Acid-Fast Stain(Ziehl-Neelsen staining) Technique: Principle, Procedure, and result

Acid-Fast Stain(Ziehl-Neelsen staining) Technique: Principle,Procedure, and result


Acid Fast staining is an important differential staining procedure or technique. It is most commonly used to identify Mycobacterium spp. techniques which was first developed by Ziehl and later on modified by Neelsen. So this method is also called Ziehl-Neelsen staining techniques. Neelsen in 1883 used Ziehl’s carbol-fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl-Neelsen staining techniques was developed.


Majority of the bacteria are stained with simple stain and Gram-stain but certain bacteria do not do so because they have waxy components of the cell wall, hence their cell wall has limited permeability. Such bacteria belong to genera like Mycobacterium and Nocardia; and are stained by acid-fast stain; the latter is used to identify Mycobacterium tuberculosis and M. leprae, the pathogens responsible for tuberculosis and laprosy, respectively.


Acid fast organisms are those which are capable of retaining the primary stain when treated with an acid (fast=holding capacity). Members of the Actinomycetes, genus Nocardia (N. brasiliensis and N. asteroides are opportunistic pathogens) are partially acid-fast. Oocysts of coccidian parasites, such as Cryptosoridium and Isospora.

Acid-fast bacteria may be defined as those cells which keep the color of the primary dye (carbol fuchsin) even after the process of decolorization by the acid-alcohol solution.Acid-fast bacteria are coated with a thick waxy material, mycolic acid, which makes the bacterial cells highly resistant to the penetration of dyes. The penetration of dye is promoted by using heat as mordant. The heat invades the dye through the waxy coat and into the cytoplasm.

ØPrimary stain : 0.3% Carbol Fuchsin – Dissolve 50g phenol in 100ml ethanol (90%) or methanol(95%). Dissolve 3g basic fuchsin in the mixture and add distilled water to bring the volume to 1 L
Ø  Decolorization solution : 25% Sulphuric acid
Ø Counter stain : 0.3% methylene blue or malachite green

  1. Make a thin smear of the material for study and heat fix by passing the slide 3-4 times through the flame of a Bunsen burner or use a slide warmer at 65-75 C. Do not overheat.
  2. Place the slide on staining rack and pour carbol fuschin over smear and heat gently underside of the slide by passing a flame under the rack until fumes appear (without boiling!). Do not overheat and allow it to stand for 5 minutes.
  3. Rinse smears with water until no color appears in the effluent.
  4. Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until the slide appears light pink in color (15-20 sec).
  5. Wash well with clean water.
  6. Cover the smear with methylene blue or malachite green stain for 1–2 minutes.
  7. Wash off the stain with clean water.
  8. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot dry).
  9. Examine the smear microscopically, using the 100x oil immersion objective.
Result and Interpretation:
  •    Acid Fast Bacilli : Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded
  •         Cells : Green (malachite green) or Blue (methylene blue)
  •         Background material : Green (malachite green) or Blue (methylene blue).

1.     heory and Practice of Histological Techniques, John D Bancroft, 6th ed, p314
2.     Dorner, W. 1926. Un procédé simple pour la colouration des spores. Le Lait 6:8-12.
3.     Schaeffer AB, Fulton M (1933). "A simplified method of staining endospores". Science. 77: 194.
4.     Endospore Stain Protocol from American Society for Microbiology website Archived 2012-06-01 at the Wayback Machine

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