Catalase test - Purpose, Principle, Biochemistry, Procedure, Result, Interpretation

Introduction to Catalase test

The biochemical test catalase test is used to determine if the test microorganism can produce the catalase enzyme which is able to decompose hydrogen peroxide to release oxygen and water.

Purpose of Catalase test

The purpose of the catalase test is to determine if microorganism is able to produce the catalase enzyme.

The enzyme catalase is a hemeprotein, a large class of metalloprotein that contains a heme prosthetic group. In catalase, the prosthetic group is made up of four atoms of trivalent iron (ferric-Fe⁺⁺⁺) per molecule which functions in oxygen reduction.

The importance of the catalase enzyme lies in its ability to protect the cell from oxidative damage by reactive oxygen species (ROS) and due to its very high turnover numbers - one catalase molecule can decompose millions of hydrogen peroxide molecules.

Catalase test-positive organisms (both aerobic and facultative anaerobes) depend on the cytochrome system for the production of catalase. If an organism lacks the cytochrome system, usually, they also do not produce catalase enzyme.

Principle, Biochemistry of Catalase test

The principle and biochemistry of catalase test can be explained on the molecular level.

In microorganisms, aerobic breakdown of sugars takes place resulting in the formation of hydrogen peroxide (H₂O₂). In the presence of gaseous oxygen, reduced flavoproteins (FPH₂) undergo electron reduction to form hydrogen peroxide and oxidized flavoprotein (FP).

FPH₂ + O₂ → FP + H₂O₂

If hydrogen peroxide, which is toxic, continues to accumulate, the bacteria will die. During the decomposition of H₂O₂ by catalase, one acts as a donor while the other acts as a substrate. The donor donates hydrogen ions to the substrate, reducing it to form a reduced substrate (H₂O) and oxygen gas (which is an oxidized donor).

H₂O₂ (acts as donor) + H₂O₂ (acts as substrate)→ (catalase enzyme) → 2H₂O + O₂

Procedure of Catalase test

The procedure of the catalase test involves the slide method and the tube method.

Slide method

1. Pick a fresh pure colony of test microorganisms (careful not to pick up any agar, especially BA) and place it on the center of a clean, grease-free slide

2. Add 1-2 drops of 3% H₂O₂ to the test organism placed on the slide with the help of a dropper

3. Observe for immediate bubble production (gas release)

Tube method

(simple tube method)

1. Add 4-5 drops of 3% H₂O₂ to the test tube

2. Pick a fresh pure colony of test microorganisms (careful not to pick up any agar, especially BA) and place it into the test tube

3. Observe for immediate bubble production (gas release)

(slant tube method)

1. Directly add 1-2 drops of 3% H₂O₂ to a fresh culture of test bacteria that has been grown in nutrient agar slant

2. Observe for immediate bubble production (gas release)

Result, Interpretation of Catalase test

The result and Interpretation of Catalase test is as follows:

Positive

Immediate gas bubble production is seen due to the release of oxygen.

Negative

No gas bubble is produced.

Catalase positive organism

The list of catalase-positive organisms are:

• Micrococcus

• Staphylococcus

• Bacillus

• Corynebacterium diphtheriae

• Listeria

• Citrobacter

• E. coli

• Enterobacter

• Klebsiella

• Shigella

• Yersinia

• Proteus

• Salmonella

• Serratia

• Nocardia

• Burkholderia cepacia

• Mycobacterium tuberculosis

• Rhodococcus equi

• Pseudomonas

• Aspergillus (mold)

• Cryptococcus (fungi)

Catalase negative organism

The list of catalase-negative organisms includes:

• Streptococcus

• Clostridium

• Enterococcus spp.