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Ice propagation in systems of biological interest. II. Effect of solutes at rapid cooling rates
Ice Propagation in Systems of Biological Interest. II. Effect of Solutes at Rapid Cooling Rates’ C. V. Lusena and W. H. Cook2 From the Division
of Applied
Biology, National Ottawa, Canada
Research Laboratories,
ReceivedNovember12, 1953 INTRODUCTION
“Snap” or “quick” freezing is generally believed to be lessinjurious to biological systems than slow freezing. Since larger ice crystals have been observed after slow than after fast cooling rates, it has been assumedthat large crystals are harmful. Some workers (7) suggest that even small intracellular crystals are injurious and that vitrification is required for storage of living materials at low temperature (7). Since the rate of heat removal is restricted by physical factors, while growth of ice crystals in distilled water is extremely rapid (lo), it is doubtful that small crystals or an amorphous phase could be obtained unless the freezing pattern was drastically affected by the structure or composition of the biological material being frozen. The relation between freezing damage and composition of intracellular solutes or extracellular suspending media has been studied by several investigators. An increase in concentration of certain solutes has been observed during the development of frost hardiness in plants (8). It also has been amply demonstrated that the addition of sugars or glycerol protects tissues and cell constituents from damage by freezing (4, 5, 9). In a model system (6), the penetration of ice through the pores of membranes was greatly reduced by solutes which acted either by depressing the freezing point or by retarding the rat3 of icecrystal growth. When concentrated gelatin jellies and sucrose solutions were rapidly cooled in the absence of membranes, supercooling occurred ahead of the advancing ice front. These observations suggested 1 Contribution issued as N.R.C. No. 3244. 0 With the technical assistance of J. M. Ross. 243
244
C.
V.
LUBENA
AND
that the kind and concentration of controlling the freezing patterns of reducing harmful effects. The effect has therefore been studied by a new METHOD
W.
H.
COOK
solute might be major factors in biological materials and thus in of splutes on freezing behavior dilatometric method.
AND MATERIrlLS
Use of a “dynamic” dilatometer, in contrast to an ordinary dilatometer in which the volume is measured after equilibrium is attained at a constant temperature, permitted the volume and temperature of the sample to be measured continuously at a constant rate of heat transfer. By plotting volume change against temperature, the temperature at which ice was formed could be determined at various rates of cooling. The dilatometer vessel (Fig. 1) consisted of brass pipe, about 3.5 cm. I.D. and 6.0 cm. height, sealed at the bottom and fitted with a screw cap with connections
0
2
4
6
CM.
FIG. 1. “Dynamic”
dilatometer.
ICE
PROPAGATION
IN
BIOLOGICAL
SYSTEMS.
11
245
to a manometer and to a filling spout with stopcock. The manometer was jacketed with circulating ice water. The vessel and connections to manometer and filling spout were surrounded by an air jacket fully immersed in an ethanol bath. The bath was adjusted to the required constantly changing temperatures by carefully balancing the flow of liquid nitrogen through a cooling coil and electricity through a heater. The rate of heat removal during freezing and of heat addition during thawing was controlled by maintaining a constant temperature differential, within &0,5”C., between the bath and the wall of the dilatometer. The temperature in the dilatometer vessel was measured with three thermocouples, one at the wall and two within the dilatometer vessel. The latter were placed in sealed hypodermic needles arranged vertically through the bottom of the vessel and extending to within 3 mm. of the interface of the solution and manometric fluid. One of these was in the center and the other halfway between center and wall. Preliminary experiments showed that the temperature recorded by the latter thermocouple was a good estimate of the average temperature of the medium. This average temperature, which is plotted in all figures, was automatically recorded every 30 sec., and the wall and center temperature every 60 sec. In each test, a 51-ml. aliquot of solution was thoroughly deaerated and frozen. If air bubbles appeared on thawing, the results were rejected. The manometric fluid was an oil (15 ml.) with a low pour point and viscosity. Because the time required to freeze the 51 ml. of solution varied with the concentration of solutes, rates of cooling were expressed as the temperature differential in degrees centigrade. A 51-ml. aliquot of water could be cooled 20°C. (from $5 to -15’C.) or heated through the same range in 75 min. at 40°C. differential, and in 150 min. at 25°C. differential. The performance of the instrument was checked by freezing distilled water. The volume expansion was within the range of reported values (2) but about 5-10% higher than the most reliable value. However, since the equipment was used to determine the temperature at which freezing occuired rather than the per cent water frozen at various temperatures, no correction was necessary. Gelatin, sucrose, and glycerol solutions of various concentrations were frozen and thawed in the dilatometer at various temperature differentials in the presence and absence of nucleating agents. Reagent-grade sucrose and glycerol (correcting for water) and Bactogelatin, containing about 2% ash, were used. Insoluble hexagonal crystals of silver iodide or calcium carbonate were added to some solutions to provide nuclei at a higher temperature. Approximately 0.25 g. of these salts was added to each aliquot. of solution, but only a small fraction remained suspended during freezing. Pure hexagonal silver iddide crystals were obtained by diluting a fivefold 0.2 M solution of silver iodide in 2 iVl potassium iodide (1). The calcium carbonate (reagent grade) was heated for 24 hr. at 470°C. RESULTS
The curves obtained by plotting volume change against mean temperature during freezing and thawing (e.g., Fig. 2) must be interpreted in relation to the method and apparatus. Section AB represents the net decrease in fluid volume during cooling. At B, nucleation started
246
C.
V.
LUSENA
AND
W.
H.
COOK
r
7
10%
5%
:E
4.0 ‘D /
L
3.0 -
FREEZING
2.0 0 ADDITION
. -THAWING
i w *
-l.OH 1 c-0
f *
Y
I
I t5 FIG.
0 2.
-5
-10 TEMPERATURE,
0 *C
I
-5
I
-10
!
- .I5
Freezing and thawing curves for 5, 10, 15, and ZOoJogelatin with and without AgI and CaCOa at 40°C. differential.
at the wall, where the temperature
was lower than the mean temperature by about, 5°C. Between B and C more nuclei were formed, and at C the rate of heat production equaled the rate of heat, removal. Between C and D most of the water froze. The estimated average temperature in this region is the “freezing temperature” of the system. From D to E the last traces of water were frozen and the contraction of the system during cooling gradually counteracted the expansion of ice formation.
ICE
PROPAGATION
IN
BIOLOGICAL
SYSTEMS.
11
217
In the thawing curves, the initial slight increase in volume, attributed to expansion of the ice, was gradually offset by some melting (EF). From F to G most of the ice melted. In the region GH the presence of residual ice was indicated by the temperature of the center thermocouple. Thawing of this ice caused a slight decrease in net volume. The difference in volume between A and H and the small discrepancies in the DEF region mere largely accounted for by the difference in in temperature of the manometric oil, which during cooling was colder and therefore more contracted than during thawing. Ot)her temperature gradients throughout the system may also have introduced minor errors. Occasional deviations from the drawn curves in the CD
FI G. 3. Freezing curves for 3 and 4 molal sucrose with and AgI at 30°C. (right) and 40°C. (left) differential.
without
248
C. V. LUSENA
AND W. H. COOK
region were caused by sudden development of an ice film along the full length of the needle recording the average temperature. With these explanations of the curves the following results can be observed in Fig. 2. In 5 % gelatin the change in volume during freezing or thawing occurred at the freezing point (F.P.) (CD coincides with FG at about -0.5”C.). With gelatin concentrations of 10, 15, and 20%, the the freezing temperature was about -3, -7, and - lO”C., respectively, although thawing still occurred at the F.P. Addition of silver iodide caused most of the solution to freeze near the F.P., while addition of calcium carbonate (a less effective nucleating agent) allowed freezing to occur at an intermediate temperature. With all gelatin concentrations tested, the freezing temperature was independent of the cooling rate within the range represented by 40°C. to 25°C. differentials. At the slow cooling rate obtained by a 2°C. differential, all jellies froze at the F.P. These results were readily reproduced as long as Bactogelatin was used, but other gelatin preparations behaved differently. In general, jellies prepared from gelatin that had been purified, ground, or heated above 60°C. froze nearer the F.P. Glycerol and sucrose solutions were also studied (Figs. 3 and 4). Thawing curves have been omitted as they did not differ significantly from the freezing curves in the presence of silver iodide. The results with 3 and 4 molal sucrose solutions at differentials of 40” and 30°C. (Fig. 3) and those with 10 and 15 molal glycerol at a 40°C. differential (Fig. 4) indicate that increasing the solute concentration depressesnot only the F.P. but also depressesthe freezing temperature below the true I
I
I IO
I
1
I -I
0
-eo
I
I
I
I
I -30
I
I
*
I
I
I5 MOLAL
MOLAL
I
-40
I -25
I
I -,
I
-4
I
-56
IEYPERIT”RE,~O. FIG.
4. Freezing
curves for 10 and 15 molal glycerol with and without AgI at 40°C. differential.
ICE
PROPAGATION
IN
BIOLOGICAL
SYSTEMS.
11
249
F.P. The results with sucrose solutions also show that increasing the rate of cooling decreased the freezing temperature. The pattern for 3 molal sucrose at a 40°C. differential is very similar to the one for 4 molal at a 30°C. differential, allowing for the difference in depression of the F.P. and amount of freezable water. This indicates that increases in either the cooling rate or the solute concentration have a similar effect on the freezing pattern. No volume increase was observed with 15 molal glycerol without silver iodide at a differential of 4O”C., i.e., a final bath temperature of about - 100°C. DISCUSSION
The results presented in a previous paper (6) show that when concentrated solutions of gelatin and sucrose were frozen rapidly, supercooling occurred ahead of the crystal front. In the present paper it is shown that this phenomenon is also dependent on nucleation since, at a fixed rate of cooling, the addition of hexagonal crystals of silver iodide or calcium carbonate caused volume expansion to occur at a higher temperature. Nucleation in ordinary aqueous solutions is heterogeneous and occurs at a “critical nucleation temperature” (C.N.T.) determined by the nature of the solution and the type of “mote” present (3). Since the C.N.T. is below the F.P., supercooling occurs initially. At slow cooling rates, the heat of crystallization is sufficient to raise the temperature to the F.P. With faster cooling, the temperature of the solution remains below the F.P. At temperatures above the C.N.T., growth of the existing crystals must be occurring sufficiently rapidly to balance the rate of heat removal. Under these conditions large ice crystals formed in living tissues might penetrate cell membranes. When the freezing temperature falls to the C.N.T., new nuclei form in the media, producing a large number of small crystals; some of these may form ahead of the existing crystal front and even beyond such structures as cell membranes (6). Since the crystals formed by new nuclei contribute to the rate of heat production, the freezing temperature can be depressed below the C.N.T. only under exceptional conditions. At very low temperatures nucleation and crystal growth are reduced and may be suppressed; therefore, some water may remain unfrozen. To obtain small crystals or an amorphous phase, it is essential that the rate of heat removal be extremely rapid or that the rate of crystal growth be retarded. Since there are practical limits to the maximum rate of heat removal that can be attained, the factors controlling rate of heat production must be considered. The rate of ice formation is determined by
250
C. V. LUSENA
AND W. H. COOK
the number of nuclei present and by the rate of crystal growth. Nucleation occurs at the C.N.T., which is determined by the “motes” present in the system (3). On the other hand, rate of crystal growth increases as the freezing temperature decreasesto about 20°C. below F.P., but is mainly affected by the kind and concentration of the solutes present (11). If high concentrations of solute are developed as ice is formed, it is possible to conceive of conditions where ice formation cannot provide sufficient heat to prevent a rapid temperature decreaseto the region where nucleation and crystal growth are suppressed. When this occurs, the small ice crystals may be interspersed with unfrozen solution. A 15 molal glycerol solution (Fig. 4) without silver iodide was sufficiently concentrated to remain unfrozen and no volume increase was evident during fast cooling to -55°C. These considerations suggest that the increase in concentration of of certain sap constituents associated with winter hardiness (8), and the addition of glycerol and sucrose (4, 5, 9) to freezing biological materials, are beneficial because they contribute to the formation of small crystals and possibly to the retention of some unfrozen water. The effect of biologically important solutes on the rate of crystal growth and nucleation will be described in a future publication. SUMMARY
In concentrated solutions of gelatin, sucrose, and glycerol, crystal formation occurred at the freezing point at slow cooling rates, but below the freezing point at rapid cooling rates. Increasing either the concentration of solute or the rate of cooling increased the difference between freezing point and temperature of freezing. Addition of hexagonal crystals of silver iodide and calcium carbonate raised the “crit.ical nucleation temperature” and thereby the freezing temperature. It is suggestedthat large ice crystals are formed when freezing occurs above the critical nucleation temperature. However, when freezing occurs at the critical nucleation temperature small ice crystals are formed, and under suitable conditions some water may not freeze. It is further concluded that the effect of solutes on nucleation and rate of crystal growth must be a major factor controlling the pattern of ice propagation. REFERENCES
R., AND MILLER, H., 2. physik. Chem. A162, 245 (1931). N. E., “Properties of the Ordinary Water Substance” (Am. Chem. Sot. Monograph No. 81). Reinhold, New York, 1940.
1. BLOCK,
2. DORSEY,
ICE
3. 4. 5. 6. 7. 8. 9. 10. 11.
PROPAGATION
IN
BIOLOGICAL
SYSTEMS.
11
251
DORSEY, N. E., Trans. Am. Phil. Sot. 38, 245328 (1948). GORHAM, P. R., AND CLENDENNING, K. A., Can. J. Research 28, 513 (1950). LOVELOCK, J. E., Biochim. et Biophys. Acta 11, 28 (1953). LUSENA, C. V., AND COOK, W. H., Arch. Biochem. and Biophys. 48,232 (1953). LUYET, B. F., AND GEHENIO, P. M., “Life and Death at Low Temperatures.” Biodynamics, Normandy, MO., 1940. SIMINOVITCH, D., AND BRIGGS, D. R., Plant Physiol. 28, 177 (1953). SMITH, A. V., POLGE, C., AND SMILES, J., J. Roy. Microscop. Sot. 71, 186 (1951). TAMMANN, G., AND B~CHNER, A., Z. anorg. u. allgem. Chem. 22, 12 (1935). TAMMANN, G., AND B~CHNER, A., Z. anorg. u. allgem. Chem. 22, 371 (1935).
of Applied
Biology, National Ottawa, Canada
Research Laboratories,
ReceivedNovember12, 1953 INTRODUCTION
“Snap” or “quick” freezing is generally believed to be lessinjurious to biological systems than slow freezing. Since larger ice crystals have been observed after slow than after fast cooling rates, it has been assumedthat large crystals are harmful. Some workers (7) suggest that even small intracellular crystals are injurious and that vitrification is required for storage of living materials at low temperature (7). Since the rate of heat removal is restricted by physical factors, while growth of ice crystals in distilled water is extremely rapid (lo), it is doubtful that small crystals or an amorphous phase could be obtained unless the freezing pattern was drastically affected by the structure or composition of the biological material being frozen. The relation between freezing damage and composition of intracellular solutes or extracellular suspending media has been studied by several investigators. An increase in concentration of certain solutes has been observed during the development of frost hardiness in plants (8). It also has been amply demonstrated that the addition of sugars or glycerol protects tissues and cell constituents from damage by freezing (4, 5, 9). In a model system (6), the penetration of ice through the pores of membranes was greatly reduced by solutes which acted either by depressing the freezing point or by retarding the rat3 of icecrystal growth. When concentrated gelatin jellies and sucrose solutions were rapidly cooled in the absence of membranes, supercooling occurred ahead of the advancing ice front. These observations suggested 1 Contribution issued as N.R.C. No. 3244. 0 With the technical assistance of J. M. Ross. 243
244
C.
V.
LUBENA
AND
that the kind and concentration of controlling the freezing patterns of reducing harmful effects. The effect has therefore been studied by a new METHOD
W.
H.
COOK
solute might be major factors in biological materials and thus in of splutes on freezing behavior dilatometric method.
AND MATERIrlLS
Use of a “dynamic” dilatometer, in contrast to an ordinary dilatometer in which the volume is measured after equilibrium is attained at a constant temperature, permitted the volume and temperature of the sample to be measured continuously at a constant rate of heat transfer. By plotting volume change against temperature, the temperature at which ice was formed could be determined at various rates of cooling. The dilatometer vessel (Fig. 1) consisted of brass pipe, about 3.5 cm. I.D. and 6.0 cm. height, sealed at the bottom and fitted with a screw cap with connections
0
2
4
6
CM.
FIG. 1. “Dynamic”
dilatometer.
ICE
PROPAGATION
IN
BIOLOGICAL
SYSTEMS.
11
245
to a manometer and to a filling spout with stopcock. The manometer was jacketed with circulating ice water. The vessel and connections to manometer and filling spout were surrounded by an air jacket fully immersed in an ethanol bath. The bath was adjusted to the required constantly changing temperatures by carefully balancing the flow of liquid nitrogen through a cooling coil and electricity through a heater. The rate of heat removal during freezing and of heat addition during thawing was controlled by maintaining a constant temperature differential, within &0,5”C., between the bath and the wall of the dilatometer. The temperature in the dilatometer vessel was measured with three thermocouples, one at the wall and two within the dilatometer vessel. The latter were placed in sealed hypodermic needles arranged vertically through the bottom of the vessel and extending to within 3 mm. of the interface of the solution and manometric fluid. One of these was in the center and the other halfway between center and wall. Preliminary experiments showed that the temperature recorded by the latter thermocouple was a good estimate of the average temperature of the medium. This average temperature, which is plotted in all figures, was automatically recorded every 30 sec., and the wall and center temperature every 60 sec. In each test, a 51-ml. aliquot of solution was thoroughly deaerated and frozen. If air bubbles appeared on thawing, the results were rejected. The manometric fluid was an oil (15 ml.) with a low pour point and viscosity. Because the time required to freeze the 51 ml. of solution varied with the concentration of solutes, rates of cooling were expressed as the temperature differential in degrees centigrade. A 51-ml. aliquot of water could be cooled 20°C. (from $5 to -15’C.) or heated through the same range in 75 min. at 40°C. differential, and in 150 min. at 25°C. differential. The performance of the instrument was checked by freezing distilled water. The volume expansion was within the range of reported values (2) but about 5-10% higher than the most reliable value. However, since the equipment was used to determine the temperature at which freezing occuired rather than the per cent water frozen at various temperatures, no correction was necessary. Gelatin, sucrose, and glycerol solutions of various concentrations were frozen and thawed in the dilatometer at various temperature differentials in the presence and absence of nucleating agents. Reagent-grade sucrose and glycerol (correcting for water) and Bactogelatin, containing about 2% ash, were used. Insoluble hexagonal crystals of silver iodide or calcium carbonate were added to some solutions to provide nuclei at a higher temperature. Approximately 0.25 g. of these salts was added to each aliquot. of solution, but only a small fraction remained suspended during freezing. Pure hexagonal silver iddide crystals were obtained by diluting a fivefold 0.2 M solution of silver iodide in 2 iVl potassium iodide (1). The calcium carbonate (reagent grade) was heated for 24 hr. at 470°C. RESULTS
The curves obtained by plotting volume change against mean temperature during freezing and thawing (e.g., Fig. 2) must be interpreted in relation to the method and apparatus. Section AB represents the net decrease in fluid volume during cooling. At B, nucleation started
246
C.
V.
LUSENA
AND
W.
H.
COOK
r
7
10%
5%
:E
4.0 ‘D /
L
3.0 -
FREEZING
2.0 0 ADDITION
. -THAWING
i w *
-l.OH 1 c-0
f *
Y
I
I t5 FIG.
0 2.
-5
-10 TEMPERATURE,
0 *C
I
-5
I
-10
!
- .I5
Freezing and thawing curves for 5, 10, 15, and ZOoJogelatin with and without AgI and CaCOa at 40°C. differential.
at the wall, where the temperature
was lower than the mean temperature by about, 5°C. Between B and C more nuclei were formed, and at C the rate of heat production equaled the rate of heat, removal. Between C and D most of the water froze. The estimated average temperature in this region is the “freezing temperature” of the system. From D to E the last traces of water were frozen and the contraction of the system during cooling gradually counteracted the expansion of ice formation.
ICE
PROPAGATION
IN
BIOLOGICAL
SYSTEMS.
11
217
In the thawing curves, the initial slight increase in volume, attributed to expansion of the ice, was gradually offset by some melting (EF). From F to G most of the ice melted. In the region GH the presence of residual ice was indicated by the temperature of the center thermocouple. Thawing of this ice caused a slight decrease in net volume. The difference in volume between A and H and the small discrepancies in the DEF region mere largely accounted for by the difference in in temperature of the manometric oil, which during cooling was colder and therefore more contracted than during thawing. Ot)her temperature gradients throughout the system may also have introduced minor errors. Occasional deviations from the drawn curves in the CD
FI G. 3. Freezing curves for 3 and 4 molal sucrose with and AgI at 30°C. (right) and 40°C. (left) differential.
without
248
C. V. LUSENA
AND W. H. COOK
region were caused by sudden development of an ice film along the full length of the needle recording the average temperature. With these explanations of the curves the following results can be observed in Fig. 2. In 5 % gelatin the change in volume during freezing or thawing occurred at the freezing point (F.P.) (CD coincides with FG at about -0.5”C.). With gelatin concentrations of 10, 15, and 20%, the the freezing temperature was about -3, -7, and - lO”C., respectively, although thawing still occurred at the F.P. Addition of silver iodide caused most of the solution to freeze near the F.P., while addition of calcium carbonate (a less effective nucleating agent) allowed freezing to occur at an intermediate temperature. With all gelatin concentrations tested, the freezing temperature was independent of the cooling rate within the range represented by 40°C. to 25°C. differentials. At the slow cooling rate obtained by a 2°C. differential, all jellies froze at the F.P. These results were readily reproduced as long as Bactogelatin was used, but other gelatin preparations behaved differently. In general, jellies prepared from gelatin that had been purified, ground, or heated above 60°C. froze nearer the F.P. Glycerol and sucrose solutions were also studied (Figs. 3 and 4). Thawing curves have been omitted as they did not differ significantly from the freezing curves in the presence of silver iodide. The results with 3 and 4 molal sucrose solutions at differentials of 40” and 30°C. (Fig. 3) and those with 10 and 15 molal glycerol at a 40°C. differential (Fig. 4) indicate that increasing the solute concentration depressesnot only the F.P. but also depressesthe freezing temperature below the true I
I
I IO
I
1
I -I
0
-eo
I
I
I
I
I -30
I
I
*
I
I
I5 MOLAL
MOLAL
I
-40
I -25
I
I -,
I
-4
I
-56
IEYPERIT”RE,~O. FIG.
4. Freezing
curves for 10 and 15 molal glycerol with and without AgI at 40°C. differential.
ICE
PROPAGATION
IN
BIOLOGICAL
SYSTEMS.
11
249
F.P. The results with sucrose solutions also show that increasing the rate of cooling decreased the freezing temperature. The pattern for 3 molal sucrose at a 40°C. differential is very similar to the one for 4 molal at a 30°C. differential, allowing for the difference in depression of the F.P. and amount of freezable water. This indicates that increases in either the cooling rate or the solute concentration have a similar effect on the freezing pattern. No volume increase was observed with 15 molal glycerol without silver iodide at a differential of 4O”C., i.e., a final bath temperature of about - 100°C. DISCUSSION
The results presented in a previous paper (6) show that when concentrated solutions of gelatin and sucrose were frozen rapidly, supercooling occurred ahead of the crystal front. In the present paper it is shown that this phenomenon is also dependent on nucleation since, at a fixed rate of cooling, the addition of hexagonal crystals of silver iodide or calcium carbonate caused volume expansion to occur at a higher temperature. Nucleation in ordinary aqueous solutions is heterogeneous and occurs at a “critical nucleation temperature” (C.N.T.) determined by the nature of the solution and the type of “mote” present (3). Since the C.N.T. is below the F.P., supercooling occurs initially. At slow cooling rates, the heat of crystallization is sufficient to raise the temperature to the F.P. With faster cooling, the temperature of the solution remains below the F.P. At temperatures above the C.N.T., growth of the existing crystals must be occurring sufficiently rapidly to balance the rate of heat removal. Under these conditions large ice crystals formed in living tissues might penetrate cell membranes. When the freezing temperature falls to the C.N.T., new nuclei form in the media, producing a large number of small crystals; some of these may form ahead of the existing crystal front and even beyond such structures as cell membranes (6). Since the crystals formed by new nuclei contribute to the rate of heat production, the freezing temperature can be depressed below the C.N.T. only under exceptional conditions. At very low temperatures nucleation and crystal growth are reduced and may be suppressed; therefore, some water may remain unfrozen. To obtain small crystals or an amorphous phase, it is essential that the rate of heat removal be extremely rapid or that the rate of crystal growth be retarded. Since there are practical limits to the maximum rate of heat removal that can be attained, the factors controlling rate of heat production must be considered. The rate of ice formation is determined by
250
C. V. LUSENA
AND W. H. COOK
the number of nuclei present and by the rate of crystal growth. Nucleation occurs at the C.N.T., which is determined by the “motes” present in the system (3). On the other hand, rate of crystal growth increases as the freezing temperature decreasesto about 20°C. below F.P., but is mainly affected by the kind and concentration of the solutes present (11). If high concentrations of solute are developed as ice is formed, it is possible to conceive of conditions where ice formation cannot provide sufficient heat to prevent a rapid temperature decreaseto the region where nucleation and crystal growth are suppressed. When this occurs, the small ice crystals may be interspersed with unfrozen solution. A 15 molal glycerol solution (Fig. 4) without silver iodide was sufficiently concentrated to remain unfrozen and no volume increase was evident during fast cooling to -55°C. These considerations suggest that the increase in concentration of of certain sap constituents associated with winter hardiness (8), and the addition of glycerol and sucrose (4, 5, 9) to freezing biological materials, are beneficial because they contribute to the formation of small crystals and possibly to the retention of some unfrozen water. The effect of biologically important solutes on the rate of crystal growth and nucleation will be described in a future publication. SUMMARY
In concentrated solutions of gelatin, sucrose, and glycerol, crystal formation occurred at the freezing point at slow cooling rates, but below the freezing point at rapid cooling rates. Increasing either the concentration of solute or the rate of cooling increased the difference between freezing point and temperature of freezing. Addition of hexagonal crystals of silver iodide and calcium carbonate raised the “crit.ical nucleation temperature” and thereby the freezing temperature. It is suggestedthat large ice crystals are formed when freezing occurs above the critical nucleation temperature. However, when freezing occurs at the critical nucleation temperature small ice crystals are formed, and under suitable conditions some water may not freeze. It is further concluded that the effect of solutes on nucleation and rate of crystal growth must be a major factor controlling the pattern of ice propagation. REFERENCES
R., AND MILLER, H., 2. physik. Chem. A162, 245 (1931). N. E., “Properties of the Ordinary Water Substance” (Am. Chem. Sot. Monograph No. 81). Reinhold, New York, 1940.
1. BLOCK,
2. DORSEY,
ICE
3. 4. 5. 6. 7. 8. 9. 10. 11.
PROPAGATION
IN
BIOLOGICAL
SYSTEMS.
11
251
DORSEY, N. E., Trans. Am. Phil. Sot. 38, 245328 (1948). GORHAM, P. R., AND CLENDENNING, K. A., Can. J. Research 28, 513 (1950). LOVELOCK, J. E., Biochim. et Biophys. Acta 11, 28 (1953). LUSENA, C. V., AND COOK, W. H., Arch. Biochem. and Biophys. 48,232 (1953). LUYET, B. F., AND GEHENIO, P. M., “Life and Death at Low Temperatures.” Biodynamics, Normandy, MO., 1940. SIMINOVITCH, D., AND BRIGGS, D. R., Plant Physiol. 28, 177 (1953). SMITH, A. V., POLGE, C., AND SMILES, J., J. Roy. Microscop. Sot. 71, 186 (1951). TAMMANN, G., AND B~CHNER, A., Z. anorg. u. allgem. Chem. 22, 12 (1935). TAMMANN, G., AND B~CHNER, A., Z. anorg. u. allgem. Chem. 22, 371 (1935).