Syto9

Bacterial cells with an initial concentration of OD600 = 0.01 in LB broth medium were transferred into glass-bottomed dishes (Cellvis, D29-14-1-N) and incubated at 25°C for 24 or 48 h to form biofilms, respectively. Subsequently, supernatant was removed and the biofilms attached on the dish were rinsed gently three times in PBS. The biofilms were then stained with SYTO 9 (Invitrogen, L13152) according to the manufacturer’s instructions, briefly, 3 μM SYTO 9 stain was added to the biofilms and incubate at room temperature in the dark for 15 min. Then it was examined using confocal laser scanning microscopy (ZEISS LSM 880) with an excitation of 488 nm. Three-dimensional images of the biofilms were generated through Z-stack imaging, utilized the Zen Black software (Zeiss, Oberkochen, Germany). Image J was subsequently used to calculate the integrated density.

Following incubation, biofilms were rinsed with 150 mM NaCl and refilled with TSB or BHI containing 5 μM Syto 9 (1:1000 dilution from a Syto 9 stock solution at 5 mM in DMSO; Invitrogen, France), a cell-permeable green fluorescent nucleic acid marker. The plate was then incubated in the dark at 30°C for 20 min to enable fluorescent labeling. Images were acquired using a Leica SP2 AOBS confocal laser scanning microscope (Leica Microsystems, France) at the MIMA2 microscopy platform¹. The excitation laser wavelength used for Syto 9 was 488 nm, and emitted fluorescence was recorded within the range 500–600 nm. Images (512 × 512 pixels) were acquired through a 63 × Leica oil immersion objective (numerical aperture, 1.4) with a z step of 1 μm and a frequency of 400 Hz. 3D projections were generated with the Easy 3D IMARIS function (Bitplane, Zurich, Switzerland). Biofilms structural parameters [biovolume (μm³), mean thickness (μm), and roughness (μm)] were extracted from image series using the PHLIP Matlab routine². Each value presented is the average of six image series acquired in three independent experiments.

Submerged biofilms were grown on the surface of polystyrene 96-well microtiter plates with a µclear^® base (Greiner Bio-one, France) enabling high-resolution fluorescence imaging as previously described [44 (link)]. An amount of 200 µL of an overnight culture in TSB (adjusted to an OD 600 nm of 0.02) was added in each well. The microtiter plate was then incubated at 30 °C for 90 min to allow the bacteria to adhere to the bottom of the wells. Wells were then rinsed with TSB to eliminate non-adherent bacteria and refilled with 200 µL of sterile TSB. The plates were incubated at 30 °C for 24 h, and 5 μM of the cell permeant nucleic acid dye SYTO 9 (diluted 1:1000 in TSB from a SYTO 9 stock solution at 5 mM in DMSO; Invitrogen, France) were added to the 200 µL culture, obtain green fluorescent bacteria. For each strain, at least 9 to 15 wells were analyzed independently.

SYTO-9 and PI stains (Invitrogen Eugen, Oregon, USA) were used to stain live and dead bacteria, respectively, using established protocols.^{57 (link)} Briefly, 1.5 μl of a 30 mM PI concentration and 1.5 μl of 3.34 mM SYTO-9 were inserted into 1 ml of 100 mM NaCl. The sample membrane was then covered with the staining mix by pipetting, incubated for 10 min in the dark and gently washed three times with 100 mM NaCl. Two independent biofilm growth experiments were carried out for each of the cellulose variants. The developed biofilms were then visualized using a CLSM (Zeiss-Meta 510, Zeiss, Oberkochen, Germany), with images collected from eight positions on each membrane (representative images are shown in Supplementary Figs. S2–S4). Image processing and determination of specific biovolume values (μm³/μm²) were conducted using IMARIS 3D software (Bitplane, Zurich, Switzerland).

For propidium iodide (PI)/Syto9 imaging, 20 μL of 100 μg/mL PI (Thermo Fisher) and 20 μL of 100 μM Syto9 (Thermo Fisher) were added to a 20-mL universal flask along with 2 mL of exponential phase culture, which had been incubated for 3.5 h. The sample was left for 15 min before being loaded into a capillary and imaged under the microscope. The dsRED channel (excitation FF01-554/23, emission FF01-609/54), and GFP channel (excitation FF01-474/27, emission FF01-525/45) were used for visualisation of the PI and Syto9 dyes, respectively.

To investigate biofilm formation by P. aeruginosa, a BioFlux microfluidics system (Fluxion Biosciences) was used following manufacturer's instructions and literature (30 (link)). Strains were grown overnight at 37°C, 200 rpm. The next day strains were sub-cultured and incubated in the same conditions for 6 h and the OD₆₀₀ adjusted to 0.05. The microfluidic plates used were BioFlux 48 well plates 0–20 dyn/cm². The microfluidic chambers in the plate were primed from the ‘in’ well with 100 μl 10% (v/v) LB until the fluid had filled the first circle of the ‘out’ well, 70 μl of the diluted strains were added to the ‘out’ well. The strains were then pumped from the ‘out’ chamber for 2 s at a speed of 2 dyn/cm² and flow was stopped for 30 min at 37°C to allow bacterial attachment. 1 ml of pre-warmed 10% (v/v) LB supplemented with 125 nM Syto-9 (Fisher scientific) was added to the ‘in’ well, and flow started again at 0.5 dyn/cm², 37°C for 14 h.
The biofilms formed were imaged using a confocal microscope (Zeiss LSM 700), at 20× magnification taking Z-stacks (∼0.92 μm) of the bottom layer of the chambers. Syto-9 excitation and emission wavelengths were set to 483 and 500 nm respectively. Biomass quantification from image stacks of biofilms was done with Comstat2 software (31 (link)) (www.comstat.dk) using default parameters.