Acid-Fast Staining


  • About
  • Principle
  • Requirements
  • Procedure
  • Observation and result


  • It was developed by Paul Ehrlich in 1882.
  • Acid fast staining is a kind of differential staining.
  • This method is used for the identification of mycobacterium and other bacteria which retain carbol fuchsin (primary stain) after the treatment of strong acid and methylene blue.


  • Mycobacteraium contain a waxy substance composed of mycolic acid in its cell wall.
  • These mycolic acid are carboxylic acid with up to 90 carbon atoms chain.
  • Mycolic acid in addition with other lipids serves as barrier and prevents the entry of dye inside the bacterial cell.
  • The dye used in this method is carbol fuchsin (lipophilic dye) which binds with the acid and lipid in the cell wall and gives red colour.
  • Binding property of the dye is related to the carbon chain length of the mycolic acid.

Requirements and reagents:

  • Bacterial culture (fresh)
  • Carbol fuchsin
  • Acid alcohol
  • Methylene blue
  • Water bath
  • Glass slide
  • Inoculating loop
  • Blotting paper
  • Microscope


  • On a clean slide prepare a smear of Mycobacterium smegmatis and Staphylococcus aureus on another slide.
  • Air dry and heat fix.
  • Pour some drops of carbol fuchsin on both the smears.
  • Place the slides in steam for 3-5 min, to avoid smear from drying add more stain time to time.
  • Cool the slide for some time and wash with distilled water.
  • Pour acid alcohol for 20-30 second to decolorize the smear or until the smear gives pink colour.
  • Wash slide with distilled water.
  • Add few drops of counter stain i.e. methylene blue to the smears for 1-2 minutes.
  • Wash and blot dry with a blotting paper.


  • Observe under the microscope and record the colour test
  • Classify the bacteria i.e. acid-fast and non-acid fast.
  • Also describe their morphology and arrangement of cells.


  • M. smegmatis cells appear red coloured indicate acid fast reaction and S. aureus appear blue colour and show non-acid reaction.


Acid-fastMycobacterium smegmatis, Mycobacterium tuberculosis
Non-Mycobacterial bacteriaNocardia

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