Downloaded from www.microbiologyresearch.org by IP: 54.157.13.203 On: Tue, 09 Feb 2016 06:19:24 Microbiology (2000), 146, 2395–2407 Printed in Great Britain Quantification of biofilm structures by the novel computer program COMSTAT Arne Heydorn, 1 Alex Toftgaard Nielsen, 1 Morten Hentzer, 1 Claus Sternberg, 1 Michael Givskov, 1 Bjarne Kjær Ersbøll 2 and Søren Molin 1 Author for correspondence : Søren Molin. Tel : 45 45 25 25 13. Fax: 45 45 88 73 28. e-mail : imsmpop.dtu.dk 1 Molecular Microbial Ecology Group, Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark 2 Department of Mathematical Modelling, The Technical University of Denmark, DK-2800 Lyngby, Denmark 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. Keywords : Pseudomonas, biofilm structure, image analysis, quantification,  INTRODUCTION In most natural, clinical and industrial settings, bacteria often grow attached to surfaces in communities known as biofilms. Biofilm-associated organisms are able to adapt to environmental changes by altering their gene expression and general physiology (Davies et al., 1993 ; Davies & Geesey, 1995 ; O’Toole & Kolter, 1998 ; Pratt & Kolter, 1998 ; Prigent-Combaret et al., 1999), including increased resistance to antibiotics (Anwar et al., 1992; Brooun et al., 2000 ; Costerton et al., 1999). One of the ways in which microbial communities adjust to environmental changes is by changing the struc- tural organization of the biofilm (Dalton et al., 1994; Woolfaardt et al., 1994 ; Van Loosdrecht et al., 1995; Møller et al., 1997 ; Nielsen et al., 2000). However, ................................................................................................................................................. Abbreviations : CSLM, confocal scanning laser microscopy ; GFP, green fluorescent protein. the underlying mechanisms for the initiation of biofilm formation and development of structural organization are only just beginning to be discovered. Attempts to describe biofilm structures quantitatively include analyses of thickness variability in Pseudomonas aeruginosa (Stewart et al., 1993), mean thickness and roughness of monoculture and binary biofilms of P. aeruginosa and Klebsiella pneumoniae (Murga et al., 1995), fractal dimension of activated-sludge biofilms (Hermanowicz et al., 1995), and density, porosity, specific surface area and mean pore radius of het- erotrophic wastewater biofilms (Zhang & Bishop, 1994). Korber and co-workers determined the minimum area size that should be investigated in order to obtain representative data of P. fluorescens biofilms (Korber et al., 1992, 1993). Lewandowski et al. (1999) defined four variables (textural entropy, area porosity, fractal di- mension and maximum diffusion distance) for analysing two-dimensional biofilm images. All four variables were 0002-4186  2000 SGM 2395