## 1 Introduction
Among the methods that are used to develop/seem to develop biofilms on a horizontal surface there are those employing CDC reactors and those using petri dishes and well plates. However, the formers require the laboratory to be equipped with CDC reactors and the latter do not prevent the biofilms from growing on the walls [11; 19]. None of these methods are suitable to accomplish the requirements of growing the biofilms on a flat surface. The first objective of this paper was the development of a robust, highly reproducible protocol for growing _Pseudomonas_ biofilms on a flat polystyrene surface under static (hydrodynamic) conditions, by investigated different factors such as area, medium concentration, level of inoculum density. Viable plate counts, biomass quantification and confocal laser scanning microscopy (CLSM) were used to study the 24-h-grown dense biofilm. The second objective was to study the development of the biofilm in time in terms of both population density and total biofilm biomass. The [PERSON] and [PERSON] (1994) model was fitted to the population density curves to determine the growth parameters. The third objective was to compare growth dynamics both in terms of viable cells and biomass obtained for biofilms growing with two different inoculum levels to better understand the influence of this factor on biofilms dynamics [23]. The fourth objective was to validate the protocol using a different bacterial strain. For this purpose, _Staphylococcus epidermidis_ was chosen, as it has a different genus, Gram-nature, shape and EPS characteristics [24; 25; 26]. The choice of polystyrene (PS) was made based on the extensive use of this material in industry because of its low cost, durability and ease to be manufactured [27; 28]. PS is largely employed in the medical industry and for biomedical applications, making it interesting for public health. It is also a polymer that is largely present in everyday life since, e.g., food containers, packaging and utensils are often made of PS. Secondly, the PS polymer constitutes a model material that is widely used in research for its inert characteristics and lack of surface chemistry. PS has recently been used in various studies concerning chemistry modification (grafting), ion implantation and nanoparticle coatings to understand microbial adhesion or to develop antimicrobial strategies [29]. Thirdly, the choice of PS was made because of its relevance in microbiological lab practices. It has been used as culture material for animal and human cells for more than 50 years, and nowadays it is commonly used for bacterial research activities [30]. The high optical clarity, the cheap cost and the thermoplastic characteristics make the material suitable for cell culture dishes and multiwell plates that are widely employed in every laboratory, pharmaceutical and hospital setting. The genus _Pseudomonas_ was chosen as a model microorganism because of its ability to easily form strong biofilms, which results in an increase of antibiotic and disinfect resistance [13; 14]. _Pseudomonas_ includes several species, among them _P. fluorescens_, which is studied for its potential in industrial applications resulting from its biosurfactants production [31]. _P. fluorescens_ is a biofilm former [32; 33]. It is commonly studied to understand the complex process of biofilm maturation [34; 35], as well as to develop techniques/technologies applicable for biofilm inactivation or removal [36]. In environmental microbiology, _P. fluorescens_ has mainly been studied for its presence in soil and surface of plants [33]. Recent study has demonstrated that it can cause infections in mammalian hosts, as well [33]. Moreover, it is also relevant in food microbiology since it is one of the main microorganisms responsible for wastage of dairy and meat products [2; 37; 38; 39]. Next to its relevance, _P. fluorescens_ has been chosen for this study because it can be easily acquired and because it is categorised as biosafety level-1 (BSL-1), which means that it does not cause disease in healthy humans.