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Catalase Test -Principle, Procedure, Results

What is the catalase test?

” It is a biological term, which is manually used to detect the production of an enzyme known as catalase in an organism.”

  • This test is a biochemical test.
  • The enzyme catalase is a particular enzyme found in all living things or living beings. It usually lives in aerobic atmosphere. It means it lives in the presence of oxygen. Either, it activates the putridity of H2O2, releasing water and oxygen.
  • Catalase is a very important enzyme in morbific organisms. Its function is to protect oxidative damage from the species of reactive oxygen in an organism.
  • The catalase enzyme negates the prophylactic effects of H2O2 because hydrogen peroxide is a toxic substance.
  • The catalase test has been used for ages. Its function is to provide the distinction of catalase-positive organisms like staphylococci from catalyst negative species e.g streptococci.
  • There are multiple uses of this test. The foremost use is that it is helpful in the hypothetical portrayal of many bacteria.
  • In the presence of oxygen, manually 3% H2O2 is required and is used.
  • In the absence of oxygen, manually 15% H2O2 is needed and is used to carry different types of reactions.

Objective of Catalase Enzyme

There are numerous objectives of the catalase enzyme.

  • It is used to discern the potentiality of various organisms to manufacture the catalase enzyme.
  • It is used to discriminate between catalase-positive and catalase-negative organisms and vice versa.
  • When the reaction is carried out, it manually gives us toxic products. One is H2O2 and the other is O2` (superoxide radical). These products can be produced in the presence and the absence of oxygen.
  • The products produced at the end of the reaction are noxious to the entire living beings. If these products are not broken down it causes cell-lysis and more catastrophic and effects.
  • There are various techniques to break down toxic substances into nontoxic substances.
  • Here the bacteria are very helpful in determining the entire reaction. H2O2 is a compound that is broken down into the water and gaseous oxygen. Then the releasing of gas bubbles occurs.
  • When catalase enzyme is manufacture it has immense uses. The foremost use is that it provides shelter to the deadly and dangerous effects of H2O2, which is assembled at the end of the aerobic procedure.
  • When the enzyme catalase is present, it can be described by appending H2O2 to the bacterium inoculum. Here the inoculum bacteria do whatever we needed. It gives us the quick release of oxygen bubbles.
  • If the catalase enzyme is not present in the entire reaction, then the bubbles will not be formed.
  • The young dominion of bacteria on agar medium, called as Blood agar.
  • For the anaerobic mechanism, the bacterial Dominion should be left unprotected further for at least 30 minutes.

Reagents and Supplies used

The reagents are listed below:

  • 30% H2O2, for bacterium Neisseria
  • 15% H2O2, for anaerobes
  • 3% H2O2, for other multiple bacteria’s.


  • The glass slides should opt.
  • Pure wooden glass sticks

Procedure of Catalase Test

There are various techniques/ methods for doing the catalase test. The first one is known as the slide method and the other method is known as the tube method.

1. Slide Method

It is also known as the drop catalase test. The slide method is one of the most influential and popular tests. This test needed comparatively a small faction of organisms.

  • Firstly, a microscope is put internally in a dish known as Petri. If a Petri dish is a choice, if not used doesn’t care. The Petri dish is used to reduce the aerosols, which might carry some pathogenic and virulent strains.
  • Secondly, a small fraction of an organism opts from a well & clean dominion for usually 18-24 hours. Then it should be placed on a microscope side.
  • Now, the most crucial step is to take blood agar when cultured thoroughly.
  • Then 3 percent of H2O2 drop onto the entire organism fraction should be applied by inculcating a dropper.
  • Lastly, we discern an appearance of bubbles against black background enough to be read thoroughly.

2• Tube Method

The second type of test is known as the tube method. The steps are listed below:

  • Firstly, we need about 4 to 5 drops of approximately 3 percent H202.
  • The above figure is added to the test tube.
  • Then we need a wooden glass stick. After that, a small faction of the organism opts from a well and clean soft corner for usually 18-24 hours. These all things should be put in a test tube.
  • The test tube can be placed against a black background.
  • Lastly, we observe the entire bubbles.

Quality Control

The quality should remain best.

  • We need catalase-positive Staphylococcus aureus.
  • Then we also need catalase-negative Streptococcus pyogenes.
  • When a test is positive, it can be described and detected as the appearance of immense bubbles.
  • If the concentration of bubbles is low, genuinely it means a weak reaction has taken place.
  • If bubbles are observed in the last 20 seconds, it means the test is negative.

Reporting Result

  • The catalase test is an important test used to distinguish between catalase-positive and catalase-negative strains.
  • Different types of bacteria can be detected through this test.
  • Bacillus is catalase-positive and Clostridium is catalase-negative.

Uses of Catalase Test

There are very helpful uses of the catalase test.

  • This test can be done in both aerobic and anaerobic mediums.
  • Different classes of bacteria are detected and demonstrated by the catalase test.
  • The different types of strains of bacteria like Clostridium which are catalase-negative are detected using this test.

Limitations of Catalase Test

There are a variety of limitations.

  • Red blood cells have an immense catalase. So, if we want to keep far away from positive results, then blood agar should not be picked with various dominions. Otherwise, the test can be done on regular basis.
  • The blood agar is a must, if not present at a given time, we need to avoid other types of agar.
  • The reagent and dominions shouldn’t be mix and shouldn’t be cultured together, otherwise, the results would be bad.
  • If we use a great percentage of H2O2, it causes severe damage to our skin. So, we need to use it as per demand.
  • If there are not enough dominions, we shouldn’t use 24 hours old dominions. If used the results would be false and restricted.
  • We should avoid the order of reverse reactions. If not, it will give us false results.

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