*2.5. Residual Antibacterial Activity of the Treated Water*

Qualitative assays were performed to assess the residual antibacterial activity of the treated water after photocatalytic degradation against the fully susceptible test organisms *Staphylococcus aureus* (MTCC code 3160) and *Escherichia coli* (MTCC code 7410) from the Microbial Type Culture Collection (MTCC, Chandigarh, India). The well diffusion method, according to the Clinical and Laboratory Standards Institute (CLSI) [17,18] was employed. All plates were prepared in 90 mm sterile Petri dishes (Tarsons, Mumbai, India) with 22 mL of Luria Bertani agar, yielding a depth of 4 mm. Test microorganism's 100 μL of inoculum suspensions (OD600-0.5, corresponding to 1.0 × 108 CFU mL−1) were poured into the agar plates when the temperature reached around 40–45 ◦C using a sterile micropipette, and homogenized thoroughly by mixing in a circular motion (pour-plate technique). After solidification, roundwells (6.0 mmin diameter) were punched into the seeded agar plates with a 6 mm cork borer. The wells were filled with 40 μL of the treated water samples (collected and filtered after regular time intervals) using a sterile micropipette. These plates were allowed to stand at 4 ◦C for 2 h and then incubated at 37 ◦C for 24 h. Three sets of simultaneous controls were used. One control was the organism control and consisted of a seeded Petri dish with no photocatalytically treated antibiotic sample. In the second control, samples were introduced in the holes of unseeded Petri dishes to check for sterility. Finally, to ensure the elimination of any solvent effect, wells filled with 40 μL of sterile double distilled water were run simultaneously as a third control. The diameters of the inhibition zones (zone of inhibition—ZOI) were measured in millimeters [19]. Each test was repeated six times and the mean values from the replicates along with standard error of mean (SEM) were calculated.
