Ziehl-Neelsen staining method is used for staining of acid fast bacilli. Acid fastness has been attributed to the high content of lipids, fatty acids and higher alcohols found in the acid fast bacteria. Of the lipids, mycolic acid, a high molecular weight hydroxy acid wax containing carboxyl groups, is acid fast even in free state. Because of these lipids ordinary aniline dye solutions do not penetrate the cell wall.
Method
The organism is not necessarily destroyed by ordinary heat fixation. Prepare, dry and fix the smear with methanol in a safety cabinet, allow to dry and then remove from the cabinet and refix with heat.
Cover the slide with strong Carbol fuchsin and heat (not boil) until stream rises. Allow the stain to react for 5 minutes. Heat should be applied intermittently to keep the stain hot.
Wash well with water after the slide cools.
Decolorise the smear with acid alcohol.
Wash in water
Counter stain with Loeffler’s methylene blue solution for 15 -20 seconds.
Wash in water, blot, dry and examine under oil immersion.
Result
Acid fast bacilli : Red
Background : Blue
Acid Fast organisms
Uses
Ziehl -Neelsen method of staining is useful in detection of acid fast organisms.
Number of bacilli in the smear may be counted and correlated with degree of infectiousness and it is important for epidemiological purpose.
This method is also suitable for staining M. Johnei after simple heat fixation of the smear.
Name of few acid fast organisms
Mycobacterium sp, Nocardia spp, Legionella sp, Rhodococcus spp, bacterial spores and oocysts of cryptosporidium .
Factors affecting acid fastness
Age of colonies
Medium in which growth occurs
Ultra violet light
Lipid content and integrity of the cell wall of the organisms
Participants