Acrylic discs were incubated with fetal bovine serum (FBS) (Gibco; Life Technologies, Carlsbad, CA, USA) in a shaker incubator (Excella E24 Incubator Shaker Series; New Brunswick Scientific, Enfield, CT, USA) at 75 rpm and 37 °C for 24 h. Then, specimens were washed once with 0.1 M PBS, and placed in 24-well plate. Dual-species biofilms were developed by adding 500 µL of each cell suspension in a well of 24-well plate. The plates were incubated aerobically in a shaker incubator at 75 rpm and 37 °C for 24 h.
After dual-species biofilm growth for 24 h, each biofilm sample was gently washed with 1000 µL of 0.1 M PBS twice for 10 s each time to remove any non-adherent cells. The samples without further exposed to test agents were used as the untreated control. For the other groups, biofilm samples were immersed in each test agent as mentioned above for 3 and 15 min, except 3 min-Compound 1 group was treated by spraying according to a previous study22 (link). Following exposure to test agents, residual biofilm samples were washed twice with 1000 µL of 0.1 M PBS.
After dual-species biofilm growth for 24 h, each biofilm sample was gently washed with 1000 µL of 0.1 M PBS twice for 10 s each time to remove any non-adherent cells. The samples without further exposed to test agents were used as the untreated control. For the other groups, biofilm samples were immersed in each test agent as mentioned above for 3 and 15 min, except 3 min-Compound 1 group was treated by spraying according to a previous study22 (link). Following exposure to test agents, residual biofilm samples were washed twice with 1000 µL of 0.1 M PBS.
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