2.3.1. With TiO2 Powder

The experiment was based on the standards JIS Z 2801 (Japanese Industrial Standard) and ISO 27447 [48,49]. TiO2 nanoparticle powder (KRONOClean7050–anatase) was suspended in 9 mL of 1/500 NB [48], and 1 mL of the bacterial suspension (Section 2.1) was added. Final concentrations were 1 g/L for TiO2 and between 8 × 104 and 2 × 105 CFU/mL for bacteria. The bacterial suspension (Section 2.1) without TiO2 was used as a control. Then, 0.4 mL of the inoculum were instilled onto a Pyrex Petri dish designed so that an external ring could receive 2 mL of a supersaturated saline solution (KNO3) to maintain 90% RH and was covered with a Pyrex lid (Figure 1). The Petri dishes were placed in a sterile flow hood and illuminated with an 8-W black-light bulb. After a few minutes, the TiO2 nanoparticles were observed to have sedimented at the bottom of the drop.

A Soybean Casein Lecithin Polysorbate 80 Medium, also known as SCDLP broth, was prepared in sterile distilled water as recommended in standard JIS Z 2801 [48], using casein peptone, soybean peptone, sodium chloride, disodium hydrogen phosphate, glucose, lecithin and Tween 80.

**Figure 1.** Schematic illustration of the deposited-drop experiments with TiO2 powder and TiO2 semi-transparent coatings (**a**) and stirring experiment (**b**).

After different contact times (2 h, 4 h, 6 h), the suspension was washed out with the appropriate amount of SCDLP broth and with sterile glass beads (*d* = 4 mm). When necessary, the washed-out suspension was diluted X times in a phosphate buffer, so that it contained 30 to 300 cells per mL. For each sample, 1 mL of the appropriate dilution was dispensed into two sterilized Petri dishes with 15 to 20 mL of trypticase soy agar (TSA) and incubated at a temperature of 36 °C ± 1 °C for 40 to 48 h. After incubation, the number of viable cells was estimated in terms of CFU. The overall procedure was also systematically carried out immediately after inoculation (*t* = 0 h) to validate the test. The antibacterial activity was then calculated as the difference between the average logarithm of the number of viable bacteria on the control without TiO2 and the average logarithm of the number of viable bacteria on the TiO2 sample:

$$A = \log\left(N\_{\text{TiO}\_2}\right) - \log\left(N\_{\text{control}}\right) = \log\left(\frac{N\_{\text{TiO}\_2}}{N\_{\text{control}}}\right) \tag{1}$$

where, A: antibacterial activity; *N*TiO2: average number of CFU on TiO2 sample at time t; *N*control: average number of CFU on control sample at time *t.*

The test was then repeated with a transparent film (9–10 cm2 ) gently placed on the inoculum before irradiation in order to increase the probability of contact between bacteria cells and TiO2 nanoparticles (forced contact).
