Background: Accurate assessment of bacterial cell viability within biofilms is vital for evaluating the efficacy of anti-biofilm agents. A common staining approach for this involves SYTO9 and propidium iodide (SYTO9/PI), however, the application of these probes often leads to an underestimation of biofilm cell viability, attributed to the presence of extracellular DNA within the biofilm, and introduces other notable limitations.

Aims: This study explores whether biofilm viability staining with the dye combination of Calcein acetyl methyl ester (Calcein-AM) plus 1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene p-Toluenesulfonate (TMA-DPH) in comparison to the conventionally used SYTO9/PI combination shows a superior correlation with the Colony forming units (CFU) determination method. Calcein-AM specifically stains metabolically active cells, providing a direct indication of viable cells, while TMA-DPH penetrates all cells and also provides insights into membrane fluidity patterns.

Methods: Biofilms of Gram-positive and Gram-negative bacterial species, treated with antibiotics, were stained with Calcein-AM/TMA-DPH and SYTO9/PI, then imaged using Confocal laser scanning microscopy (CLSM). CFU determination, the gold standard in microbiological viability assessments, was performed on the same biofilms. CLSM images were analyzed using an ImageJ-based biofilm analysis tool, comparing data to respective CFU numbers of the treated biofilms.

Results: CLSM micrographs showed divergent patterns between Calcein-AM/TMA-DPH and SYTO9/PI stainings in all bacterial species. Quantitative analysis in Gram-positive species revealed a positive correlation between Calcein-AM image analysis data and CFU, while no correlation was observed with SYTO9/PI. Work on Gram-negative bacteria is ongoing.

Conclusion: Accurate biofilm viability assessment is crucial for evaluating anti-biofilm agents. Preliminary results suggest Calcein-AM and TMA-DPH staining may provide a more precise method, emphasizing the superior efficacy of physiological state probes over DNA-binding fluorophores.