Standard Operating Procedure: Catalase Test

1. Purpose and Scope

To detect the presence of catalase enzyme in bacteria, differentiating catalase-positive organisms (e.g., Staphylococcus) from catalase-negative organisms (e.g., Streptococcus, Enterococcus).

2. Specimen Requirements

• Type: Fresh bacterial culture (18-24 hours old).
• Source: Colonies from solid media (blood agar, nutrient agar).
• Important: Do NOT use colonies from blood agar if testing for catalase (blood contains catalase - causes false positive). Use nutrient agar or TSA.
• Rejection: Old cultures (>48 hours), colonies from blood-containing media.

3. Safety and Precautions

• Wear appropriate PPE (gloves, lab coat, eye protection).
• Hydrogen peroxide is corrosive and oxidizing.
• Handle cultures in biological safety cabinet.
• Dispose of materials in biohazard waste.

4. Equipment and Reagents

• Reagent: 3% hydrogen peroxide (H₂O₂) solution.
• Store in dark bottle at 2-8°C.
• Check potency regularly (should bubble when added to catalase-positive control).
• Clean glass slide or test tube.
• Wooden applicator stick or platinum/nichrome loop (NOT iron or steel - causes false positive).
• Fresh bacterial culture on non-blood agar.
• Quality Control Organisms:
• Positive: Staphylococcus aureus ATCC 25923
• Negative: Streptococcus pyogenes ATCC 19615

5. Step-by-Step Procedure

Method 1: Slide Method (Most Common)

1. Place clean glass slide on white background.
2. Using wooden stick or platinum loop, transfer small amount of bacterial colony to slide (do not use iron/steel loop).
3. Add 1-2 drops of 3% H₂O₂ to bacterial smear.
4. Observe immediately for bubble formation (within 5-10 seconds).

Method 2: Tube Method (For Anaerobes or Small Colonies)

1. Add 1 mL of 3% H₂O₂ to small test tube.
2. Using wooden stick, transfer several colonies into H₂O₂.
3. Observe for bubble formation.

Method 3: Capillary Tube Method (For Microaerophiles)

1. Place colony in capillary tube.
2. Add H₂O₂.
3. Observe bubbles.

6. Interpretation

Positive Result

• Immediate, vigorous bubbling (effervescence).
• Indicates presence of catalase enzyme.
• Organisms: Staphylococcus, Micrococcus, Listeria, Bacillus, Corynebacterium, most aerobic and facultative anaerobic bacteria.

Negative Result

• No bubbles or weak/delayed bubbling.
• Indicates absence of catalase enzyme.
• Organisms: Streptococcus, Enterococcus, some anaerobes.

7. Quality Control

1. Test QC organisms with each new batch of H₂O₂:

• S. aureus: Should show vigorous bubbling (positive).
• S. pyogenes: Should show no bubbling (negative).

1. Check H₂O₂ potency weekly by testing against known positive control.
2. Replace H₂O₂ if negative result with positive control.

8. Special Considerations

False Positives

• Using iron/nichrome loop (use wooden stick or platinum).
• Testing colonies from blood agar (RBCs contain catalase).
• Old H₂O₂ that has decomposed.

False Negatives

• Old culture (>48 hours) - enzyme activity decreases.
• H₂O₂ too old/decomposed.
• Testing anaerobes (some produce little catalase).

Modified Catalase Tests

• Semiquantitative catalase: Measure bubble height.
• Heat-stable catalase: Heat organism at 68°C for 30 min, then test (differentiates Mycobacterium species).
• Aerobic vs anaerobic: Some organisms produce catalase only aerobically.

9. Reporting Results

• Report as "Catalase positive" or "Catalase negative."
• Include in identification workup with other biochemical tests.