Biofilm Architecture Analysis via CLSM

To determine the biofilm architecture, biofilms were grown in 24-well plates in 5-fold diluted LB medium under shaking conditions (220 rpm), with the growth medium being exchanged every 12h. Confocal laser scanning microscopy (CLSM) images were acquired using a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany). Prior to microscopy, biofilms were stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies). Quantitative analysis of the confocal laser scanning microscope images of 24-well plate-grown biofilms was performed using COMSTAT analysis (Heydorn et al., 2000 (link)). For RNA isolation, biofilms were grown at 22°C in 20-fold-diluted LB medium, using a continuous flow tube reactor system (1-m-long size 13 silicone tubing; Masterflex, Cole Parmer, Inc.) with an inner surface area of 25 cm² at a flow rate of 0.1 ml/min, as previously described (Sauer et al., 2002 (link), Sauer et al., 2004 (link), Petrova & Sauer, 2009 (link)).