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.
Table of Contents
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 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 the 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 seconds), 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).
- 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.
- 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 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.
- Catalase enzyme is present in viable cultures; do not test colonies older than 24 hours. Older cultures may give false-negative results.
- Do not perform catalase test from Mueller-Hinton agar.
Uses
- 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 helpful 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.
- 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.
- 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.
I am impressed and I learned a lot
how to differentiate between staphylococcus and streptococcus bacteria with catalase test? because almost streptococcus spp. of organisms are aerobic or microaerophilic (beta haemolytic strains).
Ravi Prasad ji
To differentiate the genus (Streptococcus or Staphylococcus), you can perform catalase test. Streptoccus spp is catalase Negative and Staphylococcus is catalase positive.
superb article..accurate and impressive
Sir i just need to know is it a journal or newspaper article because i have to write a report on it and i need a reference
Something to be careful of when doing Catalse is aerosolization of the organism…….ergo the bubbling.
Thank you so much for this. Finally have an answer for our Biochemistry :))
i tried the tube test and i got absolutely no results. but when i tried the slide test the effervescence came. why do you think this happened.
Very hard to say you exact reason on the basis of information you have provided. Please try again with fresh culture with both tube and slide test; adhering with exact protocol and share your results.
Tamara, sounds like you did not have a lot of growth in the tube and the hydrogen peroxide could have been diluted in the liquid media. Culture should be at least 24 hrs old with good growth try this protocol: http://www.microbelibrary.org/library/2-associated-figure-resource/3237-positive-tube-catalase-test
You can also drop a drop of hydrogen peroxide directly on the media if grown on TSA: http://www.microbelibrary.org/library/2-associated-figure-resource/3234-catalase-test-on-staphlococcus-aureus-streak-plate
and
http://www.microbelibrary.org/library/2-associated-figure-resource/3235-catalase-test-on-staphlococcus-aureus-streak-plate-enlarged-view
Thing to be cautious of 1). use a plastic loop or wood application since hydrogen peroxide can react with a metal loop…ie you could get a false positive., 2). some media reacts with hydrogen peroxide such as blood agar. So you could not drop the hydrogen peroxide directly on the plate. If you do the slide test make sure you do not carry over any agar (blood agar, etc) to avoid a false positive.
thank you so much for these useful information
thank you very very much
i took some useful contents in this article,,help me reference it cause i dont wanna be penalised for stealing others s work
Thank you for your comment. Here is the method of citation using APA format: Acharya, TS., (2013.Catalase test: principle, uses, procedure and results. Retrieved from http://microbeonline.com/catalase-test-principle-uses-procedure-results/
what about the use of 30% H202
While performing the catalase test on crude protein extract of drosophila species fed on a pomegranate juice supplemented diet and control, the absorbance decreased with time in the case of control but increased in the flies that recieved the polyphenol treatment. Would be great help if you could provide a possible explanation?
thank you sir for this help
Really sir you are doing a good job for all the medical lab technician.thank you sir.
Its easy to learn thank u
Very nice material easy to perform
I appreciate your efforts
Thanks.
Great information via this plate form
Sir tel me how to differentate when catalase test is performed
Thank you
thank you for the information, am impressed. kindly help me understand why the other catalases only break down H2O2 in 30s and not as the catalase in staph. could you also write on other culture media such as Todd-Hewitt broth, edward’s agar and tryptone soya broth.
Sir is there any difference in meaning between high titre sera, antisera?
Soooo.. is Moraxella Catalase positive??
Very good explanation
i am really impressed and have also learned a lot from your write up.
It made me passed excellently my microbiology end of semester exams.
hello my work on ornithobacterium rhinotracheale bacteria. I want any helpful information about its isolation and identification
I have the following question for you
1. Is all salmonella species are catalase positive
2. What are biochemical test use for the differentiation of salmonella and cetrobacter youngae
I want to check the growth of bacteria in the presence of methylene blue 0.1 %. but I am not confirmed about the formulation to make the media. I need your kind help. Thank You
Hello,Kindly let me know whether it was an error when you wrote that the positive control for catalase test is staphylococcus aureus instead of staphylococcus species.Kindly clarify, because I know coagulase test is done to identify staphylococcus aureus
The best practice is to use known strain during Quality Control procedure and most researchers prefer/use ATCC strains. If you have Staphylococcus aureus, it can also be used as a positive control for Catalase test but it’s not mandatory, you can use any species of Staphylococci for this purpose.