Sorting out bacterial viability with optical tweezers

Abstract

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.

FIG. 1

Optical trapping and fluorescent microscopy setup. The Ar⁺ laser serves to excite the dye used in the sample, while the diode laser, which works in the infrared region, serves as the trapping beam. By tilting mirror M3, the optical trap can be moved in the trapping plane. Since both the trapping beam and the beam used for excitation are directed down through the microscope objective, only the bacteria within the field of view are excited. DL, diode laser; APP, anamorphic prism pair; M1 to M6, mirrors; L1 to L4, lenses; DM1 to DM2, dichroic mirrors; MO, microscope objective; S, sample; CL, condenser lens; LS, light source; D, diffuser; FR, filter revolver; BF, blocking filter; CCD, charge-coupled device.

FIG. 2

Doubling time of E. coli bacteria at room temperature as a function of the output power of the trapping diode laser. The bacteria were held with the optical trap in the nutrient broth (NB) complex medium. For each data point, 1,000 cells were analyzed. The doubling time is an average for four generations studied directly under the microscope.

FIG. 3

Number of bacteria (in billions) per milliliter as a function of time during growth and stationary phase in NB complex medium. The same data is plotted in a linear (A) and semilog (B) scale for comparison. Cell numbers were analyzed using the two different techniques (CFU counting versus fluorescent response and total cell counts). In the figure, total counts, CFU counts, the number of green fluorescent bacteria, and the number of red fluorescent bacteria are depicted. The detection limit was 0.1% of the total number of cells at the time points shown. The sum of the counts of green and red fluorescent bacteria corresponded to total counts at all time points analyzed.

FIG. 4

Bacterial viability studied under the microscope. The bacteria were placed in a lattice using the optical tweezers. Bacteria stained fluorescent green (SYTO 9) have intact membranes, whereas bacteria stained red (PI) have damaged membranes that allow PI to enter. Since the binding affinity of PI is higher than that of SYTO 9 for binding to the DNA, bacteria with a damaged membrane will stain fluorescent red. Pictures were taken 0 min (A), 50 min (B), and 3 h (C) after the addition of fresh medium. After 50 min, the first cell divisions of bacteria stained green were visible, and after 3 h such divisions were obvious. None of the bacteria stained fluorescent red were found to be able to divide in any the experiments so far carried out.