2.8. Fatty Acid Profiling

Fatty acid profiles for bacterial samples were generated using the ‘Instant FAME’ Method following manufacturer’s instructions (MIDI Inc. Newark, DE, USA). A set of three reagents are used to extract fatty acids, derivatize to methyl esters, and separate into an organic phase prior to Gas Chromatography (GC) analysis. First, 10 mg of wet biomass was removed from the agar substrate and placed in 2 mL glass tube. Cell lysis and derivatization of fatty acids was performed by adding 250 µL of 5% potassium hydroxide, 95% methanol solution (MIDI Inc. P/N 7020-101, Newark, DE, USA). The mixture was then pulse mixed for 10 s at 3000 rpm. Next, 125 µL of analytical grade hexanes was added, followed by pulse mixing as before for 10 s. This step partitions the fatty acid methyl esters into the organic phase. Finally, 250 µL of dyed aqueous hydrochloric acid solution (MIDI Inc., P/N 7020-303) was added to the mixture to help visualize the phase boundary and facilitate transfer of the hexane layer to a new glass vial (~100 µL).

FAMEs were then analyzed with an Agilent 7890A gas chromatograph equipped with an HP-Ultra2 column (Agilent Technologies, P/N 19091B-102E) and hydrogen as the carrier gas (1.4 mL/min flow rate and 21.2 psi). The split ratio was set to 20:1. The oven temperature program started at 170 °C and increased to 288 °C at 0.5 °C/s and then increased from 288 °C to 310 °C at 1 °C/s. Identification of FAME structures and quantification of relative abundance was accomplished using the MIDI Microbial Identification Sherlock software and calibration standards (MIDI Inc., P/N 1300-C) according to the manufacturer’s protocol.