4D–F). These cords appeared to be embedded in aggregates of bacteria that did not label with Con A. The structures that labeled with Con A in other regions of the biofilm appeared diffuse and were not easily identified (data not shown). Discussion A bacterial species from an extreme environment rich in toxic compounds was isolated into axenic culture and grown in the laboratory. During the course of these studies, it was observed that the isolate produced atypical growth curves and formed a macroscopic structure tethered to the bottom of the culture tubes. These biofilms were unusual as they did not consist of the typical mucoidal material,

but were made up of well-defined solid structures. Confocal laser scanning microscopy confirmed that these mature structures contained significant NCT-501 zones of physiological Trichostatin A activity. Physical and chemical characterization of the mature biofilms was carried out and is discussed below.

When examined by light microscopy, bacterial cultures reproducibly contained similar structural motifs that were composed of viable bacteria as well as dead cells and extracellular material. At the macroscopic level, delicate flocculent material of what appeared PF 01367338 to be bacterial aggregates was enveloped by a network of fibers. Smaller fibers branched from this central core in a microscopic analogue to tree branches emanating from a trunk and surrounded by foliage (i.e., the bacterial aggregates). Each culture tube also contained one complex three-dimensional structure that resembled a parachute. At higher magnification using the confocal microscope, the thick fibers in the flocculent material appeared tightly coiled. The tightly coiled structures contained bacteria and had an affinity for fluorescently-labeled concanavalin A (conA).

These results suggest that there are specialized zones within the biofilm consisting of bacteria associated with extracellular proteins. The presence of bacterial aggregates in the biofilm that did not label with con A suggests that at least part of the extracellular material contains glycoproteins. Rapid freezing of biofilms followed by freeze substitution aminophylline and epoxy resin embedding of the specimens enabled examination of thin sections through biofilms that had been minimally disturbed [35, 36]. Cryofixation followed by freeze-substitution has been shown to be a highly effective method for preserving biofilm organization for EM examination [37]. It is well known, however, that freezing can lead to structural artifacts [38] and that highly hydrated structures such as biofilms will collapse to some extent during sample preparation that involves dehydration. These distinct features must be recognized to avoid misinterpretation of the images.