Advanced Techniques in Diagnostic Microbiology

392 A. Kilic and W. Drake
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 acidfast
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 × 10 3
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).
Traditional Culture Techniques
There are nonselective and selective mediums for culture of mycobacteria. Selective
media contains antibiotics that inhibit growth of normal flora (Adjers-Koskela
and Katila, 2003). In order to culture mycobacteria from clinical specimens, there
are various kinds of solid and liquid media such as Lowenstein–Jensen, Kirchner,
and the various Middlebrook formulations (7H9, 7H10, and 7H11) (Watterson and
Drobniewski, 2000). Specimens contaminated with normal bacterial flora such as
sputum are inoculated in a selective medium containing antimicrobial agents; sterile
body fluids are inoculated with solid and a broth media (McMurray, 2000).
The growth of solid culture media is 6 weeks or longer, whereas that of liquid
culture media is usually 7–21 days. Therefore, specimens should be cultured on
solid and liquid media (Frieden et al., 2003) at 35–37 ◦ C, with 5–10% CO 2 . All
cultures should be examined weekly for 8 weeks. The major advantages of solid
cultures are that they make it possible to examine the morphology of colonies and
visualize the pigmentation. These advantages are useful to differentiate the MTB
from other non-tuberculous mycobacteria (NTM) (McMurray, 2000).
Biochemical Tests and Morphological Features
There are various kinds of biochemical tests and morphological features for identification
of mycobacteria. Based on pigment production, mycobacteria are classified
into three groups: photochroomogens, scotochromogens, and nonchromogens.
Photochroomogens produce pigmented colonies in the light. Scotochromogens
produce pigmented colonies when grown in the dark. Nonchromogens are nonpigmented
in both light and dark, but only have light tan or buff-colored colonies
(Geo. F. Brooks, 2001; Ve’ronique Vincent, 2003). Pigmented mycobacteria are
classified as nontuberculous mycobacteria (NTM) because M. tuberculosis does
not produce pigments (Ve’ronique Vincent, 2003).