CATALASE TEST PROCEDURE
CATALASE TEST
Catalase is the enzyme that breaks down hydrogen peroxide (H2O2) into H2O and O2. The oxygen is given off as bubbles in the liquid. The catalase test is primarily used to differentiate between gram-positive cocci. Members of the genus Staphylococcus are catalase-positive, and members of the genera Streptococcus and Enterococcus are catalase-negative.
Performing The Catalase Test
1. Grow the isolate(s) to be tested for 18-24 hours on a BAP at 35-37°C with ~5% CO2 (or in a candle-jar).
2. From overnight growth on the BAP, use a disposable loop to carefully remove a colony and place it on a glass slide. • Do not transfer any of the blood agar to the slide as erythrocytes in the blood agar will cause a false-positive reaction.
3. Add 1.0 ml of 3% H2O2 to the slide and mix with the bacteria.
• H2O2 can be obtained from a commercial drug store.
• After initially opening, store H2O2 at 4°C in a tightly closed bottle as it will slowly lose potency once opened.
4. Observe the bacterial suspension on the slide immediately for vigorous bubbling.
5. It is essential to use a known positive and negative quality control (QC) strain. A Staphylococcus spp. strain can be used for a positive control and a known S. pneumoniae strain or any other streptococcal spp., i.e., S. pyogenes can be used for a negative control.
Reading the catalase test results
• The absence of bubbling from a transferred colony indicates a negative test.
• Any bubbling from a transferred colony indicates a positive test.
Procedure of Catalase test (Slide Test)
- Transfer a small amount of bacterial colony to a surface of clean, dry glass slide using a loop or sterile wooden stick
- Place a drop of 3% H2O2 on to the slide and mix.
- A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced by bubbling.
- A negative result is no bubbles or only a few scattered bubbles.
- Dispose of your slide in the biohazard glass disposal container.
Tube Catalase Test
- Add 4 to 5 drops of 3% H2O2 (Hydrogen peroxide) to in a test tube
- Using a wooden applicator stick, collect a small amount of organism from a well-isolated 18- to 24-hour colony and place into the test tube (Note: Be careful not to pick up any agar (esp if using Blood Agar).- Explanation in precaution below)
- Place the tube against a dark background and observe for immediate bubble formation (O2 + water = bubbles) at the end of the wooden applicator stick.
INSTAGRAM
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