CATALASE TEST

Catalase is an enzyme that decomposes hydrogen peroxide (H₂O₂) into water and oxygen. It is a hemoprotein with the four iron atoms in the molecule in reduced Fe³⁺  state. Most aerobic and facultative baceria except streptococci possess catalase activity.

Hydrogen peroxide is a oxidaive end product of bacterial aerobic carbohydrate metabolism. If allowed to accumulate, it is known to have lethal bactericidal effect by oxidative damage. Some bacteria produce catalase enzyme which facilitates  their cellular detoxification and protects from such damage

Purpose:

The catalase test facilitates the detection of enzyme catalase in bacteria and help to differentiate catalase positive micrococcaceae from catalase negative streptococaceae

Theory:

Recipe:

a)     3% H2O2	·      Routine Testing of aerobe
b)     15% H2O2	·      For testing of anaerobic bacteria ·      Used to differentiate strains of Clostridium (Catalase –ve)    from Bacillus (Catalase +ve)
c)     30% H2O2   (Superoxol Catalase test)	·      Used to differentiate Neisseria gonorrhoeae (100% Catalase +ve) from other Neisseria spp. (1% Catalase +ve)

Protocol: (Method variation of catalase test)

1.     Slide or Drop catalase test (Common)

2.     Tube catalase test/ Tube slant method

3.     Capillary tube method

4.     Cover slip method

5.     Semi-quantitative catalase for Mycobacterium tuberculosis

6.     Heat stable catalase test for differentiation of Mycobacterium spp.

Methods:

a)     Slide method

Well isolated 18-24 hours old colony with the help of inoculating loop or wooden applicator stick is placed on the slide and one drop of 3% H₂O₂  is added. To limit catalase aerosols  which have been shown to carry viable bacterial cells, the use of petridish to cover  the side is highly recommended.

Observation is best against a dark background and with the help of magnifying glass or 40X magnification under the microscope.

b)      Tube Catalase method

      Well isolated 18-24 hour old colony with the help of inoculating loop or wooden applicator stick is placed in a transparent tube containing few drops of 3%  H₂O_{2 .}

      The rapid and sustained appearance of bubbles or effervescence constitutes a positive test.

      Observation is best against a dark background

Positive control: Staphylococcus aureus

Negative control: Streptococcus spp.

False positive reaction

·       Bubbles formed  20-30 seconds later is not considered positive. (some enzyme other than catalase is decomposing hydrogen peroxide here)

·      If platinum inoculating loop or iron wire loop is used

·      If agar containing Red Blood Cell (RBC) is mixed with organism( catalase is present in RBC as well)

Examples of catalase positive organism:

Staphylococcus and Micrococci
Listeria, Corynebacterium diphtheriae , Nocardia
Enterobacteriaceae family
Mycobacterium tuberculosis
Aspergillus
Cryptococcus

www.microbexperts.com