Nanoscope analysis software v 1

One hundred μL of log-phase E. coli ATCC 25922 cells (OD₆₀₀ = 0.2) in an LB medium were incubated for 1.5 h at 37 °C with or without the test compounds at ten times the MIC. The samples were then centrifuged at 8000 × g for 10 min at 4 °C, and the pellet was washed twice in 100 μL of apyrogenic water. The bacteria resuspended in 50 μL of apyrogenic water were applied to mica disks and dried overnight at 28 °C before imaging. Atomic force micrographs were recorded on a Veeco Mulitmode AFM with NanoScope III controller operating in contact mode. The data were analyzed with NanoScope Analysis software v.1.40 (Veeco, USA). Scans were acquired at 25 °C at the rates of 1.0 Hz and 256 samples per line resolution. Downstream image processing and analysis were performed using NanoScope software. Height images were flattened to compensate for cell curvature, and topographical sections were used to reconstruct the surface texture in two dimensions.

S. aureus (ATCC 25923) was cultured in the brain heart infusion medium to mid-logarithmic phase and then washed twice with buffer A (20 mM Tris-HCl, pH 7.5 at 25 °C). The washed bacteria were incubated with 5 µM recombinant grass carp CXCL20b or BSA for 15 min at 37 °C. Before imaging, we washed the bacteria twice with deionized water, transferred the cells to mica disks, and dried the disks overnight at 25 °C. AFM assay was carried out as previously described. AFM images were recorded on a Veeco Mulitmode AFM with NanoScope III controller operating in contact mode [43 (link)]. The data were analyzed with NanoScope Analysis software v.1.40 (Veeco, Santa Barbara, CA, USA). Scans were acquired at 25 °C at 1.0 Hz and 256 samples per line resolution.

Bacteria were prepared for imaging as previously described [53 (link),54 (link)] (see electronic supplementary material). The data were analysed
with Nanoscope Analysis software v. 1.40 (Veeco, USA). Three
fields on each mica disc were imaged. Three-dimensional images and section
profiles were prepared using WSxM v. 5.0 software [55 (link)]. The roughness values were
measured over the entire bacterial cell surface on 3 × 3
µm² areas. The average surface root mean square (RMS)
roughness was calculated from 25 fields (300 × 300 nm²). The
data were analysed using Statistica v. 6 (StatSoft, Inc., Tulsa, OK,
USA). Statistical significance was determined by ANOVA (Tukey's honestly
significant difference test).