## 3 Results  
### Biofilm Staining  
Biomass concentration values for biofilms formed on stainless steel surfaces after 3 and 10 days are shown in Tables 3 and 4 and visually represented in Figure 1.  
Microscopic examination of the inoculated coupons shows that microorganisms were able to form biofilms on work surfaces up to \(10^{6}\) CFU/surface (Figure 2). \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{4}{c}{**10 Days Biofilm**} \\ \hline \multirow{2}{*}{**Microorganism**} & **Biomass** & **Biomass** & **Biomass (log** & **Biomass (log** \\  & **(CFU/surface) n = 3** & **(CFU/cm2) n = 3** & **CFU/surface) n = 3** & **Biomass (log** \\ \hline _Salmonella_ spp. & \(2.8\times 10^{6}\) & \(1.6\times 10^{5}\) & \(6.45\pm 0.70\) & \(5.2\pm 0.56\) \\ _L. monocytogenes_ & \(4.2\times 10^{6}\) & \(2.3\times 10^{5}\) & \(6.62\pm 0.04\) & \(5.4\pm 0.03\) \\ _Salmonella_ spp. + _L. monocytogenes_ & \(1.6\times 10^{6}\) & \(8.9\times 10^{4}\) & \(6.21\pm 0.25\) & \(5.0\pm 0.20\) \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Biomass values for biofilms after 3 days growth. Figure 1: Biomass of biofilms after 3 and 10 days, expressed as log CFU/cm2. Following optimization of biofilm growing conditions, biofilm staining tests were performed using the TBF 300 product. Both stable, strongly adhered biofilms and non-stable biofilms showing a smaller amount of biomass were stained. The product was applied as a foam over the test surface, covering the whole surface with a layer approximately 3-mm thick. A contact time of 15 min was allowed, followed by rinsing. The process is illustrated in Figure 3.  
Staining of biofilms grown over 3 days was found to be low, reflecting the fact that adherence of cells in these conditions is weak and the biofilm formed is removed from the surface with relative ease during the washing step. In this case, residual staining was only found in those areas where adhesion of microorganisms to steel is stronger, probably due to the presence of cracks or crevices. This occurs especially in edges and cutting points (see Figure 4). Figure 4: Residual staining after application of TBF 300 on a coupon where a _L. monocytogenes_ biofilm was grown for 3 days. Figure 3: Images showing the procedure for application of TBF 300 for biofilm staining, followed by rinsing and observation of residual staining. Figure 2: Microscopic image of _L. monocytogenes_ (**left**) and _Salmonella_ spp. (**right**) biofilms after LIVE/DEAD® BacLight staining and capture by confocal laser microscopy (60X magnification). Biofilms grown for 10 days showed stronger residual staining after the washing step. These biofilms are expected to contain a higher amount of biomass and show a stronger adherence to surfaces, hence allowing for increased visibility after staining (Figure 5). Coupons containing biofilm were also examined under UV light after staining with TBF 300 and washing. The dye contained in the product shows fluorescent properties under UV irradiation, which was shown to provide a better way for discrimination of biofilm-containing surfaces, as shown in Figure 6.  
These results show that TBF 300 can reliably reveal biofilm presence on stainless steel surfaces after its application in the described conditions. Staining intensity is greater as the age of the biofilm  
Figure 5: Residual staining after the application of TBF 300 on coupons where microbial biofilms were grown for 10 days. Each row corresponds to biofilms of different microorganisms: _Salmonella_ spp. (**upper row**), _L. monocytogenes_ (**central row**), mixed biofilm (**lower row**). Figure 6: Biofilm (10 days old) containing coupons under UV irradiation. Each row corresponds to biofilms of different microorganisms: _Salmonella_ spp. (**upper row**), _L. monocytogenes_ (**central row**), mixed biofilm (**lower row**). increases, as a result of a higher amount of matrix in the biofilm.