Acid Fast Staining Technique and its Details

Acid Fast Staining Technique and its Details


  • In nature, there is a variety of micro-organism each micro-organism have some special characters.
  • Most of the microorganisms are easily stained by simple staining procedures.
  • But there is some micro-organism that is not easily stained by this technique because they have a waxy covering on its surface. If anyhow they get stained they are don’t get decolourise even by strong acid.
  • Such organism require a special staining technique.
  • Acid-fast staining technique is a differential staining technique in bacteriology.
  • This staining technique was discovered by scientist Paul Ehrlich in 1883.
  • Acid-fast staining technique helps us to differentiate the organism as acid-fast and non-acid fast organisms.
  • For staining such organism Ziehl-Neelsen staining method is used. It is also called as Acid-fast staining method.


  1. Acid-fast organism- The organism that get stained by acid-fast staining technique but don’t get decolourised even by strong acid are called as an acid-fast organism.
  2. Non-acid-fast organism- The organism that easily gets stained by a staining procedure as well as decolourises easily by a strong acid are a non-acid fast organism.


  1. A clean grease free slide.
  2. A bacterial cell suspension.
  3. Staining agent- Ziehl Neelsen carbol fuchsin.
  4. Boiling water bath.
  5. Decolourising agent – Acid alcohol.
  6. Counter stain – 1% Malachite green or 0.3 % Methylene blue.


  1. Take a clean grease free slide and prepare a smear using nichrome wire loop.
  2. Air dry and heat fix the slide.
  3. The slide is flooded with ZNCF stain and placed on a boiling water bath for steaming for about 3-5 minutes.
  4. During steaming the stain is repeatedly added on the slide to avoid drying of smear.
  5. Further, the slide is treated to the decolourising agent that is acid alcohol until the stain disappears in washing.
  6. After decolourisation, the slide is given a water wash treatment.
  7. Further, the smear is flooded with the counterstain that is 1% Malachite green or 0.3 % Methylene blue for about 2 minutes.
  8. After 2 minutes the slide is washed with water, air dried and observed under oil immersion objective.

Flowchart of the Acid Fast staining procedure

Acid Fast Staining Procedure

Functions of reagents used in acid-fast staining technique

1. ZNCF ( Ziehl Neelsen carbol fuchsin)

  • It is a primary stain.
  • Many acid-fast bacteria are not stained with the common stain like carbol fuchsin because they are prepared in aqueous solution.
  • These acid fast bacteria require a stain that is prepared in phenolic stain and ZNCF stain is prepared in phenolic solution.
  • As these acid-fast bacteria have a waxy covering on their surface and phenolic stain solubilizes waxy covering and stain the cell.
  • The cells stained with ZNCF appear pink in colour.

2. Acid alcohol 

  • It is the decolourising agent.
  • It is prepared from the combination of acid that is 3% hydrochloric acid and alcohol that is 95% ethanol.

3.  1% Malachite green or 0.3 % Methylene blue

  • It acts as a counterstain.
  • It stains the decolourised cell and these cells appear green or blue in colour.


  1. Acid-fast bacteria have a waxy covering on its surface or we can say it has high lipid content in the cell wall.
  2. The cell wall of acid-fast bacteria is made up of lipids like Mycolic acid and Glycolipids.
  3. Due to these high lipid content in the cell wall, these cell wall has less permeability.
  4. So first it is necessary to increase the permeability of the cell wall so the stain can easily penetrate in the cell.
  5. The permeability of the cell wall is increased by using phenolic solution and steaming.
  6. After the permeability of the cell increases the cells get the stain.
  7. Now once the acid-fast bacteria get stained it doesn’t decolourise even by the strong decolourising agent and appear pink in colour.
  8. But the non-acid fast bacteria get decolourise easily and get stained by counterstain and appear green or blue in colour.
  9. If we use Malachite green stain cells get the stain and appear green in colour and if we use Methylene blue stain cells get the stain and appear blue in colour.


  1. Acid-fast bacteria appear pink in colour.
  2. Non-acid fast bacteria appear green or blue in colour.

Some important points

  1. The permeability of acid-fast cell is increased by phenolic stain because phenolic stain have high affinity towards the waxy covering and it is more soluble in waxy covering.
  2. For increasing the permeability we use heat steaming because steaming softens the waxy material and allow easy penetration of stain.


  1. Acid-fast staining is useful in the diagnosis of Tuberculosis and leprosy.

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