Observing growth and division of large numbers of individual bacteria by image analysis

Abstract

We describe a method that enabled us to observe large numbers of individual bacterial cells during a long period of cell growth and proliferation. We designed a flow chamber in which the cells attached to a transparent solid surface. The flow chamber was mounted on a microscope equipped with a digital camera. The shear force of the flow removed the daughter cells, making it possible to monitor the consecutive divisions of a single cell. In this way, kinetic parameters and their distributions, as well as some physiological characteristics of the bacteria, could be analyzed based on more than 1,000 single-cell observations. The method which we developed enabled us to study the history effect on the distribution of the lag times of single cells.

FIG. 1.

Setup of the flow system. a, feed flask; b, peristaltic pump; c, bubble trap; d, microscope equipped with a charge-coupled device camera; e, flow chamber; f, waste flask.

FIG. 2.

Flow chamber. (a) Top block of aluminum with inlet and outlet pipes (diameter, 1.2 mm) with two O rings; (b) polycarbonate slide with two holes (diameter, 1.2 mm); (c) polymer spacer; (d) microscope slide; (e) bottom block of aluminum.

FIG. 3.

Series of images obtained over time (a) and resultant graph (b). Taken at 5-min intervals, the images are close-up photos (magnification, ×500) of a single E. coli cell dividing during the exponential phase. More than 1,000 such images and graphs were generated in an experiment and used to create the distributions shown in Fig. 4.

FIG. 4.

Distributions of first doubling times of E. coli in different salt concentrations (1, 2, and 4% NaCl). Each culture was inoculated from the stationary phase and then grown at the ambient temperature in medium containing 10 g of tryptone per liter, 5 g of yeast extract per liter, 2 g of glucose per liter, and NaCl.

FIG. 5.

Effect of sublethal heat shock on L. innocua. Both the mean (•) and the variance (○) of the individual lag times increased with the duration of the heat shock.