Catalase is an important enzyme that breaks down reactive oxygen species (ROS) such as hydrogen peroxide, protecting cells from oxidative damage. The catalase test detects this enzymatic activity by observing bubble formation when bacterial cultures or colonies are mixed with hydrogen peroxide. A positive result indicates catalase production, allowing distinction between bacterial species - for example, Staphylococcus is catalase-positive while Streptococcus is negative. The procedure involves placing a small amount of bacterial growth on a slide and mixing with hydrogen peroxide, with bubble formation signifying a positive catalase test result.

1. Catalase test

2. Background
• Enzyme name catalase
important enzyme in protecting the cell from oxidative
damage by reactive oxygen species (ROS).
• Substrate name hydrogen peroxide
• Enzyme action breakdown of toxic H2O2
producing oxygen gas and water
catalase
• 2H2O2 2H2O + O2
• Hydrogen peroxide produce due to the aerobic
respiration of the cells and have to be breakdown to
prevent it’s toxic action on DNA and cell membrane

3. Background
• When hydrogen peroxide is added to a colony of catalase-
producing bacteria, it is broken down and the oxygen that is
produced can be seen as bubbles .
• By catalase test we can distinguish between:
– G (+ve) cocci : Staphylococcus positive where
Streptococcus is catalase negative
– G (+ve) bacilli : Bacillus is catalase positive
whereas Clostridium is catalase negative
– All enterobactrieacae (a gram negative bacilli) are
catalse positive
– Lesteria monocytogenes ( a gram positive bacilli)
are catalase positive

4. PROCEDURE
a. Place a small amount of growth from culture
onto a clean microscope slide.
If using colonies from a blood agar plate, be very
careful not to scrape up any of the blood agar— blood
cells are catalase positive and any contaminating agar
could give a false positive.
b. Add a few drops of H2O2 onto the smear. If
needed, mix with a toothpick. DO NOT use a
metal loop or needle with H2O2; it will give a
false positive and degrade the metal.

5. Interpretation
c. +ve result rapid evolution of O2 as
evidenced by bubbling.
d. –ve result no bubbles or only a few
scattered bubbles.

6. To Know
• Catalase is usually located in a cellular, bipolar
environment organelle called the peroxisome
• Louis Jacques Thénard, who discovered
H2O2 (hydrogen peroxide)
• Oscar Loew was the first to give it the name catalase
• Human catalase works at an optimum temperature
of 45 °C
• pH between 6.8 and 7.5
• one catalase molecule can convert approximately 5
million molecules[2] of hydrogen peroxide to water
and oxygen each second