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

REFERENCES: [1] Jensen PA, Scarpino P [1992]. Evaluation of eight bioaerosol samplers with bacteria. Am Ind Hyg Assoc J 53(10):660–667. [2] Sasser M [1990]. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note # 101. [3] Pendergrass SM, Jensen PA [1997]. Application of the gas chromatography-fatty acid methyl ester (GC-FAME) system for the identification of environmental and clinical isolates of the family Micrococcaceae. Appl Occup Environ Hyg 12(8):543–546. [4] Schafer M, Pendergrass SM [in preparation]. Identification and differentiation of selected members of the genus Mycobacterium by gas chromatography-fatty acid methyl ester (GC-FAME) analysis. J Applied and Environmental Microbiology. METHOD WRITTEN BY: Stephanie M. Pendergrass, DPSE, NIOSH APPENDIX. MYCOBACTERIUM CONDITIONS AND CULTURE MEDIA. For the analysis of Mycobacteria, follow the method as written with exception of the following steps. Step 6. Culture media: Middlebrook 7H10 culture media containing Middlebrook OADC Enrichment (with 0.5% glycerol).

Incubation: 35 °C in the presence of 5 to 10% CO2 for 2 to 14 days; slow growing cultures like Mycobacterium tuberculosis may require up to six weeks. Step 7. Vortex mixing: Add 3 to 5 glass beads to the mixture prior to vortexing. Step 9. Extraction: For Mycobacterium analyses, remove the top layer and add approximately 10 mg of sodium sulfate to remove any water from the FAME solution. Step 10. Transfer: The FAME solution is pipetted to a new autosampler vial. Take care not to carry over any sodium sulfate, and attach crimp cap. Step 12. Quality control: Use Mycobacterium smegmatis as a positive QC culture (SI > 0.80) for Mycobacterium analyses.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition