## 5 Conclusions  
The present work is the first study to develop (i) an easy-to-use protocol for obtaining highly reproducible biofilms, (ii) on a completely flat polystyrene surface that facilitates the biofilm handling avoiding disruption, (iii) with no need for sophisticated and expensive technologies for biofilms growth (e.g., CDC reactors). The 24-h-grown biofilms completely covered the defined area. The resulting biofilms strongly attached to the polystyrene petri dish and remained there, even after rinsing. The ability to grow biofilms on a completely flat surface has a great potential for testing antimicrobial and antibiofilm coatings/films, surface treatments and physical treatments (e.g., based on light). The use of polystyrene surfaces makes this new protocol suitable for application in every laboratory without the need for biofilm growth reactors. The protocol mimics the transition from planktonic to biofilm that happens on every kind of surface that happens after contamination of surfaces in real life. A similar protocol could be followed for different bacteria; however, the choice of _P. fluorescens_ was made based on it being BSL-1 and of crucial interest within the food industry, households and health care. The development of biofilms was studied in terms of viable cells and biomass. The use of the [PERSON] (1994) model shows a steady state after 24 h [23]. The dynamics allowed the estimation of the growth parameters and permitted a comparison between the two inoculum levels. The growth rate and initial attached population depended on the inoculum density, while the estimated maximum population in static conditions was the same for the two inoculum levels. Additionally, the total biomass development of the batch system under investigation did not follow the [PERSON] and [PERSON] (1994) model but rather a different kinetics reaching a maximum value and then decreasing to a plateau. The findings add to a growing body of research on biofilm development and confirm the importance of enumeration of both the viable cell population and quantification of the total biomass to determine when the biofilms reach a type of steady state/equilibrium. CLSM confirmed that overnight growth allowed the development of densely packed biofilms with a mature 3D structure and water channels. The biomass investigation also allowed a better understanding of the development of the biofilms of _P. fluorescens_, first on the polystyrene surface and successively at the air-liquid interface of the inoculum suspension. It was observed that an increase in the biomass did not necessarily mean an increase in the viable cell concentration within the biofilm and did not necessarily result in a thicker or more adherent biofilm. Based on that, attention should be payed to the distribution of the biofilm on the surface under investigation, in order to avoid misinterpretation. This is especially true when dealing with small surfaces, such as the wells of micro well plates, where the distribution of the biofilm is complicated to observe. The complexity of biofilm lifestyle requires an extended experimental investigation of biofilm proliferation to have a better understanding of biofilm behaviour and formation pattern. The protocol was developed on a Gram-negative bacterium, _P. fluorescens_, and validated on a Gram-positive one, _S. epidermidis_, demonstrating the wide applicability. As such, the present study explored biofilm development on widely used polystyrene surfaces, adds knowledge to the research in biofilm science and indicates the wide applicability of the developed method. Individual contributions of each of the authors to the research are the following: conceptualisation, V.A., C.S., S.A., B.H., A.C. and J.F.M.V.I; methodology, V.A., T.A. and B.H. ; software, V.A., C.S. and S.A.; validation, V.A. ; formal analysis, V.A. ; investigation, V.A. ; resources, J.F.M.V.I; data curation, V.A., writing--original draft preparation, V.A. ; writing--review and editing, V.A., C.S., S.A., T.A., B.H., A.C. and J.F.M.V.I; visualization, V.A.