ZN staining technique for Acid Fast Bacilli: Demonstration Video

It is a differential staining that divides organisms (or their structures) into acid fast (AFB) and non-acid fast (non-AFB). Original method of acid fast staining involved staining with aniline-gential violet, followed by decolorisation using strong acid. It was later improved by Ziehl and Neelsen.

  • Glass slide with fixed smear
  • Bunsen flame or spirit lamp
  • Staining reagents
  • Primary stain: Concentrated carbol fuschin
  • Decoloriser: 20% H2SO4
  • Counterstain: Methylene blue or Malachite green

Bacteria of the genera Mycobacterium and Nocardia have unusual cell walls that are waxy and nearly impermeable due to the presence of mycolic acid, and large amounts of fatty acids, waxes, and complex lipids. These organisms are highly resistant to disinfectants, desiccation and are difficult to stain with water-based stains such as the Gram stain.

  • Primary stain penetrates cell wall
  • Intense decolorization does not release primary stain from the cell wall of AFB
  • Color of AFB is based on primary stain
  • Counterstain provides contrasting background
  1. Place the slide on the rack
  2. Flood the slide and completely cover with carbol fuschin
  3. Using the metal forceps, take a piece of cotton wool soaked in alcohol, pass it through the flame and heat the slide from below until the stain emits a vapor, but do not bring to boiling point
  4. Repeat this operation twice, (within 10 minutes)
  5. Add fuschin if necessary; the slide should be covered
  6. Rinse with water, drain
  7. Apply decolorizing solution 2 min
  8. Rinse, drain
  9. Apply Methylene blue counterstain, 30 seconds
  10. Rinse, drain
  11. Air dry or blot it dry
  12. Observe under microscope
  • AFB: appears as bright red or pink rods against blue background
  • Non AFB: appears purple
  • 20% H2SO4: Mycobacterium tuberculosis
  • 5% H2SO4: Mycobacterium leprae
  • 1% H2SO4: Actinomyces and Nocardia

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