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Mycobacteriology Section

The Mycobacteriology laboratory section plays an important role in the control of tuberculosis (TB) in South Carolina by providing timely detection, isolation, identification and drug susceptibility testing of M..tuberculosis.

The Mycobacteriology laboratory’s diagnosis of new cases of pulmonary TB relies on collecting at least three consecutive, early morning, expectorated or induced sputum specimens. Sputum specimens submitted for mycobacteria culture and identification are first taken through a process called digestion and decontamination. This process destroys the non-mycobacteria. A smear of the treated concentrated sputum is stained using Truant’s fluorescent stain and examined microscopically for the presence of acid fast bacilli. Questionable fluorescent organisms may be further examined using the Zeihl-Neeslen AFB staining procedure. AFB smears are read and reported within one working day of specimen receipt.

Concentrated respiratory specimens that test AFB smear positive are also tested using the AMPLIFIED Mycobacterium tuberculosis Direct (MTD) Test (Gen-Probe, San Diego, CA). The MTD Test qualitatively detects the presence of MTB ribosomal ribonucleic acid (rRNA) and can provide clinicians with a laboratory diagnosis of TB within 48 hours of specimen receipt.

Regardless of the AFB smear or MTD test results, all concentrated sputum specimens are inoculated onto culture media. We use a solid culture medium - Lowenstein-Jensen (L-J) medium and a liquid medium - modified Middlebrook 7H9 broth for the isolation of Mycobacteria. The Lowenstein-Jensen medium is examined for growth at one, four and seven weeks. The modified Middlebrook 7H9 Broth is continuously monitored for growth during a six-week period, using an automated culture and growth detection instrument system, the MB/BacT System (Organon Teknika, Durham, NC). If growth is observed or detected in either the solid or liquid media, the ACCUPROBE Mycobacterium tuberculosis Complex Culture Identification Test (Gen-Probe), and ACCUPROBE Mycobacterium avium Complex (MAC) Culture Identification Test (Gen-Probe) are performed in sequence. Both MTB and MAC ACCUPROBE tests employ a chemiluminescent –labeled, single-strand DNA probe that is complementary to the rRNA of their respective organisms. If both of the ACCUPROBE tests are negative, additional identification methods are used. We perform high performance liquid chromatography (HPLC) analysis on mycolic acids that are extracted from the cell walls of the unidentified organism. We also perform the traditional biochemical tests on the isolate to obtain its phenotypic biochemical profile. The use of specific nucleic acid probes and HPLC mycolic acid cell wall analysis has largely eliminated the need to perform the traditional biochemical identification tests to identify mycobacteria.

ALL MTB isolates are routinely tested for susceptibility to isoniazid, streptomycin, rifampin, and ethambutol using the BACTEC 460 (Becton Dickinson Diagnostic Instruments, Sparks, MD).

Additional drug testing may be offered following a case consultation with DHEC’s TB Control Program staff.