Catalase test: Principle, Procedure, Results, Uses

The catalase test is a fundamental biochemical assay in microbiology, primarily used to identify and differentiate bacterial species based on their ability to produce the enzyme catalase. This enzyme is crucial for microorganisms that live in oxygenated environments, as it provides protection against the damaging effects of reactive oxygen species (ROS), particularly hydrogen peroxide (H2​O2​). During aerobic respiration, cells inevitably produce toxic oxygen metabolites like hydrogen peroxide and the superoxide radical (O2.−​). These ROS can cause significant oxidative damage to cellular components such as DNA, proteins, and lipids, leading to cell death.

To counter this, many aerobes and facultative anaerobes produce catalase, which rapidly neutralizes hydrogen peroxide, and often, superoxide dismutase (SOD), which converts superoxide radicals into hydrogen peroxide and oxygen. Together, these enzymes are vital for survival in aerobic conditions. Anaerobic bacteria, generally not exposed to oxygen, typically lack catalase. Beyond its metabolic role, catalase is also recognized as a significant virulence factor in certain pathogenic bacteria, such as Staphylococcus aureus, enabling them to resist oxidative killing by host phagocytic cells. The catalase test thus holds significant clinical importance in the rapid identification of bacterial pathogens, guiding diagnosis and treatment strategies.

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

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 specific Neisseria sps)

Procedure

Slide Catalase 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 at the end of the wooden applicator stick.

Results

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

Rare strains of staphylococci may be catalase-negative, and some enterococci produce a “pseudocatalase” and are weakly positive with H₂O₂.

“Pseudocatalase” in some enterococci refers to a cytochrome-based mechanism that can weakly break down hydrogen peroxide, producing a few bubbles, but it’s not true catalase. This can lead to a misleading weak positive and confusion in differentiation if not interpreted carefully.

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. Many metals (especially iron-containing ones, common in nichrome loops) can non-enzymatically catalyze the breakdown of hydrogen peroxide into oxygen and water. This chemical reaction, not mediated by bacterial catalase, would produce bubbles and lead to a false-positive result. Platinum loops are chemically inert and do not react with H2O2, making them safe for use, as are wooden or plastic sticks.

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 catalase reaction.
Red blood cells, like many eukaryotic cells, naturally contain the catalase enzyme to protect themselves from oxidative damage. If red blood cells from the blood agar medium are accidentally transferred along with the bacterial colony to the hydrogen peroxide solution, their intrinsic catalase will react with the H2O2, producing bubbles. This reaction would be mistakenly attributed to the bacterial isolate, resulting in a false-positive catalase test.

Catalase enzyme is present in viable cultures; do not test colonies older than 24 hours. Older cultures may give false-negative results.
Bacterial enzyme production, including catalase, is highest during the logarithmic (log) phase of growth when cells are metabolically active and rapidly dividing. As cultures age beyond 24 hours and enter the stationary or decline phase, their metabolic activity decreases, and enzyme production can significantly diminish. This reduction in catalase activity in older, less viable cultures can lead to insufficient bubble production, resulting in a false-negative test, even if the organism inherently produces catalase.

Do not perform catalase test from Mueller-Hinton agar.
Mueller-Hinton agar, commonly used for antimicrobial susceptibility testing, contains trace amounts of certain ingredients that can interfere with the catalase reaction. Specifically, some components or their breakdown products can non-enzymatically react with hydrogen peroxide, leading to weakly positive or equivocal results that are difficult to interpret accurately and can lead to false positives or unclear results.

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.