, 1998; Thurnheer

et al., 2004; Guggenheim et al., 2009). The biofilms were fixed in 4% paraformaldehyde (Sigma) for 1 h at 4 °C and washed once with PBS. Thereafter, the biofilm-associated microorganisms were permeabilized by exposure to lysozyme (Sigma; 70 000 U mL−1) for 2 min at room temperature and rinsed with physiological saline. FISH was carried out using a modification of a method previously described (Thurnheer et al., 2004). The biofilms were pre-incubated for 15 min at 48 °C in final hybridization buffer (0.9 M NaCl, 20 mM L−1 Tris–HCl Oligomycin A molecular weight pH 7.5, 0.01% SDS) containing 30% formamide and then placed for 3 h at 48 °C in the same solution with the oligonucleotide probes added (5 μg mL−1 for STR405 and LNA-Pging, 15 μg mL−1 for FUS664). After hybridization, the biofilms were immersed for 15 min at 48 °C in washing buffer (102 mM L−1 NaCl, 20 mM L−1 Tris–HCl 7.5, 5 mM L−1 EDTA, 0.01% SDS). Thereafter, the disks were embedded upside-down in 10 μL Mowiol mounting solution and stored at room temperature in the dark at least 6 h. Biofilms were examined using a Leica SP5® microscope (Leica, Wetzlar, Germany) fitted with

three lasers: He-Ne, argon and DPSS. Filters were set to 490–530 nm for FAM, 570–610 nm for Cy3, and 650–730 nm for Cy5. The fluorescence signal from Nutlin-3a research buy Cy5 was assigned to blue color for better differentiation from Cy3. Confocal images were obtained using a 63× (numeric aperture 1.4) oil immersion objective. Each biofilm was scanned at three random positions at the center of the disk. Z-direction series were generated by vertical optical sectioning at every position with the thickness of the slices set to 0.3 μm.

Proprietary Leica confocal software was used to acquire digital images of 1024 × 1024 pixels in size that were the average of 32 Etofibrate frames. The counts of the bacteria in the biofilm were made using image analysis software (Olympus AG, Volketswil, Switzerland) and verified manually on random views to exclude possible errors due to not counting bacteria present in bundles. The experiment was repeated twice, resulting in six disks that were scanned at three random position in the central area. Three milliliters of Columbia agar (BBL™; Becton Dickinson) supplemented with 5% human blood (Blutspendezentrum), 5 μg mL−1 hemin (Fluka, Buchs, Switzerland), and 0.5 μg mL−1 menadione (VWR International, Dietikon, Switzerland) were placed in sterile IMC ampoules and incubated anaerobically for 48 h. Specimens with the biofilms were placed in ampoules, enabling continuous contact between the biofilm and the agar. A sterile titanium disk with no biofilm on it served as the negative control. Each of the ampoules was immediately sealed under anaerobic conditions and inserted into one of the individual microcalorimeters in the 48-microcalorimeter instrument used (TAM 48®; TA Instruments, New Castle, DE).