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Section II: Procedure for primary isolation
A. Introduction
Mycobacteria Growth Indicator Tube (BBL MGIT) contains modified Middlebrook 7H9
broth base. When supplemented with MGIT Growth Supplement and PANTA, it provides an
optimum medium for growth of a majority of mycobacterial species. All types of specimens,
pulmonary as well as extra-pulmonary (except blood), can be inoculated into MGIT for
primary isolation of mycobacteria. Urine specimens have not been evaluated by BD but
other investigators have reported successful isolation of mycobacteria from urine specimens.
Mucoid specimens are expected to contain contaminating bacteria as normal flora and must
be digested (liquefaction) and decontaminated before inoculation. On the other hand,
aseptically collected body fluids or tissue biopsies do not need to be decontaminated.
However, since it is difficult to maintain sterile conditions throughout the collection of
specimens, it is recommended that all specimens be decontaminated. Aseptically collected
specimens need only light decontamination. Clinical specimens collected in large volumes
(of more than 10 ml) require centrifugation before decontamination to reduce the overall
volume and to concentrate mycobacteria present in the specimens into a smaller volume.
After decontamination, the specimen should be centrifuged again and the sediment used for
preparation of smear and inoculation for culture.
B. Important Safety Precautions
Perform all procedures, such as processing of specimens, smear preparation, inoculum
preparation, making dilutions, inoculation of media, and subculturing in a suitable biological
safety cabinet in a room dedicated for mycobacterial work. The CDC has recommended a
Biosafety Level (BSL) 2 laboratory with negative air pressure and with an appropriate
ventilation system for mycobacterial work . 2 More recently, the CDC has recommended that
work involving manipulation of TB cultures, such as DST, be done in a BSL 3 laboratory. 61
However, this work may be done in a BSL 2 laboratory providing the exhaust air from the
laboratory is discharged to the outdoors, the ventilation is balanced to provide directional
airflow into the room, access to the room is restricted when work is in progress, and the
practices and equipment recommended for BSL-3 are followed. This includes use of proper
protective gowns, gloves and respirator masks (approved by OSHA) while handling
specimens and mycobacterial cultures. International Safety Standards, along with the local
specifications, may also be followed.
Use an appropriate mycobacterial disinfectant such as Amphyl® for cleaning the work area.
The CDC states: “With so many disinfectants available, it is important to consult the product
brochures to make certain the disinfectant is bactericidal for mycobacteria.” 2
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