## 3 Results and Discussion
### Biofilm Assays on SSC
#### 3.3.2 Biofilm Logarithmic Reduction after BAC Exposure (1280 mg/L, 5 min)  
The susceptibility of biofilms formed under different BAC sub-inhibitory concentrations was determined by logarithmic reduction (the difference between Log CFU/cm\({}^{2}\) values of cellular enumeration--biofilm formation--and the corresponding Log CFU/cm\({}^{2}\) values obtained after exposure to BAC, equaling 1280 mg/L, 5 min). For strain S2-1, the presence of sub-inhibitory concentrations of BAC did not have a significant effect on biofilm susceptibility (\(p>0.05\)). However, for the BAC-R strain S2\({}^{\text{BAC}}\), the presence of a sub-inhibitory concentration of BAC (1.25 mg/L) resulted in higher resistance of the biofilm (\(p<0.05\)) (Figure 1b).  
Biofilm formation in microtiter plates has many well-known advantages and is certainly the most commonly used method for biofilm quantification. However, major drawbacks of 96-well microtiter plates are the nutrient depletion during the incubation period, and the fact that biofilm biomass is not solely formed as the result of a biofilm-forming process but also from cells sedimented to the bottom of the wells [23; 28]. In the case of the crystal violet assay, the dye binds to negatively charged surface molecules and polysaccharides in the matrix, and the assay may be influenced by the amount of expocellular polymer and by cell sedimentation, which increases with planktonic growth [30].  
Figure 1: Antibiotic of benzalkonium chloride (BAC). (**a**) Biofilm cell enumeration: comparison of the biofilms formed by cell enumeration on stainless steel coupons, after incubation for 48 h at 25 \({}^{\circ}\)C, under different sub-inhibitory BAC concentrations. (**b**) Biofilm logarithmic reduction: comparison of the log reduction, after BAC exposure (1280 mg/L, 5 min), of the biofilms formed under different sub-inhibitory BAC concentrations. The presence of an asterisk in the bar indicates significant differences from the other conditions (\(p<0.05\)). The values shown are averages of four replicates. Standard deviations are indicated.  
Biofilms developed on stainless steel were reported to be more resistant to BAC than its planktonic cell's counterparts [16; 28]. This methodology is closer to the conditions in the food industry than methods based on surface materials other than stainless steel. Biofilms formed on SSC showed a susceptibility that depended on the sensitivity of the strain and on the sub-inhibitory concentrations of BAC used to grow the biofilms. The results in this study for the strain S2\({}^{\text{BAC}}\), when compared to using the results of crystal violet staining [19], suggest that the increase in resistance may be related to a greater number of extracellular polymers in the biofilm matrix of this strain [31]. In fact, when biofilms were examined through confocal laser scanning microscopy, it was found that exposure to sub-MICs of BAC increased biofilm development by S2\({}^{\text{BAC}}\) and reduced biofilm by S2-1 [20]. It is therefore important to understand how the strain susceptibility and growth conditions differently regulate the biofilm-forming ability and the biofilm resistance of _L. monocytogenes._