Extended Data Fig. 4 | Single-cell behaviour during a glucose-to-acetate
shift through time-lapse microscopy of batch culture. a, Diagram showing
the experimental protocol (Methods section on‘Batch microscopy’). After the
medium shift from glucose to acetate, the culture was split into two identical
six-well glass-bottom plates. One was brief ly centrifuged and placed into an
incubator on a microscope for time-lapse microscopy, and phase-contrast
images were recorded. The other plate was placed in a shaker incubator as a
control, and OD 600 was monitored manually. b, Growth curves from two
biological repeats (circles and squares), obtained by monitoring OD 600 from
the control six-well plate after the media switch. The calculated lag time is
295 min, virtually identical to the batch-culture lag time that we characterized
in Fig. 1 , indicating that the environment of the six-well plate is almost identical
to that of the batch culture as far as the lag time is concerned. c, Normalized
single-cell-area traces (two biological repeats) from the other plate (n = 1,761
traces). We use cell area as a metric for biomass growth. Light blue traces show
the 1, 500 cells that crossed an arbitrary 10% threshold for increase in area
within our observation time (Methods). Red traces indicate the 261 cells that
did not cross this threshold before they became unobservable, either because
they detached from the glass or because they were were f looded by other cells.
d, Histogram showing the distribution of time required for individual cells to
increase their area by 10%. e, The percentages of cells that grew in cell area
(y-axis) by at least the amount shown on the x-axis, relative to their initial size,
are plotted. All of these data show that most cells recover after an initial lag
phase, eventually growing on acetate. Despite the relatively short observation
window of 5–6 h (beyond which the plate became too crowded by dividing cells
to allow imaging of individual cells), which is roughly equal to the batch lag
time, most cells exhibit substantial growth (e). A 10% increase in cell area is
easily detectable, and roughly 85% of cells crossed this threshold. The cells that
did cross this threshold grew continuously throughout the observation period,
exhibiting a single-cell growth curve and a lag time (c, d) that was similar to the
batch lag time. Thus, no more than roughly 15% of cells were completely growth
arrested after the shift to actetate, even during this limited observation
window. Therefore, in the lag phases studied here, the dormant
subpopulations proposed previously^12 had a negligible role in determining lag
times. (As an example, even if the roughly 15% of growth-arrested cells never
grew again, they would contribute only approximately 21 min to the total lag
time of 295 min.).