Catalase test: principle, uses, procedure and results

Catalase Test: Positive and Negative results

Catalase is an enzyme, which is produced by microorganisms that live in oxygenated environments to neutralize toxic forms of oxygen metabolites; H2O2.  The catalase enzyme neutralizes the bactericidal effects of hydrogen peroxide and protects them. Anaerobes generally lack the catalase enzyme.

Catalase mediates the breakdown of hydrogen peroxide H2O2 into oxygen and water.  To find out if a particular bacterial isolate is able to produce catalase enzyme, small inoculum of bacterial isolate is mixed into hydrogen peroxide solution (3%) and is observed for the rapid elaboration of oxygen bubbles occurs.  The lack of catalase is evident by a lack of or weak bubble production.

Catalase-positive bacteria include strict aerobes as well as facultative anaerobes. They all have the ability to respire using oxygen as a terminal electron acceptor.

Catalase-negative bacteria may be anaerobes, or they may be facultative anaerobes that only ferment and do not respire using oxygen as a terminal electron acceptor (ie. Streptococci).

Percentage of H2O2 used on catalase test:

  1. For routine testing of aerobes, 3% hydrogen peroxide is used.
  2. 15% H2O2 solution: for the identification of anaerobic bacteria
    Catalase test is used to differentiate aerotolerant strains of Clostridium (catalase negative) from Bacillus species (catalase positive).
  3. The superoxol catalase test used for the presumptive speciation of certain Neisseria organisms requires a different concentration of H2O2.

Uses Catalase Test Results 

  1. The catalase test is primarily used to distinguish among Gram-positive cocci: members of the genus Staphylococcus are catalase-positive, and members of the genera Streptococcus and Enterococcus are catalase-negative.
  2. Catalase test is used to differentiate aerotolerant strains of Clostridium, which are catalase negative, from Bacillus species, which are positive.
  3. Semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis. 
  4. Catalase test can be used as an aid to the identification of Enterobacteriaceae. Members of Enterobacteriaceae family are catalase positive.

Procedure of Catalase test (Slide Test)

  1. Transfer a small amount of bacterial colony to a surface of clean, dry glass slide using a loop or sterile wooden stick
  2. Place a drop of 3% H2O2 on to the slide and mix.
  3. A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced by bubbling.
  4. A negative result is no bubbles or only a few scattered bubbles.
  5. Dispose of your slide in the biohazard glass disposal container.

Tube Catalase Test-Procedure and Results 

  1. Add 4 to 5 drops of 3% H2O2 (Hydrogen peroxide) to in a test tube
  2. Using a wooden applicator stick, collect a small amount of organism from a well-isolated 18- to 24-hour colony and place into the test tube (Note: Be careful not to pick up any agar (esp if using Blood Agar).- Explanation in precaution below)
  3. Place the tube against a dark background and observe for immediate bubble formation (O2 + water = bubbles) at the end of the wooden applicator stick.

    Catalase Positive and Catalase Negative Reactions


You can perform Catalase Test online here

Precautions while performing catalase test

  1. Do not use a metal loop or needle with H2O2; it will give a false positive and degrade the metal.
  2. If using colonies from a blood agar plate, be very careful not to scrape up any of the blood agar as blood cells are catalase positive and any contaminating agar (carryover of red blood cells) could give a false positive.
  3. Because some bacteria possess enzymes other than catalase that can decompose hydrogen peroxide, a few tiny bubbles forming after 20 to 30 seconds is not considered as positive test.