Fluorescein isothiocyanate conjugated concanavalin a

We cultivated cells of strains K7, K7GE01, K7GE21, K7GE31, and K7GE41 until the early log phase (<5 × 10⁶) and fixed them with medium containing 3.7% (w/v) formaldehyde (Wako, Osaka, Japan). We then triple-stained cells with fluorescein isothiocyanate-conjugated concanavalin A (Sigma, St. Louis, MO, USA) for the cell wall, rhodamine-phalloidin (Invitrogen, Carlsbad, CA, USA) for the actin cytoskeleton, and 4′,6-diamidino-2-phenylindole (Sigma) for nuclear DNA, as described previously [19 (link)]. We acquired fluorescence microscopy images of the cells using a microscope (Axio Imager) equipped with a special lens (6100 EC Plan-Neofluar; Carl Zeiss, Oberkochen, Germany), a cooled-charge-coupled device camera (CoolSNAP HQ; Roper Scientific Photometrics, Tucson, AZ, USA), and appropriate software (AxioVision; Carl Zeiss). We analyzed the micrographs of the cells with image processing software designed for diploid cells (CalMorph, ver. 1.3) [20 (link)]. We obtained the morphological data of 501 traits from the single-cell data. Descriptions of each trait have been presented previously [19 (link)]. The CalMorph user manual is available at the S. cerevisiae Morphological Database (https://www.yeast.ib.k.u-tokyo.ac.jp/CalMorph/).

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).

Fluorescence staining procedures were as described previously [27 (link)]. Briefly, cells of yeast strains were cultivated until an early log phase (<5 × 10⁶) and fixed with medium containing 3.7% (w/v) formaldehyde (Wako, Osaka, Japan). We then triple-stained cells with fluorescein isothiocyanate-conjugated concanavalin A (Sigma, St. Louis, MO, USA) for the cell wall, rhodamine-phalloidin (Invitrogen, Carlsbad, CA, USA) for the actin cytoskeleton, and 4′,6-diamidino-2-phenylindole (Sigma) for nuclear DNA, as described previously [27 (link)]. Fluorescence microscopy images of the cells were acquired using a microscope (Axio Imager) equipped with a special lens (6100 Ecplan-Neofluar; Carl Zeiss, Oberkochen, Germany), a cooled-charge-coupled device camera (CoolSNAP HQ; Roper Scientific Photometrics, Tucson, AZ, USA), and appropriate software (AxioVision; Carl Zeiss). Micrographs of the cells were analyzed with image-processing software designed for diploid cells (CalMorph, version 1.3) [28 (link)] and further investigated statistically as described in the following section. Descriptions of each trait have been presented previously [27 (link)]. The CalMorph user manual is available at the S. cerevisiae Morphological Database (http://www.yeast.ib.k.u-tokyo.ac.jp/CalMorph/, accessed on 22 August 2022).