Biofilm formation
Biofilm formation for B. pseudomallei strains (Bp1026bΔasd , Bp82, and JW270) and B. thailandensis E264 was monitored using both a scanning electron microscope (SEM) and spectrophotometric analysis of bound crystal violet (CV) in a 96 well polystyrene plate (Corning, CLS3628BC) as described by O’Toole et al [71]. For the SEM, JW270 was grown on a coverslips and each coverslip is taken out and processed at 12, 24, 34, 48 and 72 hours. To process the pellicle, the bacteria is grown in 2ml of broth with coverslip dropped in a 6well plate. After 72 hours, a wide tip pipette is used to gently suction off media from the sides of the well making sure to leave the pellicle intact. Removal of the media collapses the pellicle on the coverslip. The coverslip is washed and fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M sodium cacodylate buffer for 1 hour. Samples were washed, post fixed with 1% osmium tetroxide, and alcohol dehydrated (30%, 50%, 75%, 85%, 95%, 3 changes of 100% and a single 1:1 with 100% ethanol and hexamethyldisilzane) for 10 minutes each. Sample was left to dry in 100% HMDS for 24 hours under the hood and imaged with Carl Zeiss Sigma VPFE microscope.
For biofilm assay using CV, 106 bacterial cells were added to 10ml of LB broth or LB broth supplemented with DAP/adenine-thiamine and 9 replicates of 200µl per strain were cultured in six 96 well plates for 1-3 days. Three plates each were incubated either at 37°C or at RT. The absorbance (OD600nm) of the plates were read daily and three wells of each strain were serially diluted and plated to determine bacterial counts and viability in a biofilm. Spent media in the 96-well plate were discarded and wells gently washed twice with 200µl of 1x PBS. One hundred microliters of 0.1% crystal violet solution was used to stain biofilm for 15mins. The biofilm was washed twice with 1x PBS and air-dried in a biosafety cabinet. Biofilm is extracted using 200µl of 30% acetic acid and the bound cells are quantified at an optical density of 550 nm (OD550nm) (Molecular devices, SpectraMax M5).