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American Cancer Society MASTAA
Conference of Cryobiology
To be published in "Federation Proceeding
+
THE FREEZING AND THAWING OF ISOLATED CELLS*
Peter Mazur
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee
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I.
INTRODUCTION
should best
Colls oushote survive low-temperature exposure best when they are
frozen and thawed under conditions that minimize damage to their macro-
molecular solutes and their structural components. Although this must be
true ultimately, it is often not true when judged by commonly accepted
criteria of damage. Thus, cells that cytologically appear to be badly
distorted
Hottubed and damaged while frozen are often viable after thawing, whereas
cells that appear cytologically normal when frozen are dead after thaving.
Cells can survive freezing under conditions that alter isolated enzymes,
but
cor conversely, they can be killed under conditions that produce no
detectable alteration in the structure or activity of a particular enzyme.
One obvious reason for these discrepancies is the complexity of a
functioning cell relative to the functions or structures of its constituents.
The cumulative consequences of undetected alterations in the constituents
could easily prove lethal. Or conversely, alterations observed in an
isolated constituent might either not occur in a cell or have trivial
consequences if they do occur.
-
:
- .'-
,
*
*
DETI,
.
The cell also differs from its constituents in another way. It is a
compartment in which the intracellular solution 18 separated from the
l as we shall see
external solution by a semi-permeable membrane, and this compartmentation
plays an important role in freezing damage (Mazur, 1963 a,b; 1964 a,b).
As cells are cooled
below 0°C, they become subject to three
phenomena: the temperature falls, ice crystals form, and liquid water 18
removed, thus raising the concentration of solute. There are a priori
reasons why each phenomenon could be injurious, and for a few cells, there
18 evidence as to which 18 responsible for injury. Although the three
phenomena generally occur simultaneously, it 18 possible to separate their
effects and determine experimentally the contributions that each makes to
freezing injury.
II. THE EFFECTS OF LOWERELD TEMPERATURE-THERMAL SHOCK
Slow cooling without ice formation is not damaging to most cells, but
rapid chilling to temperatures above or below 0°C can sometimes be decidedly
injurious. Such injury 18 referred to as thermal shock. The injury can
--
-
be extensive. Thus, thermal shock kills nearly 90% of bull spermatozoa and
.
.
.-
.
.
..
TANTRA
over 99.9% of låg phase cells of Pseudomonas pyocyanea (Smith, 1961,
w
ww
.
p. 440; Gorill and McNe11, 1960).
The cause of the damage remains uncertain. Lovelock (1954) has
suggested that it involves damage to 11 poprotein complexes in cell membranes.
Smith (1961, p. 440) reported that lecithin added to the medium protects
spermatozoa against thermal shock, a fact that suggests that a suddin fall
in temperature may cause a 1068 of lecithin from the plasma membrane.
Strange and Dark (1962) found that the permeability barriers of thermally-
shocked cells of Aerobacter aerogenes are damaged.
Injury from sudden chilling, however, is far from a universal
occurrence. In fact, it appears to occur only in certain species and under
certain quite specific conditions. Bull sperma, for example, are much more
susceptible to thermal shock than hyman sperma (Smith, 1961, pp. 1635).
1ų (Sherman 1963)
Cells of Escherichia coli are highly susceptible in the early stages of
logarithmic growthy but are aïmost completely resistant in late låg phase
and in stationary phase (Meynell, 1958) (Fig. 1).
If thermal shock was a major fator in the death of frozen-thawed cells,
damage should occur regardless of whether or not the suspension contains ice. .
This has not been found to be the case in the few instances in which direct
comparisons have been made between the survival of cells in supercooled
media and in frozen media at the same temperature. As shown in Table I,
about 100% of cells of stationary phasex yeasty and E. coli survive super-..
cooling to -10° to -15°C whereas only about half that many or less survive
when the suspending fluid 18 Prozen.
My guess is that thermal shock is not a major contributor to the
freezing injury of most isolated cells, but this must remain only a guess
until the necessary experiments have been performed on a wider variety of cells.
III. OFFECTS OF SOLUTE CONCENTRATION AND DEHYDRATION
At some temperature below 0°C, the water in a suspension of cells
will begin to freeze. As it freezes, pure ice separates out of solution, and
concomittantly, the solutes in the residual liquid solution become
increasingly concentrated.
If the solution 18 ideal, one can calculate the concentration at any
given temperature from Raoult's law
popote - .(2-x2)
.
and from the integrated form of the Clausius-Clapeyran equation
-----...
-4
.
7
tn p/P. -
(2)
when p and P. are the vapor pressures of the water in the solution and of
pure water at temperatura T, X, and yo are the mole fractions of water and
solute, T. 18 the freezing point of pure water, and Le and R are the molar
heat of fusion and the gas constant (Mazur, 19646). .
In other words
to ķy - to(3-)
(3)
By making several assumptions which are approximately valid for
:
very dilute solutions (Glasstone, 1946, p. 645), Equation (3) can be
simplified to
(T.-T)
m =
Keyin
Greek nu
(4)

when m is the molal concentration at temperature T, K the molal freezing
point constant (about 1.86 for water), and Ý the number of species into which
the solute dissociates.
There are two important conclusions to be drawn from Equations (3)
and (4). The first is that an ideal nonelectrolyte solution will become
concentrated by 1 molal unit for each 2 degree drop in temperature below its
18
freezing point, and a med salt like Naci will concentrate by 1 molal unit
for each 4 degreo fall in temperatura. The second conclusion is that the
concentration 18 fixed at a given temperature (assuming the external
pressure to be constant). A corollary to this 18 that the osmolal
concentrations of solutes inside and outside the cell at a given temperature
must became equal, given sufficient time for equilibration. Of course,
the accuracy which these conclusions will apply to real solutions will depend
on how closely real solutions approximate ideal behavior.
.
As more and more ice forms in a solution, the fraction of unfrozen
water decreases. Again, if the solution 18 ideal, that fraction (q) is
approximately
hu
(5)
where mí 18 the concentration of the solution before it is frozen
(Mazur, 1963a; 19640).
As the continued fall in temperature causes more and more water to be
withdrawn from the solution und deposited as ice, the solution will eventually
become saturated with respect to solute, and a further fall in temperature
will cause all of the solute to precipitate and all of the water to be
converted to ice. If only one solute 18 present, this complete solidification
will occur at a specific temperature, the eutectic point. If many solutes
are present, each will precipitate out at its eutectic point (neglecting
supersaturation), but some liquid water will remain until the temperature
has dropped below that of the lowest eutectic point.
There are many reasons why increasing concentrations of intra- and
extracellular solutes might prove lethal. High concentrations of electrolytes
can cause modifications in the secondary and tertiary structure of macro-
molecules, they can remove lipids from cell membranes (Lovelock, 1954), and
they can cause large changes in pH as various species of solutes precipitate
out below their eutectic points (van den Berg, 1959).
Furthermore, the increasing concentration of cellular reactants could
conceivably increase the velocity of some biochemical reactions more than
they are slowed by the decrease in temperature (Butler and Bruice, 1964).
For example, Washin (1962) believes that increased concentration of thymine
accounts for the fact that the induction of thymine dimers by UV Irradiation
proceeds readily in frozen solutions but not in liquid solutions.
10
It is perhaps less to be expected that deleterious effects could be
produced by a reduction in g; that 18, by reducing the amount of liquid
water around solutes. But there is some evidence that this may in fact be the
case. Lovel.ock (1957), for example, found that the extent of denaturation
of plasma B lipoprotein is not correlated with the concentration of salts
in the suspending medium, but that it is correlated with the number of degrees
the solutions are cooled below the eutectic point of the electrolytes
Asahina
present (Fig. 2). Similarly, Ashanine (1962) found that the temperature at
which sea urchin eggs are killed correlated well with the eutectic point of
the suspending medium.
Levitt (1962) has recently developed a theory of freezing injury that
is based on the removal of water. He suggests that the dehydration brings
linked
structural proteins into contact, as a result of which they become bridged by
the conversion of sulfhydryl groups into ait sulfide bonds. If the ss bonds
are stronger than the aggregate of hydrogen bonds and hydrophobic interactions
that determine normal protein structure, the proteins could become denatured
either during freezing, or more likely, during thawing as the sudden
reappearance of water produces stresses on the disulfide-linked molecules.
.
.
.
.
Although this hypothesis is attractive, the experimental evidence to
support it has not yet been obtained.
IV.
INJURY FROM ICE FORMATION
The third consequence of lowered temperature 16 the formation of ice
crystals, and it is here that the properties of cells as semi-isolated
compartments become especially important. As already mentioned, if we specify
the temperature, we fix the equilibrium concentration of solutes and the
fraction of unfrozen water both inside and outside the cell. But specifying
the temperature tells nothing about when the ice has frozen, or in what form
it has frozen.
As discussed in detail elsewhere (Mazur, 1964 a, b), the protoplasm in
intact cells tends to remain supercooled above about -10°C even when the
extracellular medium is frozen. This immediately establishes a higher vapor
pressure inside the cell than outside, a situation that is thermodynamically
unstable. There are two ways to reestablish equilibrium: supercooled water
can either flow out of the cell and freeze externally, or it can freeze within
the cell.
The factors that influence the outflow of water are indicated by
the following two equations (Mazur, 19630):
to pg/p-
e
Het toczy/
KART ta py/Pe
.. KART
In these equations, Pr and P. are the intracellular and extracellular vapor
pressures, Te 18 the normal freezing point of the cell contents,
and x
are the mole fractions of water in the cell at temperatures T and Tp, V
18 the volume of intracellular supercooled water, t 18 time, k 18 the
permeability constant of the cell for water, mitte A 18 the area of the cell
surface, and y, 18 the molar volume of water.
Equation (6) states that if the internal solute concentraiion remains
constant (1.e.,the right-hand term 18 zero), the ratio of the internal to
external vapor pressures will increase with decreasing temperature. In
other words, the greater the supercooling (AT), the greater the ratio of
the vapor presoures.
Aut according to Equation (7), an increase in the ratio of Pe/P
increases the rate at which water leaves the cell. The water loss will raise
the concentration of intracellular solutes, which in turn will reduce xq,
and reduce the ratio of Da/P.

If we know the relation between temperature (T) and time (t) (1.e.,
the cooling velocity), these two mutually dependent equations can be combined
to give an expression that gives the amount of supercooled water in a cell
(V) as a function of temperature and several parameters (Mazur, 19630 ),
That expression 18 a 2nd order differential equation:
porte-am as - [(for + 2) 25*5) - varet nga nje )
In this equation B 18 the conling velocity (assumed to be uniform) and
18 the temperature coefficient of the permeability constant (KỂ 18 the
known permeability constant at temperature Tg).
to
The Numerical solutions for this equation for yeast cells cooled at
I
are
various velocities to shown in Fig. 3. The curve labeled "ES" shows the
water contents of celis cooled infinitely slowly. Such cells would continuously
maintain vapor pressure equilibrium with the external ice by dehydration. It
can be seen that the water contents of yeast cooled at 1°C/min remain close to
the equilibrium values at all temperatures. However, at higher cooling rates,
the water-content curves begin to diverge more and more from equilibrium.
This means that at higher cooling velocities, the cell water is not able to leave
the cell sufficiently rapidly to eliminate the vapor pressure differential.
.
.
.
la
14
..
'.
.
1
.
This is the same thing as saying that the intracellular water becomes
.
.
supercooled. The extent of supercooling is given by the horizontal distance
between a point on the curve and the equilibrium curve.
The more the water 18 supercooled, the more probablk it becomes that
.
It will freeze (Mazur, 19640).
Thus, if yeast 18 cooled at lºc/min, the
cellula- water does not become supercooled and so cannot freeze. But if,
for example, the cells are cooled at 100°c/min their protoplasm becomes
extensively suporcooled l
ost and the probability of intracellular freezing
becomes high.
Some general conclusions can be drawn from the solutions to Equation (8).
1)
It cells are cooled sufficiently slowly, they will dehydrate and will
not freeze intracellularly. 2) If they are cooled sufficiently rapidly, they
idu dehydrate le86, and they will freeze intracellularly. 3) Numerical
values can be assigned to "sufficiently slowly" and "sufficiently rapidly'
1 values for the required parameters are available. 4) The important
biological parameters are (a) the surface to volume ratio of the cell and
hence the site of the cell, and (b) the absolute permeability of the cell
to water as a function of temperature. The greater the surface to volume ratio
.
15
and the higher the permeability, the higher the cooling rate required to
produce intracellular ice. 5) The cooling velocities required to produce
intracellular ice vary over at least a 5000-fold range for different cells.
I from the curves in, Lone con
Thus, Fig. 4 predicth that exthrocytes will have to be cooled at about
I according to Fig 3
5000°c/min before they wi11 freeze internally whereas yeast wiù freeze at
F-4)
cooling velocities agove 1° to 10°c/min fest.
One final aspect of ice formation needs mentioning. Although increasing
the cooling velocity increases the likelihood of intracellular ice, the
resulting ice crystals become progressively smaller and less perfect. Small
hence, are thermodynamically unstable (Mazur, 19640). It 18 possible,
therefore, that if cells are cooled very rapidly, the resulting intracellular
ice crystals will be so small as to be innocuous. In such a case, the warming
velocity could have a profound effect on survival. If the cells are warmed
slowly, the unstable crystals may be converted to crystals of damaging size;
bis 17 the cells are warmed rapidly, the unstable crystals weila melt before
have
they had a chance to grow.
This conversion of smaller crystals to larger ones
18 termed recrystallization and it has been observed in water (Meryman, 1957),
Wh
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.
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.
16
1
.
gels (MacKenzie and Inyet, 1962) and various cells and tissues (Rey, 1957;
:
*
:-
Menz and Ivyet, 1961; and Rapatz and Luyet, 1961). Meryman's electron
..
.
micrographs of the recrystallization of water are shown in Fig. 5. A possible
11
.
:
hypothesis to explain the relation between crystal size and injury has been
presented elsewhere (Mazur, 1964b). It suggests a mechanism whereby
modifications which reduce the surface energies of ice crystals could rupture
structural components of the cell.
V. CAUSES OF INJURY IN SPECIFIC CELLS
Two cells that have been used extensively in freezing studies are
yeast and red blood cells. I would like to mention some of the factors that
affect their survival and show how they relate to the physical-chemical
phenomena just discussed.
A. Yeast
· Fig. 6 shows the results of experimental determinations of the survival
Evo
(dashed curve), survival 1o' seen to increase to a maximum as the cooling
velocity 1s increased from 0.05 to 10-10°C/min; it then decreases as the
.
.
.
17
cooling velocity is further increased, reaching a minimum at some 500 to
1000°C/min. Finally, survival increases once again with cooling rates above
1000°C/min. When the cells are warmed slowly instead of rapidly (dotted
curve), the picture is different. Survival drops precipitously as the
.
cooling velocity rises above 1°C/min, and it continues to drop even at very
high cooling velocities. In fact, only about 10-5 % of the cells survive the
sequence of cooling at 300°C/min or more followed by slow warming at 2°C/min.'
The solid curve shows the extent to which the cellular water 18
supercooled at -15°C as the cooling velocity 18 increased. This curve was
obtained from the numerical solution to Equation (8) (Fig. 3). One notes
that the cooling velocities at which survival begins to drop ( 1 to 10°C/min)
nearly coincide with the calculated cooling velocities at which the cells begin to
contain supercooled water at -15°C. Since supercooled water at -25°C 18
likely to freeze, the coincidence of the survival and supercooling curves
Bugtests a causal relation; it suggests that survival begins to drop when
cooling 18 mare rapid than 10°C/min because intracellular ice forms in an
Increasing proportion of the cells.
!
.!
18
The photomicrographs support this conclusion. The left and center
micrographs depict cells frozen at the indicated rates and substituted
with ethanol while frozen. As predicted by Equation (8), the cells cooled
at 1°C/min are shrunken. The magnitude of the reduction in volume indicates
they have lost at least 90% of their water (Mazur, 19610; 1963a). Since
the residual 10% of the water in yeast has been shown by calorimetric
measurements to be incapable of freezing (Wood and Rosenberg, 1957; Souzu,
Nei, and Bito, 1961), it appears likely that these cells contain little if
any intracellular ice. On the other hand, the cells cooled at about 100°C/min
twice that of the slowly cooled cells
are muce or less shrunken. Their volume 18 senter for ko mmet fototomicrograph
IM
Boston Bokhandelight. Thus, they must contain more than 10% of their normal water
content and that excess must be frozen (Mazur, 1963a). Nei (1960) has observed
a similar relation between the volume of yeast and the cooling velocity.
.
The over-all shape of the survival curve can be accounted for in terms
of two factors: solute concentration and intracellular freezing. Survivals
are low with very slow cooling because cooling atp sayx. 0.05°C/min produces long
exposures to the concentrating solutes. Increasing the cooling velocity
shortens the exposure time and so survivai rises. But when the cooling
19
velocity exceeds 1°C/min, the beneficial action of shortening the exposure
time 18 countered by the appearance of intracellular ice in the cells, and
at that point survival begins to drop. Between 10° and 500°C/min, an increasing
proportion of the cellular water freezes and the crystals are large enough to :
be immediately deleterious to most cells; hence, survivals are low regardless
of whether warming 18 rapid or slow. But when the cooling velocity exceeds
1000°C/min, the ice crystals that form in the cell are small. They remain
small and relatively innocuous 11 warming is carried out rapidly, but they
grow to damaging size if warming is carried out slowly.
B. Red Blood Cells
Gehenis, Rapatz, and Luyet (1963) and Luyet, Rapatz, and Gehenio (1963)
have determined the percentage of red cells that remain unhemolyzed after
blood 18 frozen at various cooling velocities. Their results are shown by the
А
dembed curve in Fig. 7. None survive when cooling 18 below 1000°c/min, but
E
t
recovery increases abruptly when cooling is between 1000° and 3300°C/min
and then drops abruptly with still higher cooling velocities. The cotted and
( Bonde,
sob Gurves show the 'extent of supercooling of the cell water at -10° and
-15°C respectively. These curves were calculated from the numerical solutions
shown in Fig. Die
Once again, as with yeast, the cooling velocity at which
...
.
20
20
survival begins to drop is very close to the cooling velocity at which the
chief
cells begin to contain supercooled water. The wing difference between the
red cells and the yeast 18 the numerical value to the "critical" velocity.
It 18 3000 to 5000°C/min for the red cells and 1° to 10°C/min for yeast. The
difference 18 accounted for by the high permeat 1lity of the red cell to water,
the low temperature coefficient of the permeability constant, and the high
surface to volume ratio of the cell (Mazur, 19636).
The explanation of the shape of the survival curve appears to be the
same as for yeast. Cooling velocities below the. "critical" are harmful
because they expose the cells for too long a period to concentrated electrolytes..
Cooling velocities above the "critical" are harmful because they induce
. In support of these conclusions,
intracellular freezing. Lovelock (1953) has demonstrated that red cells can
be hemolyzed by exposures to concentrated electrolytes of a seconds.onetesa. and.
-Head Rapatz, Nath, and Luyet (1963) have shown that, cooling velocities exceeding
several thousand degrees per minute. produce "holes" inside freeze-substituted
and freeze-dried red calls, and these holes presumably represent the prior
location of intracellular ice crystals (Fig. 8).
.
The shapes of the survival curves and the marked difference between the
values of the "critical" cooling velocities for yeast and red cells emphasizes
(unqualified,
the danger in 888igning significance to the words "slow" and "rapid". Thus,
a statement that "rapid cooling is more harmful than slow" would be true for
yeast between lº and 1000°C/min, but not between 8
and 1°C/or between
Tain
1000º and 10,000°C/min, and it would be true for red blood cells only
above 3000°C/min.
VI. A TWO-FACTOR HYPOTHESIS OF A
FREEZING
INJURY
A good portion of freezing injury in yeast and red blood cells seems appears
to be due to the combined effects of exposure to concentrated solutes and
the formation of large intracellular crystals.
This also appears to be true

on the basis of more fragmentary evidence in a number of species of micro-
organisms (Mazur, 19640). This two-factor hypothesis is also consistent with
the fact that the survival of a number of mammalian cells 18 optimal at
around 1°C/min and with the fact that survivals tend to be enhanced by rapid
warming and thawing. However, proof or disproof of the applicability of the
hypothesis to specific cells requires a good deal more quantitative information
...
on the effects of temperature and cooling and warming rates than 18
generally available. Making predictions on the basis of numerical solutions
to Equation (8) requires knowing the permeability of the cell to water and the
temperature coefficient of the permeability constant, but values for these
two parameters are known for only a few 1solated cells, and for very few tissues.
Unquestionably, the two factors are not the only factors involved in
freezing injury. Sherman (1964) has recently reported that ascites tumor
are
cells can survive' intracellular ice formation. Lindeberg and Lode (1963)
- although
have evidence that stationary phase cells of E. coliy although resistant to
they
thermal shock in dilute physiological solutions, become damaged by rapid
chilling at sub zero temperatures when immersed in solutions of sodium chloride
80 concentrated as not to freeze. Other exceptions will doubtless be mentioned
by others at this conference.
Even if this two-factor hypothesis turns out to have rather broad validity,
1t still does not explain the basic causes of injury. Because of time limitations,
I have touched only briefly on a few of the explanations that have been offered
to account for the deleterious effects of concentrated solutes, dehydration,
and intracellular ico formation. (These and other theories are discussed in
more detail elsewhere (Mazur, 1964b)]. The validity and general
applicability of these explanations still remain to be determined.
.
T
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.
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-
':
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40. Weiser, R. 8. and Clarice M. Osterud. J. Becteriol. 50: 413-439,
1945.
41. Wood, Thomas H. and Alburt M. Rosenberg. Biochim. Biophys. Acta 25: 78-87,
1957.
TOOTNOTS
"Research sponsored by the U. 8. Atomic Energy Commission under contract
with the Union Carbide Corporation.
44 Sc
TABLE I
Survival of Moroorganisms in Supercooled and Frozen Suspending
Media* anten telefona iltangenting
Organism
Tamperature
% Survival in
supercooled frozen
media media
Referenco
Escherichia coli
-9
95
Welber and Ostelka (1945)
(T-11)
Sato(1954) (T-14]
--
--------
Saccharomyces cerevisiae
-13
100
100
33-6748 Araki and Nej (1962)
33-6
*
Arapi and Nei(1962)
"(T-1, F4]
Mazur (1961) (T-II, III)
-15
90
24-37
24-3
* Welber and Ostematic used 1% peptone as the suspending medium. The others used
distilled water.
t** The value depended on the cooling velocity.
,
.
FIGURE LEGENDS
T
26
ES
Figure 1. Relationship between the growth phase of cells of
Escherichia coli and susceptibllity to thermal shock.
(a ) - Samples of suspension were removed from the
culture at the indicated times after inoculation and cooled
rapidly by dilution in Ringer's solution at 4°C. Survival
was determined by plate count. (o ) - Viable count
of unchilled samples. The point bearing the arrow was
derived from samples which yielded no colonies; it represents
what the survival would have been if one colony has been
formed. Redrawn from Meynell (1958) by permission of the
J. Gen. Microbiology.
Figure 2. Relationship between the extent of denaturation (turbidity)
of 8 lipoprotein after being frozen for 15 hours at -40°C
and the eutectic points of suspending solutions containing
the indicated salts. From Lovelock (1957), by permission
of the Royal Society (London).
Figure 3. Calculated percentages of intracellular water (v/v.) remaining
in yeast cells as a function of temperature and cooling
velocity. The curve "Eq" represents the equilibrium water
content. The other curves are the numberical solutions to
Equation 8. The values for the parameters are given in
Mazur (1963), Fig. 20) except for b, the temperature coefficient
of the permeability constant. The value of b used here 18
2
i
-
.
..
2
.
L
s
7
'
..
..
.
0.065 wherous the oarlier culoulations used 0.0325. The former
corresponds approximately to an activation energy for the
promeation of water of 9600 cal/mole, which is the value measured
by Hempling (1960) in ascites cells.
Figuro '4. Calculatod percentagon of intracellular water remaining in
human red blood cells as a function est temperature and cooling
velocity. From Mazur (1963), Fig. 50) by permission of the
Rockefeller Institute Press (J. Gen. Physiol. 47: 347-369).
Figure 5. Recrystallization of ice. Electron micrographs of vacuum
evaporated replicas made from ice films produced in high vacuum.
• From Meryman (1957) by permission of the Royal Society (London).
Figure 6. Effect of cooling velocity on the survival, morphology,
and extent of supercooling of cells of the yeast Saccharomyces
-)
water, cooled to -30°C or below and warmed either rapidly (--
vity or slowly (----). The curves are logarithmic means of
data from Mazur (1961a), Araki and Nei (1962), Doebbler and
Rinfret (1963), Moor and Münlethaler (1963), and Green and Mazur
(unpublished data). The photomicrographs are from Mazur (1961b).
The left and center micrographs are of cells substituted with
cold ethanol after slow and rapid freezing. The right hand light
micrograph 18 of untreated cells. Ultra-rapidly cooled yeast
appear normal morphologically when examined by the electron
microscopex (Mundkur, 1960, Moor and Mihlethaler, 1963). The
solid curve shows the extent of supercooling of the cellular
water at -15°C, and was derived from Figure 3.
-
...
-
-----
--
----
-
.
.
.
Figure 7. The recovery (100 - % hemolysis) and extent of supercooling of
mammalian erythrocytes as a function of cooling velocity. The
recoverie. (Curve A) are from date of Gehonio, et . (1963)
and Luyet, et al. (1963). Curves B and C show the extent of
supercooling in cells at -10°C and -15°C respectively and
were calculated from Figure 4. The unlabeled solid and dotted
curves show for comparison the survival and extent of supercooling
of yeast.
Figure 8.
Electronmicrographs of bovine blood cooled to -150°C in a 20-4
layer and substituted with ethanol at -78°C. From Rapatz et al,
1963 by permission of Biodynamica.
2
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