Catalase Test

Catalase test in one of the biochemical analysis that generally comes into use for the identification of the organisms, whether they are catalase producers or not. It is primarily used to distinguish between the two gram-positive bacteria, namely Staphylococcus and Streptococcus species. Streptococcus bacteria are mostly facultative anaerobes (may produce catalase), while few are obligate anaerobes (cannot produce catalase).

Staphylococcus bacteria are aerobic and contain catalase enzyme to breakdown the reactive oxygen forms like superoxide and hydrogen peroxide. Thus, catalase functions as an “Antioxidant” that breakdown the toxic oxygen reactive species inside a cell to produce non-toxic products like H2O and O2.

Content: Catalase Test

  1. Definition
  2. Overview
  3. Principle
  4. Test Reagent
  5. Procedure
  6. Test Results
  7. Uses
  8. Limitations


Catalase test can define as a biochemical analysis of microorganisms that generally differentiates among the two different forms of bacteria, namely aerobic and anaerobic, based on the oxygen requirement. Aerobic bacteria require oxygen for their growth, and metabolizes oxygen into many reactive oxygen species, like superoxide anion, peroxides, hydroxyl radicals etc. The reactive oxygen species if remain within the cells, it can lead to oxidative damage in the cell organelles, DNA etc.

Catalase enzyme present within the organisms that grow in an oxygen-rich environment and protects the microorganism from the oxidative stress by the oxygen reactive forms. Anaerobic bacteria include two sub-groups like facultative and strict anaerobes. Facultative anaerobes can grow in the presence or absence of oxygen, and it may also contain catalase enzyme, but obligate anaerobes lack such enzyme.

Overview of Catalase Test

Before moving onto the theory of catalase test, we must be aware of specific terms that we must go through this article.


It is an enzyme that is ubiquitous, or present in almost all living organisms. Catalase enzyme belongs to the class of “Peroxidases” that functions as an antioxidant agent. It comprises of four polypeptide chains, which breaks down toxic hydrogen peroxide into non-toxic oxygen and water. Thus, a catalase enzyme works as a detoxifying agent by defending a cell in counter to oxidative damage by hydrogen peroxide.

Hydrogen peroxide

It is a toxic by-product, which forms as an outcome of cellular respiration. The peroxide ion of H2O2 acts as strong oxidizers, which oxidizes protein, membrane lipids etc. that ultimately cause cell apoptosis or death, if not removed quickly. It forms by the reaction of superoxide and hydrogen ion, by the enzymatic activity of Superoxide dismutase.

Principle of Catalase Test

The principle of the catalase test is based on the rapid detection of catalase presence, which becomes evident by the formation of copious gas bubbles. Catalase positive organisms can detoxify the toxic effect of  H2O2 by the catalytic activity of catalase. Oppositely, catalase-negative microorganisms cannot decompose H2O2 due to lack of catalase. The catalase test involves the mixing of inoculum with hydrogen peroxide.

hydrogen peroxide breakdown by catalase

If the test organism contains catalase, then they will degrade H2O2 into H2O and O2 (in the form of bubbles). If the test organism lacks catalase, then there will be no formation of gas bubbles.

Test Reagent

The catalase test only uses a single reagent (hydrogen peroxide). 3% of hydrogen peroxide is needed to check the presence of catalase in aerobically cultured bacteria. 15% of hydrogen peroxide is required to check the presence of catalase in anaerobically cultured bacteria.

  • For the preparation of a 3% solution of H2O2: Dissolve 3 grams of hydrogen peroxide in 100 ml of distilled water.
  • Similarly, for the preparation of a 15% solution of H2O2: Dissolve 15 grams of hydrogen peroxide in 100 ml of distilled water.


The catalase test can be performed by two standard methods, namely the tube and slide method.

Tube Method

It involves the following sequential steps:

  1. First, sterilise the test tubes by either using autoclave or hot air oven.
  2. Then under sterile condition, pour 1-3ml of hydrogen peroxide into the tubes.
  3. After that, take 24 hours of bacterial inoculum via a sterilized inoculating loop.
  4. Dip the inoculating loop straight into the test tube containing H2O2
  5. Observe the tubes for instant bubbling.

Slide Method

It involves the following sequential steps:

  1. First, sterilise glass slides by either using autoclave or hot air oven.
  2. Then under sterile condition, take 24 hours old bacterial inoculum via a sterilized inoculating loop.
  3. Prepare a bacterial smear onto the glass slide.
  4. Add 1-2 drops of H2O2 to the top of the bacterial smear.
  5. Observe the glass slide for the formation of bubbles.

Test Results

Positive result: It is evident by the formation of effervescence or copious gas bubbles.
Example: Corynebacterium diphtheria, mycobacterium tuberculosis, Rhodococcus equi, Staphylococcus sp, Listeria sp etc.

Negative result: In a negative test, there will be no formation of oxygen bubbles.
Example: Streptococcus sp, Enterococcus sp etc.
test results of tube and slide catalase test

Uses of Catalase Test

  • Catalase test marks the existence of the catalase enzyme in the microbial sample.
  • It differentiates between catalase-positive and catalase-negative organisms according to their genera as well as speciation.
  • It also uses as a possible method for the identification and differentiation of different organisms belonging to the Enterobacteriaceae family.
  • Catalase test also distinguishes between the aerobic and obligate anaerobic bacteria based on the production of catalase enzyme.
  • It also differentiates between the aerotolerant strains of Clostridium sp (non-catalase producers) from the Bacillus sp (catalase producers).

Limitations of Catalase Test

  • To perform a catalase test, a test organism incubated for 18 to 24 hours should be used.
  • Hydrogen peroxide should be freshly prepared while experimenting, as it is a volatile compound.
  • Test organism inoculated from the blood agar media frequently gives false-positive results, as the RBCs are catalase-positive cells.

Therefore, the catalase test is a prevalent method to differentiate between different groups (Aerobic and anaerobic), species based on the production of catalase enzyme.

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