Microscopic Techniques

Acid-fast staining is a fast, cheap, and convenient method for direct detection of mycobacteria from clinical specimens (Inderlied, 2004). Although microscopy provides preliminary information, it is not an adequate method for differentiating

MTB from other Mycobacterium species. Also, microscopic techniques are not suitable for examination of specimens, such as urine, which are contaminated with nonpathogenic bacteria (Gray, 2004).

In microscopy, Ziehl-Neelsen (ZN), Kinyoun's stain, and fluorochrome stain, methods are used. It is necessary that a reference laboratory report results of acid-fast stain within 24 h of receiving the specimens. In this method, due to mycolic acid-rich cell wall, carbol fuchsin dye is retained after washing with acid alcohol. This method is advised by WHO and IUATLD (Frieden et al., 2003). An alternative method is a fluorochrome stain made of auramine-rhodamine, which stains mycolic acids in the AFB cell wall. To visualize one AFB, approximately 5 x 103 bacilli per ML sputum should be present. In smear examination, it is necessary to assess 300 fields before reporting a specimen as AFB negative (Tenover et al., 1993). With respect to culture, microscopy specificity is 99%, and sensitivity is 25-75%. In some patients who receive antituberculosis therapy, it is possible to have positive smears and negative cultures, which reflect nonviable bacilli (McMurray, 2000).

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