Catalase test

 

Aim

To determine the ability of microorganisms to degrade hydrogen peroxide by producing the enzyme catalase.

Principle

During aerobic respiration, microorganisms produce hydrogen peroxide.  Accumulation of these substances will result in death of the organism unless they can be enzymatically degraded.  These substances are produced when aerobes, facultative anaerobes, and microaerophiles use the aerobic respiratory pathway, in which oxygen is the final electron acceptor, during degradation of carbohydrates for energy production.  Organisms capable of producing catalase rapidly degrade hydrogen peroxide as follows:

2H₂O₂                                                  2H₂O    +    O_{2 ↑}

(Hydrogen peroxide)                        (Water)        (Free Oxygen)

 

Catalase production can be determined by adding the substrate H₂O₂ to the culture of the organism to be tested.  If catalase is present, the chemical reaction is indicated by bubbles of free oxygen gas.  This is a positive catalase test.  The absence of bubble formation is the negative catalase test.

Requirements

24 hr nutrient agar cultures of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

3% Hydrogen peroxide, Bunsen burner, inoculating loop, glass marking pencil etc.

 Procedure

1.  A loopful of cuture is placed on a clean glass slide and 1-2 drops of 3% hydrogenperoxide is added.

2.  Mixed well and immediately observed for the bubble formation.

 

Observations

On addition of hydrogen peroxide Bacillus, Staphylococcus, Pseudomonas and E.coli cultures produced bubbles within minutes, indicating the formation of free oxygen gas.  Streptococcus sp. produced no characteristic reaction.

 Result

Bacillus, Staphylococcus, Pseudomonas sp. and E.coli are catalase positive whereas Streptococcus sp. is catalase negative.