), 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 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 it into the test tube (Note: Be careful not to pick up any agar (especially if using Blood Agar).
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.

Results

• Catalase positive reaction: Evident by immediate effervescence (bubble formation)
• Catalase negative reaction: No (effervescence) bubble formation or a few bubbles after 20 seconds.

Catalase Positive Organisms: Staphylococci, Micrococci, Bacillus species, Propionibacterium acnes, Helicobacter pylori, Haemophilus influenzae, Moraxella species, Neisseria species, Pasteurella species, Brucella species

Catalase Negative Organisms: Streptococci (For example, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes), Enterococcus species, Clostridium species (For example, Clostridium perfringens, Clostridium septicum, Clostridium tertium), Haemophilus ducreyi, Ekinella corrodens, Kingella species

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 before using them.
A. Staphylococcus aureus ATCC 25923- catalase-positive
B. Streptococcus pyogenes ATCC 19615- catalase-negative

S. aureus secretes catalase and superoxide dismutase, which inhibit organism destruction by the myeloperoxidase system of phagocytic cells.

Precautions/Limitations

1. Rare strains of staphylococci may be catalase-negative, and some enterococci produce a “pseudocatalase” and are weakly positive with H₂O₂.
2. 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.
3. If using colonies from a blood agar plate, be careful not to scrape up any of the blood agar as red blood cells are catalase-positive. The presence of any contaminating agar (carryover of red blood cells) could give a false positive.
4. Catalase enzyme is present in viable cultures; do not test colonies older than 24 hours. Older cultures may give false-negative results.
5. Do not test from Mueller-Hinton agar.

Uses

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 helpful 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. Shigella dysenteriae types 1, 3, 4, 6, 9, 11, and 12 and S. boydii type 12 are catalase-negative, whereas other species of Shigella, EIEC, and STEC are catalase-positive.
7. 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. American Society for Microbiology
• Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society for Microbiology. https://doi.org/10.1128/9781555818814
• Mandell GL. Catalase, superoxide dismutase, and virulence of Staphylococcus aureus. In vitro and in vivo studies with emphasis on staphylococcal–leukocyte interaction. J Clin Invest. 1975 Mar;55(3):561-6. doi: 10.1172/JCI107963. PMID: 1117067; PMCID: PMC301784.