WEBCLS Procedure Manual
CATALASE TEST
AUTHOR: WEBCLS
ADOPTED: April 11, 2001
PURPOSE:
The catalase reaction is used in the identification of gram positive cocci (differentiate between Streptococcus and Staphylococcus species) and some gram positive bacilli.
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.
SPECIMEN:
Organisms used for testing must be isolated and not contain mixed flora.
REAGENTS / MATERIALS:
3% Hydrogen Peroxide
Inoculating loop and/or wooden applicator sticks
Clean glass slides

Reagent Storage:
  • Store in a tightly sealed amber or brown colored bottle and store at 2o-8oC or room temperature. DO NOT FREEZE OR OVERHEAT.
  • The expiration date is shown on the bottle. DO NOT use beyond the expiration date.
  • Do not use the product if there is evidence of dehydration, deterioration, contamination, or color change.
QUALITY CONTROL:
Frequency/Use:
The activity of the hydrogen peroxide must be confirmed each day of use.

Acceptable Limits / Corrective Action:
Staphylococcus aureus-ATCC 33592 as a Positive Control
Enterococcus faecalis-ATCC 29212 as a Negative Control.

If unexpected QC results are observed:
  1. Review test method for procedural errors. Confirm that all reagents tested are within the acceptable usage date. Repeat testing.
  2. If repeat testing does not produce acceptable results, report discrepancy to the Microbiology Supervisor or designee.
  3. Replace unacceptable reagent with a new vial and/or lot number. Repeat testing.
DO NOT REPORT PATIENT TEST RESULTS UNTIL ALL QC DISCREPANCIES ARE RESOLVED.

Recording:
Record QC in Daily QC Log.

PROCEDURE:
  1. Using an inoculating loop or a wooden applicator stick, transfer a small amount of the colony to be tested (pure culture colonies), from the agar to the surface of a clean, dry glass slide.
  2. Immediately place 1 drop of 3% hydrogen peroxide on to the organism on the slide.
  3. Observe the slide for immediate formation of bubbles indicating oxygen production.

REPORTING RESULTS:
Positive test: Rapid and sustained production of gas bubbles; indicating the presence of catalase.
Weakly Positive test: Few and somewhat sustained production of gas bubbles, indicating the presence of catalase.
Negative test: No bubbling indicates no catalase present.

LIMITATIONS:
Interferences:
  • Colonies from 24-hour cultures are recommended as older colonies may show false negative results.
  • Colonies from blood containing agar may show a weakly positive result (a few bubbles elaborated slowly), due to the catalase enzyme found in red blood cells. This reaction should not be confused with a truly positive catalase reaction; (copious bubbles elaborated quickly).
  • Some bacteria produce a peroxidase that catalyzes a breakdown of hydrogen peroxide causing the reaction to be weakly positive; (a few bubbles elaborated slowly). This should not be confused with a truly positive reaction.
  • Do not add organism to reagent, particularly if iron-containing inoculating loops are used. Iron containing loops will cause false positive test results if exposed to hydrogen peroxide.
  • Do not drop peroxide directly onto the blood agar plates since erythrocytes possess catalase activity and can produce false positive results.
REFERENCES:
  • Bailey and Scott. Diagnostic Microbiology, 9th ed. St. Louis, Mo., Mosby. 1994.
  • Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I. Washington DC. ASM Press. 1992.
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