Bacteria growing in vitro form biofilms with reproducible macroscopic features Initially, axenic cultures of the bacterial isolate propagated exponentially, but the optical density of the growth medium started to decline significantly 24 h following inoculation [see Additional file 1]. The drop in planktonic bacterial numbers, estimated by optical density, coincided with the formation of macroscopic opaque structures in the bottom of the culture tube. These structures had a diaphanous, gossamer appearance [see Additional

file 2] and consisted of a dense, fibrillary core, with interdispersed white flocs I-BET-762 molecular weight that usually were anchored firmly to the bottom of the tube when grown as standing cultures; in shaking cultures, the material was commonly detached from the bottom of the tube. It was concluded that the structures in the bottom of the tubes were biofilms. Examination of the mature (between 1 and 3 weeks old) hydrated biofilms in a dissecting microscope revealed macroscopic features that were reproducible from culture to culture. An aggregation of delicate flocs of opaque material made up the bulk of the biofilm volume (Fig. 1A and 1B). Tethered to this construct via a thin cord was a parachute-like CFTRinh-172 ic50 appendage DMXAA in vivo approximately 2 mm in diameter (Fig. 1C) that consisted of material resembling fibrous sheets (Fig. 1D). While each culture only contained one of these highly unusual parachute-like

structures, they were consistent macroscopic biofilm features

when P. fluorescens EvS4-B1 was grown in minimal media. Glutaraldehyde fixation of the biofilms led to rapid dissolution of the flocculent material and slowly dissolved the fibrous, string-like core. The parachute-like appendage was the only biofilm component that remained after aldehyde fixation and subsequent staining and dehydration. Figure 1 P. fluorescens EvS4-B1 biofilms (21 days) contain macroscopic 3-dimensional structures. (A) Gentle disruption of the biofilm revealed a fragile mass of amorphous material connected to a parachute-like structure. (B) The structures were either well-defined packets (arrowheads) or aggregated flocs (asterisk) anchored to a fibrillary core (arrow). (C) The parachute-like structure was made up of 5 or 6 compartments. next (D) Backlighting highlighted the fibrous nature of the parachute-like structure (arrow). Scale bars = 1.5 mm. Biofilms formed by the bacterial isolate have a complex ultrastructural morphology P. fluorescens EvS4-B1 biofilms were prepared for SEM analysis using cryomethods. Conventional aqueous cross-linking and contrasting agents, such as glutaraldehyde and osmium tetroxide, were not used because of the structural disruption we observed under the dissection microscope as described above. Low magnification SEM examination of the prepared biofilms revealed unique structural features (Fig. 2). Running through the biofilm were cords of twisted material (Fig. 2A).