Evaluation of Streptococcus mutans Biofilm Maturation Using Live/Dead Staining

Objectives: Streptococcus mutans biofilm formation is an important virulence property associated with the ability to form dental plaque leading to the initiation of caries. The objective of this study was to evaluate two fluorescent vitality stains, calcein-AM/propidium iodide (PI) and SYTO9/PI, for their ability to accurately evaluate the dynamics of S. mutans biofilm maturation.
Methods: S. mutans overnight cultures were normalized and seeded on 24-well tissue-culture plates for growth in the presence 0-3% sucrose for up to 48 hours. Following growth, biofilms were subjected to vital fluorescent staining using calcein-AM/PI or SYTO9/PI followed by crystal violet (CV) staining for total biofilm. Fluorescence was measured using a microplate reader. Additionally, viable bacteria within biofilms were enumerated by colony counting for comparison with live/dead and crystal violet staining. Data were subjected to analysis of variance with Tukey test (α=0.05) using GraphPad Prism.
Results: Calcein-AM/PI effectively measured biofilm characteristics at 24 hours of growth as compared to colony count, but did not accurately represent vital biofilm-associated cells in a more mature biofilm at 48 hours. Fluorescent staining with SYTO9/PI positively correlated with colony counts performed on biofilms at all growth periods. SYTO9/PI along with CV staining at different time points revealed growth characteristics of the biofilm based on inoculum and sucrose concentration.
Conclusions: The ability to form a biofilm, a virulence property of S. mutans, is often measured using CV staining. Our results show that the use of live/dead staining prior to crystal violet staining can provide further details as to the dynamics of biofilms and may be more helpful when comparing virulence of strains or monitoring efficacy of therapeutics. Additionally, the choice of live/dead stain is important with regards to the maturity of the biofilm as calcein-AM was unable to accurately stain older biofilms.