Ziehl-Neelsen’s acid fast staining

This is a special type of staining method for few selective groups of bacteria which
are having mycolic acid on their cell wall.

Certain bacteria like Mycobacterium sp. do not stain readily by simple staining or even Gram’s Method of differential staining. It is because of high lipid (mycolic acid) content of their cell wall. Their staining is facilitated by heat. Once stained, they retain the colour of the primary stain (concentrated carbol fuchsin) even when treated with strong decolourizer. These organisms are designated as acid fast.

Reagents (composition):
a. Concentrated carbol fuchsin
b. Decolourizer (Ethanol (95%) 97 ml and Concentrated HCL 3 ml)
c. Loeffler’s alkaline methylene blue
1. A portion of formalin preserved tuberculous lung.
2. Staining solutions – a, b, c.

1. Cut a piece of tuberculous lung and make an impression smear upon a microscopic
slide. Dry the smear and fix over the flame.
2. Flood the smear with concentrated carbol fuchsin. Apply heat from bottom of the
slide but do not allow boiling. Stain should not dry on the slide. Keep it for 3-5
3. Wash with running tap water.
4. Decolourize the smear with decolourizer till no stain comes out.
5. Wash with running tap water.
6. Counter stain with Loeffler’s alkaline methylene blue for about 15-20 seconds and
then wash again with running tap water.
7. Dry it and examine under oil immersion lens (100X).
Acid-fast bacteria will show intense red colour against blue coloured tissue debris.