Members of the bacterial genus Mycobacterium contain large amounts of lipid (fatty) substances within their cell walls. These fatty waxes resist staining by ordinary methods. Because this genus contains species that cause important human diseases (the agent of tuberculosis is a Mycobacterium), the diagnostic laboratory must use special stains to reveal them in clinical specimens or cultures.
When these organisms are stained with a basic dye, such as carbolfuchsin, applied with heat or in a concentrated solution, the stain can penetrate the lipid cell wall and reach the cell cytoplasm. Once the cytoplasm is stained, it resists decolorization, even with harsh agents such as acid-alcohol, which cannot dissolve and penetrate beneath the mycobacterial lipid wall. Under these conditions of staining, the mycobacteria are said to be acid fast (see colorplate 9). Other bacteria whose cell walls do not contain high concentrations of lipid are readily decolorized by acidalcohol after staining with carbolfuchsin and are said to be nonacid fast. One medically important genus, Nocardia, contains species that are partially acid fast. They resist decolorization with a weak (1%) sulfuric acid solution, but lose the carbolfuchsin dye when treated with acid-alcohol. In the acid-fast technique, a counterstain is used to demonstrate whether or not the fuchsin has been decolorized within cells and the second stain taken up.
The original technique for applying carbolfuchsin with heat is called the Ziehl-Neelsen stain, named after the two bacteriologists who developed it in the late 1800s. The later modification of the technique employs more concentrated carbolfuchsin reagent rather than heat to ensure stain penetration and is known as the Kinyoun stain. A more modern fluorescence technique is used in many clinical laboratories today. In this method, the patient specimen is stained with the dye auramine, which fluoresces when it is exposed to an ultraviolet light source. Because any acid-fast bacilli take up this dye and fluoresce brightly against a dark background when viewed with a fluorescence microscope, the smear can be examined under 400 × (high-dry) magnification rather than 1,000 × (oil-immersion) magnification. As a result, the slide can be screened more quickly for the presence of acid-fast bacilli (see colorplate 9).
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