Pc 2

Generally, biofilm formation could reach a relatively stable state after 72 h inoculation in microtiter plates.^{16,26,31 (link)} To understand the chlorine resistance of the five different bacteria, the 72 h incubated biofilms were used in chlorination experiments. The chlorination experiments were performed in 250 μL PBS (pH 7.0) free from chlorine. Briefly, NaClO (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) was diluted with PBS to create a working solution with chlorine concentrations of 0.3 mg L⁻¹, 0.6 mg L⁻¹, 1 mg L⁻¹, 2 mg L⁻¹, 4 mg L⁻¹ and 10 mg L⁻¹, which were calibrated using a residual chlorine meter (PC II, HACH, USA). The negative control comprised of only 250 μL of PBS, and the positive control was each species of biofilm cultured in 250 μL of PBS without chlorine. Biofilm chlorination experiments were conducted in a shaking incubator at 150 rpm at 25 °C to ensure sufficient contact between the working solution and biofilm. Fresh working solution was gently changed every 20 min during the chlorination period of 1 h. Wells were rinsed two times with 250 μL 0.5% (w/v) NaS₂O₃ to stop the chlorination reaction, and then rinsed one time with sterilized PBS to remove non-adherent and loosely attached planktonic bacteria in each well. The plates were then air-dried at a clean bench for 30 minutes for further analyses.