Quantification of biofilm structures by the novel computer program COMSTAT

The structural organization of four microbial communities was analyzed by a novel computer program, COMSTAT, which comprises ten features for quantify ing 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 th e COMSTAT program of four variables describing biofilm structure - mean thi ckness, roughness, substratum coverage and surface to volume ratio showed t hat the four Pseudomonas strains represent different modes of biofilm growt h. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies, which were eventually succee ded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum and formed flat, uniform biofilms. P. aureofaciens re sembled P. aeruginosa, but had a stronger tendency to form micro-colonies. Finally, the biofilm structures of P. fluorescens had a phenotype intermedi ate 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 conce ntration. Moreover, biofilm roughness increased with lower citrate concentr ations, whereas surface to volume ratio increased with higher citrate conce ntrations.