Appendix

11
Growth in Liquid Medium
Growth Curve
Cells growing in a batch (shake flask) culture generally experience
four distinct growth phases: a lag phase, an exponential (log) phase,
a stationary phase, and a death phase. Cells in the lag phase, in which
an aliquot of cells from an older culture has been transferred into
fresh medium, do not grow right away. These cells must adjust to
the new medium before growth will begin at a rapid rate. The length
of the lag phase is dependent on a number of factors: age and genotype
of the inoculum, temperature, nutrient levels of both the old and new
media, aeration, and the concentration of toxins that may have been
formed in the old medium. The lag phase of Saccharomyces cerevisiae
at 30~ in YEPD lasts for about 3 hours. For Escherichia coli at
37~ the lag phase is usually 10-60 minutes.
Once the cells begin growing rapidly, they are said to enter the
exponential, or logarithmic (log), growth phase. Cells in this phase
are growing rapidly and, unlike cells in the lag and stationary phases,
most cells are in the same physiological state. The growth rate during
log phase is dependent on the nutrient level and aeration of the
medium. Oxygen is frequently the limiting factor in yeast cultures,
and the cultures must be shaken rapidly for sufficient 02 to dissolve.
The doubling time of S. cerevisiae grown in YEPD at 30~ may be
between 90 and 100 minutes, while a 200-minute doubling time
may be expected in minimal medium. The presence of an introduced
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215

plasmid may slow down growth since plasmid replication requires

energy and metabolites. At 37~ a typical E. coli strain doubles in
20-30 minutes in rich medium and in 50-60 minutes in minimal
medium. The growth rate constant (/~) serves to define the rate of
growth of a culture during balanced growth: /~ = In 2 + g (mean
doubling time or generation time). For further discussion of this
concept and derivation of this formula, see Mandelstam et al. (1982).
As nutrients within the flask are consumed and inhibitory products accumulate, the growth rate slows and eventually stops as the
culture enters the stationary phase. Cells in a stationary phase culture
are not all in the same physiological state; some are dividing while
others are dying. Only the overall population size remains constant.
As more nutrients are depleted, more cells die than are produced,
and the culture enters the death phase.

Aeration
Escherichia coli and Saccharomyces cerevisiae can grow both aerobically or anaerobically, although anaerobic growth does not occur
with all carbon sources. With glucose, anaerobic growth occurs, but
at about 10% of the rate of aerobic growth. At a density of approximately 107 cells/ml in liquid medium, oxygen cannot diffuse from
the atmosphere to the cells fast enough for aerobic growth, and unless
the medium is shaken or directly aerated by bubbling, growth of a
culture slows considerably at this cell density. Even with aeration,
the oxygen supply is inadequate above a cell concentration of
2-3 x 109 cells/ml (E. coli)or 2-3 x 108 cells (S. cerevisiae).

Inoculation and Subculture

Since many cells in an inoculum obtained from a slant culture are
dead, most experiments begin by inoculating'a small volume of
growth medium and then growing the cells overnight. In the morning,
cell growth will have stopped and the cell density will usually be

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Appendix 11

2-3 x 109 cells/ml for E. coli in rich medium and 2-4 x 108 cells/
ml for S. cerevisiae in rich medium. TO obtain actively growing cells
for any particular experiment, the overnight culture is diluted 10-100
times and regrown for several hours, at which time the cells are
usually in exponential growth. Also, if an exponentially growing
culture is rapidly chilled to below 8~ (by shaking a growth flask in
ice water), stored at 4~ and then rewarmed rapidly to the original
temperature at which the culture had been growing, growth will
resume without a lag.

Reference
Mandelstam, J., McQuillen, K., and Dawes, I. (1982). "Biochemistry
of Bacterial Growth," 3rd Ed. Halsted Press, Oxford, UK.