Sorting out bacterial viability with optical tweezers

We have developed a method, using laser, optical tweezers and direct micros copic analysis of reproductive potential and membrane integrity, to assess single-cell viability in a stationary-phase Escherichia coli population. It is demonstrated here that a reduction in cell integrity, determined by usi ng the fluorescent nucleic acid stain propidium iodide, correlated well wit h a reduction in cell proliferating potential during the stationary-phase p eriod studied. Moreover. the same cells that exhibited reduced integrity we re found to be the ones that failed to divide upon nutrient addition. A sma ll but significant number of the intact cells (496 of 7,466 [6.6%]) failed to replicate. In other words, we did not find evidence for the existence of a large population of intact but nonculturable cells during the stationary -phase period studied but it is clear that reproductive ability can be lost prior to the loss of membrane integrity, In addition, about 1% of the stat ionary-phase cells were able to divide only once upon nutrient addition, an d in a few cases, only one of the two cells produced by division was able t o divide a second time, indicating that localized cell deterioration, inher ited by only one of the daughters, mag occur. The usefulness of the optical trapping methodology in elucidating the mechanisms involved in stationary- phase-induced bacterial death and population heterogeneity is discussed.