## 4 Conclusions  
In this study, two novel tools commercially available for validation of hygiene control in food industries have been evaluated. Both detection of microbial contamination at very low levels and identification of biofilm presence in a rapid and easy way are very significant for microbiological control in food industries. With such tools, a quality supervisor can react immediately to hygiene results and optimize cleaning and disinfection procedures to achieve total elimination of microbial contamination. These new techniques can be of great help to complement current methodologies for  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Microbial Concentration (CFU/mL)** & **Contact Slide** & **FreshCheck** \\ \hline \(10^{6}\) & \(>\)300 & Green \\ \(10^{5}\) & \(>\)300 & Green \\ \(10^{4}\) & \(>\)300 & Green \\ \(10^{3}\) & 52 & Green \\ \(10^{2}\) & - & Green \\ \(10^{1}\) & 2 & Green \\ \(0\) (control) & - & Purple \\ \hline \hline \end{tabular}
\end{table}
Table 6: FreshCheck capacity to detect different levels of microbial contamination of _L. monocytogenes_ in comparison with Contact Slide.  
Figure 8: Surfaces after application of FreshCheck showing a negative result (**upper,** no contamination) and a positive result (**lower,** presence of microorganisms).  
improved assessment of hygienic status in food industries. Their speed and ease of use allows their use before starting the production processes, contributing to guarantee food safety of food products that reach the market.  
TBF 300 was able to visibly stain biofilms at least 3 days old for the microorganisms tested. Cells' attachment to surfaces occurs within a few hours, with mature biofilms starting to be formed between 48-72 h depending on the environmental conditions [7; 28]. Therefore, TBF 300 can be used for the identification of spots where hygiene procedures are not effective, allowing contamination to accumulate over time and leading to the formation of microbial biofilms. The identification of such spots reveals deficiencies in the hygiene plan and corrective measures can be planned and implemented accordingly.  
Biofilm detection is complemented with the use of FreshCheck for generic identification of residues after cleaning and disinfection. The results of this study show that FreshCheck can react with viable microorganisms at very low levels of contamination, so that the efficacy of the hygiene process can be evaluated on the spot with high sensibility. ATP-bioluminescence methods are usually employed for this purpose because of their sensitivity, speed and ease of use. However, they require purchasing expensive equipment for reading bioluminescence results. In contrast, FreshCheck avoids the need for dedicated equipment and is therefore a cheaper approach that can potentially provide more relevant results in the presence of stressed cells without requiring special equipment.  
Research on hygiene validation methodologies continues to advance with the aim of providing faster, easier and more informative tools for detection of contamination in food processing environments. The combined use of these tools can help quality supervisors to continuously improve their control on hygiene procedures, with more information on the characteristics of the contamination found and thus potential sources of contamination and corrective measures. Microbial contamination, in the form of non-attached cells or biofilms, is of high concern for food safety, and most control methods available are either non-specific or require long processing times. The two novel tools evaluated in this study improve the process of hygiene validation and prevention of food contamination.