Catalase peroxidase test for MTB

Catalase Peroxidase test for MTB

Principle:

Catalase is an intracellular, soluble enzyme that splits hydrogen peroxide into water and oxygen. The oxygen bubbles into the reaction mixture to indicate catalase activity. Virtually all mycobacteria possess catalase enzymes, except for certain isoniazid–resistant mutants of M. tuberculosis and M.bovis.

Mycobacteria possess several kinds of catalase that vary in heat stability. Quantitative differences in catalase activity can be demonstrated by the 68⁰C test at pH 7 (indicates loss of catalase activity due to heat). Drug susceptible strains of M. tuberculosis lose catalase activity when heated to 68⁰C for 20 minutes. For these tests, cultures on LJ should be used.

Controls:

Check reagents by testing extract from an uninoculated tube of medium (negative control).

1.  1. 0.067M phosphate buffer solution, pH 7.0

Solution 1:

Na₂HPO₄, anhydrous :9.47 g 

Distilled water: 1 litre

Dissolve disodium phosphate in water to make a 0.067 M solution.

Solution 2: 

KH₂PO₄: 9.07 g 

Distilled water :1 litre 

Dissolve in water to make 0.067 M KH₂PO₄ solution.

Mix 61.1 ml of Solution 1 with 38.9 ml of Solution 2. Adjust the pH to 7. 

2. Hydrogen peroxide, 30% solution

 Store in the refrigerator.

3. Tween-80, 10% 

Tween- 80:10 ml 

Distilled water: 90 ml 

Mix Tween-80 with distilled water and autoclave at 121^o C for 10 minutes. 

Allow to cool. Store in the refrigerator.

4. Complete catalase reagent (Tween-peroxide mixture):

Immediately before use, mix equal parts of 10% Tween-80 and 30% hydrogen peroxide. 

Use 0.5 ml reagent for each strain to be tested.

Procedure:

Image

Results and interpretation:

If bubbles appear (due to the production of oxygen gas), the bacteria are catalase positive. 

If no bubbles appear, the bacteria are catalase negative.

Resource

Revised National TB Control Programme Training Manual for Mycobacterium tuberculosis Culture & Drug susceptibility testing.

Assessment