Scancoat six

The morphological properties of the biofilms on the PVC and PS plates were examined with confocal laser scanning microscope (CLSM) and SEM but it was then impossible to conduct similar analysis on the biofilms developed on Al plates. Biofilms for CLSM were first stained with fluorescein isothiocyanate-conjugated concanavalin A (Sigma, United States) at 1 mg/mL and then visualized under a CLSM (LSM7 DUO 710, Carl Zeiss, United States) at 40× magnification. For each biofilm, 3 replicates were used for taking Z-stack images. The stacked CLSM images were then analyzed for microbial cell density, biovolume, mean thickness, roughness, and coverage using the image quantification tool PHLIP34 (link). For SEM, biofilm samples were dehydrated in serial concentrations of ethanol (30, 50, 70, 85, 95, and 100%; 15 min each), critical-point dried (CPD-2, Pelco, United States), and sputter coated with gold (Scancoat Six, Edwards, United Kingdom) prior to examination under a SEM (JSM-6700F, JEOL, Japan).

The presence of the biofilms on the surface of the wooden shelves was analyzed by scanning electron microscopy (SEM) using the FEI ESEM Quanta 200 apparatus (FEI Co., Hillsboro, OR, USA) at the Department of Engineering of University of Palermo (Italy). The splinters were dehydrated (82 ) and dried (10 (link)) before being mounted on an aluminum holder. All the samples were sputter coated with a thin layer of gold (83 (link)) under argon atmosphere for 90 s (Scancoat Six; Edwards, Crawley, United Kingdom) in order to avoid electrostatic charging under the electron beam.

Samples for SEM analysis were prepared as previously described with slight modification (Kong et al., 2018 (link); Boudjemaa et al., 2019 (link)). Preformed biofilms on a copper strip surface were treated with elasnin (5 μg/ml) or TSBG for 6 h followed by overnight fixation with 4% (v/v) glutaraldehyde under 4°C. Thereafter, biofilms were dehydrated in a graded ethanol series (30, 50, 70 and 90% v/v with distilled water and thrice with 100% ethanol for 10 min each step), followed by air drying. Samples were then gold-coated using a gold coater Scancoat Six (Edwards, Irvine, CA, United States) and observed using SEM (JSM-6390, JEOL, Akishima, Tokyo, Japan).

C. watsonii cells were harvested in exponential phase, 2 hours after the beginning of the dark period, onto 0.4 µm polycarbonate membranes, and incubated overnight into a fixative with adjusted osmolarity (3% glutaraldehyde in 0.1M cacodylate pH 7.4, NaCl 1.75%). Membranes were then washed, post-fixed for 1 h with 1% osmium tetroxide in 0.1M cacodylate buffer with 1.75% NaCl, and then dehydrated with graded increasing concentrations of ethanol (50, 70, 96, 100%) and critical point dried (CPD 7501, Quorum Technologies). Finally, membranes were mounted on stubs, gold-sputtered (Scancoat Six, Edwards) and observed with a conventional SEM (Scanning Electron Microscope, Cambridge Stereoscan S260). Pictures were analysed with ImageJ software [41] (link) in order to determine cell diameters and biovolumes. Due to experimental constraints, cell diameters and biovolumes were determined on four cultures (dFe = 3.3, 13.3, 43.3 and 403.3 nM).

For scanning electron microscopy, brain embryos were dissected in PBS and fixed in 2% PFA/2.5% glutaraldehyde. Fixed samples were treated with 2% osmium and washed several times in ultrapure water, dehydrated in a graded series of ethanol concentrations and prepared for scanning electron microscopy using the critical point procedure (CPD7501, Polaron). Their surfaces were coated with a 20 nm gold layer using a gold spattering device (Scancoat Six, Edwards). Samples were observed under a Cambridge S260 scanning electron microscope at 15 keV.
For transmission electron microscopy, dorsal E12.5 telencephalons were dissected in PBS then fixed in a 2% PFA/1% glutaraldehyde in 0.1 M phosphate buffer for 3 h. After rinsing in PBS, the tissue was postfixed in 1% osmium tetroxide for 30 min on ice, protected from light, with shaking. The tissue blocks were then dehydrated in 50% and 70% ethanol baths for 7 min each then stained in 1% uranyl acetate in methanol. After the final dehydration, the samples were immersed for 40 min in a graded series of ethanol/Epon solutions (2:1, 1:1, 1:2 ratios), then in pure Epon. The samples were mounted in Epon blocks for 48 h at 60°C to ensure polymerization. Ultrathin sections (70 nm) were cut sagittally on an ultra-microtome (Ultracut E; Leica) and analyzed with a transmission electron microscope (Technai 12, Philips).