Scanning electron microscopy (SEM)

The same protocols as described in the preparation of the bacterial suspension and the broth microdilution assay were followed to prepare the histone H5-treated bacteria and the untreated control cells for SEM. After a 3-hour incubation of the 1:1 (v/v) ratio of planktonic bacteria and histone H5 (or 1:1 (v/v) ratio of planktonic bacteria and sterile H₂O for the untreated control cells), 100 µL of this solution was filtered directly through an Isopore™ polycarbonate membrane filter (0.2 μM pore size, EMD Millipore Co Cork, IRL) which was placed in a Swinny Stainless Steel 13 mm Filter Holder (EMD Millipore, MA, USA) connected to vacuum suction. The filter was then removed and placed onto a Kimwipe™ (Kimberly-Clark™ Professional Kimtech Science™) in a glass cell culture dish. The filters were fixed with 5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.5 (VWR, Radnor, PA, USA) overnight at 4 °C. The fixative was removed and the samples were dehydrated using sequential ethanol washes of 20, 40, 60, 80, 90, 95, and twice in 100% for 10 minutes each. Filters were then treated with 1:2 - hexamethyldisilizane (HMDS):100% ethanol, 2:1 - HMDS:100% ethanol and 100% HMDS (twice) for 10 min each (Sigma Aldrich, Oakville, ON, Canada). Filters were air dried overnight in the fume hood, sputter coated with gold to ~10–15 nm thickness and viewed using a Tescan Vega-II XMU VPSEM instrument at the Carleton University Nano Imaging Facility (Ottawa, Ontario, Canada).