Background

Bacterial biofilms, complex structures attached to surfaces, are matrices composed by proteins, DNA, polysaccharides and water networks, in which cells are embedded (Costerton et al., 1995). Pseudomonas aeruginosa is a versatile bacterium that can be found in terrestrial and aquatic environments, or as human pathogen, either as free cells or as cells in robust biofilms. P. aeruginosa biofilms represent a serious problem because of the adverse effects on human health and industry (Nickel et al., 1985; Gibson et al., 1999; Willcox et al., 2001; Ramsey and Wozniak, 2005; Rajasekar et al., 2010; Mulcahy et al., 2014) and their high resistance to antibacterial agents (Mah and O’Toole, 2001; Mah et al., 2003). Because of their resistance and robustness, P. aeruginosa biofilms represent a model for biofilm studies (Ciofu and Tolker-Nielsen, 2019). The development of P. aeruginosa biofilms is regulated by a complex genetic program; in addition, biofilm formation is modified by environmental factors (O’Toole et al., 2000; Di Bonaventura et al., 2007; Ben Said et al., 2011; Gambino and Cappitelli, 2016; Pezzoni et al., 2018).

Factors related to the high resistance of biofilms include impaired diffusion of antibacterial compounds, reduced sensitivity due to slow growth rate of cells in biofilms, emergence of resistant bacterial phenotypes, presence of antioxidant products in the biofilm matrix, among others (Pezzoni et al., 2014; Hall and Mah, 2017). Strategies employed to combat biofilms include chemical and physical treatments such as application of disinfectants, antibiotics and ultrasound (Bridier et al., 2011; Wu et al., 2014; Gnanadhas et al., 2015). The high resistance of biofilm cells to commonly used disinfectants and the risk to human health and the environment by the use of increasing bactericide doses prompted to redirect research to safer strategies, such as the use of photocatalytic techniques, proposed as inexpensive, safe and effective (Dalrymple et al., 2010; Gamage McEvoy and Zhang, 2010; Pezzoni et al., 2020). On the other hand, since adhesion of microorganisms to surfaces depends on the surface topography and roughness, preventing biofilm formation by tuning surfaces at nanoscale level is a major opportunity in the development of antibiofilm strategies (Katsikogianni and Missirlis, 2004; Pezzoni et al., 2017).

In order to deepen in the understanding of biofilm properties and/or evaluate the effectiveness of antibacterial treatments, several techniques have been employed to evaluate the amount of live bacteria in biofilms (Welch et al., 2012). Among them, two known methods are the counting of the number of colony forming units (cfu) and the live/dead staining (Merritt et al., 2005; Smith and Hunter, 2008; Tsvetanova, 2020). Both methods provide valuable but different information. In the cfu method, a viable cell is one capable to form a colony. On the other hand, the live/dead staining evaluates membrane integrity. The live/dead staining method employs the fluorescent stains SYTO 9 and Propidium iodide (PI), and viability is evaluated by fluorescence microscopy. PI is a red dye that only enters cells with permeabilized cytoplasmic membrane, while SYTO 9 is a green dye that stains all types of cells. According to this criterion, green cells (intact membrane) are live and red cells (disrupted membrane) are dead. While the colony count method requires biofilm disruption and a subsequent step of cell culture to visualize individual colonies, the live/dead method can be applied on entire biofilms and allows us to evaluate the morphological aspects of the biofilm. Since the two methods are based on different criteria, the data obtained from them will not be identical but they will follow the same tendency upon a given antibacterial treatment (Berney et al., 2006; Bosshard et al., 2010; Pezzoni et al., 2014 and 2020). In this work, we detailed how to apply both methods to P. aeruginosa biofilms.