Biofilm Formation and Scanning Electron Microscopy

Overnight cultures were diluted 1:10 in fresh LB medium and were added to 48-well microtiter plates containing autoclaved Aclar fluoropolymer film (Electron Microscopy Sciences, Hatfield, PA). Biofilms were grown for 48 h at 37 °C, shaking at 50 rpm, and prepared for SEM using cationic dye stabilization methods.^{57 (link),58 (link)} Briefly, Aclar membranes containing biofilm growth were washed three times in 0.2 M sodium cacodylate buffer, and submerged in primary fixative (0.15 M sodium cacodylate buffer, pH 7.4, 2% paraformaldehyde, 2% glutaraldehyde, 4% sucrose, 0.15% alcian blue 8 GX) for 22 h. Samples were washed three more times prior to a 90 minute treatment with secondary fixative (1% osmium tetroxide, 1.5% potassium ferrocyanide, 0.135M sodium cacodylate, pH 7.4). After three final washes, biofilms were chemically dehydrated in a graded ethanol series (25, 50, 70, 85, 95 [2×] and 100% [2×]) before CO₂-based critical point drying. Aclar membranes were attached to SEM specimen mounts using carbon conductive adhesive tape and sputter coated with ~ 5 nm iridium using the Leica ACE 600 magnetron-based system. Biofilms were imaged using a Hitachi S-4700 field emission SEM with an operating voltage of 2 kV.