aeruginosa cells were diluted 1 : 100 with an overnight culture in LB and cultured with and without indole derivative (1 mM) at 37 °C for 12 h with shaking at 250 r.p.m. Cell culture (100 μL including cells and culture supernatant) was added into diluted human red blood cells that had been separated previously by centrifugation at 900 g for 5 min, washed with phosphate-buffered saline (PBS) buffer three times and diluted at 3% of red blood cells in PBS buffer. For hemolytic activity, the mixture was incubated
at 37 °C for 1 h with shaking at 250 r.p.m. The supernatant was collected by centrifugation at 1600 g for 10 min and the optical density was measured at 543 nm. Except for the pyoverdine assay, overnight cultures were diluted 1 : 100 after growth in LB medium and incubated with indole derivatives (1 mM) or DMSO as a control. The 2-heptyl-3-hydroxy-4(1H)-quinolone selleck chemical (PQS) assay was adapted from Attila et al. (2008): after growth for 12 h, culture supernatants were extracted with acidified ethyl acetate and analyzed by thin layer chromatography. The pyocyanin assay of P. aeruginosa was adapted from Essar et al. (1990): after growth for 12 h, culture supernatants were extracted with chloroform and analyzed spectrophotometrically.
The rhamnolipid assay of Wilhelm et al. (2007) was adapted: after growth for 12 h, culture supernatants Nivolumab were assayed for rhamnolipids using the orcinol colorimetric assay. The pyochelin assay was adapted from Gupta et al. (2011): after growth for 12 h, culture supernatants were assayed for pyochelins using the nitrite-molybdate reagent. The pyochelin concentration was measured
spectrophotometrically at 310 nm. At least two independent experiments were conducted. For the pyoverdine assay, minimal succinate medium Cobimetinib datasheet and 0.5 mM indole derivatives were used due to growth delay with 1 mM. After 12 h growth in the minimal medium, the pyoverdine concentration was measured spectrophotometrically at 405 nm (Stintzi et al., 1998). To examine the polymeric matrix production, SEM was carried out following a protocol outlined in the literature (Lee et al., 2011). Briefly, a nylon filter was cut into 0.5 × 0.5 cm pieces and placed in 96-well plates with 300 μL of cells with an initial turbidity of 0.05 at 600 nm. The cells and the nylon filters were incubated together to form biofilm cells at 37 °C for 24 h without shaking. Afterwards the cells were fixed with glutaraldehyde (2.5% in the final concentration) and formaldehyde (2% in the final concentration) and incubated at 4 °C overnight. The biofilm cells grown on the nylon filters were examined using an S-4100 scanning electron microscope (Hitachi, Japan) at a voltage of 15 kV and magnifications ranging from ×2000 to ×10 000. Protease activity was determined using skim milk agar plates (Quiblier et al., 2011) containing 5 g of nonfat dry milk (skim milk) and 0.5 g of Bacto-agar in 50 mL of distilled water. Culture supernatants (30 μL) of P.