Catalase Test: Purpose, Principle, Method, Interpretations, Precautions

Hydrogen peroxide H₂O₂ made one of the oxidative final product of aerobic carbohydrate metabolism. If allowed to accumulate it is lethal for bacteria. Chemically catalase is hemoprotein. Similar in structure to hemoglobin. Except that the four iron atoms in the molecule are in the oxidize form (Fe₃⁺) rather than the reduced form (Fe₂⁺). Negative in all streptococci, most aerobic and facultative bacteria.

Uses or Purpose:

• Catalase test differentiate between micro-organisms who produce catalase enzyme, (Staphylococci) and non catalase producing micro-organisms, (Streptococci).
• The morphologically similar Enterococcus or Streptococcus (catalase negative) and Staphylococcus (Catalase positive) can be differentiated using the catalase test.
• This test is valuable in differentiating between aerobic and obligate anaerobic micro-organisms.
• Semi-quantitative catalase test is used for the identification of Mycobacterium tuberculosis.
• This test also differentiate between catalase negative bacteria who are aero tolerant (Strain of Clostridium), Catalase positive bacteria (Bacillus spp).
• Catalase test may also use in the identification of Enterobacteriaceae spp.
• Also to differentiate members of Microcaccaceae from members of the Streptococci

Principle:

• Production of hydrogen peroxide to oxygen and water is by an enzyme Catalase which act as catalyst in this reaction.

• An organism is bringing it into contact with hydrogen peroxide which is being tested for catalase production.
• If the bacteria is Catalase producer, Oxygen bubbles production can be seen.

Requirements:

• Hydrogen peroxide (H₂O₂) concentration 3%
• Sterilized wooden stick or Glass rod
• Inoculum or culture plate
• Slide if using the slide method
• Test tube if using the Tube method

Method:

Slide Method:

• With the help of a wireloop or sterilized wooden stick, remove colony from culture plate and place on the surface of a clean and dry slide.
• Mix with a small drop of 3% hydrogen peroxide H₂O₂ on the glass slide already having a bacterial colony.
• Production of oxygen bubbles can be observed.

Tube Method:                                                                              

• Take 2 to 3 micro liter of the (H₂O₂) solution into a test tube.
• With the help of a sterilized wooden stick or a glass rod, remove several/many colonies of the bacteria which is being used for test and mix with the hydrogen peroxide (H₂O₂) solution.
• Observe for immediate oxygen bubbles.

Results:

• Active bubbling …………………………..   Positive
• No bubbles ……………………………   Negative

Controls:

• Positive …………………………………….   Staphylococcus spp
• Negative ……………………………………    Streptococcus spp

Precautions of Catalase Test:

• The test micro-organisms must not be selected from blood agar culture plate. Because RBCs have enzyme catalase and its existence will responsible for a false positive results.
• Culture plate must be 18 – 24h old or 1 day.
• H₂O₂ should be fresh because it is unstable.
• Iron wire loop should not be used.
• Some type of micro-organisms produce an enzyme (Peroxidase) that is responsible for catalyzes and breakdown of H₂O₂ which is further affecting the reaction and give weak positive. This must not be compared with a true positive results.
• Do not add micro-organism in the solution, predominantly, if iron containing inoculating wireloops are used in the test. Because iron containing wireloops can give false positive results if they are expose or mix with hydrogen peroxide (H₂O₂).