Microbiology Lab Tests: Hemolysis, Fermentation, Catalase & Coagulase

Course & Session Overview

• Course: BIOL-107 (Microbiology Lab)
• Week: 8
• Experiments scheduled for today:
  • Sheep Blood Agar (SBA) Hemolysis
  • Phenol Red Fermentation (PRF) Broths (three sugars)
  • Coagulase Test
  • Catalase Test (performed last)
• Coming up next week:
  • Pre-Lab 9 (online submission)
  • Lab Quiz 6

Hemolysis on Sheep Blood Agar (SBA)

• Purpose: Detect production of exotoxins (hemolysins) that lyse red blood cells (RBCs).
• Key reactions:
  • Alpha (α) hemolysis
  • Partial RBC destruction
  • Greenish or olive coloration around colonies
  • Beta (β) hemolysis
  • Complete cell lysis
  • Clear, transparent zone surrounding growth
  • Gamma (γ) hemolysis
  • No hemolysis
  • Agar remains red and opaque
• Representative organisms tested:
  • Staphylococcus aureus (SA)
  • Staphylococcus epidermidis (SE)
  • Pseudomonas aeruginosa (PA)
• Interpretive significance: Degree of hemolysis aids in differentiating closely related species and assessing virulence (e.g.
  β-hemolytic pathogens often more aggressive).

Phenol Red Fermentation (PRF) Broths

• Purpose: Differentiate organisms based on ability to ferment specific carbohydrates and to detect accompanying gas production.
• Carbohydrates tested today:
  • Glucose
  • Sucrose
  • Lactose
• Indicators & read-outs:
  • Phenol Red pH indicator
  • Yellow ⇒ acidic end-products (fermentation)
  • Fuchsia / hot pink ⇒ alkaline end-products (peptone utilization)
  • Durham tube inside each broth traps any evolved gas bubbles.
• Tube allocation: Three tubes per organism (one tube per sugar).
• Representative organisms:
  • Escherichia coli (EC)
  • Pseudomonas aeruginosa (PA)
  • Staphylococcus aureus (SA)
• Decision logic:
  • Acid only → yellow, no gas in Durham
  • Acid + gas → yellow + visible bubble
  • Alkaline → fuchsia, no gas
  • No change → original red

Coagulase Test

• Purpose: Detect the enzyme coagulase, which converts fibrinogen → fibrin, forming a protective clot that helps bacteria evade phagocytosis.
• Read-outs:
  • Positive = visible agglutination / clot formation
  • Negative = suspension remains fluid, no clumps
• Representative organisms:
  • Staphylococcus aureus (coagulase-positive)
  • Staphylococcus epidermidis (coagulase-negative)
• Clinical relevance: Differentiates pathogenic S. aureus from less-virulent coagulase-negative staphylococci (CoNS).

Catalase Test

• Purpose: Detect ability to detoxify reactive oxygen species by producing catalase.
• Reaction: \mathrm{2H2O2 \xrightarrow{catalase} 2H2O + O2 \uparrow}
• Procedure: Add a drop of \mathrm{H2O2} to a fresh colony on a slide or plate.
• Read-outs:
  • Positive = immediate, vigorous bubbling (release of \mathrm{O_2})
  • Negative = no bubbles
• Timing note: Must be performed last to avoid aerosolizing organisms before other tests are complete.
• Organisms: All strains in today’s lab are subjected to this assay.

Organism–Test Matrix (Abbreviations)

• SA = Staphylococcus aureus
• SE = Staphylococcus epidermidis
• PA = Pseudomonas aeruginosa
• EC = Escherichia coli

Legend: A = Acid (yellow), G = Gas, – = No change

Administrative & Practical Reminders

• Submit Pre-Lab 9 online before next session.
• Lab Quiz 6 will cover today’s methods, expected results, and interpretation guidelines.
• Always observe proper biosafety: perform catalase last to minimize risk, and dispose of blood-containing media in biohazard waste.