Standard Operating Procedure Subject Catalase Test Index Number Lab-3380 Section Laboratory Subsection Microbiology Category Departmental Contact Sarah Stoner Last Revised 9/18/2019 References Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers for Medicare and Medicaid Services (CMS) and/or COLA. Applicable To Employees of Gundersen Health System Laboratory, Gundersen Tri-County, Gundersen St. Joseph, Gundersen Boscobel Hospital, and Gundersen Palmer Lutheran Hospital Laboratories. Detail PRINCIPLE: The breakdown of hydrogen peroxide into oxygen and water is mediated by the enzyme catalase. When a small amount of an organism that produces catalase is introduced into hydrogen peroxide, rapid elaboration of bubbles of oxygen, the gaseous product of the enzyme’s activity, is produced. CLINICAL SIGNIFICANCE: This test is used as an aid in distinguishing between Staphylococci and Streptococci. All members of the genus Staphylococcus are catalase (+), where as members of the genus Streptococcus are catalase (-). Listeria monocytogenes {catalase (+)} can be distinguished from beta-hemolytic streptococcus {catalase (-)}. Most Neisseria sp. are catalase (+). Catalase can also help distinguish Bacillus sp. {catalase (+)} from Clostridum sp. {mostly catalase (-)}. SPECIMEN: Isolates preferably grown on non-blood containing media not older than 24 hours old. REAGENTS AND MATERIALS: 1. 3% hydrogen peroxide (from stock bottle). Store 2o – 25o C. Do not freeze or overheat. Light sensitive, store in brown bottle. 2. Clean microscope slide or glass test tube 3. Wooden applicator stick EQUIPMENT/INSTRUMENTATION: N/A QUALITY CONTROL: Each new lot and shipment or once a month, perform QC on reagent with stock organisms of S aureus (positive) and Beta strep group A (negative). Record results on quality control chart. If test gives unexpected results, repeat using different stock organisms or try a new bottle of 3% hydrogen peroxide. Record corrective action taken. Refer to Lab-3255 Quality Control-Microbiology Department. Page 1 of 2 Standard Operating Procedure Implementation IMPLEMENTATION: SLIDE METHOD: 1. Carefully remove colony(s) from agar with wooden applicator stick and smear them on a clean microscope slide. 2. Place a few drops of 3% hydrogen peroxide onto the smear, and immediately observe for the release of oxygen (bubbles). TUBE METHOD: 1. Place a few drops of 3% hydrogen peroxide into a glass test tube. 2. Carefully remove colony(s) from agar with wooded applicator stick and submerge into the test tube of hydrogen peroxide, and immediately observe for the release of oxygen (bubbles). PROCEDURAL NOTES: N/A CALCULATIONS: N/A LIMITATIONS: 1. DO NOT perform catalase tests on colonies older than 24 hours because the enzyme is present in viable cultures only. Older cultures may give false-negative reactions. 2. Take care not to transfer blood-containing media to the glass slide since catalase is found in red blood cells. A technologist can distinguish the very weak reaction of contaminating RBC by performing a control catalase test with a small amount of the blood containing agar. If the reaction from the colony is much stronger than that from the agar along, the test can be considered positive. 3. Some bacteria produce peroxidase that catalyzes the breakdown of hydrogen peroxide. The catalase test may thus appear to be weakly positive (a few bubbles slowly elaborated). This reaction should not be confused with a truly positive catalase test. REVIEW AND CHANGES: This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as the maximum review date. Review will be done by the Technical Leader, Medical Director or designated person. Changes require retyping document or form and review by the Medical Director. REFERENCES: 1. Bailey & Scott’s Diagnostic Microbiology, Ellen Jo Baron, PhD, Lance R. Peterson, MD, Sydney M. Finegold, MD, SM(AAM), 9th edition, Mosby-Year Book, Inc., Saint Louis, Missouri 1994 pp. 100- 101. 2. Clinical Microbiology Procedures Handbook, Henry D. Isenberg, Editor in Chief, ASM, Washington, DC, 1992 pp. 1.20.4-1.20.5. Page 2 of 2 .