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
214
Growth in Liquid Medium 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 prod-
ucts 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
aerobi- cally 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 approxi- mately 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
216 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.
