Acid-fast stain (Ziehl-Neelsen method) is a useful differential staining procedure that specifically stains all members of the genera mycobacteria. The procedure utilizes heat and phenol (carbolic acid) to help the penetration of the dye, basic fuchsin, to the inside of mycobacterial cells, which are impermeable to basic rosaniline dyes in routine stains such as the Gram stain. The high lipid and wax content of the mycobacterial cell walls is thought to be the reason for such impermeability.The stain is the gold standard procedure for diagnosis of tuberculosis and leprosy. Being unassociated with the human flora (except Mycobacterium smegmatis found in human smegma), finding of acid-fast bacilli in human specimens such as sputum and nasal scrapings is strongly indicative of an active infectious process, namely of tuberculosis and leprosy. Acid-fast pathogens other than mycobacteria include very few genera such as the bacterium Nocardia and the fungus Cryptosporidium. Ziehl-Neelsen stain can also be used for the primary identification of these other acid-fast pathogens.
This flash animation graphically illustrates the steps for staining acid-fast bacilli by the Ziehl-Neelsen procedure. While illustrating the step-by-step procedure, a time-lapse screen shows the events which take place in the smear at high magnification.