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.
- See SPECIAL PRECAUTIONS
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.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
