5 g/L YE broth at 1.9 ml/min (residence time 185 m). A diagram of
the CDC reactor system as it was used for this study is available from the manufacturer at http://www.biosurfacetechnologies.com. After 24 h of culture under these conditions, one coupon holders was again replaced HSP990 order aseptically, and examined by epifluorescence microscopy. After 48 h of continuous culture, all remaining biofilm coupons were removed and examined by epifluorescence microscopy. Viability Staining The biofilms on disks in batch culture were examined by epifluorescence microscopy using the BacLight viability staining kit (L-7012, Invitrogen). Staining was performed by covering the inward face of the glass coupon in the stain mix in a sterile 12 well plate, and washing with sterile water after the appropriate time. Five minutes with a
concentrated stain mix (1.5 μl of each stain per ml) was found to be sufficient. Stained glass coupons were mounted on cleaned glass slides, and observed by epifluorescence microscopy using an Axioplan 2 microscope (Carl Zeiss, NY) equipped with appropriate filter sets (41002, 41017, Chroma Technologies), and an Xcite-120 illuminator (Exfo Life Sciences, Ontario, Canada). Images were captured using an SBIG 1402-XME (Santa Barbara Instruments, Santa Barbara, CA mounted on a 1× NU7026 ic50 c-mount adapter, with a 0.2 second exposure. The monochrome images were captured using the CCDops software supplied with the camera. Captured images were merged using ImageJ http://rsb.info.nih.gov/ij/. The camera ccd was cooled maximally for all fluorescence imaging (20°C below ambient). Whole image contrast and brightness enhancement was used to optimize for publication only. Visible light
imaging Still images from swarming plates and time lapse movies were captured with a CoolSnapFX (Roper Scientific) cooled ccd camera using ImagePro MC Express on a Zeiss Axioplan 2. Biofilms were examined using 1% Crystal Violet as a simple stain. Color images were captured using a Kodak DC290 digital camera, using the Kodak image capture software provided. Macroscopic colony images and wetting Tenoxicam agent images were collected using a Fuji FinePix 5700 digital camera. Colonies were photographed using a black velvet cloth to damp reflection. To capture images of the wetting agent, the plate was illuminated using diffuse reflected light, and angled to capture the refractive quality of the layer. For all microscopy, calibration images were captured with all microscope lenses of a stage micrometer, and Image J was used for measurement and scaling. Results Swarming motility Our laboratory developed a swarming agar plate based on previous growth and swarming experiments in V. paradoxus and P aeruginosa. Our swarming agar used for initial studies used 0.5% agarose to solidify the plate, the freshwater media (FW) base previously used by Leadbetter and Greenberg , with 0.2% glucose as a carbon source. Previous work in P.