Catalase Test Principle and Procedure

In this catalase test principle and procedure post we have briefly explained catalase test principle, objectives, requirements, catalase test in microbiology procedure, uses and limitations.

Catalase Test Principle and Procedure

Catalase test in microbiology is a biochemical test for aerobic bacteria that measures catalase enzyme synthesis in the bacterium. Hydrogen peroxide is produced by aerobic bacteria that use oxygen. Their electron transport mechanism converts reduced flavoprotein to oxidised flavoprotein and hydrogen peroxide by transferring electrons from reduced flavoprotein to oxygen.

Hydrogen peroxide is harmful to bacteria’s enzymatic action. The creation of catalase, an enzyme that transforms hydrogen peroxide to water and oxygen, allows for survival in the face of this antimetabolite. Catalase test in microbiology is used to distinguish between staphylococci (catalase-positive) and streptococci (catalase-negative) (catalase-negative).

Catalase-negative bacteria can be anaerobes or facultative anaerobes, meaning they only ferment and do not respire when oxygen is used as a terminal electron acceptor. Normally, 3 percent Hydrogen peroxide is used to detect catalase in aerobic organisms, while 15 percent Hydrogen peroxide is used to detect catalase in anaerobes.

Objectives

Catalase test in microbiology is a crucial tool for determining if gram-positive bacteria are staphylococci or streptococci. Catalase is a hydrogen peroxide-converting enzyme that produces water and oxygen gas. Catalase test in microbiology is simple to carry out by mixing bacteria and H2O2.

Principle

Catalase is a protein that aids in the conversion of hydrogen peroxide (H2O2) to oxygen and water. To determine whether a bacterial isolate can manufacture catalase enzyme, a little inoculum of the isolate is mixed into a 3 percent hydrogen peroxide solution and the quick development of oxygen bubbles is observed. A lack of or insufficient bubble creation indicates a lack of catalase.

2H2O2 → 2H2O+ O2 (gas bubbles)

Requirements

Reagent

1. 30% H2O2 for Neisseria

2. 15% H2O2 for anaerobes bacteria’s

3. 3% H2O2 for Aerobic bacteria’s

Supplies

1. Glass Slide

2. Glass Sticks

3. Inoculation loop

Catalase Test in Microbiology

Slide or Drop Method

1. In a petri dish, place a microscope slide. Keep the petri dish cover handy (it’s optional to use the petri dish, however it helps to limit catalase aerosols). Collect a little amount of organism from a well-isolated 18- to 24-hour colony and transfer it onto the microscope slide using a sterile inoculating loop (No agar must be picked up with the colony).

2. Place 1 drop of 3 percent H2O2 onto the organism on the microscope slide with a dropper or Pasteur pipette. The rapid development of oxygen (within 5-10 seconds) as demonstrated by bubbling is a favourable result. No bubbles or a few scattered bubbles are a negative result.

catalase test

Image Source: American society for microbiology

Test Tube Method

1. In a test tube, add 4 to 5 drops of 3% H2O2. Collect a tiny amount of organism from a well isolated 18- to 24-hour colony with an applicator and transfer it into the test tube. Place the tube against a dark background and look for instant bubbliness or bubble creation at the end of the wooden applicator stick (O2 + water Equals bubbles).

2. Positive reactions are characterized by immediate bubbliness or the creation of bubbles. To view faint positive reactions, use a magnifying glass or a microscope. A catalase-negative reaction is one in which no bubbles develop because there is no catalase enzyme to hydrolyze the hydrogen peroxide.

Image Source: American society for microbiology

Tube Slant Method

1. Add 1.0 ml of 3 percent H2O2 to a densely infected pure culture cultured on a nutrient agar slant for 18 to 24 hours. Place the tube against a dark background and watch for bubbles to appear right away. Positive reactions are immediately manifested by the creation of bubbles. A catalase-negative reaction is one in which no bubbles occur.

catalase test

Image Source: American society for microbiology

Results

Positive catalase reaction: bubbliness or bubble production occurs immediately or within 20 seconds in catalase test in microbiology.

Catalase negative reaction: no bubbles or a few bubbles after 20 seconds (no catalase enzyme to hydrolyze the hydrogen peroxide) in catalase test in microbiology.

Applications

1. Because hydrogen peroxide (H2O2) is highly unstable, it should be checked daily before use in catalase test in microbiology.

2. Catalase test in microbiology requires growth from an 18-24 hour culture. Catalase activity declines with age, resulting in a false-negative reaction.

3. The activity of catalase is determined by the aerobic process. Before a Catalase test in microbiology, anaerobically incubated organisms must be exposed to ambient oxygen for at least 30 minutes. If this step is skipped, false-negative findings may arise.

4. A positive catalase reaction in anaerobic organisms can take up to a minute after the reagent is added.

5. Some Aerococcus and Enterococcus species strains can produce a weak catalase or pseudocatalase response.

Limitations

1. Members of the genus Staphylococcus are catalase-positive, while those of the genera Streptococcus and Enterococcus are catalase-negative.

2. Catalase test in microbiology can be used to help identify Enterobacteriaceae bacteria. Catalase is found in the Enterobacteriaceae family of bacteria.

3. Due to superoxol, Neisseria gonorrhoeae creates a heightened elaboration of bubbles not found in other members of the genus.

Further Readings

Reference