Xt microscope control

The cell morphology of E. coli biofilms present on GLA and SIL coupons before and after 6 h of antibiotic treatment was assessed by SEM. Prior to observations, biofilm samples were fixed using 3% wt. glutaraldehyde in cacodylate buffer pH 7.2 [41 ] for 10 min and exposed to an ethanol dehydration series of 50, 60, 70, 80, 90, and 2 × 100% (v/v) ethanol, followed by a chemical dehydration series of 100% ethanol + hexamethyldisilazane (HMDS, Ted Pella, USA) at 50, 60, 70, 80, 90, and 2 × 100% (v/v) HMDS [42 (link)], for 5 min at each concentration. The bare surfaces were also subjected to the same dehydration treatment. All the coupons were then dried for 1 day and sputter-coated with a palladium-gold thin film [43 (link)]. The bare surfaces and the biofilm samples were viewed with a SEM/EDS system (FEI Quanta 400FEG ESEM/EDAX Genesis X4M, FEI Company, USA) in high-vacuum mode at 15 kV. In the case of biofilm samples, twenty images from three independent coupons were analysed before and after the antibiotic treatment. The microscope software (xT Microscope Control, FEI Company, USA) was used to determine the cell length by measuring 100 randomly selected cells in each condition.

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) was performed at the Manawatu Microscopy and Imaging Centre, Massey University, Palmerston North, New Zealand. SEM was performed on an aliquot of sample 3 (see section on Quinella-enriched samples, above) fixed to a Formvar grid (Sigma-Aldrich, St. Louis, MO, USA), stained with 2% (w/v) uranyl acetate and examined with a FEI Quanta 200 scanning electron microscope (Philips Electron Optics, Eindhoven, The Netherlands). Part of sample 3 was prepared for TEM by washing the cell pellet three times in sterile water, resuspending in modified Karnovsky’s fixative (2% [w/v] paraformaldehyde and 3% [w/v] glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2) for embedding in resin (Procure 812; ProSciTech, Qld, Australia), and thin sections made using an EM UC7 ultra-microtome (Leica Microsystems, Wetzlar, Germany). TEM used a Tecnai G2 Biotwin transmission electron microscope (FEI, Hillsboro, OR, USA). Electron microscopy used XT Microscope Control and TUI version 4.5 software (FEI).