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Introduction of Catalase Test

Catalase test is used to differentiate those bacteria that produce this enzyme, such as staphylococci, from non-catalase producing bacteria such as streptococci.

Principle of Catalase Test

Catalase acts as a catalyst in the breakdown of hydrogen peroxide into nascent oxygen and water. This nascent oxygen causes bubbling. An organism is tested for catalase production by bringing it into contact with hydrogen peroxide. Bubbles of oxygen are released if the organism is a catalase producer.

Requirements for Catalase Test

• Test organism
• 3% Hydrogen peroxide  (H₂O₂)
• sterile bamboo sticks
• Test tubes
• Slides ( for slide method)
• Quality control (QC) strains for negative control and positive Control

Procedure of  Catalase Test

1. Pour 2–3 ml of the hydrogen peroxide solution into a test tube.
2.  Using a sterile bamboo stick or a glass rod remove several colonies of the test organism and immerse them in the hydrogen peroxide solution.
3. Look for immediate bubbling as shown in figure

Result Interpretation of  Catalase Test

Active bubbling – Positive

No bubbles – Negative

 Quality Control 

Positive  control (PC): Staphylococcus aureus (ATCC25923)

Negative  control (NC): Streptococcus pyogenes (ATCC 19615)

Uninoculated (UN): No organism only 3% H₂O₂

Precautions

1. Care must be taken when testing a bacterium cultured on a medium containing blood because catalase is present in red blood cells. If any of the blood agar is removed from the organism, a false-positive reaction may occur.
2. A Nichrome wire loop is not recommended to pick up the colonies.
3. Performing the test on a slide is not recommended because of the risk of contamination from active bubbling. When the rapid slide technique is used, the hydrogen peroxide solution should be added to the bacterial suspension after placing the slide in a Petri plate. The plate should then be covered immediately, and the preparation observed for bubbling through the lid.
4. The culture should not be more than 24 hours old.

Further Readings

1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University press.
2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
3. Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
6. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.