Scanning confocal laser microscopy (SCLM) was used to visualize fully hydrated microbial biofilms. The improved rejection of out-of-focus haze and the increased resolution of SCLM made it preferable to conventional phase microscopy for the analysis of living biofflms. The extent of image improvement was dependent on the characteristics of individual biofilms and was most apparent when films were dispersed in three dimensions, when they were thick, and when they contained a high number of cells. SCLM optical sections were amenable to quantitative computer-enhanced microscopy analyses, with minimal interference originating from overlying or underlying cell material. By using SCLM in conjunction with viable negative fluorescence staining techniques, horizontal (xy) and sagittal (xz) sections of intact bioMflms of Pseudomonas aeruginosa, Pseudomonasfluorescens, and Vibrio parahaemolyticus were obtained. These optical sections were then analyzed by image-processing techniques to assess the distribution of cellular and noncellular areas within the biofilm matrices. The Pseudomonas biofllms were most cell dense at their attachment surfaces and became increasingly diffuse near their outer regions, whereas the Vibrio biofilms exhibited the opposite trend. BioMflms consisting of different species exhibited distinctive arrangements of the major biofilm structural components (cellular and extracellular materials and space). In general, biofilms were found to be highly hydrated, open structures composed of 73 to 98% extracellular materials and space. The use of xz sectioning revealed more detail of biofilm structure, including the presence of large void spaces within the Vibrio biofilms. In addition, three-dimensional reconstructions of biofilms were constructed and were displayed as stereo pairs. Application of the concepts of architectural analysis to mixed-or pure-species biofilms will allow detailed examination of the relationships among biofilm structure, adaptation, and response to stress.Biofilms are organized multicellular systems with structural and functional architecture which influence metabolic processes, response to nutrients, resistance to antimicrobial agents, predation, and other factors. Structural studies of microbial biofilms and their formation have been performed by using light microscopy to examine wet mounts (16,23), by using transmission and scanning electron microscopy (12)(13)(14)(15)24), and by developing conceptual models (12). Electron microscopy techniques are laborious and can produce artifacts resulting from sample preparation and limit three-dimensional (3D) reconstruction of biofilms. Light microscopy used in conjunction with computer-enhanced microscopy (CEM) is an effective tool, but it is best applied during the early phases of biofilm development (7,16,19,20). Scanning confocal laser microscopy (SCLM) offers the promise of detailed visualization of thick microbiological samples in cases in which application of traditional phase or fluorescence microscopy is limited. SCLM allows eliminat...

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Pseudomonas aeruginosa 579 biofilms formed on dialysis membranes retarded piperacillin diffusion.

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The peptidoglycan of Escherichia coli K-12 strain AB264 was isolated by treating whole cells with sodium dodecyl sulfate and was purified by deoxyribonuclease, ribonuclease, and trypsin treatment. Like the peptidoglycan of Bacillus subtilis, this peptidoglycan proved able to bind substantial amounts of metallic ions from aqueous solution. In particular, most metals of the transition I series were bound from solution in amounts greater than or equal to 1 mumol/mg dry weight peptidoglycan.