Fig. 5

Size and fluorescence of the VBNC-induced cells of V. campbellii BAA-1117™ and V. japonicus UU24. Cells were incubated at 30 °C to induce resuscitation in the presence of different hydrogen peroxide concentrations, i.e. oxidative stress. Black line: cells incubated in the presence of 0.007 mM hydrogen peroxide. Green line: cells incubated in the presence of 0.02 mM hydrogen peroxide. Purple line: cells “resuscitated” at 30 °C and inserted to indicate the hypothetical shift in case of resuscitation. If resuscitation had occurred, black and green lines would have been merged with purples ones. A, B and C V. campbellii BAA-1117™. While the resuscitated cells increased in cell size and DNA quantity and decreased in green fluorescence when stained with PI (please check Fig. 1), stressed cells preserved the phenotypic features of VBNC cells. A Size (FSC-A). The size of stressed cells did not increase as that of resuscitated cells. B Green fluorescence (FL1-A) of cells stained with SYTO™ 9. The quantity of DNA in stressed cells did not increase. C Green fluorescence of cells stained with propidium iodide. The fluorescence of stressed cells did not decrease. D V. japonicus UU24; green fluorescence of cells stained with propidium iodide. While in resuscitated cells a big population has lost the fluorescence, in stressed cells, this population is much smaller. While cells stressed with 0.007 mM hydrogen peroxide were still able to resuscitate and grow, cells stressed with higher concentrations of hydrogen peroxide are not. Size and DNA quantity did not show in this strain any appreciable difference between resuscitated, VBNC and stressed cells