The importance of combining methods to assess Candida albicans biofilms following photodynamic inactivation

•Methylene blue in oral formulation was able to reduce candida albicans virulence after PDI.•Assays may present different responses, depending on the time point measured.•Sodium dodecyl sulfate interacts with crystal violet and makes the method unfeasible. Background: Methylene blue (MB)-mediated photodynamic inactivation (PDI) has shown good results in killing Candida spp. Although MB solutions are commonly used, new formulations have been designed to improve PDI. However, chemical substances in the formulation may interfere with the PDI outcome. In this sense, different methodologies should be used to evaluate PDI in vitro. Herein, we report different methodologies to evaluate the effects of PDI with an oral formulation (OF) containing 0.005% MB on Candida albicans biofilm. Methods: Biofilms were treated using the MB-OF, with 5 min pre-irradiation time and exposure to a 640 nm LED device (4.7 J/cm2). PDI was evaluated by the XTT reduction test, counting the colony forming units (CFU), a filamentation assay, crystal violet (CV) staining, and scanning electronic microscopy (SEM). Results: PDI was able to reduce around 1.5 log10 CFU/mL, even though no significant differences were noted in metabolic activity in comparison to the control immediately after PDI. A significant decrease in yeast to hyphae transition was observed after PDI, while the biofilm exhibited flattened cells and a reduced number of yeasts in SEM. The CV assay showed increased biomass. Conclusion: MB-OF-mediated PDI was effective in C. albicans biofilms, as it significantly reduced the CFU/mL and the virulence of surviving cells. The CV data were inconclusive, since the OF components interacted with the CV, making the data useless. Taken together, our data suggest that the association of different methods allows complementary responses to assess how PDI mediated by a formulation impacts biofilms.