DOI: 10.6084/m9.figshare.3468608.v1
title: Fatty Acid Methyl Ester (FAME) profiling dataset for Bacillus ACT spores prepared in different growth medium formulations

description: Five different <i>Bacillus </i>organisms were used for this dataset: <i>Bacillus</i> <i>cereus</i> T-strain, donated by the Counterterrorism and Forensic Science Research Unit, Quantico VA , <i>Bacillus</i> <i>cereus</i> 14579 (ATCC#14579), <i>Bacillus</i> <i>thuringiensis</i> HD1 (ATCC# NR-610), <i>Bacillus</i> <i>thuringiensis</i> HD522 (ATCC# NR-611), <i>Bacillus</i> <i>anthracis</i> Sterne BA690 (ATCC# NR-9399), and <i>Bacillus</i> <i>anthracis</i> Sterne 34F2 (LLNL A0517) . Organisms were handled using Biosafety Level 2 procedures. All cultures of <i>Bacillus</i> were maintained at 30 °C on Trypticase Soy Agar (TSA) (30 g Trypticase soy broth (Becton Dickinson, Franklin Lakes, NJ), 15 g agar (American BioAnalytical, Natick, MA)). Broth starter cultures were made by inoculating single colonies of <i>Bacillus</i> into 125 mL of Trypticase Soy Broth (TSB). Starter cultures were incubated for 16 to 18 hours at 30 °C and 225 rpm in an orbital shaker. To induce sporulation, 1 mL of starter culture was added to 250 mL of sporulation medium. <i>Bacillus</i> spores were prepared in three different medium recipes, G medium (G), G medium supplemented with 8 gL<sup>-1</sup> of meat peptone (GPep), and G medium supplemented with 8 gL<sup>-1</sup> tryptone (GTryp). All sporulation cultures were incubated at 30 °C and 275 rpm in an orbital shaker. The cultures were monitored throughout the incubation period and were harvested when the proportion of spores in the cell population reached ≥ 90%. Cells were harvested between 24 and 48 hours depending on the species/strain and medium composition.After culturing, spores were harvested by centrifugation at 3,000 x g for 15 minutes at 4 °C. Following harvesting, all spores were washed three times in cold, ultrapure water (18.2 MΩ∙cm) followed by an overnight wash at 4 °C on a rocking platform. Spore preparations were microscopically confirmed to contain ≥ 98% phase-bright spores, washed two more times, and stored at 4 °C prior to analysis. At least three replicate spore preparations of each species/strain were grown for all media. <b>Gas chromatography and fatty acid profiling.</b> Fatty acid profiles for <i>Bacillus</i> spores were generated using a modified protocol of the Instant FAME (iFAME) Identification System developed by MIDI, Inc. for low quantity samples, &lt; 3.0 mg (Sherlock MIS Operating Manual, September 2012). This protocol was chosen because it requires minimal starting material, nominal processing time (&lt; 15 minutes per sample, and has been previously validated for other biodefense applications. <br> Approximately 1.5 mL of the spore suspension was pelleted by centrifugation in a mini-centrifuge (Labnet International, Inc., Edison, NJ) for one minute at room temperature. The supernatant was removed prior to fatty acid extraction and methyl ester generation. The organic layer (~100 µl) was removed and added to new 2 ml glass vials. The vials were then loaded onto an Agilent Technologies 7890A Gas Chromatography (GC) System for FAME profiling. Two replicate GC samples from each spore preparation were included in analysis for a total of 106 samples. GC FAME profiling was performed with the MIDI Microbial Identification Sherlock software according to the manufacturer’s instructions. MIDI No. 1300-AA Calibration Standard for the Sherlock Rapid Method was used for identification and quantification of fatty acid peaks.