Page:NIOSH Manual of Analytical Methods - 0801.pdf/3

AEROBIC BACTERIA by GC-FAME: METHOD 0801, Issue 1, dated 15 January 1998 - Page 3 of 4

b. Harvest approximately 40 mg (either by weighing into test tube or harvesting an amount about the size of a half moon on the platinum loop) of the pure cultured bacteria from the 3rd quadrant (or quadrant with confluent growth). c. Place into a 13-mm × 100-mm test tube and cap. NOTE: A harvest of 40 mg should ideally correspond to approximately a total area count of 300,000 as measured on the GC chromatogram. 7. To each test tube, add 1 mL of saponification reagent and tightly cap. a. Vortex 30 seconds, then place in a 100 °C water bath for 5 min. b. Remove from water bath, vortex for 30 seconds, and replace in the water bath for 25 min to complete the saponification process. 8. Cool test tubes in a water bath (room temperature). a. Add 2 mL of methylation reagent and cap tightly. b. Vortex for 30 seconds and place an 80 °C water bath for exactly 10 min. 9. Cool the test tubes in an ice bath for several minutes. a. Add 1.25 mL of extraction reagent and cap tightly. b. Place the test tubes in the hematology mixer and mix end over end for 10 min. c. Remove the bottom layer by pipetting and add 3 mL of basic wash mixture. Mix end over end for 5 min. 10. Remove the top layer (except for Mycobacterium analyses) by pipetting, transfer to an autosampler vial, and attach a crimp cap. NOTE: If no definitive separation occurs, add several drops of saturated sodium chloride solution and agitate to facilitate separation. CALIBRATION AND QUALITY CONTROL: 11. Calibrate daily with a fresh solution of the MIDI FAME calibration standard. The system automatically recalibrates after every ten injections. 12. Use Xanthomonas maltophilia as a positive QC culture (SI > 0.90). Other bacterial cultures such as Bacillus subtilus, Pseudomonas aeruginosa, Micrococcus roseus, and Mycobacterium smegmatis (SI>0.80) serve as suitable blind QC cultures. MEASUREMENT: 13. Set gas chromatograph according to manufacturer’s recommendations and to conditions given on page 0801-1. Inject a 2-µL sample aliquot with an autosampler. 14. The FAME profile generated for each unknown bacteria analyzed is electronically compared to a computer generated library containing the fatty acid profiles of over 5,000 bacteria. Bacterial identifications are generated for each sample and ranked in order based upon similarity indices. NOTE: Identification is based on comparison with a profile library; therefore, sample identification is not definitive. The similarity index (SI) indicates how closely the sample compares to known bacteria in the library collection. EVALUATION OF METHOD: Approximately 500 analyses of bacterial cultures comprising 40 different genus and 80 plus species were completed in the evaluation of this method [3,4]. Overall accuracy of the GC-FAME-MIS in this evaluation was > 98%. Correct identification of Mycobacterium cultures was highly dependent upon the addition of glycerol to the Middlebrook 7H10 culture media.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition