Eight well chamber coverglass slides

Biofilms were formed by NTHi cells cultured within chambers of eight-well-chamber coverglass slides (Thermo Scientific, Waltham, MA) as described previously (48 (link)). Briefly, biofilms were formed by NTHi cells cultured within chambers of eight-well-chamber coverglass slides (Thermo Scientific, Waltham, MA) using mid-log-phase NTHi cultures. Bacteria were inoculated at 4 × 10⁴ CFU in a 200-μL final volume per well and incubated at 37°C with 5% CO₂ for 24 h, with the growth medium replaced after 16 h. To visualize biofilms, the biofilms were stained with LIVE/DEAD BacLight stain (Life Technologies) and fixed overnight in fixative (1.6% paraformaldehyde, 2.5% glutaraldehyde, and 4% acetic acid in 0.1 M phosphate buffer [pH 7.4]). Fixative was replaced with saline before imaging with a Zeiss 980 Meta laser scanning confocal microscope. Images were rendered with Zeiss Zen software. Z-stack images were analyzed by COMSTAT2 (49 (link)) to determine biomass (μm³/μm²), average thickness (μm), and roughness (Ra).