Figure 4.

Farnesol disrupts established E. faecium biofilms. (A) The 24 h old established biofilms exposed to farnesol for 24 h have reduced cell viability, indicative of biofilm disruption and cell killing. Data are represented as the mean ± SD (n = 3). (B) Flattened views of three-dimensional stacked Live/Dead images of 24 h old established E. faecium biofilms after 24 h exposure to farnesol. Green indicates live cells (SYTO^® 9 staining), while red indicates dead cells (propidium iodide staining). Scale bars are 20 μm. (C) Quantitative evaluation of fluorescence intensity by Photoshop^® from the images in (B), with data representing the mean ± SD (n = 3). (D) Quantitative evaluation of fluorescence intensity by Comstat2 from the images in (B) (biomass (µm³/µm²) and average thickness (µm)), using Otsu thresholding [31]. Ctrl indicates the ethanol control corresponding to the same volume of ethanol used to carry farnesol. Ctrl_3 = 10%, Ctrl_6 = 20%, and Ctrl_10 = 33.3% of ethanol. * p < 0.0001 against each of other groups.