*2.2. Antibacterial Activity of TiO2 in the Dark*

TiO2 nanoparticles (KRONOClean 7050) were suspended in 1/500 NB [48] at the concentration of 13.9 g/L. Eleven milliliters of the suspension were then deposited onto a sterile Petri dish, so that the total area of the inside part of the dish was covered. The Petri dishes were placed in a sterile flow hood for air drying until the water had totally evaporated. A film of TiO2 was visible at the bottom. Then, 11 mL of the inoculum (between 8 × 104 and 2 × 10<sup>5</sup> cells/mL) were deposited on the TiO2 film, and the Petri dishes were covered with a lid [48]. After a fixed time (0 and 24 h), the lid was removed, the bottoms of the Petri dishes were gently scraped with a plastic loop in order to remove any adhered cells and 1 mL of the suspension was collected and diluted in phosphate buffer. Control samples were studied in Petri dishes without TiO2.

One-mL quantities of the appropriate dilutions were then dropped into distilled sterile water and filtered on cellulose ester filters (ࢥ = 0.45 ȝm) in order to separate bacterial cells from nanoparticles. The filters were then deposited on trypticase soy agar 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 CFU/mL.
