A Reproducible Method for Growing Biofilms on Polystyrene Surfaces: Biomass and Bacterial Viability Evolution of Pseudomonas fluorescens and Staphylococcus epidermidis

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.