Basic Microbiology Practical No. 6

Basic Microbiology - Practical No. 6

• Intended Learning Objectives (ILOs):
  • Students will be introduced to selective and differential media and learn to define both types.
  • Students will differentiate between lactose and non-lactose fermenting bacteria on MacConkey agar.
  • Students will understand the significance of catalase and coagulase tests, and their principles.
  • Students will perform and interpret catalase and coagulase tests.
  • Students will understand the importance of Oxidase and Urease tests, and their principles.
  • Students will perform and interpret Oxidase and Urease tests.
  • Students will be introduced to the API-20E test and learn to interpret its results.

Precautions

• Wear appropriate personal protective equipment (PPE) for the lab procedure.
• All specimens should be treated as infectious.
• Follow all standard operating procedures (SOPs) designed for each test.
• Record results immediately.

Catalase

• Catalase is an enzyme that breaks down hydrogen peroxide (H₂O₂) into oxygen (O₂) and water (H₂O).
• Most aerobic and facultative anaerobic bacteria have catalase activity.
• This test helps identify unknown bacteria.
• Do not use metal instruments with H₂O₂ or take colonies from blood agar, as this may give false positive results due to metal degradation.

Coagulase

• Staphylococcus aureus produces coagulase, which clots plasma using two mechanisms (free and bound).
• This enzyme is useful in differentiating S. aureus from other coagulase-negative staphylococci.

Oxidase

• This test identifies organisms that produce cytochrome c oxidase.
• A chromogenic reducing agent in the reagent changes colour when oxidized.
• If the organism produces cytochrome oxidase, the reagent turns blue or purple within 15 seconds.

Urease

• Urea agar (developed by Christensen in 1946) is used to differentiate enteric bacilli.
• The urease test determines the ability of an organism to split urea via the production of the enzyme urease.
• Hydrolysis of urea produces ammonia and CO₂.
• Formation of ammonia leads to alkalinization, which is detected by phenol red colour change.
• Weakly positive organisms may take several days to produce a colour change from light orange to magenta.

API 20E Test

• API 20E is a multi-test system useful for identifying bacteria (e.g. enteric bacteria, non-fastidious Gram-negative rods).
• It employs 20 miniaturized biochemical tests.
• Time, cost, and space are key factors in clinical laboratory usage of this system.

Catalase Test (Procedure)

• Using a clean microscope slide, add a drop of H₂O₂.
• Use a loop to pick up bacteria from a solid medium and place it in the drop.
• Observation: Look for rapid gas bubble production (positive) or no gas bubbles (negative).

Coagulase Test (Procedure)

• Use a slide and inoculate it.
• Place one drop of citrated mammalian plasma on the slide and mix.
• Observe immediate clumping; this indicates a positive result.
• Alternatively, use a tube method for this test to distinguish between free and bound coagulase.

Oxidase Test (Procedure)

• Use sterile filter paper and add oxides reagent.
• Then add bacterial colonies to the filter paper
• Observe for a colour change to purple within 10-30 seconds.

Urease Test (Procedure)

• Inoculate a urea slant with the test organism.
• Incubate at the appropriate temperature for 24-72 hrs and observe for colour change to pink/red.