Influence of environmental stress on distributions of times to first division in Escherichia coli populations, as determined by digital-image analysis of individual cells

Abstract

The distributions of times to first cell division were determined for populations of Escherichia coli stationary-phase cells inoculated onto agar media. This was accomplished by using automated analysis of digital images of individual cells growing on agar and calculation of the "box area ratio." Using approximately 300 cells per experiment, the mean time to first division and standard deviation for cells grown in liquid medium at 37 degrees C and inoculated on agar and incubated at 20 degrees C were determined as 3.0 h and 0.7 h, respectively. Distributions were observed to tail toward the higher values, but no definitive model distribution was identified. Both preinoculation stress by heating cultures at 50 degrees C and postinoculation stress by growth in the presence of higher concentrations of NaCl increased mean times to first division. Both stresses also resulted in an increase in the spread of the distributions that was proportional to the mean division time, the coefficient of variation being constant at approximately 0.2 in all cases. The "relative division time," which is the time to first division for individual cells expressed in terms of the cell size doubling time, was used as measure of the "work to be done" to prepare for cell division. Relative division times were greater for heat-stressed cells than for those growing under osmotic stress.

FIG. 1.

Results of three replicate analyses showing distributions of times to first cell division following inoculation of cells grown to stationary phase at 37°C and subsequently grown at 20°C on agar. %N, percentage of analyzed cells.

FIG. 2.

Influence of preinoculation heating on distribution of times to first division. Stationary-phase cells grown at 37°C were heated at 50°C for the stated times prior to inoculation and analysis during growth at 25°C.

FIG. 3.

Effect of heating time at 50°C on mean times to first division (closed symbols) and the coefficient of variation of division times (open symbols). Stationary-phase cells grown at 37°C were heated prior to inoculation and growth at 20°C.

FIG. 4.

Influence of NaCl in the growth medium on the distribution of times to first division. Stationary-phase cells grown at 37°C were inoculated on agar medium containing the stated concentrations of NaCl (wt/vol), and the division times were determined during growth at 20°C.

FIG. 5.

Effect of NaCl in the growth medium on mean times to first division (closed symbols) and the coefficient of variation of division times (open symbols). Stationary-phase cells grown at 37°C were inoculated onto agar medium containing the stated concentrations of NaCl and incubated at 20°C.

FIG. 6.

Mean RDTs (closed symbols) and coefficients of variation (open symbols) for cultures subjected to heating at 50°C prior to inoculation (A) and to growth in the presence of NaCl (B). The RDTs were calculated as time to first division for individual cells divided by the doubling time of the cell two-dimensional area.

FIG. 7.

Mean cell two-dimensional areas at division for cultures subjected to heating at 50°C prior to inoculation (A) and to growth in the presence of NaCl (B).