Catalase Test: Procedure, Significance, and Bacterial Identification Made Easy

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Introduction

The Catalase Test is a rapid and efficient diagnostic tool for identifying bacterial species. By detecting the presence of the enzyme catalase, this test distinguishes catalase-positive bacteria, like Staphylococcus, from catalase-negative organisms, such as Streptococcus. Widely used in clinical microbiology, it provides valuable insights for bacterial identification.

Catalase is an enzyme that catalyzes the hydrogen peroxide (H2O2) decompose into water and oxygen. Since some bacteria release catalase and some don’t, the test is used in microbiological laboratories to characterizeand identify between bacteria.

Principle

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.

Required reagents and consumables

A. Hydrogen peroxide reagent

  1. 30% For Neisseria
    Caution is needed since 30% H2O2 is very corrosive to the skin. If you get into touch with it, thoroughly rinse with 70% ethyl alcohol instead than water.
  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.

B. Consumable

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

Catalase Test Procedure: A Step-by-Step Guide

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

Interpretation of Catalse Test results

  1. A positive test results in the appearance of bubbles instantly.
  2. Few bubbles appear in a weak reaction.
  3. After 20 seconds, a negative test indicates no or just a few bubbles.
Catalase Test Resultx
Catalase test

Catalase Test: Practical Considerations and Common Errors

  • 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.
  • Do not test from Mueller-Hinton agar.
  • False-positive results can be obtained by selecting colonies with certain metal bacteriological loop materials; platinum loops do not.
  • 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.
  • If you apply the reagent to the colony in the wrong order, you’ll get false-negative results.
  • 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.
  • 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.

References:

  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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