Differential Stains Lecture

Introduction to Differential Stains

• Differential Stains (also called Diagnostic Stains)
  • Unlike simple stains, they provide more information by answering questions about the specimen.

Gram Stain

• Importance: First stain used on unknown organisms to determine cell wall type.
• Cell Wall Types:
  • Gram-positive: Thick peptidoglycan cell walls.
  • Gram-negative: Thin peptidoglycan wall with lipid outer membrane.
• Staining Results:
  • Purple organisms: Gram-positive.
  • Red/Pink organisms: Gram-negative.
• Note: Refer to separate video for detailed Gram stain procedure.

Acid-fast Stain

• Purpose: Identifies organisms with waxy cell walls (Mycolic acid).
• Organisms:
  • Mycobacterium (e.g., M. tuberculosis - tuberculosis, M. leprae - leprosy)
  • Nocardia
• Staining Process:
  • Preferred stain: Kinyoun's carbol fuchsin.
  • Decolorizing agent: Acid alcohol.
  • Counterstain: Methylene blue.
• Staining Results:
  • Red organisms: Acid-fast positive.
  • Blue organisms: Acid-fast negative.

Spore Stain

• Purpose: Identifies spore-forming organisms.
• Organisms:
  • Clostridium (e.g., C. difficile, C. tetani, C. botulinum)
  • Bacillus (e.g., B. anthracis - anthrax)
• Staining Process:
  • Stain: Malachite green for spores.
  • Counterstain: Safranin for vegetative cells.
• Staining Results:
  • Green spores indicate spore presence.
  • Vegetative cells do not stain green.

Flagella Stain

• Purpose: Identifies presence of flagella for motility.
• Process:
  • Uses a mordant to adhere stain and thicken flagella for visibility.
• Remark: Flagella is a complex structure, covered in separate bacterial anatomy modules.

Capsule Stain

• Purpose: Identifies bacterial capsules, using a negative stain approach.
• Process:
  • Capsule itself cannot be stained.
  • Staining involves coloring the background and cell, leaving the capsule clear.
• Staining Results:
  • Clear areas around organisms indicate presence of capsules.
  • Important as a negative stain method: Background is stained, not the capsule.

Conclusion

• These stains help in identifying key characteristics of microorganisms, aiding in diagnosis and understanding of their structural features.