Fatty Acid Methyl Ester (FAME) profiling dataset for Bacillus ACT spores prepared in different growth medium formulations
Five different Bacillus organisms were used for this dataset: Bacillus cereus T-strain, donated by the Counterterrorism and Forensic Science Research Unit, Quantico VA , Bacillus cereus 14579 (ATCC#14579), Bacillus thuringiensis HD1 (ATCC# NR-610), Bacillus thuringiensis HD522 (ATCC# NR-611), Bacillus anthracis Sterne BA690 (ATCC# NR-9399), and Bacillus anthracis Sterne 34F2 (LLNL A0517) . Organisms were handled using Biosafety Level 2 procedures. All cultures of Bacillus 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 Bacillus 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. Bacillus spores were prepared in three different medium recipes, G medium (G), G medium supplemented with 8 gL-1 of meat peptone (GPep), and G medium supplemented with 8 gL-1 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.
Gas chromatography and fatty acid profiling. Fatty acid profiles for Bacillus spores were generated using a modified protocol of the Instant FAME (iFAME) Identification System developed by MIDI, Inc. for low quantity samples, < 3.0 mg (Sherlock MIS Operating Manual, September 2012). This protocol was chosen because it requires minimal starting material, nominal processing time (< 15 minutes per sample, and has been previously validated for other biodefense applications.
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.
