cells are chilled (divalent cations such as magnesium,
rate, with rapid warming offering the best chance for
survival (Mackey 1984).
tially protect against the effect of chilling and have
We would anticipate that the cooling rate microor-
been shown to mediate recovery), cell number (loss
ganisms would encounter during snowmaking would
be very rapid (>100C/min)* and thus the effect of
of viability is greater the smaller the cell population),
rate of cooling, and temperature range over which
freezing at this rate would be less than at slower rates.
cooling takes place. During rapid cooling, permeabil-
However, we also anticipate that the warming and cool-
ity changes in the cell membrane are caused by a
ing rates in the snowpack would be relatively low. This
phase transition in the membrane lipids from a liquid
slow freezethaw cycling may have more of an effect
crystalline to a gel state (MacLeod and Calcott 1976).
than the initial freezing process.
Slow cooling allows a lateral phase separation of the
Storage death and susceptibility of various
lipids and proteins of the membrane, whereas rapid
bacterial species
cooling "fixes" these components in a random, disor-
dered state, resulting in membrane leakiness (Mackey
Several studies have shown that in addition to the
1984).
death of cells on initial freezing, there is usually fur-
ther death during frozen storage. Usually, death occurs
Freezethaw damage
rapidly in the early stages followed by a slowing of the
Both the rates of cooling and warming affect sur-
rate until, in the later stages, numbers remain almost
vival of cells that have been frozen and thawed. Differ-
constant, with greater survival at lower temperatures
ent cooling and warming rates produce different kinds
(Mackey 1984). According to Mazur (1966), death rates
of damage (MacLeod and Calcott 1976). Damage var-
are low or zero when storage is at temperatures of
70C or below, while temperatures between 60C and
ies depending on the chemical composition of the freez-
0C decrease the survival of most species with time.
ing medium, especially the presence of NaCl (MacLeod
and Calcott 1976). The type and strain of organism, its
The rate of the decrease in survival depends on the spe-
phase of growth when frozen, and the temperature and
cies, the storage temperature, the nature of the freezing
duration of frozen storage are also important factors
medium, and in some cases the cell concentration
(Mackey 1984). The initial number of bacteria can also
(Mazur 1966, MacLeod and Calcott 1976). Death is
affect survival, with high concentrations having a pro-
presumed to be mainly due to continued exposure to
tective effect (Mazur 1966). Resistance of bacteria to
concentrated solutes (Mackey 1984).
freezing varies widely; cell shape and differences in
According to Mackey (1984), bacteria vary widely
membrane fatty acids and proteins have been found to
in their response to frozen storage. They found that fe-
affect cryosensitivity (Mackey 1984).
cal streptococci and Staphylococcus aureus survived
Most cell types, whether procaryotes or eukaryotes,
well under most conditions, whereas Vibrio
have an optimum cooling rate for survival that varies,
parahaemolyticus, Yersinia enterocolitica,
depending on the water permeability of the membrane
Campylobacter jejuni, and vegetative cells of
and on the surface-to-volume ratio of the cell (Mackey
Clostridium perfringens declined in numbers by as
much as 102 to 105 within a few weeks at 20C, and
1984). For many bacterial species, maximum survival
occurs at cooling rates between 6 and 11C per min
other organisms such as Salmonella species and E. coli
are of intermediate resistance, with their survival highly
(Mazur 1966, MacLeod and Calcott 1976, Mackey
dependent upon the composition of the frozen medium.
1984). Mazur (1966) proposed that, at slow cooling
McCarron (1965) studied the survival of six bacte-
rates, ice crystals form extracellularly, thus concentrat-
rial species in ice at subfreezing temperatures (2C,
ing the solutes in the extracellular solution, thereby
20C). Bacteria included three gram-negative rods (E.
causing the cell to dehydrate. Solute concentrations
coli, Aerobacter aerogenes, Serratia marcescens), two
inside and outside the cell then reach levels that can
gram-positive cocci (Micrococcus roseus and Sarcina
cause denaturation of proteins and breakdown of mem-
lutea), and one gram-positive, sporeforming rod (Ba-
branes. At more rapid cooling rates (above this opti-
cillus subtillis). McCarron found that more than 90%
mum), the temperature is reduced at a faster rate than
of the bacteria were inactivated in the first two days
water can flow through cell membrane. This results in
but that the remaining cells persisted for several months.
the ice nucleation in intracellular water. At very rapid
rates of cooling (>100C/min), ice crystal growth is
M. roseus (one of the gram-positive cocci) and the
retarded or prevented and survival again is greater.
However, very small ice crystals may grow and cause
damage if these cells are warmed slowly. Hence sur-
*Personal communication, Scott Barthold, Sno.matic Con-
vival of ultrarapid cooling is dependent on warming
trols and Engineering, Inc., Lebanon, New Hampshire, 1999.
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