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Quantification of biofilm structures by the novel computer program comstat
The structural organization of four microbial communities was analysed by a novel computer program COMSTAT which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria Pseudomonas putida P. aureofaciens P. fluorescens and P. aeruginosa tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure – mean thickness roughness substratum coverage and surface to volume ratio – showed that the four Pseudomonas strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum and formed flat uniform biofilms. P. aureofaciens resembled P. aeruginosa but had a stronger tendency to form micro-colonies. Finally the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0·03 mM 0·1 mM or 0·5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover biofilm roughness increased with lower citrate concentrations whereas surface to volume ratio increased with higher citrate concentrations.
Experimental reproducibility in flow-chamber biofilms
The structural organization of microbial communities is influenced by many factors e.g. nutrient composition shear stress and temperature. This paper presents a general method for quantitative comparison of biofilm structures and assessment of experimental reproducibility between independent biofilm experiments. By using a novel computer program COMSTAT biofilm structures of Pseudomonas aeruginosa and an isogenic rpoS mutant were quantified. The strains were tagged with the green fluorescent protein (GFP) and grown in flow chambers with a defined minimal medium as substrate. Three independent rounds of biofilm experiments were performed and in each round each of the two variants was grown in two separate channels. Nine image stacks were acquired in each channel 146 h after inoculation. An analysis of variance model incorporating the factors experiment round bacterial strain channel number and image stack number was used to analyse the data calculated by COMSTAT. Experimental reproducibility was verified by estimating the magnitude of the variance of the effects round () and the interaction between bacterial strain and round (). Mean thickness of the wild-type and rpoS mutant biofilms was estimated at 6·31 μm (SE 0·81 μm) and 16·85 μm (SE 0·87 μm) respectively.