Published: 5th Sep 2025, 15:22

The biggest smiles hide the biggest secrets, and so do the microbes with the tiniest bodies. You wish you could read their next move? Well, it’s quite fascinating that it is not impossible to do so. How? Have you heard of biochemical tests? These tests often play a crucial role in unravelling the microbe’s true intentions and identities. Amongst all the biochemical tests, Catalase test is one of the quickest, easiest, and reliable assays that can reveal a bacterium’s ability to deal with oxidative stress. But beyond the bubbling glass slides, Catalase-510 (2000-5000 Unit/Mg) has more roles in its movie than you think. It is often used in industrial biotechnology, molecular biology, and even clinical diagnostics.

In this blog, let’s explore the principle, procedure, interpretation, applications, and commercial uses of Catalase in detail. Get your lab coats and glass slides ready to pop the bubbles of information with Catalase and Catalase Test.

What is The Catalase Test?

Biochemical tests are widely used to identify the bacterial nature at the physiological level and their biochemical tendencies. One such test is the catalase test.

Catalase is an enzyme that catalyzes the breakdown and decomposition of the highly reactive oxidizing agent— Hydrogen Peroxide (H₂O₂) into non-harmful products like water and oxygen, and helps bacterial cells overcome oxidative stress.

Hydrogen Peroxide is one of the most toxic byproducts of aerobic metabolism and can cause oxidation of cellular components, leading to cellular damage. This reaction is marked vital due to Hydrogen peroxide’s high oxidizing potential. If hydrogen peroxide is allowed to accumulate in the cells, it can lead to lethal outcomes without the production and activity of catalase. 

Key Reaction:

2 H₂O₂ → 2 H₂O + O₂ ↑

The oxygen released appears as visible bubbling or effervescence and is a telltale sign of a positive catalase reaction.

The Principle Behind The Catalase Test:

Organisms living in the presence of oxygen, for example, Aerobes or Facultative anaerobes, follow pathways of aerobic metabolism and often produce toxic byproducts related to Reactive Oxygen Species (ROS). These reactive oxygen species include strong oxidizing agents like hydrogen peroxide, superoxide radicals, and hydroxyl radicals. Hence, survival in such environments is only possible through the possession of detoxifying enzymes to decompose such reactive species. One such enzyme is Catalase. Catalase is one of these detoxifying enzymes that specifically targets Hydrogen Peroxide, a strong oxidizing agent. It rapidly breaks down hydrogen peroxide to give out oxygen and water, which are both non-toxic cellular products. The evolution of gas bubbles, therefore, becomes an observable result indicating a positive catalase activity. While Catalase does target other reactive oxygen species directly, it still plays a major role by eliminating one of its major sources of cellular oxidative stress.

The test is widely popular and useful in differentiating Gram-positive cocci: 

• Staphylococcus spp. are catalase-positive
• Streptococcus spp. and Enterococcus spp. are catalase-negative.

Procedure For Catalase Test:

There are quite a few ways to perform the catalase test. Depending on the bacterial culture type and lab resources, it can be done through three simple methods.

1. Slide Test (Rapid and Common)

Material Required: 

• 3% Hydrogen Peroxide
• Clean glass slide
• Sterile wooden stick or disposable loop
• Bacterial colony (preferably, 1-2 days old)

Procedure:

• Take a drop of 3% Hydrogen Peroxide on a clean glass slide.
• Use a sterile loop to transfer a colony from an agar plate.
• Mix it well with the drop of hydrogen peroxide.
• Observe for immediate bubbling.

Precautions:

• One shall avoid using colonies directly from blood agar, as the catalase from the Red blood cells may interfere with the results and give false positives.
• One shall not use any metal loops, as they can catalyze the breakdown of hydrogen peroxide and mimic a positive result.

2. Tube Method (For Microbes in Liquid Suspension)

Procedure:

• 1-2 mL of hydrogen peroxide is added to a small test tube.
• Introduce a loopful of bacterial suspension into it.
• Observe for bubbling along the sides of the test tube.

3. Plate Method (Directly on Agar)

• A drop of hydrogen peroxide is added directly onto a single colony on the plate surface.
• This method must not be performed on Blood and Chocolate Agar plates.

Interpretation of Results:

Note: To validate the catalase test results, always run positive and negative controls.

Catalase-Positive and Catalase-Negative Organisms:

A bacterium ability to produce catalase reveals its important characteristics and helps in its classification. Some of the catalase-positive and negative bacteria are listed below.

Catalase-positive organisms include:

• Some of the gram-positive bacteria, such as Staphylococcus spp., Micrococcus spp., Listeria monocytogenes, Corynebacterium spp., and Bacillus spp. 
• Some of the gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., Salmonella spp., and Shigella spp.

Catalase-negative organisms include:

• Bacteria such as Streptococcus spp., Enterococcus spp., most species of Clostridium, and Lactobacillus spp. 
• One noteworthy point is that some of the Enterococcus strains is that they might give out a weak catalase-like activity due to the presence of a pseudocatalase, but are still classified as catalase-negative organisms.

Real-World Applications of Catalase

The catalase test is not just limited to the laboratories, but has many practical applications in multiple sectors.

1. Clinical Microbiology

Used for rapid differentiation of the pathogenic cocci:

• Important for the diagnosis of Staphylococcal and Streptococcal infections.
• Identifying catalase-positive aerobes and anaerobes.

2. Molecular Biology

• Catalase is linked with important molecular studies of apoptosis, oxidative stress, and ROS signalling pathways, and oxidative DNA damage.

3. Food and Dairy Industry

• Used to remove Hydrogen peroxide from milk, cheese, and other dairy products that might be added during the sterilization process.

4. Textile Industry

• It is employed during bleaching processes to eliminate residual hydrogen peroxide after fabric treatment.

5. Environmental and Industrial Biotechnology

• Applied in bioremediation and wastewater treatment for the decomposition of hydrogen peroxide.

Why Choose Catalase by TM Media For The Catalase Test?

1. Catalase by TM Media is commonly used in:

• Laboratory research in cell biology and its ability to overcome stress induced by Reactive Oxygen Species.
• Studies related to oxidative damage in proteins, lipids, and nucleic acids.
• Industrial removal of Hydrogen peroxide in various processing environments.

2. It is commercially available catalase with the following product specifications:

Frequently Asked Questions (FAQs)

Q1. What is Catalase?

A. The metabolism in aerobic organisms leads to the production of reactive oxygen species, such as Hydrogen Peroxide. The accumulation of these can lead to the oxidation of cellular components and potentially result in cell death. Hence, catalase is responsible for the detoxification of cells by neutralizing hydrogen peroxide by catalyzing the reduction of Hydrogen Peroxide to Water and Oxygen. 

Q2. What gas is released during the Catalase Test?

A. Oxygen is released during the Catalase Test. As hydrogen peroxide is broken down, Water and Oxygen are formed. Formation of bubbles when performing the test is what confirms the release of oxygen.

Q3. Can the catalase test be done inside test tubes?

A. Yes, the catalase test can be performed in test tubes. The procedure for the same is mentioned below:

• Prepare Hydrogen Peroxide Solution
• Take the Test Tube and add about 1–2 mL of hydrogen peroxide into a clean test tube.
• Collect the Bacterial Sample using a sterile wooden stick or disposable loop
• Drop the bacteria into the test tube containing H₂O₂.
• Observe the Reaction

Q4. Can I perform a catalase test using the colonies from blood agar?

A. No, it is not recommended to perform the catalase test using the colonies from blood agar.  Blood agar contains Red blood cells, which have a catalase activity of their own. This can lead to false positives. Hence, it is recommended to use colonies from nutrient agar or tryptic soy agar.