In addition, Otto and Silhavy described increased expression of Cpx-regulated genes upon 1 h contact of Escherichia coli with artificial surfaces as compared to planktonic cells maintained in suspension; surprisingly, this regulation was observed with stationary phase cells in contact with a SR-58611A hydrophobic surface only. Lately, Li and co-workers showed, in Caulobacter crescentus, that formation of physical contact between the bacterium and an artificial surface triggered ‘‘just-in-time’’ adhesin production. These results suggest that bacterial cells possess a ‘tactile’ machinery which signals formation of surface contact. However, the functional responses put forward in these experiments have also been shown to be upregulated in stationary phase cell populations and in bacteria subjected to various external stresses – e.g. nutrient deprivation, medium pH or osmolarity changes – raising the question of the direct relationship of these signals with formation of surface contact. Here we develop an experimental approach aimed at addressing this question in a configuration which enables simultaneous detection of permanent physical contact and relevant biological activity at the single cell level. The principle of the experiments consisted in using dispersed surfaces in the form of micrometric latex particles as an adhesive substrate brought into contact with GFP-expressing bacterial cells in suspension so as to generate a microsystem in which adherent cells co-exist with single planktonic and aggregated cells. The system can then be characterized using flow cytometry, enabling multi-parametric short-time-scale analysis of the mixture. To detect the impact of initial adhesion on cell metabolic activity, we used a fluorescent marker of bacterial respiration, a tetrazolium ion the fluorescence of which can be directly related to cell metabolic activity. The experiments were performed in an E. coli strain constitutively expressing GFP and curli – a surface multimeric protein structure that fosters surface attachment and self-association. The results indicated that bacterial metabolic activity was affected by formation of a single micrometric contact at the cell surface, either with a synthetic surface or with another cell, as early as the first ten minutes of permanent contact formation, suggesting that bacteria have developed an efficient and fast sense of touch. Interestingly, we observed that both cell-cell and cell-synthetic substrate contact triggered a similar metabolic drop. The implications of these findings on the potential existence and possible nature of a bacterial sense of touch will be discussed below. Clarification of these questions will be useful for a better understanding of the physiological shift induced by bacterial cell development on surfaces, a longstanding concern in microbiology. In order to expose the early bacterial cell response to adhesion, we implemented a strategy consisting of using dispersed surfaces as the adhesive substrate and flow cytometry multiparametric analysis. This approach carried out using GFP-expressing bacteria provides large statistics data sets and time resolution on the order of seconds to determine cell-surface adhesion kinetics. To achieve quantitative monitoring of cell respiration at a single cell level, we first searched for a means of taking advantage of the multiparametric nature of flow cytometry to design a two-color approach to bacterial respiration. For this purpose, we introduced into the experiment a fluorescent marker of bacterial respiration, a tetrazolium ion the fluorescence of which can be directly related to its level of reduction in the electron transfer chain of the bacteria, and thus to the metabolic activity of the cells under study. In its oxidized form, the CTC dye is a non-fluorescent molecule. In contrast, when the compound is reduced via the cell membrane electron transfer chain – in competition with molecular oxygen – it is converted into a water-insoluble product exhibiting characteristic red fluorescence whose intensity inside the bacterial cell reports the amount of reduced product and thus the cell respiration level, as previously reported. In principle, it is possible, using flow cytometry, to measure the individual cell capacity to reduce CTC and to obtain distribution of this property over a perfectly dispersed cell population. Yet, as soon as cells assemble, the single cell information is lost, except if an internal standard enables counting the number of individuals in the assemblage. For this purpose, we analyzed in parallel the flow cytometry profiles of an exponentially growing E. coli cell population constitutively expressing GFP and incubated with CTC. Exponentially growing bacteria cultivated at 37uC under stirring were incubated with 5 mM CTC for 30 min just before the flow cytometry test. In parallel, aliquots of the same culture were incubated with Sytox red as a dead cell marker. Incubation time was chosen on the basis of CTC reduction kinetics in cells that indicated that a pseudo-plateau had been reached at that time. The same series were prepared with cells taken out of the incubator and left at room temperature for 2 h before the test and with cells previously fixed in 3.7% formaldehyde solution in PBS for 1 h at room temperature. FCM dot plots and histograms of GFP, CTC and Sytox red fluorescence intensities collected in their respective emission channels, i.e. FL1, FL3 and FL4, versus forward scattering, are shown in Fig. 1. Subcellular debris was removed from the analysis by gating the data to an SSC+ region, corresponding to SSC values higher than 10. Then, we defined for each fluorescence channel a threshold value delimiting positive and negative regions with respect to the considered marker and we determined the corresponding FCM parameters, cell percentage and fluorescence intensity.