In-Depth Notes on Bacterial Media and Testing Techniques

Selective vs. Differential Media

• Selective Media:

  • Contains ingredients targeting and inhibiting certain bacteria, allowing others to grow.
  • Results: Growth vs. No Growth.

• Differential Media:

  • Aims to distinguish different types of bacteria based on metabolic and phenotypic capabilities.
  • Must include substrates for differentiation and indicators for visible changes (+/-).

• Media can be selective, differential, or both.

  • Examples: MSA (Mannitol Salt Agar) and EMB (Eosin Methylene Blue Agar) are both selective and differential.

Mannitol Salt Agar (MSA)

• Type: Selective and Differential Medium.

• Selectivity: High salt concentration inhibits non-halotolerant species.

• Differentiation: Based on fermentation of mannitol (sugar).

  • Only Staphylococcus aureus can ferment mannitol, producing acid.
  • pH indicator phenol red detects acid production (red: neutral, yellow: acidic).

• Examples of Results:

  1. Staphylococcus epidermidis: Growth, no yellow halo (non-fermenter).
  2. Escherichia coli: No growth (non-halotolerant).
  3. Staphylococcus aureus: Growth with yellow halo (fermenter).
  4. Pseudomonas aeruginosa: No growth (non-halotolerant).

Eosin Methylene Blue Agar (EMB)

• Type: Selective and Differential Medium.
• Selectivity: Dyes Eosin Y and Methylene Blue kill Gram positive bacteria.
• Differentiation: Based on lactose fermentation.
  • Lactose fermentation yields pink/purple colonies; vigorous fermenters show metallic green sheen (typically E. coli).

Phenylethyl Alcohol Agar (PEA)

• Type: Selective Medium for Gram-positive organisms.
• Function: Inhibits Gram-negative bacteria by interfering with DNA synthesis.
• Commonly for Staphylococcus and Streptococcus.

Carbohydrate Utilization Tests

• Overview: Test the ability to ferment different carbohydrates, often yielding acid and sometimes gas.
  • Methods:
  • Phenol Red Broth: Contains phenol red (pH indicator) and a carbohydrate.
    • Red indicates neutral, yellow indicates acid production.
    • Durham tube captures gas (if produced).
  • Starch Hydrolysis Plate: Tests for starch breakdown.
    • Iodine added for detection; absence of brown/black indicates positive hydrolysis.

Citrate Test

• Medium: Contains citrate as sole carbon source.
• Function: Test ability to utilize citrate; corresponds to growth or no growth.
• Indicator: Bromothymol blue changes color based on pH (green: negative, blue: positive).

Catalase Test

• Function: Assesses presence of catalase enzyme that breaks down hydrogen peroxide (H2O2).
• Positive Result: Bubbles appear when hydrogen peroxide is added.

Oxidase Test

• Purpose: Identify if bacteria possess cytochrome c oxidase.
• Procedure: Reagent added; if positive, colonies turn dark purple.
• Read within 1-2 minutes to avoid false positives.

Fluid Thioglycollate Medium (FTM)

• Differential medium establishing an oxygen gradient to classify bacteria based on respiration needs:
  • Obligate Aerobes: Require O2.
  • Obligate Anaerobes: O2 is toxic.
  • Microaerophiles: Require lower O2 concentrations.
  • Facultative Anaerobes: Prefer aerobic but can respire anaerobically.
  • Aerotolerant Anaerobes: Do not need O2 but are not harmed by it.

Blood Agar Plate

• Purpose: Differentiates Streptococcus species based on hemolytic activity.
• Types of Hemolysins:
  • Beta: Complete lysis.
  • Alpha: Partial lysis.
  • Gamma: No lysis (negative).
  • Different tests can further identify species such as Streptococcus pyogenes and Streptococcus pneumoniae.

Indole Test (SIM Media)

• Purpose: Tests for tryptophan breakdown by tryptophanase.
• Procedure: Kovac’s reagent added after incubation – color change confirms presence of indole.

Kirby-Bauer Disc Diffusion Assay

• Method: Assesses antibiotic effectiveness against bacteria.
• Procedure involves streaking culture, applying antibiotic discs, and measuring zones of inhibition after incubation.