Catalase Test: Introduction, Principle, Procedure, Result Interpretation, and Precautions

Introduction of Catalase Test

The catalase test is the most common in bacteriology and is commonly used to differentiate those bacteria that produce this enzyme, such as staphylococci, from non-catalase producing bacteria such as streptococci. It is two types depending on the testing methods and they are slide and tube methods. Here we discuss both methods separately.

The list of some catalase-positive and negative bacteria is as follows-

Catalase Positive Bacteria	Catalase Negative Bacteria
Staphylococci (Staphylococcus aureus, S. epidermis)	Streptococci (Streptocoocus pyogenes, S. agalactiae, S. pneumoniae, Enterococcus)
Micrococci (Micrococcus luteus, M. roseus)	Gardenella vaginalis
Enterobacteriaceae (E. coli, Klebsiella, Proteus, Enterobacter, Citrobacter, Morgenella, Hafnia)	Fusobacterium species
Corynebacterium diphtheriae	Shigella dysenteriae type I
	Kingella kingae
	Eikenella corrodens

Principle of Catalase Test

Catalase is an enzyme that acts as a catalyst in the breakdown of hydrogen peroxide into nascent oxygen and water. This nascent oxygen causes bubbling. An organism is assayed for catalase production by bringing it into contact with hydrogen peroxide (H₂O₂). Bubbles of oxygen are released if the test organism is a catalase producer.

2 H₂O₂ → 2 H₂O + O₂

Requirements for Catalase Test

• Test organism/ bacteria
• 3% Hydrogen peroxide  (H₂O₂)
• Sterile bamboo sticks
• Test tubes (for tube method)
• Slides (for slide method)
• Coverslip optional
• Quality control (QC) strains for negative control and positive control

Procedure of  Catalase Test for Tube Method

• Pour 2–3 ml of the hydrogen peroxide solution into a test tube.
• Taking a sterile bamboo stick or a glass rod remove numerous colonies of the test organism and immerse them in the hydrogen peroxide solution.
• Observe immediate bubbling as shown in the footage.
• Follow the same steps for positive and negative controls.

Procedure of  Catalase Test for Slide Method

• Take a clean and grease-free glass slide.
• Put a drop of 3% hydrogen peroxide on it.
• Pick up a pure isolated colony of the test organism using a coverslip and immerse it in H₂O₂.
• Observe immediately bubble as shown in the figure.
• Follow the same steps for positive and negative controls.

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 during the testing of 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 causing improper reporting.
2. A nichrome wire loop is also omitted to pick up the colonies.
3. Performing the test on a slide is not recommended since 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 on a Petri plate. The plate should then be covered immediately, and the preparation observed for bubbling through the lid.
4. The bacterial growth 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. 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.
3. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
4. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
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.