Acid fast staining was developed in Ehrlich in 1882 and was later successively modified by Ziehl and Neelsen. Its main application is in the staining and demonstration of acid fast bacilli in sputum smears. Acid fastness in the property of certain bacteria (mycobacteria), their structures (spores) or protozoal forms (oocyts of cryptosporidium) by virtue of which, they resist decolourization by weak mineral acids following staining by an intense dye (strong carbol fuchsin). Most common of the acid fast staining techniques is the Ziehl-Neelsen technique.

 Principle of acid fast staining (in mycobacteria):

The waxy mycolic acid containing cell wall of mycobacteia are relatively impermeable to ordinary staining techniques. They can be stained by aniline dyes using drastic measures such as application of heat and phenol. Heat softens the wax in the cell wall and allows the stain (basic fuchsin) to enter. The fuchsin dye is more soluble in phenol than in water or alcohol. Phenol in turn is more soluble in lipids or waxes, thus the dye-phenol mixture enters the cell. Once stained, it resists decolourization by weak mineral acid (20% H2SO4). This is due to the fact that phenol-dye mixture is more soluble in waxes of the mycobacteria than the acid or alcohol. This way phenol acts as a mordant. While the mycobacteria retain the primary stain (pink), the background material gets decolourized and takes up the counterstain (methylene blue).

 Components of Ziehl-Neelsen stain:

  • Primary stain: Strong carbol fuchsin (consists of basic fuchsin and carbolic acid [phenol])
  • Decolourizer: 20% sulfuric acid (H2SO4) or acid alcohol
  • Counterstain: Loeffler’s methylene blue or malachite green

 Procedure of ZN staining:

The sputum specimen is smeared uniformly in a small area over a new, scratch-free glass slide and fixed.
The slide is placed on the staining rack and is flooded by strong carbol fuchsin solution.
The slide is intermittently heated (for ~5 minutes) from underneath using a spirit lamp until fumes arise. Care must be taken not to boil the solution or drying of the slide.
The slide should be washed in water and decolourized by 20% H2SO4 until the slide is almost colorless or pale pink. Decolourization generally requires contact with sulfuric acid for a total time of at least 10 minutes.
The smear is then washed and counterstained with Loeffler’s methylene blue solution for 1-2 minutes.
The slide is washed again and dried.

Structures that are acid fast:
Mycobacteria, Actinomyces in tissue sections, cultures of Nocardia, oocysts of Cryptosporidium, Isospora  and bacterial spores

Modifications of acid fast staining:

  • Use of alcohol as secondary decolourizer
    • After primary decolourization with sulfuric acid, the smear may be treated with 95% alcohol as secondary decolourizer. M.tuberculosis is both acid fast and alcohol fast, while saprophytic mycobacteria are only acid fast.
  • Use of acid-alcohol as decolourizer
    • Instead of using 20% sulfuric acid as decolourizer, 3% HCl in 95% alcohol may be used. This also differentiates tubrcle bacilli from saprophytic mycobacteria. It is especially used in diagnosis of renal tuberculosis.
  • Modifications in the percentage of sulfuric acid
    • 5% H2SO4 for M.leprae
    • 1% H2SO4 for Actinomyces in tissue
    • 0.5% H2SO4 for cultures of Nocardia
    • 0.25-0.5% for spores and for oocysts of Cryptosporidium and Isospora
  • Cold methods: These are modifications where heating is not employed. Increasing the concentration of phenol and increasing the duration of staining substitutes heating.
    • Kinyoun’s modification
    • Gabbett’s modification

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  Last edited in June 2006