Environmental Engineering Reference
In-Depth Information
Timer
Cold
Warm
Power Bar
N.O.
Valves
N.C.
Valves
Sample
Fig. 2.1 Diagram of the cryocycler designed to automatically subject microbial cultures to
freeze-thaw cycles. Solid lines show the flow pattern in the 'power-off ' state with the valves in
position to circulate cold ethylene glycol (e.g. -188C) through the jacketed sample chamber.
Dotted lines show the flow pattern during the warm (e.g. 58C) 'power-on' state (adapted
from [27])
jacketed glass chamber that is filled with ethylene glycol. Typically, triplicate
samples are subjected to a freeze-thaw regime consisting of a 2 h cycle at
temperatures below the freezing point and just above the melting point (e.g.,
-18 and 58C with average warming rates of 0.58C/min and cooling rates of
1.08C/min, although other protocols have also been used). Aliquots that are
removed periodically during the cycling are then monitored for surviving cells.
It is important to note that occasionally, cultures that originated as single
isolates and used as controls can supercool rather than freeze at temperatures
close to 08C. Thus to ensure that all samples freeze at the same temperature, a
few sterilized AgI crystals should be added to the cultures at the start of the
experiments.
Experiments using the cryocycler show that the viability of the various soil
consortia is dramatically reduced by multiple freeze-thaw cycles, with cell
numbers typically decreasing by five orders of magnitude after 48 cycles
(Fig. 2.2). The decrease in viability does not simply result from a reduction in
numbers since the phenotype of the colonies suggests that there is a shift in the
complexity of the populations. Early experiments on the cryocycler demon-
strated that there was a differential susceptibility to freeze-thaw treatments
depending upon the species and the origin of the communities. For example,
when 10 8 cells of Escherichia coli or Pseudomonas chlororaphis were subjected
to freeze-thaw cycling, none remained viable after 24 and 48 cycles, respec-
tively. Survivors of this stringent regime, in several experiments using different
starting populations, included species of Chryseobacterium, Pseudomonas, and
Buttiauxella. Recovered viable species often had interesting properties (see the
following sections).
 
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