A cell suspension consisting of 106 cells/ml was incubated with various concentrations of antibiotics or AgNPs, or combinations of AgNPs with an antibiotic for 4 h at 37°C. After incubation, bacteria were harvested at the indicated time points and 100-μl aliquots were taken from each sample to determine the number of colony-forming units (CFUs). Experiments were
performed with various controls including a positive control (AgNPs and MHB, without inoculum) and a negative control (MHB and inoculum, without AgNPs). All samples were plated in triplicate and values were averaged from three independent experiments. The experiments with sublethal concentrations of antibiotics or AgNPs, or combinations of AgNPs and antibiotics, were performed for 4 h at 37°C. Determination selleck of biofilm activity using the tissue culture plate method
(TCP) selleck compound This assay was performed to determine the ability of AgNPs to inhibit biofilm activity. The assay is based on colorimetric measurements of the crystal violet incorporated by sessile cells [22, 23]. Briefly, individual wells of sterile, 96-well flat-bottom polystyrene TCPs were filled with 180 μl of a single bacterial species (1 × 106/ml). After culturing for 24 h, different concentrations of AgNPs were added. The cell culture plates were then incubated for 4 h at 37°C. For combination experiments, bacteria were treated with sublethal concentrations of antibiotics, or individual antibiotics in combination with AgNPs. After incubation, the media were removed and the wells were washed three times with 200 μl sterile distilled water to remove non-BLZ945 adherent bacteria. The wells were air dried for 45 min and 200 μl per well of a 0.1% (v/v) crystal violet solution in water were added for 45 min. The wells were then washed five times with 300 μl of sterile distilled water to remove excess stain. The dye incorporated by the adherent cells was solubilized with 200 μl of 95% (v/v) ethanol. The absorbance of each well was
measured at 595 nm using a microtiter ELISA reader. The absorbance difference between treated and control wells was considered as an index of bacterial adherence to the surface and thus the activity of biofilms. RANTES The percentage inhibition of biofilm activity was calculated using the following equation: [1 - (A595 of cells treated with AgNPs/A595 of non-treated control cells)] × 100 [24]. Experiments were performed in triplicate. The data are expressed as means ± SD. Measurement of reactive oxygen species (ROS) generation An assay for superoxide anions was carried out according to the manufacturer’s instructions (In Vitro Toxicology Assay Kit, (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)-carbonyl]-2H-tetrazolium inner salt (XTT) based, catalog number TOX2), was purchased from Sigma-Aldrich, USA. All test strains were grown in MHB.