Catalase Test: Purpose, Principle, Procedure, Results & Interpretation
Rapid biochemical test for differentiating catalase-positive organisms (Staphylococci, Micrococci) from catalase-negative species (Streptococci, Enterococci). Essential in clinical microbiology for preliminary identification.
🧪 Theory
Catalase decomposes H₂O₂ → H₂O + O₂. Immediate bubbling = positive.
📋 Purpose
Differentiate Staphylococcus (+) from Streptococcus (-). Speciation of anaerobes & gram positives.
⚙️ Procedure
Slide, tube, or slant method. Use 3%–15% H₂O₂. Read immediately.
📊 QC
Positive: S. aureus; Negative: S. pneumoniae
Purpose of Article
This test detects the catalase enzyme present in most cytochrome-containing aerobic and facultative anaerobic bacteria. Streptococcus and Enterococcus species are exceptions. Yeast such as Cryptococcus neoformans is catalase positive and can be presumptively identified. This UK SMI should be used in conjunction with other UK SMIs.
Information History
In 1893, Gottstein brought attention to bacterial catalase. ~30 years later, McLeod and Gordon published the first classification scheme based on catalase. Initial methods were cumbersome; techniques by Gagnon and Thomas were later streamlined.
Purpose
The catalase test facilitates detection of the enzyme catalase in bacteria. Essential for differentiating catalase-positive Micrococcaceae from catalase-negative Streptococcaceae. Also valuable in speciation of certain gram positives and anaerobes.
Uses: Differentiate Staphylococcus (+) from Streptococcus (-); Bacillus (+) from Clostridium (-); aids in Enterobacteriaceae differentiation.
Introduction
The catalase test detects the presence of catalase enzyme by decomposition of hydrogen peroxide to release oxygen and water: 2 H₂O₂ → 2H₂O + O₂. The reaction is evident by rapid formation of bubbles. Hydrogen peroxide is formed by some bacteria as an oxidative end product; catalase decomposes it.
Theory
Catalase expedites the breakdown of hydrogen peroxide (H₂O₂) into water and oxygen: 2H₂O₂ + Catalase → 2H₂O + O₂. This reaction is evident by the rapid formation of bubbles.
RECIPE
For aerobes: 3% H₂O₂ (store refrigerated in dark bottle).
For anaerobes: 15% H₂O₂ solution.
Superoxol (Neisseria): Refer to additional recommendations.
Principle of Catalase Test
The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. Presence is evident when a small inoculum is introduced into hydrogen peroxide, and rapid elaboration of oxygen bubbles occurs. Lack of catalase is evident by lack of or weak bubble production. Culture should be 18–24 hours old.
Bacteria thereby protect themselves from the lethal effect of hydrogen peroxide accumulated as an end product of aerobic carbohydrate metabolism.
Objectives of Catalase Test
- To detect the ability of organisms to produce the catalase enzyme.
- To differentiate catalase-positive organisms like micrococci and staphylococci from catalase-negative organisms like streptococci.
Uses of Catalase Test
- Differentiate catalase-positive Micrococcaceae/Staphylococcaceae from catalase-negative Streptococcaceae.
- Differentiate aerobic and obligate anaerobic organisms.
- Presumptive differentiation among Enterobacteriaceae.
- Differentiate aerotolerant Clostridium (negative) from Bacillus (positive).
- Speciation of certain gram-positive organisms like Aerococcus.
Reagents and Equipment
Hydrogen peroxide reagent: 30% for Neisseria, 15% for anaerobes, 3% for other bacteria.
Supplies: Glass slide, sterile wooden/glass sticks, platinum loops/wires, test tubes, bijoux bottles.
Media: Discrete bacterial/yeast colonies on solid medium (avoid blood agar).
PROCEDURE – Catalase Test Methods
Sources: ASM, UK SMI, Standard Laboratory Protocols
Slide (Drop) Method
- Place a clean glass slide inside a petri dish (optional but recommended to contain aerosols).
- Using a sterile wooden applicator stick or platinum loop, collect a small amount of organism from a well-isolated 18–24 hour colony.
- Place the organism onto the slide. Avoid picking up agar (especially from blood agar – RBCs contain catalase and may cause false positives).
- Add 1 drop of 3% H₂O₂ directly over the organism.
- Observe immediately for bubble formation against a dark background.
- Positive: Immediate effervescence (bubbles). Negative: No bubbles or very weak reaction.
Tube (Bottle) Method
- Add 4–5 drops (or 1–2 ml) of 3% H₂O₂ into a clean test tube or bijoux bottle.
- Using a sterile wooden applicator stick, collect a small amount of organism from an 18–24 hour colony.
- Place the organism into the tube, or rub the colony on the inside wall just above the liquid surface, then tilt to allow H₂O₂ to cover the colony.
- Observe immediately for bubble formation against a dark background.
- Positive: Immediate effervescence (bubbles). Negative: No bubbles.
Tube (Slant / Agar Slant) Method
- Add 1.0 ml of 3% H₂O₂ directly onto an 18–24 hour heavily inoculated pure culture grown on a nutrient agar slant.
- Replace the cap (if applicable) and observe immediately for bubble formation.
- Positive: Immediate effervescence (bubbles). Negative: No bubbles.
Special Variations
- For anaerobes: Use 15% H₂O₂ solution. Expose cultures to air for 30 minutes prior to testing.
- Superoxol test (Neisseria): Use 30% H₂O₂. N. gonorrhoeae produces immediate vigorous bubbling.
- Semiquantitative / heat-stable catalase: Used for Mycobacterium speciation (different protocol – not covered here).
15. Algorithm: Catalase Test
Quick Reference – Step Diagram
18–24h pure colony
3% (or 15% for anaerobes)
Immediate bubbling?
Catalase present
Catalase absent
Result Interpretation and Examples
| Reaction | Observation | Examples |
|---|---|---|
| Positive | Immediate copious bubbles | Staphylococcus, Micrococcus, Bacillus, Enterobacteriaceae, Pseudomonas |
| Negative | No or very few bubbles | Streptococcus, Enterococcus, Clostridium (aerotolerant) |
Reporting result: Catalase test separates staphylococci (+) from streptococci/enterococci (-). Bacillus (+) vs Clostridium (-).
Quality Control
Positive control: Staphylococcus aureus ATCC 33592 / NCTC 6571
Negative control: Enterococcus faecalis ATCC 29212 / Streptococcus mitis NCTC 10712
Fungi: Cryptococcus neoformans NCPF 3168 (+), Candida albicans NCPF 3281 (-)
Hydrogen peroxide is unstable; quality control check daily or prior to use.
Technical information/limitations
5.1 Interpretation of results
Media containing whole RBCs will contain catalase and give false positive. Enzyme present only in viable cultures; use 18–24h cultures.
5.2 False reactions
Weak catalase/pseudocatalase may be produced by some Aerococcus and Enterococcus strains. Anaerobes should be exposed to air for 30 min prior to testing.
5.3 Quality control
Hydrogen peroxide is unstable and must be refrigerated. Avoid light exposure.
Precautions of Catalase Test
- Do not take organisms from blood agar (RBCs contain catalase).
- Culture should be 18–24 hours old.
- Hydrogen peroxide must be fresh.
- Iron wire loop should not be used (false positive).
- Some bacteria produce peroxidase (weak positive).
- Do not add organism to reagent if using iron-containing loops.
Limitations of Catalase Test
- RBCs contain catalase; avoid blood agar.
- Do not test from Mueller-Hinton agar.
- Metal loops (except platinum) may yield false positives.
- Older cultures (>24h) may give false negatives.
- Reversing order of reagent may cause false negatives.
- Some S. aureus may appear negative by drop method; repeat with tube method.
- 30% H₂O₂ is caustic; if contact occurs, wash with 70% ethyl alcohol, not water.
SAFETY
Catalase testing can be hazardous due to bacteria-laden aerosols. Work must be performed in a microbiological safety cabinet. Hydrogen peroxide is highly corrosive; appropriate PPE must be worn. Follow local COSHH and risk assessments.
COMMENTS AND TIPS
Always use strict aseptic techniques. Perform under biosafety hood if possible. Do not use this procedure for Mycobacteria testing. Superoxol test (Neisseria gonorrhoeae) produces immediate vigorous bubbling.
🤖 Catalase Test Interpretation Tool
Select the observed reaction to quickly interpret your Catalase Test result.
❓Fequently Asked Questions (FAQs)
A rapid biochemical test that detects catalase enzyme by observing oxygen bubbles.
Fresh 3% hydrogen peroxide (or 15% for anaerobes).
Immediate bubbles indicate catalase production (e.g. Staphylococcus).
No bubbles indicate absence of catalase (e.g. Streptococcus).
Yes, RBCs contain catalase and may cause false positives.
Use 18–24 hour cultures for accurate results.
Biochemical Tests MCQs with Answers
References and Sources
- Biochemical Tests for the Identification of Aerobic Bacteria (2016). Clinical Microbiology Procedures Handbook, 3.17.1.1–3.17.48.3. DOI:10.1128/9781555818814.ch3.17.1 ASM Clinical Microbiology Procedures Handbook – (accessed on July 01, 2026)
- American Society for Microbiology. Catalase Test Protocol (ASM PDF). ASM.org Catalase Test Protocol – (accessed on July 01, 2026)
- MicrobiologyInfo. Catalase Test: Principle, Uses, Procedure and Interpretation. MicrobiologyInfo.com – (accessed on July 01, 2026)
- International Journal of Scientific Research (IJSR). Catalase Test Study Article. IJSR.net – (accessed on July 01, 2026)
- Dalynn Biologicals. Catalase Test Reagent Technical Sheet. Dalynn.com – (accessed on July 01, 2026)
- Clinical Science Info. Catalase Test Overview. ClinicalSci.info – (accessed on July 01, 2026)
- Diagnostics Game (Weebly). Case Study: Catalase Test. DiagnosticsGame Weebly – (accessed on July 01, 2026)
- NIOS (National Institute of Open Schooling). Microbiology Lesson 11 PDF. NIOS.ac.in – (accessed on July 01, 2026)
- Europe PMC. Catalase-related microbiology research article. EuropePMC.org – (accessed on July 01, 2026)
- Centers for Disease Control and Prevention (CDC). Request Streptococcal Testing CDC.gov – (accessed on July 01, 2026)
- Quizlet. Catalase Test Flashcards and Review Notes. Quizlet.com – (accessed on July 01, 2026)
- MicrobeNotes. Catalase Test- Principle, Procedure, Types, Results, Uses. Microbe Notes – (accessed on July 01, 2026)
- MicrobeOnline. Catalase test: Principle, Procedure, Results, Uses Microbe Online – (accessed on July 01, 2026)
- Hardy Diagnostics. Catalase Reagent Technical Information. HardyDiagnostics.com – (accessed on July 01, 2026)
- Iowa State University. Catalase Test Video Demonstration. Iowa State University Microbiology Video – (accessed on July 01, 2026)
- Michigan State University. Catalase Test Virtual Lab Simulation. MSU Virtual Lab – (accessed on July 01, 2026)
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