CATALASE TEST - Biochemical Tests for Bacterial Identification

 AIM:

     To identify whether the organism is capable of producing the enzyme Catalase.

PRINCIPLE:

     Hydrogen peroxide is a Reactive oxygen species (ROS) which is lethal to cells, catalase in bacteria protects the cell from ROS. Catalase is an enzyme present in microorganisms which breakdown Hydrogen peroxide into water and nascent oxygen.

             3%   2H₂O_{2 ----------}^Catalase_{------------->}  2H₂O   +   2(O)

Test can be performed in 2 ways:

    Slide catalase test

    Tube catalase test

REAGENTS:

•  3% freshly prepared hydrogen peroxide

PROCEDURE:

Slide catalase test

1. Take a clean glass slide and divide it into 3 circles using white pencil/marker and mark it as Positive control, Test and Negative control respectively.
2. Add 2-3 drops of 3% Hydrogen peroxide on marked areas.
3. Positive control and Negative control ATCC cultures were inoculated onto the respective areas using tooth stick.
4. Then the test organism was inoculated onto the slide.
5. Check for effervescence/bubble formation immediately within 60secs.
6. Results were interpreted.

Tube catalase test

1. Take 3 test tubes and mark each as Positive control, Test and Negative control respectively.
2. Add 3-5 drops of 3% Hydrogen peroxide to all the test tubes.
3. Inoculate PC and NC into the respective tubes using wooden sticks.
4. Inoculate Test organism into the test tube using wooden stick.
5. Note for effervescence/bubble formation immediately within 60secs and interpret the results.

INFERENCE:

 

    Positive control (ATCC 25923 S.aureus) - Brisk effervescence/bubble formation was observed immediately.

    Negative control (ATCC 29212 E.fecalis) - No bubble formation/effervescence was appreciated even after 60secs.

 

The controls were satisfactory. Now we can interpret the result of our test organism.

    Test - Brisk effervescence/bubble formation was immediately observed.

 

EXAMPLES:

 

CATALASE POSITIVE ORGANISMS	CATALASE NEGATIVE ORGANIMS
Staphylococcus aureus	Streptococcus sp.
Enterobacteriaceae	Enterococcus sp.
Mycobacterium sp.	Clostridium sp.
Vibrio cholerae	Shigella dysenteriae type 1

 

 

NOTE:

            Test colony should not be taken from the Blood agar as it may produce False Positive results due to the presence of peroxidase enzyme in RBCs.