Catalase Test


Bacteria that produce the enzyme catalase break down hydrogen peroxide into water and gaseous oxygen, allowing gas bubbles to escape. Most bacteria can be identified using this test.


A.Hydrogen peroxide reagent

  1. 30% For Neisseria
    30 percent H2O2 is highly caustic to the skin, so use caution. If you come into contact with it, wash with 70% ethyl alcohol instead of water right away.
  2.  15% For anaerobes
  3.  3% For other bacteria (purchase or dilute 30% 1:10 in deionized water prior to use)
    NOTE: 30% Reagent can be used for all tests, but it is more hazardous.
  4. Store at 2 to 8C.

1. Glass slide
2. Sterile wooden sticks or plastic or platinum loops or wires


A. Touch the middle of a well-isolated colony that has been isolated for 18 to 24 hours to a clean glass slide.
   1. Be sure colony is visible to the naked eye on slide.
   2. If colony is from BAP, use care not to pick up blood.
B. Place 1 drop of peroxide reagent on the slide and watch for effervescence immediately.
     1. If possible, use a magnifying glass.
     2. Place your hand over a dark background to make the bubbles pop.
C. Discard slide into sharps container


A. A positive test results in the appearance of bubbles instantly.
B. Few bubbles appear in a weak reaction.
C. After 20 seconds, a negative test indicates no or just a few bubbles.

Catalase test


A.RBCs contain the catalase enzyme. Using colony from blood-agar will result in false-positive results. If picking up or expanding the colony is tough, repeat the test with CHOC, which does not interfere with the assay.
B. Do not test from Mueller-Hinton agar.
C. False-positive results can be obtained by selecting colonies with certain metal bacteriological loop materials; platinum loops do not.
D. Do not test colonies older than 24 hours because the enzyme is only present in viable cultures. It’s possible that older cultures would produce false-negative findings.
E. If you apply the reagent to the colony in the wrong order, you’ll get false-negative results.
F. This method can show that certain strains of S. aureus are catalase negative. Further testing to classify these troublesome species can be done with the aminolevulinic acid (ALA) test.
G. Reimer and Reller suggest streaking a CHOC plate as for disk susceptibility testing and inserting a dot of viridans group streptococci to confirm the lack of catalase for Gardnerella vaginalis (Streptococcus sanguis ATCC 35557). The absence of catalase is confirmed by a strong zone of inhibition around a dot of viridans group streptococci.


  1. Isenberg, H. D., & American Society for Microbiology. (1992). Clinical microbiology procedures handbook. Washington, D.C: American Society of Microbiology.
  2. Mahon, C.R., Lehman, D.C., Manuselis, G. (2014). Textbook of Diagnostic Microbiology (5th ed.). New York: Saunders.
  3. Tille, Patricia M., author. (2014). Bailey & Scott’s diagnostic microbiology. St. Louis, Missouri :Elsevier
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