Fig. 1.

Single-cell approach to study viable but non-culturable (VBNC) cells. Schematic illustrating the steps carried out to distinguish VBNC cells from susceptible non-lysed (SNL), susceptible lysed (SL), and persister (P) cells. a A 2-μL aliquot of a stationary phase E. coli BW25113 culture was loaded in the lateral channels of the mother machine device. b Between t = 0 and t = 3 h, ampicillin was injected in the mother machine device at a concentration of 25 × the minimum inhibitory concentration in Lysogeny broth (LB) progressively lysing the majority of bacteria (SL cells, leftmost channel). c Between t = 3 and t = 24 h, LB was injected in the microfluidic device, P bacteria started to grow and eventually gave rise to progeny (third channel from the left). d At t = 24 h, a live/dead staining assay was performed by flowing in the microfluidic device SYTO9 and propidium iodide, distinguishing VBNC bacteria staining green (second channel from the left) from SNL bacteria staining red (rightmost channel). Representative bright-field (e–f) and fluorescence images (g) of the four phenotypes illustrated above, before (e) and after (f, g) drug treatment. The weak red staining in the channel containing the SL bacterium was due to cell debris barely visible in the corresponding bright-field image. Scale bar, 5 μm