Page:NIOSH Manual of Analytical Methods - 0801.pdf/2

 AEROBIC BACTERIA by GC-FAME: METHOD 0801, Issue 1, dated 15 January 1998 - Page 2 of 4

REAGENTS:

EQUIPMENT:

1.	Sodium hydroxide pellets (NaOH),* reagent grade. 2.	Methanol,* GC/HPLC grade. 3.	Hydrochloric acid (HCl),* 6 N. 4.	Sodium chloride (NaCl), reagent grade. 5.	Hexane,* GC/HPLC grade. 6.	Methyl-t-butyl ether (MTBE),* GC/HPLC grade. 7.	Sodium sulfate, ultrapure grade. 8.	TSA nutrient agar. 9.	Granulated agar. 10.	TSBA agar. Dissolve 30 g trypticase soy broth and 15 g granulated agar to 1 L deionized water. 11.	Saponification reagent. Dissolve 45 g NaOH in 150 mL methanol and 150 mL deionized water. 12.	Methylation reagent. Mix 325 mL 6 N HCl with 275 mL methanol. 13.	Extraction reagent. Mix 200 mL hexane with 200 mL methyl-t-butyl ether. 14.	Basic wash solution. Dissolve 10.8 g NaOH in 900 mL deionized water. 15.	Saturated NaCl solution. 16.	MIDI FAME calibration solution (MIDI, Inc., Newark, DE)

1.	Sampler: Andersen impactor, 15 × 100-mm culture plates containing TSBA culture media. 2.	Sampling pump, 28.3 L/min, with flexible tubing. 3.	Gas chromatograph, flame ionization detector, Ultra-2 capillary column, and microbial identification system (MIS) (page 0801-1). 4.	Water baths, 80 °C, 100 °C, and room temperature. 5.	Ice bath. 6.	Vortex mixer, test tube. 7.	Hematology mixer. 8.	Test tubes, screw cap, 13-mm × 100 mm. 9.	Incubator with humidity adjustment (100%), set at 28 ± 1 °C. 10.	Glass beads, 3-mm. 11.	Glass pipettes, disposable. 12.	Dispensette bottles, 4. 13.	Platinum innoculating loop, 4-mm. 14.	Autoclave. 15.	Autoclave biohazard bags. 16.	Bactoincinerator. 17.	Refrigerant packs.

SPECIAL PRECAUTIONS: Sodium hydroxide is caustic and may cause burns. Hydrochloric acid causes severe burns. Methanol, hexane, and methyl-t-butyl ether are flammable. Methanol is toxic by ingestion. Handle all bacterial cultures in approved biosafety cabinet, level II minimum. Wear appropriate eye protection, rubber gloves, and lab coat/apron. SAMPLING: 1.	Calibrate each pump with a representative sampler in line. 2.	Attach sampler to pump with flexible tubing. 3.	Sample at an accurately known flow rate at 28.3 L/min for a total sample size of 50 to 300 L. 4.	Remove culture plates from sampler, cover, and pack securely for shipment (media side up). NOTE:	Keep samples cool, but protect from freezing. SAMPLE PREPARATION: 5.	Isolate individual bacteria by pure culture technique. NOTE: See APPENDIX for Mycobacterium conditions. 6.	Select a single pure colony from unknown field samples and innoculate the method-specific TSBA agar using the quadrant streaking technique. NOTE:	Identification with the clinical library of the FAME system requires incubation on blood/ chocolate agar plates at 35 °C. a.	 Incubate at 28 °C for 24 to 48 hours.
 * See SPECIAL PRECAUTIONS

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition