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Hydrogen peroxide is often used as a topical disinfectant in wounds, and the bubbling that is seen is due to the evolution of O2 gas. H2O2 is a potent oxidizing agent that can wreak havoc in a cell; because of this, any cell that uses Oxygen (O2) or can live in the presence of oxygen (O2 ) must have a mechanism to get rid of the peroxide. One of those ways is to make catalase.
Catalase test is used to differentiate those bacteria that produces the enzyme catalase, such as staphylococci, from non-catalase producing bacteria such as streptococci. Normally 3% H2O2 is used for the routine culture while 15% H2O2 is used for detection of catalase in anaerobes.
Principle of Catalase Test
As stated above,The enzyme catalase initiates the breakdown of hydrogen peroxide into oxygen and water. Hydrogen peroxide being lethal to bacteria,production of the enzyme catalase serves as a defence mechanism and properly help identifying catalase postive bacteria from catalase negative bacteria from the production of air bubbles when the inoculum is mixed with Hydrogen Peroxide or not.
PROCEDURE
TUBE METHOD
- Pour 1-2 ml of hydrogen peroxide (H2O) solution into a a clean test tube.
- With the help of a sterile wooden stick or a glass rod, take several colonies of the 18 to 24 hours test organism and immerse in the hydrogen peroxide solution.
- Then observe for immediate bubbling or not.
Slide Method
- Transfer a small amount of colony growth in the surface of a clean, dry glass slide using a loop^or sterile wooden stick.
- Place a drop of 3% H2O2 in the glass slide.
- Observe for the evolution of oxygen bubbles.
RESULTS
OBSERVATION AND INTERPRETATION
Positive: Observe active bubbling
Negative: No or very few bubbles produced.
List of Catalase Positive bacteria
Staphylococci, Micrococci, Listeria, Corynebacterium diphtheriae, Burkholderia cepacia, Nocardia, the family Enterobacteriaceae (Citrobacter, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella, Serratia),Pseudomonas, Mycobacterium tuberculosis, Aspergillus, Cryptococcus, and Rhodococcus equi.
List of Catalase Negative bacteria :
Streptococcus and Enterococcus spp
NOTE
Always remember to do both Positive and Negative control for better and reliable results
For positive control,you can use Staphylococcus aureus– ATCC 33592
Negative control Enterococcus faecalis– ATCC 29212
Precautions of Catalase Test to avoid false positive Results
- Hydrogen peroxide must be fresh as it is very unstable.
- Don't use Iron wire loop for this test.
- Some bacteria produce a peroxidase that catalyzes a breakdown of hydrogen peroxide causing the reaction to be weakly positive; (a few bubbles elaborated slowly). This should not be confused with a truly positive reaction.
- do not add organism to reagent but reagent to organism, particularly if iron-containing inoculating loops are used. Iron containing loops will cause false positive test results if exposed to hydrogen peroxide.
- The test organisms should not be taken from blood agar culture. Red Blood cells contain catalase and their presence will give a false positive test.Culture should be 18 to 24 hours old.
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