Microbiology Lab: Acid Fast Stain

Introduction

• Presenter: Kevin Tow Coff
• Topic: Acid Fast Stain from Experiment 4
• Purpose: To identify acid-fast organisms

What is an Acid-Fast Organism?

• Characteristics:
  • Bacteria with wax-like, lipid-dense cell walls
  • Cell wall contains mycolic acid and phenolic lipids
  • Hydrophobic and resistant to:
    • Disinfectants
    • Dry conditions
    • Gram stains

Importance of Acid-Fast Stain

• Resistance to Gram Stain:
  • Gram stains ineffective for acid-fast organisms due to lipid-rich cell walls
• Special Staining Technique Needed: Acid-fast stain

Examples of Acid-Fast Bacteria

• Mycobacterium tuberculosis
  • Causes tuberculosis
• Mycobacterium leprae
  • Causes leprosy
• Nocardia
  • Another genus of acid-fast organisms

Staining Procedure

Steps

1. Heat Fixing:
   • Prepare smear and heat fix
2. Primary Stain:
   • Use carbol fuchsin
   • Apply heat (Fisher burner) to penetrate cell wall
   • Critical step: heating is essential to drive stain into cells
3. Decolorization:
   • Use acid alcohol
   • Acid-fast organisms remain red; non-acid-fast organisms become colorless
4. Counterstain:
   • Use methylene blue
   • Non-acid-fast organisms appear blue; acid-fast organisms remain red

Absence of Mordant

• No iodine used, unlike Gram stain

Visual Identification

• Acid-Fast Organisms: Red or fuchsia (e.g., bacilli like Mycobacterium)
• Non-Acid-Fast Organisms: Blue

Key Points to Remember

• Genera:
  • Mycobacterium
  • Nocardia
• Diseases:
  • Tuberculosis
  • Leprosy
• Color Results:
  • Red indicates acid-fast
  • Blue indicates non-acid-fast
• Critical Step: Heating to penetrate carbol fuchsin into cell walls

Conclusion

• Acid-fast stain differentiates organisms not identifiable by Gram stain
• Essential for identifying bacteria causing tuberculosis and leprosy
• Remember: Red = Acid-fast, Blue = Non-acid-fast

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