Polymer coverslip

A logarithmic-phase bacterial culture in LB at an optical density at 600 nm (OD₆₀₀) of 0.5 was inoculated onto an Ibidi μ-Slide, the surface of which is an Ibidi polymer coverslip (catalog number 80606; Ibidi). At the indicated times from 5 to 60 min postattachment, the channel was rinsed twice in phosphate-buffered saline (PBS) to remove nonadherent cells, and 200 μL of TRIzol was added to the channel. TRIzol was collected and stored at −80°C for RNA isolation (Fig. 1). Three independent biological replicates were collected for each time point. For time points with low bacterial adhesion, multiple samples were pooled to generate sufficient RNA for library generation.
Coupons of silicone, glass, and polycarbonate plastic were purchased from Biosurfaces Technologies (catalog numbers RD128-Si, RD128-GL, and RD128-PC). Coupons were placed into a 24-well plate and conditioned in LB for at least 10 min prior to bacterial addition. LB was removed and replaced with 2 mL of a logarithmic-phase bacterial culture in LB (OD₆₀₀ of 0.7). At the indicated time points, the coupon was rinsed twice in PBS and placed into 1 mL of TRIzol. TRIzol was collected and stored at −80°C for RNA isolation. Three independent biological replicates were collected for each surface condition. On surfaces with low bacterial adhesion, multiple samples were pooled to generate sufficient RNA for library generation.

An aliquot of 1000 μL supersaturated solution of potassium iodide (KI) and Rhodamine B was placed in a sterile 35 mm µ-dish with a polymer cover slip (Ibidi). Imaging was conducted on an Abberrior STEDYCON microscope (excitation laser line at 594 nm) at high excitation power and the fastest acquisition setting, using a fixed 512 × 512 pixel area. SymPhoTime 64 (Picoquant) was used to extract the decay curve by measuring the fluorescence readout for 30 seconds, with care taken to avoid saturation effects. Picoquant instructions for IRF extraction were followed.