Lecture 6: Staining Specimens for Bright Field Microscopy

Introduction to Staining

• Most microorganisms appear colorless under a standard light microscope due to their composition (mostly water).
• Staining improves contrast between the specimen and background, making cells visible.

Types of Stains

1. Simple Stains
   • Use one basic dye.
   • Highlight entire organism, revealing shapes and structures.
2. Differential Stains
   • React differently with various bacteria.
   • Distinguish between cell types.
3. Special Stains
   • Color and isolate specific microbe parts (e.g., endospores, flagella).

Staining Process

• Direct mixing with dye is ineffective due to excess unbound dye.
• Fixing the Cells:
  • Air dry and heat fix cells to the slide.
  • Use flame or slide warmer to coagulate proteins, sticking cells to the slide.
  • Avoid overheating to prevent distortion.
• Staining:
  • Use dyes (usually positively charged) that bind to negatively charged cell components like DNA, proteins, cell membrane.
  • Rinse and pat dry after staining.

Differential Staining

• Gram Stain
  • Distinguishes bacteria based on cell wall differences.
  • Gram-positive: Stains purple-blue, sensitive to penicillins and cephalosporins.
  • Gram-negative: Stains reddish-pink, more resistant to antibiotics.
  • Important in medical microbiology for initial bacterial classification.
• Acid Fast Stain
  • Identifies bacteria with mycolic acid in cell walls (e.g., Mycobacterium tuberculosis).
  • Acid-fast bacteria: Retains reddish-pink carbolfuchsin stain.
  • Non-acid-fast bacteria: Stains blue after counterstain.

Special Stains

• Flagella Stain
  • Uses carbolfuchsin dye and mordant to make flagella visible.
• Endospore Stain
  • Stains endospores green within pink rod-shaped bacteria.
• Capsule Stain
  • Detects gelatinous capsules indicating virulence.

Practical Application

• Example of Gram stain kit: Contains crystal violet, safranin, iodine (mordant), and acetone alcohol mixture (decolorizer).

Suggested Reading

• Chapter 3 related to this lecture.

Check Your Understanding

• Self-assessment questions provided to understand key concepts.