Staining Methods
The best known and distinctive property of the genus, Mycobacteria, depends upon their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol. Once stained the cells resist decolorization with acidified organic solvents and are therefore called ACID FAST. Although the ability to retain arylmethane dyes such as carbol fuchsin and auramine-rhodamine after washing with alcohol or weak acids is a primary feature of this genus it is not entirely unique to the genus. Other bacteria which contain mycolic acids, such as Nocardia, can also exhibit this feature. The exact method by which the stain is retained is unclear but it is thought that some of the stain becomes trapped within the cell and some forms a complex with the mycolic acids. This is supported by the finding that shorter chain mycolic acids or mycobacterial cells with disrupted cell walls stain weakly acid-fast.
Fluorochrome staining has some advantages and disadvantages over Ziehl-Neelsen staining. One advantage is that the smear is examined under a lower magnification with a dry objective allowing a much larger area of the smear to be examined in a shorter time. One of the disadvantages of fluorochrome staining is that organisms apparently dead, or rendered non-cultivable by chemotherapy may still fluoresce positive. This disadvantage is caused by the superiority of the fluorochrome stain (auramine rhodamine) over the carbol fuchsin stain to bind intensely with the mycolic acids. Thus in fluorescence microscopy, more bacilli are stained than would normally be when stained by the Ziehl-Neelsen method.
