Page:NIOSH Manual of Analytical Methods - 0900.pdf/2

MYCOBACTERIUM TUBERCULOSIS, AIRBORNE: METHOD 0900, Issue 1, dated 15 January 1998 - Page 2 of 4 REAGENTS:

 Filter stripping solution containing 1% Triton X-100 in 10 mM Tris-HCl, pH 8.0. Alternately, the Sputum Wash solution (Roche) may be used. Roche reagent kit, the AMPLICOR Mycobacterium tuberculosis test, containing various solutions and controls. Bleach (5.25% sodium hypochlorite). 

See SPECIAL PRECAUTIONS

EQUIPMENT:

 Sampler: polytetrafluoroethylene (PTFE) filter, 37-mm, 1.0-µm pore size, with cellulose support pad in plastic, three-piece filter cassette (Costar #130810). Sampling pump, 4 L/min or greater, with flexible connecting tubing. Petri dishes, polystyrene, 50-mm (Gelman #7242, or equivalent). <li>Forceps and/or tweezers.</li> <li>Disposable gloves.</li> <li>Platform clinical rotator.</li> <li>Serological pipets, sterile, disposable, 2.0-mL.</li> <li>Microcentrifuge tubes, 2.0-mL.</li> <li>Microcentrifuge.</li> <li>Heating block or water bath.</li> <li>PCR thermocycler and accessories.</li> <li>Microplate reader.</li> <li>Microplate washer.</li> <li>Microplate incubator.</li> <li>Aerosol barrier pipettor tips or positive displacement micropipettors.</li> <li>Sample bags, size 10.2 × 15.2 cm.</li> </ol>

SPECIAL PRECAUTIONS: Entering rooms contaminated with airborne TB is a health risk. Appropriate respiratory protection should be worn [6]. Filter samples should be opened in a biosafety cabinet. Once the samples are heated for the specified time period, there is little risk to the laboratory worker conducting the analysis. The TB PCR method is extremely sensitive. Therefore, segregation of the various laboratory activities is essential in order to prevent the generation of false positives.

SAMPLER ASSEMBLY:

<ol> <li>Assemble each filter cassette in a clean environment wearing disposable, preferably powder free or low powder, gloves.</li> <ol style="list-style-type: lower-alpha;"> <li>With forceps, insert support pad and PTFE filter into lower section of cassette.</li> <li>Attach middle and top cassette sections and insert plugs.</li> <li>Place shrinkable sealing band around cassette and air dry.</li> <li>Place each filter cassette into a sample bag for transport (one sampler/bag).</li> </ol> </ol>

SAMPLING:

<li>Calibrate each pump with a representative filter cassette in line.</li> <li>Sample closed-face at a flow rate of 4 L/min or greater. The sampling time period depends on the suspected concentration. If the concentration is unknown, sample for long time periods (hours). NOTE:	If the airborne particle concentration is unknown, assume the particles are present at a low concentration.</li> <li>Reinsert cassette plugs, place each filter cassette in a sample bag (one cassette/bag), and pack securely for shipment. Ship at ambient temperatures, according to CDC guidelines for shipment of human pathogens (42 CFR Part 72).</li> </ol> NIOSH Manual of Analytical Methods (NMAM), Fourth Edition