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.