by K Reiner · Cited by 95 — Enzyme-based tests play a crucial part in the identification of bacteria. In. 1893, a publication by Gottstein brought attention to bacterial catalase
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American Society for Microbiology © 2016 1 Catalase Test Protocol | | Created: Thursday, 11 November 2010 Author Karen Reiner Information History In order to survive, organisms must rely on defense mechanisms that allow them to repair or escape the oxidative damage of hydrogen peroxide (H 2O2). Some bacteria produce the enzyme catalase which facilitates cellular detoxification. Catalase neutralizes the bactericidal effects of hydrogen peroxide (13) and its concentrat ion in bacteria has been correlated with pathogenicity (8). Enzyme -based tests play a crucial part in the identification of bacteria. In 1893, a publication by Gottstein brought attention to bacterial catalase, making it one of the first bacterial enzymes to be described (6, 9). Some 30 years later, McLeod and Gordon (9) developed and published what is thought to be the first bacterial classification scheme based on catalase production and reactions (6). Initial methods of catalase detection were cumbersom e, labor -intensive, time -consuming, and required specialized equipment (6). Over the years, the techniques first described by Gagnon et al. (6) and particularly those of Thomas (12) have been modified and streamlined, thus greatly simplifying the performan ce of this test. Purpose The catalase test facilitates the detection of the enzyme catalase in bacteria. It is essential for differentiating catalase -positive Micrococcaceae from catalase -negative Streptococcaceae . While it is primarily useful in differe ntiating between genera, it is also valuable in speciation of certain gram positives such as Aerococcus urinae (positive) from Aerococcus viridians (negative) and gram -negative organisms such as Campylobacter fetus , Campylobacter jejuni , and Campylobacter coli (all positive) from other Campylobacter species (7, 8). Some have reported its value in the presumptive differentiation among certain Enterobacteriaceae (11). The catalase test is also valuable in differentiating aerobic and obligate anaerobic bacteri a, as anaerobes are generally known to lack the enzyme (8, 9). In this context, the catalase test is valuable in differentiating aerotolerant strains of Clostridium , which are catalase negative, from Bacillus , which are catalase positive (8). Theory The c atalase enzyme serves to neutralize the bactericidal effects of hydrogen peroxide (13). Catalase expedites the breakdown of hydrogen
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American Society for Microbiology © 2016 2 peroxide (H 2O2) into water and oxygen (2H 2O2 + Catalase 2H 2O + O 2). This reaction is evident by the rapid formation of bubbles (2, 7). RECIPE For routine testing of aerobes, use commercially available 3% hydrogen peroxide (2, 7). Store the hydrogen peroxide refrigerated in a dark bottle. For the identification of anaerobic bacteria, a 15% H 2O2 solution is necessary (1). In this context, the catalase test is used to differentiate aerotolerant strains of Clostridium , which are catalase negative, from Bacillu s species, which are positive (8). The superoxol catalase test used for the presumptive speciation of certain Neisse ria organisms requires a different concentration of H 2O2. Refer to the fiAdditional Recommendationsfl section for details. PROTOCOL There are many applications and method variations of the catalase test. These include the slide or drop catalase test, the t ube method, the semiquantitative catalase for the identification of Mycobacterium tuberculosis, the heat -stable catalase used for the differentiation of Mycobacterium species, and the capillary tube and cover slip method (7). One of the most popular methods in clinical bacteriology is the slide or drop catalase method, because it requires a small amount of organism and relies on a relatively uncomplicated technique. This protocol delineates the procedure for the qualitative slide and tube catalase met hods, which are primarily used for the differentiation of staphylococci and streptococci. Slide (drop) method Place a microscope slide inside a petri dish. Keep the petri dish cover available. The use of a petri dish is optional as the slide catalase can be properly performed without it. However, to limit catalase aerosols, which have been shown to carry viable bacterial cells (4), the use of a petri dish is strongly recommended. Using a sterile inoculating loop or wooden applicator stick, collect a small amount of organism from a well -isolated 18- to 24 -hour colony and place it onto the microscope slide. Be careful not to pick up any agar. This is particularly important if the colony isolate was grown on agar containing red blood cells. Carryover of red b lood cells into the test may result in a false -positive reaction (5, 7). Using a dropper or Pasteur pipette, place 1 drop of 3% H 2O2 onto the organism on the microscope slide. Do not mix. Immediately cover the petri dish with a lid to limit aerosols and ob serve for immediate bubble formation (O2 + water = bubbles). Observing for the formation of bubbles against a dark background enhances readability. Positive reactions are evident by immediate effervescence (bubble formation) (Fig. 1). Place microscope sli de over a dark background and use a magnifying glass or microscope to observe weak positive reactions.
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American Society for Microbiology © 2016 3 If using a microscope, place a cover slip over the slide and view under 40x magnification. No bubble formation (no catalase enzyme to hydrolyze the hydro gen peroxide) represents a catalase -negative reaction (Fig. 1). Quality control is performed by using organisms known to be positive and negative for catalase. Note: If a platinum inoculating loop is used, do not add 3% H 2O2 to the slide before the organ ism, as the platinum wire in the loop may produce a false -positive result. This is not the case with nichrome wire. FIG. 1. Slide catalase test results. (Top) The positive reaction was produced by Staphylococcus aureus ; (bottom) the negative reaction was produced by Streptococcus pyogenes . Tube method (10) Add 4 to 5 drops of 3% H 2O2 to a 12 x 75 -mm test tube (10). Using a wooden applicator stick, collect a small amount of organism from a well -isolated 18 – to 24 -hour colony and place into th e test tube. Be careful not to pick up any agar. This is particularly important if the colony isolate was grown on agar containing red blood cells. Carryover of red blood cells into the test may result in a false -positive reaction (5, 7). Place the tube ag ainst a dark background and observe for immediate bubble
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American Society for Microbiology © 2016 4 formation (O 2 + water = bubbles) at the end of the wooden applicator stick. Positive reactions are evident by immediate effervescence (bubble formation) (Fig. 2A). Use a magnifying glass or microsco pe to observe weak positive reactions. No bubble formation (no catalase enzyme to hydrolyze the hydrogen peroxide) represents a catalase -negative reaction (Fig. 2B). Quality control is performed by using organisms known to be positive and negative for catalase. A
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American Society for Microbiology © 2016 5 B FIG. 2. Tube catalase test results. (A) The positive reaction was produced by Staphylococcus aureus ; (B) the negative reaction was produced by Streptococcus pyogenes . Tube (slant) method Add 1.0 ml of 3% H 2O2 directly onto an 18 – to 24 -hour heavily inoculated pure culture grown on a nutrient agar slant (7). Place the tube against a dark background and observe for immediate bubble formation. Positive reactions are evident by immediate effervescence (bubble formation) (Fig. 3A). N o bubble formation (no catalase enzyme to hydrolyze the hydrogen peroxide) represents a catalase -negative reaction (Fig. 3B).
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American Society for Microbiology © 2016 8 Bartlett Publishers, Inc., Sudbury, MA. REVIEWERS This resource was peer -reviewed at the ASM Conference for Undergraduate Educators 2010. Participating reviewers: Victoria Akpata Kuwait University, Safat, Kuwait Ned Barden Massachusetts College of Pharmacy, Boston, MA Benita Brink Adams State College, Alamosa, CO Esperanza Cabrera De La Salle University, Manila, Philippines Gina Cano -Monreal Texas State Technical College, Harli ngen, TX Russell Cossaboom University of Michigan ŠFlint, Flint, MI Jonathan Davis Doña Ana Community College, Las Cruces, NM Stella Marie Doyungan Texas A&M University ŠCorpus Christi, Corpus Christi, TX Cornelius Joel Funk John Brown University, Siloam Springs, AR Robert Fultz Sam Houston State University, Huntsville, TX Hygia Guerreiro Escola Bahiana de Medicina e Saúde Pública, Salvador, Brazil Anne Hanson University of Maine, Orono, ME Jan Hudzicki University of Kansas Medical Center, Kansas Cit y, KS Parisa Jazbi Folsom Lake College, Folsom, CA
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American Society for Microbiology © 2016 9 D. Sue Katz Rogers State University, Claremore, OK Archana Lal Independence Community College, Independence, KS Donald Lehman University of Delaware, Newark, DE Min -Ken Liao Furman University, Greenvil le, SC Hilda Merchant Oakland Community College, Farmington Hills, MI Amy Miller Raymond Walter College, University of Cincinnati, Mason, OH Oluwatoyin Osunsanya Muskingum University, New Concord, OH Gary Patterson College of the Marshall Islands, Majuro, MH Todd Primm Sam Houston State University, Huntsville, TX Johana Melendez Santiago Hillsborough Community College, Plant City, FL Patricia Shields University of Maryland, College Park, MD Sheridan Shupe Delaware Technical & Community College, Georgetown, DE Ann Stewart -Akers South University, Columbia, SC Erica Suchman Colorado State University, Ft. Collins, CO Dan Trubovitz Miramar College, San Diego, CA Ann Vernon St. Charles Community College, Cottleville, MO Cuc Kim Vu St. Catherine Un iversity, Minneapolis, MN
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