Biofilm formation The influence of NOS-derived NO on biofilm formation was tested by investigating the morphology and fine structure of spot colonies grown on MSgg fortified with 1.5% Protein Tyrosine Kinase inhibitor agar. Additionally, the amount of vegetative cells and spores in biofilms grown on the liquid-air

interface (‘pellicles’) in MSgg medium was quantified. Both agar and medium were supplemented with sterile filtered (0.2 μm, Spartan, Millipore, Schwalbach, Germany) 100 μM L-NAME, 75 μM c-PTIO or 130 μM Noc-18 after autoclavation. Colony morphology was investigated in 6-well microtiter plates (Nunclon Surface, Nunc, Denmark) and colony fine structure was investigated in Petri dishes (Sarstedt, Nümbrecht, Germany). The wells of the microtiter plates were filled with 6 mL and the Petri dishes with 25 mL MSgg agar. After the agar dried for ~ 16 h at room temperature (RT), 5 μL of a LB-grown overnight culture was spotted on the agar surface, dried open for 10 min in a laminar flow hood, and incubated at 26°C. Fine structure of 3 days old colonies was visualized

by illuminating the sample with an external light source (swan neck lamp, KL 1500 electronic, Schott, Mainz, Germany) and capturing reflected light with a DS-Q1-MC CCD camera (Nikon, Japan) mounted on a light microscope (DM RA2, Leica, Solms, Germany) equipped with Leica 5× HKI-272 NA0.15 HC PL Fluotar lens. Whole colony morphology was documented with a digital camera after 4 days of growth. Pellicle formation was quantified in glass test

tubes containing 25 mL MSgg medium. MSgg tubes were PCI-34051 ic50 inoculated with 25 μL of mid-exponential phase culture and incubated for 7 days at 26°C without agitation. Directly after the inoculation 980 μL medium was removed from the tube and subjected to NO staining with CuFL as described above. During the course of biofilm formation 3 vials of each treatment per day were sacrificed for determination Montelukast Sodium of viable cell and spore counts. Biofilms were homogenized in the MSgg medium by sonication (Labsonic U, B. Braun, Melsungen, Germany) for 10 min at ~ 40 W on ice. The cells were plated on LB agar, and incubated 24 h at 26°C to determine the number of colony forming units (cfu). Spore counts were determined from the same samples by subjecting a part of the homogenates to pasteurization for 20 min at 80°C in a water bath prior to plating. O2 and NO concentrations in the biofilm incubations were measured with microsensors as previously described [43, 44]. Swarm expansion assay Swarm experiments were conducted as described by Kearns and Losick [13]. Briefly, cells grown in LB at 37°C to the mid-exponential phase were harvested by centrifugation (15 min, 4000 RCF, 15°C) and re-suspended in phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4) containing 0.5% ink. Swarm plates were prepared in Petri dishes (diameter = 8.5 cm) by pouring 25 mL LB fortified with 0.