Eight well chambered coverglass slides

Biofilms were formed by NTHi cultured within chambers of eight-well-chambered cover glass slides (Thermo Scientific, Waltham, MA) as described previously (60 (link)). Briefly, mid-log-phase cultures of NTHi strains were diluted with sBHI. NTHi strains were inoculated at 4 × 10⁴ CFU in 200 μl total volume per well, and slides were incubated at 37°C with 5% atmospheric CO₂. Biofilms were grown for up to 24 h, with the growth medium replaced after 16 h when applicable. To visualize, 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 510-Meta laser scanning confocal microscope. Images were rendered with Zeiss Zen software.

Biofilms were formed by NTHI cultured within chambers of eight-well-chambered coverglass slides (Thermo Scientific, Waltham, MA) as described previously (51 (link)). Briefly, mid-log-phase cultures of NTHI strains were diluted with buffered sBHI that contained 100 mM HEPES (Fisher BioReagents), adjusted to pH 7 or 100 mM TAPS (Sigma-Aldrich), adjusted to pH 9. NTHI cells were inoculated at 4 × 10⁴ CFU in a 200-μl total volume per well and slides were incubated at 34 or 37°C, as indicated, with 5% atmospheric CO₂. Biofilms were grown for either 16 or 24 h, with the growth medium replaced after 16 h. To visualize, 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 510 Meta-laser scanning confocal microscope. Images were rendered with Zeiss Zen software.