Sterile cell scraper

To prepared biofilms, MRSA was inoculated at approximately 5 × 10⁵ CFU/mL in 6-well plates (Corning, New York, NY, USA) containing 22 mm × 22 mm plastic slides (VWR) and CAMHB with 2% NaCl at 37°C for 96 h without shaking. CAMHB containing 2% NaCl with 4 μg/mL methicillin was combined with MIC or double MIC of BPE and incubated at 37°C under shaking at 120 rpm for 1 and 24 h. For numerical analysis, MRSA cells were recovered with sterile cell scraper (VWR, PA) from the plastic surface and plated on BP.
For visualization, treated biofilms on plastic slides were stained with FilmTracerTM LIVE/DEAD Biofilm Viability Kit (Life Technologies, OR). Slides were viewed at room temperature using a Zeiss AxioObserver. Z1 inverted microscope, and images were acquired using the Zeiss Axiovision Rel 4.6 software with the Zeiss Axiocam HRC camera. All exported images were processed in Microsoft Office Picture Manager (Version 2010). RNA was also extracted from the bacterial cells of treated MRSA biofilms on plastic slides and used for quantitative RT-PCR assay.

The ability of EHEC to form biofilms on glass surfaces in the absence or presence of CFCSs and/or BPEs was performed following the method previously described [16 (link)]. Briefly, 100 µl of EHEC, containing approximately 5 × 10⁵ CFU/ml, was inoculated in triplicate in wells of 6-well plates (Corning, USA) containing 22 × 22 mm² glass slides. Wells containing LB broth (control) or LB broth supplemented with different concentrations of BPEs (0.1 mg/ml GAE or 0.5 mg/ml GAE or 1.0 mg/ml GAE), or CFCSs with BPEs were incubated for 48 without shaking at 37 °C. Then, the glass slides were rinsed with PBS for five times and bacterial cells were recovered using sterile cell scraper (VWR, USA) from the glass surface and enumerated.

The human embryonic stem cell (hESC) line WA09 (H9) was obtained from WiCell. The ES cell line was maintained in feeder-free conditions using mTESR1 (StemCell Technologies), and passaged onto Matrigel (Corning)-coated tissue culture dishes every 4–5 days (Ludwig et al., 2006 (link)). For passaging, H9 cells were treated with Gentle Cell Dissociation Reagent (StemCell Technologies) for 6 min and then lifted from the plate using sterile cell scrapers (VWR). The split ratio was 1:50. The passage number of the cells used in this study was between 50–60.