monitoring of the growth and viability of surface-adhering
using the eGFP/3 μM PI assay. (a) Time series of surface-adhering E. coli on bare glass substrates. The dual staining assay (6 μM SYTO® 9 / 30 μM PI) decreases bacterial viability on untreated glass substrates after incubation times longer than 1 hour as E. coli (AAEC191A) incubated with 6 μM SYTO® 9 containing medium failed to replicate (inset). In contrast, eGFP-expressing E. coli (AAEC191A pHis-GFP) that were incubated with 3 μM PI were able to replicate and grow on the glass surface. (b) Viability of E.coli (AAEC191A pHis-GFP) on antimicrobial DMOAC-coated glass surfaces as monitored by eGFP/PI fluorescence microscopy. Pre-exposure of the DMOAC surfaces to fetal bovine serum (FBS) completely blocked the antimicrobial activity. Microscopy images show the overlay of the SYTO® 9 / eGFP and PI fluorescence channels, i.e. differentiating live (green) from dead bacteria (red). 3 independent fields of view from different experiments were analyzed containing a total of 125–250 surface attached bacteria for each condition. Error bars represent the standard deviation. Scale bar 20 μm.