🧪 Station 4 — Performance and Interpretation of Bacterial Biochemical Tests
🎯 Task
You are provided with a bacterial culture and the materials required for one biochemical test. Perform the test using the appropriate technique, observe the reaction, and report the result as positive or negative.
⚡ Model Station Answer What to say in the exam
Catalase: I add hydrogen peroxide to the test colony; immediate bubbling indicates a positive reaction.
Coagulase: I test the organism with plasma; visible clumping on the slide or clot formation in the tube indicates a positive reaction.
Oxidase: I apply a fresh colony to oxidase reagent; prompt development of a dark purple colour indicates a positive reaction.
I compare with the appropriate control where provided and report the demonstrated test as positive or negative.
🔎 Stepwise Procedure / Approach
- Prepare safely. Perform hand hygiene, wear appropriate PPE, confirm the labelled culture and reagents, and work using aseptic technique.
- Select a suitable colony. Use a fresh, well-isolated colony and avoid unnecessarily transferring culture medium with the organism.
- Catalase test: Place the test colony on a clean slide or other surface supplied at the station and add a drop of 3% hydrogen peroxide.
- Read catalase immediately. Rapid production of oxygen bubbles = catalase positive; absence of bubbling = catalase negative. Avoid contaminating the test with blood-containing agar when possible because this may interfere with interpretation.
- Coagulase test: For the slide method, prepare a smooth bacterial suspension in saline, retain a saline suspension as the auto-agglutination control, and mix plasma with the test suspension. Visible clumping indicates a positive slide coagulase reaction.
- If the tube method is supplied: inoculate the organism into the provided plasma and incubate according to the station protocol. Formation of a stable plasma clot indicates a positive tube coagulase test. Auto-agglutination in the slide control makes the slide result unreliable.
- Oxidase test: Transfer a fresh colony using a suitable wooden or plastic applicator to oxidase reagent paper/disc or the test system provided. Avoid an inappropriate reactive metal wire for this step.
- Read oxidase promptly. Development of a dark blue-purple colour within the reagent’s specified reading interval—commonly within seconds and generally no later than about 30 seconds—indicates an oxidase-positive reaction. A delayed colour change should not be interpreted as positive.
- Complete the station. State the test performed and report positive or negative clearly, then dispose of contaminated material safely and perform hand hygiene.
🖼️ Visual Learning
Catalase, Coagulase & Oxidase — Technique and Result Recognition
🎥 Practical Video
Watch the practical performance and visual interpretation of the three biochemical tests, with emphasis on the reactions commonly demonstrated in an MBBS microbiology practical station.
📝 Important Viva Questions
Q1. What is the principle of the catalase test?
Answer: Catalase breaks hydrogen peroxide into water and oxygen. The liberated oxygen produces visible bubbles in a positive test.
Q2. Which important organisms are commonly differentiated by the catalase test?
Answer: It is commonly used to differentiate Staphylococcus species, which are catalase positive, from Streptococcus and Enterococcus, which are catalase negative.
Q3. What does the coagulase test detect?
Answer: It detects coagulase activity. A positive reaction causes bacterial clumping in the slide method or plasma clot formation in the tube method.
Q4. Which clinically important Staphylococcus is classically coagulase positive?
Answer: Staphylococcus aureus is classically coagulase positive.
Q5. Why is a control suspension used during the slide coagulase test?
Answer: It identifies spontaneous auto-agglutination. If the control itself clumps, the slide test cannot be interpreted reliably.
Q6. What is the principle of the oxidase test?
Answer: It detects bacterial cytochrome c oxidase. Oxidation of the reagent produces a rapid dark blue-purple colour in a positive reaction.
Q7. Why must the oxidase test be read promptly?
Answer: The reagent can undergo oxidation on standing, so a delayed colour change may give a false-positive interpretation. Read the result within the specified test interval.