1. If the plate is refrigerated, it should be allowed to warm up to room temperature and then incubated for 15 min at 37°C before performing the test. Pick a loopful of colonies from a not-too-old pure culture plate and place on a clean glass slide. Do not take your colonies from a blood agar plate. Blood contains catalase; therefore a false positive reaction would be obtained.
2. Add one or two drops of 3% H2O2 and wait 10-15 seconds to observe.
-- Positive test: immediate bubbling (O2 formed).
-- Negative test: no bubbling.
a. When doing the slide test, always add organism to the slide first and then add the reagent since platinum used in the inoculation needle may produce a false positive result. Nichrome wire does not cause bubbling.
b. H2O2 is very unstable when exposed to light. H2O2 decomposition also increases as temperature increases due to dissolved oxygen. Therefore it is important to keep this reagent in the refrigerator at all times when not in use and to shake before it is used.