Acid fast staining procedure

Acid fast staining procedure is used for the demonstration of acid fast microorganisms mainly Mycobacterium tuberculosis.The poperty of acid fastness is due to the presence of certain lipids (especially mycolic acid) in the cell wall. The commonly used acid fast staining procedure is Zheil Neelsen technique.

Materials required:

  • Zheil Neelson carbol fuschin
  • 20% sulphuric acid
  • methylene blue 0.5%


  • Heat fix the smear
  • Flood the slide with ZN carbol fuschin
  • Steam the stain by heating the slide intermittently for 7-10minutes; taking care to prevent boiling or drying up
  • Decolourise with 20% sulphuric acid, alternate acid and water till the smear is pale pink
  • wash under tap water
  • counter stain with methylene blue for one minute
  • wash under tap water
  • dry with blotting paper and view under microscope

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