Time-lapse microscopy methods were used to monitor growth, survival and death of Salmonella enterica serotype Agona individual cells on solid laboratory medium (tryptone soy agar) in the presence of various salt concentrations (0.5%, 3.5%, 4.5% and 5.7% NaCl). The results showed a highly heterogeneous behavior. As NaCl concentration increased, the distribution of the first division time was shifted to higher values and became wider. The mean first division time increased from 1.8 h at 0.5% NaCl to 5.48 h, 16.2 h, and 35.9 h at 3.5%, 4.5% and 5.7% NaCl, respectively. The concentration of NaCl in the growth medium also affected the ability of the cells to divide. The percentage of cells able to grow decreased from 88.9% at 0.5% NaCl to 66.5%, 32.8%, and 6.9% at 3.5%, 4.5% and 5.7% NaCl, respectively. In the latter case (5.7% NaCl), 74 cells out of 406 cells tested (18%) died with mean time to death 5.03 h and standard deviation 6.70 h. To investigate the effect of the behavior of individual cells on the dynamics of the whole population, simulation analysis was used. The simulation results showed that the simultaneous growth, survival and death of cells observed under osmotic stress can lead to a total population behavior known as the "Phoenix" phenomenon. The simulation findings were confirmed by validation experiments using both viable counts and time lapse microscopy. The results of the present study show the high heterogeneity of individual cell responses and the complexity in the behavior of microbial populations at conditions approaching the boundaries of growth.

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Since biofilm development represents a crucial issue within industrial, clinical and domestic sectors, innovative technologies/approaches (e.g., light technology for inactivation, antibiofilm coatings) are required to eradicate them. In this multidisciplinary scenario, protocols for the development of biofilms are necessary, particularly, in laboratories (not specialised in biofilm science) lacking in sophisticated devices for their growth. A protocol was developed for growing Pseudomonas fluorescens (Gram-negative) biofilms on wide, flat, polystyrene surfaces within 24 h. Several factors, such as inoculum level, area size and growth medium concentration, were investigated. Biofilm development was studied via viable cells and biomass quantification. A comparative analysis between kinetics and growth parameters, estimated using the Baranyi and Roberts model, was conducted at different inoculum levels (104 and 107 CFU/mL). The inoculum levels did not influence the final population within the 24-h-grown biofilms, but they influenced the total biomass development, which followed different kinetics. Confocal laser scanning microscopy confirmed that overnight growth allowed for development of a densely packed biofilm with its 3D structure. The developed protocol was validated for Staphylococcus epidermidis (Gram-positive). The present work is the first study to develop an easy-to-use protocol to obtain highly reproducible biofilms, on flat polystyrene surfaces, with no need for sophisticated technologies.

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