In the last few years, a suggested explanation for the long-term survival of biofilm population is related to the presence of a small fraction of the population that expresses a particularly recalcitrant physiology and a dormant state of high tolerance upon the application of stress. Such phenotypic variants might include the differential expression of efflux pumps. This project tried to find out the actual role of efflux pumps and other genomic expression in biofilm recalcitrance. In order to further understand the nature of bacterial chronicity, initially the project started with testing the possibility of using CellTrackers® blue 7-amino-4-chloromethylcoumarin (CMAC) and Acridine Orange to monitor the efflux pump activity in the wild type E. coli AG 100, AacrAB mutant 100A and A acrR mutant 100B. Instability and non-specificity of CMAC, the concentration dependent dimerization of Acridine Orange, the failure to distinguish between the efflux activity in 100A and 100B strains, and the strong likelihood of multiple efflux systems necessitated to change the approach and to use DNA transcript micro-arrays technologies to examine efflux pumps activity together with the effect of growth as a biofilm. Different models of perfused biofilm fermenters were investigated. The in-line filter fermenter and the Sorbarod biofilm device showed ability of growth rate control, enough biomass for RNA extraction, but the biofilms formed in both models were heterogeneous. The re-designed Sartorius 47 mm in-line biofilm fermenter succeeded to provide sufficient bacterial biomass samples of growth rate controlled biofilm (c. 1.4-1.9xl09 cfu/membrane) and its spontaneously dispersed daughter cells (c. l.OxlO7cfu/ml). E, coli AG100 was grown at p 0.25 hr'1 and 0.07 hr"1. Planktonic cells growing in chemostat chamber at the same growth rates were used as control. Triplicate samples of planktonic, biofilm and spontaneously dispersed daughter cells were collected immediately in RNA protect reagent®. RNA extraction was earned out using Qiagen® RNAase mini kit, 4 pg was the total RNA yield per membrane filter. DNA transcript micro-arrays were performed using the anti-sense Affymetrix® gene chip of E. coli kl2. ORFs (736) were found to be significantly and differentially expressed either between planktonic and biofilm at the same growth rate or between biofilm and its correspondely newly formed daughter cells. Of these were intergenic regions (36.5%) and hypothetical proteins (27%). The reminders (268 ORFs) are responsible for efflux pump transporter, motility, attachment, DNA replication and cell division, cold shock proteins, heat shock protein, anaerobic respiration, lipopolysaccharide metabolism, prophages, cpx regulon, sigma 54, outer membrane proteins, rpoS, toxin Anti-toxin, general stress response, and other miscellaneous ORFs. It was noticed that ABC efflux pump transporters were found to be up-regulated at the outer layer of the biofilm community. On the other hand, secondary transporters were up-regulated in deeper layers. This could support the theory that H+ pockets are more predominant in deeper layeisof the biofilm consortium which provides the H+ antiporter efflux system with H+ as fuel for efflux process. Outer membrane proteins OMP and/sA both participate in bacterial resistance, were found to be dominant in slow growing cells. This confirms the theory that slow growing cells are more resistant. Finally, SirA might play a major role in biofilm dispersion.