FIG 7.

Inhibition of biofilm formation by QC. (A) NBC099 cells were grown on glass coverslips under aseptic and humid conditions in the presence or absence of QC. Degrees of biofilm formation were assessed in terms of cell viability in biofilm cultures with the LIVE/DEAD cell viability kit (Life Technologies). Live SYTO 9-stained cells were visualized by LSCM with an FITC (excitation and emission wavelengths of 480 and 500 nm) filter. (B) Cells grown on glass slides were washed with PBS and stained with 0.2% CV solution for 5 min at room temperature. After the cells were dried, biofilm formation was visualized by differential interference contrast microscopy and photographed. (C) NBC099 cells were grown in a 96-well flat-bottom microplate in the presence or absence of QC for 48 h. The medium was then discarded, and the cells were sterilized by adding 70% ethanol to each well for 1 min and washed twice with distilled water. Cells were stained by adding 0.2% CV solution for 15 min. After the microplate was dried, isopropanol containing 0.04 N HCl and 0.25% sodium dodecyl sulfate was added and the A₅₉₀ was measured. Results are expressed as percentages of inhibition relative to the control. Each value is the mean result ± the standard deviation of three independent experiments. (D and E) Exponentially growing NBC099 cells that had been exposed to QC (D) and farnesol (E) for 48 h were harvested, washed, fixed in 2.0% glutaraldehyde, and then postfixed in 1% osmium tetroxide buffer. After dehydration in a graded ethanol series, the cells were embedded in spur resin and sections were cut on an Ultramicrotome. The sectioned grids were stained with saturated solutions of uranyl acetate and lead citrate and examined by SEM. (F) Sensitivity of QC-treated NBC099 biofilms to FCZ. NBC099 biofilms were grown in the absence or presence of QC. After 72 h, the biofilms were exposed to FCZ for 36 h. Cell viability was assayed by staining with the LIVE/DEAD Cell Viability kit. PI-stained areas are dead cells, and SYTO 9-stained areas are live cells, as analyzed by LSCM.