## 3 Results
### Reproducibility and Confocal Laser Scanning Microscopy Images  
The monitoring of the evolution of the biomass and the population density showed a steady state after 24 h for biofilms obtained with inoculum level of \(10^{7}\) CFU/mL. A stationary phase is observed for the population density growth curve, and a plateau is observed (after the peak) in the optical density measurements. For this reason, the reproducibility of the obtained biofilm was studied specifically after 24 h of growth. The mean value of the optical density on ten independent replicates demonstrated to be \(4.95\pm 0.43\). Viable plate counts of 20 independent replicates resulted in an average population density of \(17.78\pm 0.44\). These data demonstrated a good reproducibility of the biofilm that is obtained after 24 h when following the developed protocol.  
The protocol yielded a fully mature biofilm, as demonstrated by confocal laser scanning microscopy. After fluorescent staining, 24-h-grown biofilms were visualised with CLSM. The estimated biofilm thickness was approximately 73 \(\upmu\)m. The three CLSM images (in Figure 6a-c), corresponding to half the depth of the biofilm (approximately 38 \(\upmu\)m), show the complex architecture of the biofilms. The blue dye (CFW) makes it possible to visualise the polysaccharides of the EPS, specifically 1-3 or 1-4 \(\upbeta\)-glycosic bonds between saccharides [6]. This represents the biofilm matrix. The green dye (SYTO9) is a nucleic acid stain that diffuses through cellular membranes; consequently, it binds to the DNA of both viable and dead cells. In fact, it also stains the extracellular DNA that is part of the EPS. The PI is an intercalating agent that stains the dead cells, penetrating their damaged membrane. The CLSM images served as a way of understanding the 3D structure, and the different components within the biofilm. Figure 4 represents the combination of the three laser channels (CWF, SYTO9 and PI) at the same depth of 38 \(\upmu\)m. The structure of the biofilm is displayed in the images (Figure 6a-d), where the  
Figure 6: Confocal laser scanning microscopy images of a z-stack in the centre of the biofilm: biofilm stained with (**a**) calcofluor white (CFW) solution displaying the EPS; (**b**) SYTO9 displaying the alive cells and extracellular DNA; (**c**) propidium iodide (PI) displaying the dead cells; (**d**) overlapping of the three channels of excitation for CFW, SYTO9 and PI; (**e**) z-stack 3D reconstruction of 24-h-grown biofilm. The bars represent 25 \(\upmu\)m in (**a**), (**b**) and (**c**), 100 \(\upmu\)m in (**d**).  
black circular spots represent the water channels. They clearly differ in the dimension of the pores and are essential for the transportation of nutrients. Figure 6c displayed larger dark areas, indicating that the dead cells are in lower quantity and are clumped together. Figure 6e displays a 3D reconstruction of the biofilm using z-stack acquisition. As can clearly be seen, the top part of the biofilms was mainly covered by EPS to protect the cells from external factors (blue), while the middle and bottom part, growing in contact with the polystyrene, was mainly composed by densely packed cells (green).