The sections represent regions of biofilm containing structured networks of fibers and sheets, but few bacteria. (A) The walls consisted of thin laminar structures (arrowhead) with globular material (arrow) accumulating in branching regions; CFTRinh-172 molecular weight scale bar = 500 nm. (B) In other regions of the biofilm, the wall-like structures had different thicknesses. The thin walls (arrowhead) were attached to SC79 purchase thicker walls (arrow); scale bar = 500 nm. (C) Different wall morphologies consisted of thin, straight walls (arrowhead) branching from thicker walled structures (arrows); scale bar = 500 nm. (D) The thicker walls were composed of globular amorphous masses (arrows) covered in part

by a distinct coating (arrowheads); scale bar = 200 nm. (E) and (F) The different components of the thicker walls consisted of globular masses (arrows) separated by and covered with thin coatings (arrowheads); scale bar = 500 nm. Biofilms are chemically heterogeneous Hydrated biofilms from multiple cultures were combined taking care to minimize the inclusion of spent media without disturbing the fragile structures. No further handling of the biofilms was carried out prior to freeze-drying in order to preserve the chemical integrity of the structures. Physical or chemical treatments of the samples learn more such as centrifugation, filtration, extraction, and ion exchange chromatography have the potential to significantly alter the biofilm

composition, thus biasing the results of the chemical analysis. The method described here is simple, convenient, minimally invasive, and is designed to provide representative samples for compositional analysis. Hydrated biofilms (0.9189 g) afforded 15.6 mg of dry material (16.0 17-DMAG (Alvespimycin) HCl mg g-1) consisting of biofilm and spent media, where-as spent media free of biofilm (1.9255 g) afforded 10.8 mg of dry material (5.6 mg g-1). Assuming that the dry material makes up a negligible proportion (1.7% in the case of biofilm plus media) of the mass of the hydrated sample, the media contribution to the mixed sample was estimated as 5.2 mg (0.9189

× 5.6), or 33% [(5.2/15.6) × 100%]. Background contributions from spent media to the chemical sample make-up were subtracted from the mixed biofilm-media samples according to eq. 1. This simple relationship was employed throughout to estimate biofilm composition. Results of the biofilm chemical analyses are summarized in Table 1. Table 1 Biofilm chemical composition. Analyte Analysis method Mass concentration (μg mg-1)a Calcium ICP-AES 29.9 Magnesium ICP-AES 10.1 Total proteins UV absorption 490 Total proteinsb Folin reaction (Lowry assay) 240 Acidic polysaccharidesc Phenol-sulfuric acid reaction 79 Neutral polysaccharidesc Phenol-sulfuric acid reaction 67 Nucleic acids UV absorption 46 DNA DAPI-fluorescence 5.4 aDry material. bMeasured as BSA. cMeasured as dextrose monohydrate. The principal IR absorption bands of the mixed biofilm/media sample are presented elsewhere [see Additional file 1].