## 2 Materials and Methods
### Biofilm Formation  
The study of the formation and structure of the biofilms was carried out by CLSM using a previously described procedure [16]. CLSM combined with a quantitative analysis of the images that were obtained enabled numerical data to be collected for the structural parameters of biofilms, such as biovolume, facilitating the comparison of different biofilms by means of statistical analyses.  
The strains were incubated in TSB for 24 h at 37 \({}^{\circ}\)C. Dilutions were then performed in the same culture broth until a concentration of \(10^{6}\) cfu/mL was achieved. A volume of 125 \(\upmu\)L was added to wells of Corning(tm) 96-Well Half Area High Content Imaging Glass Bottom Microplates (Fisher Scientific Ltd., Dublin, Ireland), and to wells of microtiter plates with a graphene monolayer coated bottom (Graphene Headquarters, San Sebastian, Spain). A volume of 250 \(\upmu\)L was added to wells of Nunc(tm) MicroWell(tm) 96-Well Optical-Bottom Plates with Polymer Base (Thermo Scientific(tm), SLU, Waltham, MA, USA), and to wells of microtiter plates with food safe epoxy resin coated bottoms (Resinence(r), Mormant-sur-Vernisson, France).  
The microtiter plates were then incubated for 1 h at 12 \({}^{\circ}\)C to allow the microorganisms to adhere, and the wells rinsed once with 150 mM sodium chloride (NaCl, Sigma-Aldrich Co., Saint Louis, MO, USA) to eliminate any bacterium that were not adhering. A volume of 125 \(\upmu\)L of sterile TSB was then added in the case of the glass and graphene surfaces, and 250 \(\upmu\)L in the case of the polystyrene and resin. The plates were then incubated under static conditions without culture medium renewal for 120 h at 12 \({}^{\circ}\)C.  
Two dyes were used, SYTO9 and propidium iodide (PI). The first emits green fluorescence during CLSM imaging and is used to identify living microorganisms with an intact membrane. The second emits red fluorescence, which stains dead bacteria with damaged membranes. A volume of 2.0 \(\upmu\)L of a 1:1 mixture of SYTO9 (stock 3.34 mM in DMSO) and PI (stock 20 mM in DMSO) fluorescent dyes from the BacLight Bacterial Viability Kit (Invitrogen, Carlsbad, CA, USA) was added to 1000 \(\upmu\)L of TSB, and a measured quantity of the resultant solution was added to each well: 125 \(\upmu\)L for the glass and graphene, and 250 \(\upmu\)L for the polystyrene and resin. The plate was then incubated in the dark at 37 \({}^{\circ}\)C for 20 min to enable the fluorescent labelling of the bacteria.  
CLSM imaging was performed using a Zeiss LSM 800 Airyscan confocal laser scanning microscope with ZEN 2.3 software (Carl Zeiss, Jena, Germany). Channel mode visualization was performed using the \(63\times(0.8\) NA) objective with oil immersion. Table 1 shows the microscope parameters used.  
\begin{table}
\begin{tabular}{c c c} \hline  & \multicolumn{2}{c}{**Dye**} \\ \hline
**Parameter** & **SYTO 9** & **Propidium Iodide** \\ \hline Pinhole & 44 \(\upmu\)m & 44 \(\upmu\)m \\ Laser Wavelength & 488 nm: 0.20\% & 561 nm: 0.20\% \\ Excitation Wavelength & 483 nm & 305 nm \\ Emission Wavelength & 500 nm & 617 nm \\ Detection Wavelength & 450–560 nm & 560–700 nm \\ Imaging Device & LSM 800/GaAsP-Pmt2 & LSM 800/Airyscan \\ Detector & GaAsP & Airyscan \\ Detector Gain & 700 V & 700 V \\ \hline \end{tabular}
\end{table}
Table 1: Stains and microscope parameters used in studying _Listeria_ spp. biofilms.  
Three stacks of horizontal plane images (\(512\times 512\) pixels corresponding to \(126.8\times 126.8\)\(\upmu\)m) with a z-step of 1.0 \(\upmu\)m were acquired for each well from three different randomly chosen areas. Three independent experiments were performed for each strain and surface material. Thus, a total of 324 CLSM images were obtained (9 strains \(\times\) 4 surface materials \(\times\) 3 replicates \(\times\) 3 surfaces in each well). The original Zeiss files (CZI format) were imported into the IMARIS 9.1 software package (Bitplane, Zurich, Switzerland) to analyse the images [16].