Advanced Techniques in Diagnostic Microbiology

23. Diagnosis of Mycobacterium tuberculosis 391
specimen collection. However, by traditional culture methods, TB identification
and drug susceptibility testing can take 4–6 weeks.
Gastric Lavage
Other specimens that may yield a diagnosis include the CSF and gastric lavage
(Gray, 2004). Gastric lavage for swallowed sputum is generally collected from
young children who do not have suitable sputum. Because Mycobacterium cannot
survive for a long period in acidic gastric washing, gastric lavage should be sent to
a laboratory promptly in 10% sodium carbonate. Gastric lavage specimen should
be collected before breakfast on three separate occasions (Inderlied, 2004).
Cerebral Spinal Fluid
Cerebral spinal fluid (CSF) requires high-volume aspirates to successfully stain and
culture MTB. Typically, microbiology labs require at least 5 ML for a TB culture.
Urine
Midstream urine specimens should be collected in a sterile plastic container (wax
free) with a leak-proof cap for three early mornings. Blood specimens should be
collected in a sodium polyanethol sulfonate (SPS) tube and must be treated with
lytic agent such as deoxycholate and should be concentrated by centrifugation
before inoculating the media. Fluids should be processed promptly and inoculated
into a liquid growth medium as well as a solid medium. Biopsy specimens should
be immediately sent to the lab (Inderlied, 2004).
It can be expected that specimens collected from nonsterile sites will be contaminated
by normal microflora. Therefore, in order to reduce contamination by
normal flora, decontamination processes are necessary. The most commonly used
mucolytic agents for sputum specimens are freshly-prepared N-acetyl-L-cysteine
(NALC) and dithiothreitol (DTT or sputolysin). NALC and sodium hydroxide are
generally used for digesting and decontaminating because they kill most bacteria
and fungi. Following decontamination, specimens are neutralized with buffer and
concentrated by centrifugation. Because swab specimens do not generally contain
sufficient materials for culture, 1 g of tissue or 10 ML of fluid are preferred
(Inderlied, 2004).
Conventional Methods for Identifying MTB
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