## 1 Introduction
Ideally, these methods should be (i) fast, so that they can be used on site after cleaning and disinfection and before production resumes; (ii) easy to use, so that they do not require specialized personnel or equipment; (iii) non-expensive, accessible to industries of all sizes; and (iv) specific, so that more detailed information on the characteristics of the contamination can be obtained. In particular, detection of biofilms is critical due to the potential risk for food safety in production environments [17]. Conventional microbial sampling techniques such as swabing or ATP bioluminescence do not provide information on the presence of a biofilm. Confirmation of the presence of a microbial biofilm usually requires microscopic techniques, which are not useful for routine controls. In the past few years, there has been growing interest in the use of dye-based reagents to stain the biofilm matrix and thus reveal their presence [18]. Among these, TBF 300 (Betelgeux-Christeyns, \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Technique** & **Target for Detection** & **Cost per Sample \({}^{\star}\)** & **Ease of Use \({}^{\ddagger}\)** & **Specificity \({}^{\ddagger}\)** \\ \hline ATP bioluminescence & ATP molecules contained in living cells & Moderate & High & Moderate \\ Contact plating & Viable microorganisms & Low & High & High \\ Swabbing & Viable microorganisms & High & Low & High \\ Test strips & Residues of detergents or disinfectants & Low & High & High \\ Visual inspection & Visible residual soiling & Low & High & Low \\ \hline \hline \end{tabular} \({}^{\star}\)_Low:_ no or inexpensive materials (<1 \(\ell\)/sample) required. _Moderate:_ low cost for fungibles (<3 \(\ell\)/sample) but expensive equipment required. _High:_ Dedicated facilities and personnel required. \({}^{\star}\)_Low:_ requires personnel with specific training. _High:_ Basic training required. \({}^{\ddagger}\)_Low:_ Limited discrimination of contamination types. _Moderate:_ Discrimination between organic/biologic contamination and inorganic contamination. _High:_ A specific type of contamination is detected.  
\end{table} 
Table 1: Summary of techniques and methodologies currently employed for validation of hygiene in food production environments. Valencia, Spain) has been available in the market since 2011 as a rapid tool for specific identification of biofilms [18; 19]. TBF(r) 300 is based on Rhodamine B as a staining agent, which is a counterstaining dye and is reported to reveal the total extent of biomass, independent of its activity [20]. This product is applied as foam or sprayed onto sample points, where it is allowed to act for a few minutes and then rinsed. The composition of TBF 300 includes surfactants and auxiliary components to enhance foam performance and stability of the dye. The permanence of residual staining after the washing step is normally associated to the presence of biofilms on the surface [21; 22]. This product is recommended for application on metallic surfaces due to the risk of permanent staining of porous or some plastic surfaces, which would lead to false positives. A more recent tool of great interest for assessing the hygiene status of surfaces is FreshCheck (Fresh Check UK). This product is based on Chromeazurol S (CAS), a chromophore containing iron (Fe\({}^{3+}\)) that changes its color when the iron ion is removed from its structure [23]. Most microorganisms produce siderophores with high iron binding affinity, so they will sequester iron from CAS and thus produce a visible change in color [24]. The change in color can also occur in the presence of other residues usually found in the food industry, such as residual detergents or food soiling [25]. FreshCheck is sprayed onto test surfaces and results are obtained within 30 s, in the form of a color change easily visible with the naked eye. FreschCheck is purple when applied on clean surfaces and it changes to a range of colors (usually green or orange) when reacting with residual contamination.