Sorting out bacterial viability with optical tweezers.
We have developed a method, using laser, optical tweezers and direct microscopic 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 using the fluorescent nucleic acid stain propidium iodide, correlated well with a reduction in cell proliferating potential during the stationary-phase period studied. Moreover, the same cells that exhibited reduced integrity were found to be the ones that failed to divide upon nutrient addition. A small 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 stationary-phase cells were able to divide only once upon nutrient addition, and in a few cases, only one of the two cells produced by division was able to divide a second time, indicating that localized cell deterioration, inherited by only one of the daughters, may occur. The usefulness of the optical trapping methodology in elucidating the mechanisms involved in stationary-phase-induced bacterial death and population heterogeneity is discussed.
Ericsson
M
M
Department of Physics, Chalmers University of Technology and Göteborg University, Göteborg, Sweden.
Hanstorp
D
D
Hagberg
P
P
Enger
J
J
Nyström
T
T
D004926:Escherichia coli / Q000254:growth & development / Q000502:physiology*; D005456:Fluorescent Dyes; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000379:methods; D055095:Optics and Photonics; D011419:Propidium; D011933:Reagent Kits, Diagnostic
D005456:Fluorescent Dyes; D011933:Reagent Kits, Diagnostic; D011419:Propidium