## 4 Discussion
This may be because TSBS has lower nutrients such as amino acids, proteins, and minerals that allow the adhesion and formation of a biofilm; however, TSBA can also contain other nutrients such as saturated, polyunsaturated, and monounsaturated fatty acids that can influence the formation of a biofilm. [PERSON] et al. [47] reported that biofilm formation by _Salmonella_ Typhimuriumium was higher when grown in lettuce lysates with low sodium than when grown in high-nutrient LB broth. Likewise, the aggregative fimbraise of _Salmonella_ spp. are expressed in response to nutrient limitation, under conditions of low osmolarity and low growth temperature, as well as in the stationary phase of growth [48]. Furthermore, in our study, we incorporated TSBA and TSBS in order to simulate the accumulation of organic materials in washing water, as surfaces that come into contact with freshly cut food produce are associated with a poor sanitization process in the commercial fresh produce industry. In fact, food is rich in nutrients and suitable for the growth and reproduction of pathogens; in solid or viscous food, bacteria can easily adhere to the surface of food materials and food processing equipment and can eventually form a biofilm [49]. Hence, operating procedures for cleaning and disinfection must be implemented to prevent the formation or maturation of a biofilm, an accumulation of organic material that can affect the hygienic state of a surface [15]. Our results of epifluorescence microscopy reveal that the biofilm structure is made up of microcolonies of metabolically active cells with the presence of EPS or the organic matter from TSBA or TSBS medium (Figure 2A-F). In addition, acridine orange is intercalated with the RNA, indicating metabolically active cells [24; 25]. SEM analysis of biofilms formed by representative mono-species _Salmonella_ showed that cells were linked to each other and embedded in dense EPS (Figure 3A,B). However, the components that make up the EPS depend on the microbial species and in particular on their genetics and metabolic pathways, and the concentration of these can vary according to the various carbon or nitrogen sources in the culture medium where the biofilm develops [50]. Therefore, the EPS are the biofilm structure, and these can influence bacterial attachment and transport mechanisms indirectly by impacting both the cell surface hydrophobicity and the surface charge [51]. This can be relevant from a food safety perspective as in this study, the estimation of biofilm formation by _Salmonella_ serotype strains shows they have the capacity to form biofilms on PP and polystyrene. Our results agree with studies by other researchers who showed that _Salmonella_ is able to form biofilms on polyethylene bags, PVC pipes, plastic surfaces, polystyrene, stainless steel, etc. [16; 37; 46; 52]. Hence, biofilms are reservoirs that may be present on food processing and food contact surfaces, subsequently acting as a potential source of direct and indirect contamination by the introduction of foodborne pathogens into the fresh produce industry. _Salmonella_ serotype biofilms can cause negative impacts in the fresh produce industry, such as reduced operational efficacy in heat exchangers, accelerated metal corrosion, and resistance to the disinfectant used in sanitation procedures. Our results emphasize the importance of incorporating other techniques, such as confocal laser scanning microscopy (CLSM) to determine the components that make up the EPS of biofilms, for implementing strategies for the prevention and removal of biofilms in the fresh produce industry. However, further study is needed to better understand how medium nutrient composition, environmental factors, and strain characteristics influence adherence and biofilm formation by _Salmonella_ serotypes.