Growing reproducible biofilms with respect to structure and viable cell counts

Abstract

We have developed a new method of growing 4-day-old biofilms that are reproducible, with respect to viable cell number and biofilm structure. To demonstrate the utility of the method, we grew biofilms composed of Pseudomonas aeruginosa (ATCC#700829), P. fluorescens (ATCC#700830) and Klebsiella pneumoniae (ATCC#700831), 18 times in flat-plate reactors under well-defined conditions of: flow rate, nutrient concentration, temperature, inoculum and growth rate. The resulting 4-day-old biofilms were approximately 2 µm thick and exhibited a high degree of reproducibility. The number of viable cells that accumulated per unit surface area and the biofilm areal porosity were reproduced within 10% error. We have also quantified other parameters characterizing biofilm structure using biofilm-imaging techniques: fractal dimension, textural entropy and diffusion distance as auxiliary parameters characterizing the reproducibility of biofilm accumulation. As a result of analysis, we have introduced a new parameter to better quantify and characterize the number of viable cells in biofilms, "specific number of viable cells" (SNVC). This parameter is the viable cell number normalized with respect to the surface area covered by the biofilm and with respect to the biomass of the biofilm. This new descriptor represents the dynamics of biofilm accumulation better than the traditionally used colony-forming unit (CFU) per surface area covered by the biofilm because it accounts not only for the surface coverage but also for the biofilm thickness.