Catalase Test: Principle, Procedure, Results, and Uses

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Catalase Test

Many of the biochemical tests are identified and performed in the laboratories and in experiments to differentiate a characteristic of a microbe and they are differentiated from other organisms. One such biochemical test is catalase test.

Catalase test is mostly used to detect the production of catalase enzymes in the aerobic organisms.

What is Catalase Test?

Catalase enzyme is one of the common enzymes which is found in all living beings which survive in oxygen and it also catalyses the decomposition of hydrogen peroxide, releasing water and oxygen.

Catalase is considered as one of the important enzymes in the pathogen organisms as it helps in protecting the organism from oxidative damages from the reactive oxygen species.

Catalase enzyme also results in neutralising the bactericidal effects of the hydrogen peroxide, and its concentration in bacteria has been correlated depending upon the pathogenicity of the organism.

Catalase test is being used from olden days in order to differentiate the positive catalase organisms from the negative organisms like streptococci.

Catalase positive organisms includes Staphylococci. This test is very much useful in identifying the characterises of most of the bacteria.

In aerobic conditions only 3% H2O2 is used, whereas in case of anaerobic conditions 15% of H2O2 is used.

Catalase Test Purpose

 The main aim of this test is to detect the ability of the organism to produce the enzyme catalase.

 To differentiate the catalase positive organisms from the catalase negative organisms.

Catalase Test Principle

The metabolic activity of the aerobic and the facultative anaerobic micro-organisms has the capability to produce toxic by-products like Superoxide radical and hydrogen peroxide.

Where these products are toxic to the organisms and they may also result in lysis of the cell if the cell is not broken.

Considering the pathogenic organisms, different mechanisms are used in order to break the products into non-toxic substances.

The bacteria which is capable of synthesising the enzyme catalase, hydrolyses the hydrogen water into gaseous oxygen and water that results in the liberation of gas bubbles.

Thus, the production of this enzyme, catalase protects the organism from suffering those lethal effects of the hydrogen peroxide which is accumulated at the end of the aerobic metabolism.

The presence of this enzyme can be detected by adding hydrogen peroxide in the bacterial inoculum, which thus results in the liberation of rapid air bubbles.

If the enzyme is absent then there will be no liberation of such air bubbles.

Micro Organisms Tested

The organisms that can be used for catalyse test includes the colonies of young bacteria, which are left in a culture of 18 hour or less than that, and mostly agar media is preferred here.

In case of anaerobes the colonies should be exposed to the air for about 30 minutes prior to the test.

Catalase Test Reagents

• Hydrogen peroxide Reagent

• But the reagent of hydrogen peroxide various for each organism, where as it is 30% for Neisseria, 15% for anaerobes, 3% for other bacteria. 

• Glass slide

• Sterile glass sticks

• Sterilised Platinum loops or wires

Catalase Test Procedure

The procedure for catalase test includes more than one method. The methods used are slide or drop catalase test, tube method, heat-stable catalase test, Semiquantative catalase test, Capillary tube method and cover -slip method.

Here the Heat-stable catalase test is mostly used for differentiating the species of Mycobacterium, the semi-quantative catalase method is mostly used for identifying the species, Mycobacterium tuberculosis.

From all these methods, the test which are most commonly performed in clinical bacteriology includes slide or drop catalase method, as this method requires only a small number of organism and it also works relatively as an uncomplicated technique.

1. Slide Method

To perform this slide method, first the microscopic slide is placed inside a petri dish. But it is also not necessary that the slide must be placed inside a petri dish as it is used only to limit the catalase aerosols, that carries viable bacterial cells.

Then a small amount of organism is collected from a well isolated colony from an 18 to 24-hour culture using the inoculating loops and it is placed on the microscopic slide.

No agar should be picked up along with the colony when the culture is taken from the blood agar.

About one drop of 3% of hydrogen peroxide is added upon the organism that is placed on a microscopic slide, using a Pasteur pipette or a dropper.

Then we can observe the formation bubbles which is formed against a dark background.

2. Tube Method

In tube method about 4 to 5 drops of 3% of hydrogen peroxide is added into a test tube.

Then the small amount of organism is picked up using an inoculation loop from an 18 to 24-hour colony and it is placed into the test tube.

Then the test tube is placed against the dark background and we can observe the bubbles that are formed immediately.

Catalase Test Quality Control

The organisms listed below are tested foe positive and negative results.

 If the result is positive then it denotes the presence of Staphylococcus aureus.

 If the results are negative then it denotes the presence of Streptococcus pyogenes.

Catalase Test Result

 The positive test is usually determined by appearance of immediate bubbles

 Whereas if only one or two bubbles are appeared, then it indicates the weak reaction

 The negative test is indicated by no bubbles are only few bubbles.

Catalase Test Citations


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