2.3.2. With TiO2 Semi-Transparent Coating

The deposited-drop experiment was repeated with semi-transparent coating formulated using TiO2 nanoparticles as an antibacterial product: TiO2 powder (KronoClean 7050, KRONOS/Société Industrielle du Titane, Paris, France) and TiO2 dispersion (Kronos type 7454, trial product, KRONOS/Société Industrielle du Titane, Paris, France). The coating formulation included water and acrylic based on the work of Martinez *et al.* [46], as shown in Table 1. Sterilized cover-glasses (26 × 76 mm2 ) were covered with the coatings by instilling 1 mL, so that the total area of each glass was coated. The cover-glasses were then placed under a sterile flow hood for air drying. After drying, the semi-transparent coatings with TiO2 powder (STC-SP (with silicates), STC-P) were gently sanded with fine sandpaper in order to prevent the possible inclusion of nanoparticles in the binder. The semi-transparent coatings with TiO2 in aqueous suspension (STC-A) were pre-aged by irradiating them with UV light (2.5 W/m2 ) for 80 h. The amount of TiO2 was estimated at 2.5 mg/cm2 for samples coated with TiO2 powder (STC-SP, STC-P) and 0.63 mg/cm2 for samples coated with TiO2 aqueous suspension (STC-A). In order to evaluate the possible inclusion of nanoparticles in the binder of STC-A, samples were also prepared with water and TiO2 aqueous suspension, without acrylic resin (STC-A2). For each test sample, corresponding controls were prepared in the same way with water and acrylic resin, but without TiO2.


**Table 1.** Formulation of semi-transparent coatings. STC: semi-transparent coating.

The inoculation suspension (of Section 2.1) was diluted to make the concentration of inoculum 8 × 104 to 2 × 10<sup>5</sup> CFU/mL. The coated cover-glasses were placed over the internal ring of the Pyrex Petri dishes shown in Figure 1. Relative humidity was maintained with 2 mL of the supersaturated saline solution (KNO3) deposited in the external ring of each dish. Then, 0.4 mL of the inoculum were instilled on each coated cover-glass, and a transparent plastic film was applied, spreading the inoculum over a surface area of 10 cm2 . The Petri dishes were then covered with a Pyrex lid (Figure 1), placed in a sterile flow hood and illuminated with an 8-W black-light bulb.

After different contact times (2 h, 4 h, 6 h), the cover-glasses were recovered with sterile pliers and placed in plastic Petri dishes for wash-out. The wash-out of bacteria cells and the following procedures for CFU counting were repeated as in Section 2.3.1.
