Mature Biofilm Cell Viability Assay

The effect of two thiazolidione derivatives on cell viability in mature biofilms (24 h) was determined using the Live/Dead Bacterial Viability method (Live/Dead BacLight, Molecular Probes, USA) with SYTO 9 and propidium iodide (PI) dyes to stain live and dead cells within mature biofilms [36 (link)]. Overnight cultures of E. faecium EF16M64 was grown in cell-culture dishes (Fluorodish, FD35–100) with TSBG medium and incubated at 37 °C for 24 h. Planktonic bacteria were then removed and discarded, and fresh TSBG containing the derivatives monotherapy (at MIC concentrations) or the derivatives combined with a final concentration of 8 × MIC of ampicillin and daptomycin were added and incubated at 37 °C for a further 72 h, exchanging the media every 24 h for fresh media containing appropriate derivatives. After staining, mature biofilms were observed under a confocal laser scanning microscope (CLSM, Leica) with oil-immersion objective. IMARIS 7.0.0 software (Bitplane) was used to edit and analyze the original images. Green and red fluorescence represented the viable and dead cells, respectively. The PI/total percentage, representing the proportion of dead cells within the mature biofilm, was estimated using ImageJ software (Rawak Software Inc., Stuttgart, Germany). This assay was performed in triplicate and similar results were obtained.