Analysis of spatial variability within mot⁺and mot⁻pseudomonas fluorescensbiofilms using representative elements

The spatial variability for most measurable parameters contained within biofilms is very large. Therefore a procedure for determining statistically representative regions of analysis is desirable. Scanning confocal laser microscopy, a computer‐controlled xy stage, and fluorescence exclusion staining were used to obtain a series of optical thin sections of biofilms formed by motile (mot+) and nonmotile (mot−) Pseudomonas fluorescens on the surfaces of glass flow cells. Based on a representative elementary area (REA) analysis procedure, the images were used to construct montages large enough to encompass the range of variation in biofilm cell area. The minimum area of analysis required to be representative varied with depth in the biofilm and between the strains used. Biofilms consisting of mot− P. fluorescens were variable. Thus, large area (REA ≥ × 105‐μm−) were required for statistically valid comparisons of cell distribution. REAs for the mot+ biofilm reflected a more uniform distribution of cells at all depths (REA >6 x 104um"); however, significant differences between the mean cell areas at the 0, 8 and 16 μm sampling depths were still observed (range = 4.8 ± 3.1 to 15.1 ± 7.8 μm‐50 μm−2). Phenotypic heterogeneity was evident in mot− biofilms in the form of cell filaments, which were absent in the mot− biofilms. Filament formation in pseudomonads is a response to oxygen limitation, suggesting microscale variation of this parameter within the biofilm. The existence of microenvironments was further supported by the observation that the concentration of fluorescein molecules within cell masses in the mot− biofilm reached only 57% of the macroenvironment fluorescein concentration. In addition, topographical analyses showed that mot+ biofilms were significantly deeper than mot”; biofilms (42 ± 19 μm vs 19 ± 10 μm, respectively). This factor, in conjunction with an increase in the amount of cell material and associated polymers at all biofilm depths may have resulted in hindered diffusion of oxygen. These studies also confirm that cellular motility plays an important role during cell positioning and formation of biofilm microenvironments, which in turn may induce changes in cell morphology and metabolism. These observations may be relevant where predictive models have been based on unrepresentative biofilm regions or assumptions that the biofilm was isotropic.