2B). When specimen preparation led to breaks in this structure,

the biofilm core was exposed (Fig. 2C) and consisted of small numbers of bacteria embedded AZD2014 in a matrix of fibers and particulate matter aggregating on the fibers (Fig. 2C). In other parts of the biofilm, the fibers were more apparent and formed irregular, net-like structures (Fig. 2D). At higher magnification it was possible to see that the fibers were organized into ordered networks of periodic nets. These nets contained few bacteria (Fig. 2E) and were covered by thin sheets of material similar to that observed around the bacteria embedded in the particulate matter (Fig. 2F). Figure 2 Scanning electron micrographs of P. fluorescens EvS4-B1 biofilms (14 days) prepared using cryomethods. (A). Fibrillary structures appeared to be made up of twisted fibers (arrow) scale bar = 1 μm. (B). Flat sheets of material (arrowhead) also were observed. Some of the sheets seemed to be wrapped around other structures (arrow); scale bar = 20 μm. (C) The inside core of the “”wrapped”" structures consisted of bacteria, [B], embedded in an extracellular matrix of particulate matter and a thin sheet of material

(arrow); scale bar = 1 μm. (D) The outer sheet (arrowheads) enveloped an inner core consisting of fibers forming irregular network-like structures (arrow); scale bar = 10 μm. (E) The Ku-0059436 chemical structure network consisted of fibers arranged in a periodic pattern. The bacteria (arrows) were two to three times larger than the spaces in the network; scale bar = 2 μm. (F) A sheet of material, [S], covered the fiber

network and was attached to it. The fibers were associated with bacteria, [B], and particulate matter, [P]; scale bar = 2 μm. The ultrastructures observed by SEM are not artifacts resulting from sample preparation The transmission electron Acetophenone microscopy (TEM) images of the embedded biofilms (Fig. 3) are consistent with the corresponding SEM data (Fig. 2) and therefore validate the ultrastructural organization observed in the SEM suggesting that they did not result from sample preparation. The honeycomb-like structures, as well as the morphology of the partitions, are clearly visible using both techniques. The structures appeared to have two types of walls. Either it was thin with a smooth surface, or it was thicker and made up of globular structures (Fig. 3D–F). The thicker walls, although smooth on the surface, were of variable thickness giving them a bumpy appearance (Fig. 3D–F). The section staining revealed separations between the components of the thicker walls and globular masses separated by thin sheets (Fig. 3E–F). No obvious freezing damage due to ice crystal formation was observed suggesting that the EM data presented here are of real ultrastructural features in the biofilms and are not the result of eutectic crystallization. Figure 3 Transmission electron microscopy images of P. fluorescens EvS4-B1 biofilms (21 days).