Ziehl Neelsen staining

This differential staining method was introduced by Ehrlich in 1882 and was subsequently modified by Ziehl and Neelsen independently. This staining method is useful in staining Mycobacteria in clinical specimens. Mycobacterial cell wall is made up of a waxy material (mycolic acid) that normally does not allow ordinary stains to enter the cell. The staining technique comprises of a primary stain, a decolouriser and a counterstain. The primary stain, which is typically concentrated (strong) carbol fuchsin is made by dissolving the dye basic fuchsin in phenol. Basic fuchsin dissolves better in phenol than in water. Heating the slide softens the waxy material of cell wall and phenolised dye readily enters the cell. Once stained by this method, these bacteria do not readily decolorize by weak mineral acids. Such bacteria are called acid fast bacteria. The non acid fast structures in the smear are then visualized by counterstaining with methylene blue solution. The acid fast bacilli appear pink in colour.

The procedure adopted in our institution is as follows:
The smear is flooded entirely with concentrated carbol fuchsin solution and heated using a spirit lamp from beneath. The heating should be intermittent and should not be intense to boil the solution or dry it completely. Typically, flaming must be stopped once fumes arise and allowed to cool. The solution is then poured off and washed in gentle stream of running tap water. The smear is then covered with few drops of 20% sulfuric acid and allowed to act for 1-2 minutes and then washed in tap water. The process of decolourisation may be repeated until the smear is faintly pink or almost colourless. The smear is then washed in water and counterstained with methylene blue solution and allowed to act for 30 seconds. The slide is then washed in water and dried with blotting paper and observed under oil immersion objective.

A positive sputum sample typically contain pink coloured, rod shaped bacteria that are slightly curved, sometimes branching, sometimes beaded in appearance, present singly or in small clumps against a blue background of pus cells and epithelial cells.

For more information and commonly asked questions, visit www.microrao.com/staining.htm

Photo of acid fast bacilli in sputum smear