HOFFMAN, HEINER (New York University, New York, N.Y.), AND MICHAEL E. FRANK. Time-lapse photomicrography of cell growth and division in Escherichia coli. J. Bacteriol. 89:212-216. 1965.-Photomicrographs at 15-see intervals of cells growing at 37 C disclosed that in a cell with a generation time of 21.0 miin the processes of furrowing, cross-wall formation, and cell separation are completed within 2.5 min after the division furrow first becomes clearly visible. Among a large number of cultivations examined, only a few cells late in one microculture at 43.5 C failed to separate once the cross wall was completed. Measurements of cell lengths during a 5-min period, extending from just before to just after division, showed that elongation of the cell is a discontinuous process, although the growth rate over the 5-min period is exponential. At the time of cell division, it appears that the synthesis of cell-wall material is diverted entirely into formation of the cross wall. The introduction of phase-contrast microscopy greatly improved the possibilities for the direct cytological investigation of single-cell growth of living bacteria in relation to cytokinesis. Nevertheless, very few studies of this problem have been carried out. Among the better known earlier attempts is that of Mason and Powelson (1956), who were primarily interested in nuclear division. They took photomicrographs at 2-min intervals, with bright phase-contrast optics, of microcultures on a gelatin medium wAith a refractive index designed to disclose greater internal detail than seemed possible with agar. Although welldefined "nuclear figures" were found in Escherichia coli cells, the cell wall could not be seen, and Mason and Powelson were unable to establish when the transverse septum completely separated the sister cells. MIore recently, Schaechter et al. (1962) employed a modified version of the technique in a study which timed the period required for division of bacterial nuclei (nuclear interdivision time). Photographs were taken at intervals ranging between 1 and 3 min. From measurements of cell lengths, these authors concluded that E. coli cells increased in length at all times during the division cycle without noticeable discontinuities. In the present investigation, we have re-examined the problem of cell growth in relation to cytokinesis in E. coli by means of dark phase optics rather than bright. The study was carried out primarily on the basis of one micr-ocolony which was photographed at 15-see intervals and a second which was photographed at 3-min intervals. Analysis of the photomicrographs revealed some previously undescribed aspects of the time required for certain stages in the division process, but failed to confirm some of the observations of Schaechter et al. (1962) in regard to cell growth. MATERIALS AND METHODS The orgainism used was rough-phase E. coli ATCC 8677. The Fleminig microculture technique, in which a dry film of smeared broth culture on a cover slip is overlaid with agar, was used for growing the microcoloniies. A 37 C "rapid-sequence" cultivationi was photographed at 15-sec iintervals; the record of this cultivation has been used in an earlier paper (1lhoffman and Frank, 1964b), which describes some details of the techniques used (see also Hoffman and Frank, 1961, 1963a). The second cultivation, photographed at 3-min intervals, was incubated at 43.5 C in a Zeiss heatiing stage rather thani in the microscope inicubator box used for the first culture. Measuremeiits of cell lenigth were made with a draftsman's caliper to the closest 0.5 mm from photographic prints at 7,OOOX for the rapid-sequenice cultivation and at 3,750X for the 43.5 C cultivation.