Correlative Imaging and super resolution microscopy studies reveal complexities in determining live-dead state of bacteria

Imaging techniques are widely used to determine the physiological state of bacterial cells and provide an important platform for antibacterial evaluation in biofilms research. The commercial counter-staining SYTO 9 – propidium iodine kit is a popular choice for viability studies, with cell membrane damage due to antimicrobial action leading to replacement of the SYTO 9 dye with propidium iodine. This study investigates the live-dead state of cells in early-stage Staphylococcus aureus biofilms using correlative Fluorescence Microscopy (FM), Scanning Electron Microscopy (SEM) and super resolution Structural Illumination Microscopy (SIM). Correlative imaging data obtained at the single-cell level show that the physiological states of individual bacterial cells indicated by the SYTO 9 – propidium iodine counterstain in FM does not correlate directly with the detailed cell morphology observed by SEM. In addition, SIM was used to map sub-cellular distributions of SYTO 9 – propidium iodine dyes within single cells and revealed greater complexity than hitherto assumed, with 4 different cell-states identified, including double-stained ones and those where SYTO-9 is bound to substances at the cell perimeter. With this knowledge, we present ternary plots to illustrate the significant impacts of this complex staining behaviour on underestimation of cell-membrane damage due to antimicrobial actions and, thus, overestimation of bacterial survival rate in biofilms research.