Catalase Test- Scope, Principle, Procedure, Uses and Results and Interpretation

 

Scope

To detect the catalase enzyme present in most cytochrome-containing aerobic and facultatively anaerobic bacteria.   It is used to differentiate those bacteria that produce an enzyme catalase, such as staphylococci, from non-catalase producing bacteria such as streptococci.

Principle

Catalase is an enzyme that breakdown hydrogen peroxide (H2O2) into water and oxygen. The catalase is a hemoprotein, similar in structure to hemoglobin, except that the four iron atoms in the molecule are in the oxidized (Fe3+), rather than the reduced (Fe2+), state. Excluding the streptococci, Enterococci, and a few other genera, most aerobic and facultative bacteria possess catalase activity.

Hydrogen peroxide forms as one of the oxidative end products of aerobic carbohydrate metabolism. If allowed to accumulate, it is lethal to bacterial cells. Catalase converts hydrogen peroxide into oxygen and water as shown by the following reaction:

2H2O2 → 2H2O + O2 (Gas bubbles Appear)

The catalase test is most commonly used to differentiate members of the Micrococcaceae and Staphylococci which are positive, from members of the Streptococcaceae.

Materials and Reagents

 Hydrogen peroxide 3% stored in a brown bottle under refrigeration

 An 18- to 24-hour culture of the organism to be tested

Procedure

Tube Method

Pour 1-2 ml of hydrogen peroxide solution into a test tube.

With an inoculating needle or a wooden applicator stick, transfer growth from the center of a colony to immerse in the hydrogen peroxide solution.

Observe for immediate bubble formation.

Slide Method

With an inoculating needle or a wooden applicator stick, transfer growth from the center of a colony to the surface of a glass slide.

Add one drop of 3% hydrogen peroxide and observe for bubble formation.

Results and Interpretation

Positive:  Active bubbling - Catalase produced.

Negative: No release of bubble - No Catalase produced.

The rapid and sustained appearance of bubbles or effervescence constitutes a positive test. Because some bacteria possess enzymes other than catalase that can breakdown hydrogen peroxide, a few tiny bubbles forming after 20–30 seconds is not considered a positive test. In addition, catalase is present in red blood cells; so care must be taken to avoid carryover of red blood cells with the colony material.

Slide Method

Tube Method

Quality Control

The hydrogen peroxide reagent must be tested with positive and negative control organisms each day or immediately before unknown bacteria are tested.

Positive control: Staphylococcus aureus

Negative control: Streptococcus species

 Limitations

Media containing whole red blood cells will contain catalase and could therefore give a false positive result.

The enzyme is present in viable cultures only.  Do not perform on cultures over 24 hours old.  Older cultures may give false-negative reactions.

A weak catalase or pseudocatalase reaction may be produced by some strains of Aerococcus and Enterococcus species due to presence of peroxidases.

Hydrogen peroxide is unstable and should be stored in a spark proof fridge.  Avoid any undue exposure to light.

Nichrome loop can react with hydrogen peroxide to produce false-positive reactions.  Hence, use only wooden or plastic applicators for this test.

Caution: Hydrogen peroxide is an irritant.

Uses of Catalase Test

It is used to differentiate aerotolerant strains of Clostridium, which are catalase negative, from Bacillus species, which are positive.

The morphologically similar Enterococcusor Streptococcus and Staphylococcus can be differentiated using the catalase test.

Also valuable in differentiating aerobic and obligate anaerobic bacteria.

Semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis.

Catalase test can be used as an aid to the identification of Enterobacteriaceae.

Reference

Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott’s Diagnostic Microbiology, 12th Ed., Mosby Elsevier.

Winn et al. Koneman’s Color Atlas and Text book of Diagnistic Microbiology. 6th Ed., Lippincott Williams & Wilkins, Wolters Kluwer.