# Resistance of \(L\). _monocytogenes_ and \(S\). Typhimurium
## 3 Results and Discussion  
Influence of the Biofilm Age on the Total Biofilm Mass and the Cell Density of the Untreated biofilms  
The total biofilm mass and the cell density of the untreated _L. monocytogenes_ biofilms following 1, 2, 3, 7, and 10 day(s) of incubation at 30 \({}^{\circ}\)C have been determined and the obtained results have been presented in Figures 2a and 3a, respectively. Based on Figure 2a, it can be concluded that the total biomass of the _L. monocytogenes_ biofilm does not change until three days of incubation. However, as the biofilm ages even more (i.e., following seven and 10 days of incubation), the OD values have increased significantly. Since the crystal violet stains both the matrix and the cells [29], this indicates a higher production of EPS matrix by the biofilm-associated cells and/or an increased cell density. The increased cell density can, however, not be confirmed based on the results obtained in Figure 3a. The _L. monocytogenes_ cell density initially remains (for both media) constant until three days of incubation. However, following seven and 10 days of incubation, the cell density has decreased significantly for both media. Consequently, the increased total biomass following seven and 10 days of incubation is the result of an increased production of EPS matrix. It is, however, important to notice that there is a high variability in OD for the seven and 10 days old _L. monocytogenes_ biofilms (Figure 2a), which can be explained based on visual observations when performing the crystal violet staining. The 7 and 10 days old biofilms were less strongly attached to the surface, resulting in some parts of the biofilm that got rinsed off prior to the staining. Consequently, these detached parts were not included anymore in the quantification procedure. For _S._ Typhimurium, similar experiments were performed, i.e., the total biofilm mass and the cell density of the (untreated) biofilms following 1, 2, 3, 7, and 10 day(s) of incubation at 25 \({}^{\circ}\)C were determined and the obtained results have been presented in Figures 2b and 3b, respectively. In contrary to the results that were obtained for _L._ monocytogenes, no significant differences can be observed between the OD values obtained for all different _S._ Typhimurium biofilm ages (Figure 2b), meaning that the total biofilm mass does not change as a function of the biofilm age. Consequently, the amount of produced EPS matrix and the number of cells are not expected to change as function of the _S._ Typhimurium biofilm age. Nevertheless, in Figure 3b, it can be observed that the cell density prior to CAP treatment does change as function of the biofilm age. Until 3 days of incubation, the biofilm age does not significantly influence the cell density prior to CAP treatment. However, upon further incubation (i.e., following seven and 10 days of incubation), the cell density (prior to CAP treatment) has decreased significantly compared to the one-day old (reference) biofilm, although not significant for the selective medium following 10 days of incubation. The decrease in cell density following seven and 10 days of incubation is not expected since the total biofilm mass of the untreated _S._ Typhimurium biofilms does not change as function of the biofilm age (Figure 2b). The (significantly) lower cell densities for the seven and 10 days old _L. monocytogenes_ and \(S\). Typhimurium biofilms can be the result of biofilm-associated cells that have died due to starvation, i.e., following seven and 10 days of incubation, many of the available nutrients have been consumed. However, it could also be the result of detachment of (some parts of) the biofilm and cells becoming planktonic again [30].