ORIGINAL ARTICLE Measurement of biofilm formation by clinical isolates of Escherichia coli is method-dependent P. Naves 1,2 , G. del Prado 1 , L. Huelves 1 , M. Gracia 1 , V. Ruiz 1 , J. Blanco 3 , V. Rodrı´guez-Cerrato 1 , M.C. Ponte 1 and F. Soriano 1 1 Department of Medical Microbiology and Antimicrobial Chemotherapy, Fundacio ´ n Jime ´nez Dı´az-Capio, Madrid, Spain 2 Unit of Exact and Technological Sciences, Universidade Estadual de Goia ´ s, Ana ´ polis, Brazil 3 Laboratorio de Referencia de E. coli (LREC), Department of Microbiology and Parasitology, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain Introduction Biofilms are microbial communities that grow attached to surfaces and ⁄ or interfaces; they are embedded in a fre- quently self-produced matrix of extracellular polymeric substances, and exhibit an altered growth rate and gene transcription (Donlan and Costerton 2002). The medical and environmental impacts of microbial biofilm have led to an expanding investigation of the biology and regula- tory mechanisms of biofilm formation and dispersal (Jef- ferson 2004; Pru ¨ß et al. 2006). Several factors have been implicated in the development of a model biofilm system, such as media composition, temperature, presence of antimicrobial agents, the causal organism, quantity of inoculum, hydrodynamics forces, and characteristics of the substrata (roughness, chemistry and conditioning films) (Donlan and Costerton 2002). Because of diffusion limitations inherent to biofilm structure that result in local variations in pH, nutrient and oxygen availability and concentrations of microbial metabolites, the gene expression is heterogeneous (Hancock and Klemm 2007). In the laboratory, four general systems have been routinely used to study bacterial biofilm formation: (i) biofilm grown in flow cells devices analysed by confocal scanning laser microscopy; (ii) batch cultures under static conditions, generally in microtitre plates, visualized using a nonspecific dye; (iii) floating biofilms or liquid-air pelli- cles; and (iv) images of bacterial colonies on surface of agar-solidified media (Branda et al. 2005). Besides, bio- film studies are confounded by the intrinsic limitations of the in vitro biofilm models and techniques available to study the roles of the involved genes (Jefferson 2004). Keywords biofilm, Escherichia coli, methodology, microtitre plate, static assay. Correspondence Francisco Soriano, Department of Medical Microbiology and Antimicrobial Chemotherapy, Fundacio ´ n Jime ´nez Dı´az- Capio, Madrid, Spain. E-mail: fsoriano@fjd.es 2007/1794: received 8 November 2007, revised 19 December 2007, accepted 22 January 2008 doi:10.1111/j.1365-2672.2008.03791.x Abstract Aims: In this study, we have evaluated the impact of methodological approaches in the determination of biofilm formation by four clinical isolates of Escherichia coli in static assays. Methods and Results: The assays were performed in microtitre plates with two minimal and two enriched broths, with one- or two-steps protocol, and using three different mathematical formulas to quantify adherent bacteria. Different biofilm formation patterns were found depending on the E. coli strain, culture medium and reading optical density on one- and two-steps protocol. Strong or moderate biofilm formation occurred mostly in minimal media. The mathe- matical formulas used to quantify biofilm formation also gave different results and bacterial growth rate should be taken into account to quantify biofilm. Conclusions: Escherichia coli forms biofilms on static assays in a method- dependent fashion, depending on strain, and it is strongly modulated by cul- ture conditions. Significance and Impact of the Study: As verified in the studied E. coli strains, biofilm formation by any organism should be cautiously interpreted, consider- ing all variables in the experimental settings. Journal of Applied Microbiology ISSN 1364-5072 ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 585–590 585