practical exercises

Ziehl-Neelsen (Acid Fast) staining technique


The cell wall of Mycobacterium sps typically contain waxy substance (mycolic acid) that makes it impervious to staining by aqueous staining solutions. These bacteria can not be stained by simple stains or even by Gram staining. These bacteria can however be stained by drastic measures, and once stained can not be readily decolourized by weak mineral acids. Hence, these bacteria are called acid fast bacilli and the staining method is called acid fast staining. It is also a type of differential staining method.

Acid fast staining was introduced by Ehrlich in 1882. It was subsequently modified by Ziehl and Neelsen. There are two types of acid fast staining: hot method and cold method. Ziehl-Neelsen is a hot method of acid fast staining. The components of Ziehl-Neelsen stain include primary stain (strong/concentrated carbol fuchsin), decolourizer (20% H2SO4) and counterstain (Loeffler's methylene blue).

Strong carbol fuchsin solution is basic fuchsin dissolved in phenol (carbolic acid). Heating the slide helps to soften the waxy material on the bacterial cell wall. The waxy material is hydrophobic to aqueous solution but not to phenolic solution of basic fuchsin. Hence strong carbol fuchsin is able to stain the cell. Upon staining, they tend to resist decolourization by 20% H2SO4 (sulphuric acid). The background is then stained by Loeffler's methylene blue.

One of the main applications of acid fast staining is the diagnosis of pulmonary tuberculosis. Sputum sample obtained from the patient is used to make thin and uniform smear on glass slide. The readymade smear is provided to you.

Requirements: Smear on glass slide, staining rack, spirit lamp, Strong carbol fuchsin, 20% H2SO4, Loeffler's methylene blue, blotting paper, immersion oil, microscope.

Keep the slide with the smear facing upwards on the staining rack. Flood the entire slide with strong carbol fuchsin solution. Heat the slide with gentle waving from below using lighted spirit lamp until fumes arise from the stain. Do not let the stain solution boil or dry out. Pour more stain if required. Allow the fumes to disappear and repeat the process of heating the slide two more times. Allow the slide to cool down. Pour off the stain and decolorize the smear using 20% H2SO4 for at least one minute. Wash the slide under gentle stream of running tap water. Examine the slide for signs of proper decolourization. The smear must be almost colourless or faintly pink. Repeat the process of decolourization until the smear get properly decolourized. Wash the smear and place the slide back on the rack. Clean the other side of the slide with 20% H2SO4, if it is stained as well. Cover the smear (NOT the entire slide) with few drops of Loeffler's methylene blue and allow it to act for a minute. Was the slide in tap water and dry it using blotting paper. Place a drop of immersion oil on the smear and observe it under 100x objective.

Pink coloured, slightly curved bacilli in singles or in small clumps seen against a blue background of epithelial cells and pus cells.

The given smear contains one of the acid fast bacilli (May be Mycobacterium tuberculosis).

gram stain

Note: You will have search many fields to spot Acid Fast Bacilli.

For more information on Frequently asked question in Staining techniques, click here

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