Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at −196°C and −20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (−196°C and −80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at −20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

The Rejoining Time of Chromatid Breaks Induced by Gamma Radiation in Vicia faba Root Tips at 3 °C

The observation was made previously that the reduction in radiosensitivity in Vicia faba (as measured by postirradiation root growth) by prolonging the exposure time from about 10 minutes to 24 hours is much less marked at 3°C. than at 19°C. If chromosome damage is mainly responsible for the reduced root growth, this observation might be explained by a smaller drop in the "two-hit" aberration component, resulting from an increased time for which breaks are available for rejoining at 3°C. This hypothesis was tested by comparing chromatid aberration frequencies in root meristem cells produced by 105 rads of ⁶⁰Co γ rays, given at dose rates of 19.4 and 0.073 rads per minute. Beans were maintained in aerated water at 2°C. prior to and during irradiation, and at this temperature the rate of development of cells was such that the two different exposure times both occupied a period during which the cell sensitivity was approximately constant. Immediately subsequent to irradiation, the roots were returned to 19°C. and examined cytologically. All chromatid aberrations were less frequent after low dose rate treatment, but only the chromatid interchange reduction was significant. The average time for which breaks are available for reunion, calculated from Lea's G function, was found to be 12 hours (95 per cent C.L. 6 to 24 hours).     

Potassium Chloride as Stomatal Osmoticum in Allium cepa L., a Species Devoid of Starch in Guard Cells

K⁺ and Cl⁻ contents of guard cells and of ordinary epidermal cells were determined in epidermal samples of Allium cepa L. by electron probe microanalysis; malate contents of the same samples were determined by enzymic oxidation. KCl was, in general, the major osmoticum in guard cells, irrespective of whether stomata had opened on leaves or in epidermal strips floating on solutions. The solute requirement varied between 50 and 110 femtomoles KCl per micrometer increase in aperture per pair of guard cells. Stomata did not open on solutions of K iminodiacetate, presumably because its anion could not be taken up. Stomata opened if KCl or KBr was provided. Taken together, the results indicate that the absence of starch from guard cells deprived them of the ability to produce malate in amounts of osmotic consequence and that the presence of absorbable Cl⁻ (or Br⁻) was necessary for stomatal opening.     

Induction of Freezing Tolerance in Spinach during Cold Acclimation

Spinach (Spinacia oleracea L.) seedlings, grown in soil or on an agar medium in vitro, became cold acclimated when exposed to a constant 5°C. Plants subjected to cold acclimation, beginning 1 week postgermination, attained freezing tolerance levels similar to that achieved by seedlings that were cold acclimated beginning 3 weeks after sowing. Seedlings at 1 week of age had only cotyledonary leaves, while 3-week-old seedlings had developed true leaves. Plants grown in vitro were able to increase in freezing tolerance, but were slightly less hardy than soil-grown plants. These results suggest that spinach, a cool-season crop that begins growth in early spring when subzero temperatures are likely, can undergo cold acclimation at the earliest stages of development following germination. Axenic seedlings, grown in vitro, were used to develop a noninjurious radiolabeling technique. Leaf proteins were radiolabeled to specific activities of 10⁵ counts per minute per microgram at 25°C or 5 × 10⁴ counts per minute per microgram at 5°C over a 24 hour period. The ability to radiolabel leaf proteins of in vitro grown plants to high specific activities at low temperature, without injury or microbial contamination, will facilitate studies of cold acclimation.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

OSMOTIC PROPERTIES OF THE EGG CELLS OF THE OYSTER (OSTREA VIRGINICA)

Investigations of the osmotic properties of oyster eggs by a diffraction method for measuring volumes have led to the following conclusions: 1. The product of cell volume and osmotic pressure is approximately constant, if allowance is made for osmotically inactive cell contents (law of Boyle-van''t Hoff). The space occupied by osmotically inactive averages 44 per cent of cell volume. 2. Volume changes over a wide range of pressures are reversible, indicating that the semipermeability of the cell during such changes remains intact. 3. The kinetics of endosmosis and of exosmosis are described by the equation, See PDF for Equation, where dV is rate of volume change; S, surface area of cell, (P-P_e), the difference in osmotic pressure between cell interior and medium, and K, the permeability of the cell to water. 4. Permeability to water during endosmosis is 0.6µ³ of water per minute, per square micron of cell surface, per atmosphere of pressure. The value of permeability for exosmosis is closely the same; in this respect the egg cell of the oyster appears to be a more perfect osmometer than the other marine cells which have been studied. Permeability to water computed by the equation given above is in good agreement with computations by the entirely different method devised by Jacobs. 5. Permeability to diethylene glycol averages 27.2, and to glycerol 20.7. These values express the number of mols x 10^–15 which enter per minute through each square micron of cell surface at a concentration difference of 1 mol per liter and a temperature of 22.5°C. 6. Values for permeability to water and to the solutes tested are considerably higher for the oyster egg than for other forms of marine eggs previously examined. 7. The oyster egg because of its high degree of permeability is a natural osmometer particularly suitable for the study of the less readily penetrating solutes.     

Studies on the effects of subzero temperatures on the viability of spores of Aspergillus flavus. I. The effect of rate of warming

1. The survival of spores of Aspergillus flavus suspended in distilled water and cooled rapidly to –70 to –75°C. was found to depend primarily on the rate of subsequent warming of the frozen suspension. Only 7 per cent of the spores germinated following slow warming at 0.9°C. per minute, whereas about 75 per cent germinated following rapid warming at 700°C. per minute. 2. Viability was dependent on the rate at which the suspensions warmed from –70 to 0°C. (subzero warming), but was not dependent on the rate of thawing of the frozen water in which the spores were suspended. 3. The logarithm of the percentage of germination appeared to be a linear function of the logarithm of the rate of subzero warming when spores were warmed at rates ranging from 0.12 to 1000°C. per minute. 4. The lethal effects of slow warming from –70 to 0°C. were more pronounced between about –20 and 0°C. than between –70 and –20°C. In the former range of temperatures, the percentage of germination decreased sharply as slow warming progressed towards 0°C. 5. Slow warming from –70 to 0°C. was more harmful to the spores than was a 1 or 2 hour exposure to constant temperatures between –70 and 0°C. 6. Slow warming was found to be more harmful than rapid warming when spores were suspended in horse serum, 0.16 molal sodium chloride, or 0.29 molal sucrose as well as in distilled water.     

Cellular osmotic phenomena during stomatal movements ofCommelina communis

Summary The accuracy of most of the published values for guard cell osmotic pressures is disputed and it is considered that many values are grossly in error. Since most of the values were obtained from incipient plasmolysis experiments limitations of the technique were investigated. It was concluded that it is not possible to use the incipient plasmolysis method for accurately determining guard cell osmotic pressures since all concentrations of plasmolytica (concentrations down to 0.1 M sucrose or calcium nitrate were used) bring about incipient plasmolysis depending on the period of time the tissue is immersed in the plasmolytica. In other words, the concentration of a plasmolyticum at which incipient plasmolysis occurs continues to decrease as the plasmolysing time increases. Furthermore, the time taken for incipient plasmolysis to occur varies according to the solutes in the plasmolyticum and the extent of stomatal aperture.A reason for the changing values of guard cell osmotic pressures was the loss of K⁺, and to a lesser extent, Cl^–, Ca²⁺ and Na⁺, and sugars and organic acids from the tissue during exposure to graded concentrations of plasmolytica (sucrose and calcium nitrate). A good correlation between loss of solutes from the epidermal tissue and decrease in guard cell osmotic pressure was not observed, however.Histochemical tests for K⁺ support the view that leakage of K⁺ from the guard cells occurs while the tissue is immersed in the plasmolytica except when high concentrations of sucrose (2.0 M) and calcium nitrate (greater than 1.0 M) were used and then leakage was minimal. However, these high concentrations of plasmolytica caused cell damage.The osmotic relationships of the various cell types within the epidermis ofCommelina communis were investigated during stomatal movements. Although absolute values for the osmotic pressures of the various cell types could not be evaluated it was apparent from the rates of changes of the osmotic pressures that when stomata closed guard cell osmotic pressures decreased while epidermal and subsidiary cell osmotic pressures increased to almost the same values as the guard cells.     

The Effects of Streptomycin, Desiccation, and UV Radiation on Ice Nucleation by Pseudomonas viridiflava

Streptomycin (100 micrograms per milliliter), desiccation (over CaSO₄), and ultraviolet radiation (4500 microwatts per square centimeter at 254 nanometers for 15 minutes) reduced ice nucleation activity by Pseudomonas viridiflava strain W-1 as determined by freezing drops of the bacterial suspensions. Highest residual ice nucleation activity by dead cells was obtained by desiccation, although no freezing above −3.5°C was detected. The rate and extent of loss of ice nucleation activity following streptomycin and ultraviolet treatment was affected by preconditioning temperature. At 21°C and above, loss of activity by dead cells was rapid and irreversible.     

Enzymic and substrate basis for the anaplerotic step in guard cells

From the maximum rate of malate accumulation in Vicia faba L. guard cells during stomatal opening the maximum rate of organic anion synthesis is calculated to be 200 millimoles per kilogram dry weight per hour. A minimum estimate for the phosphoenolpyruvate (PEP) carboxylase-catalyzed reaction in guard cells is 650 millimoles per kilogram dry weight per hour which is significantly higher than in any other leaf tissue. The apparent K_mpep of the guard cell enzyme is 60 μm at pH 8.7, but is probably higher at lower pH. The concentration of PEP in guard cells was 270μm (=2.2 × 10⁻¹⁵ moles/guard cell pair) during anion synthesis. These results support the possibility that the carboxylation of PEP is the anaplerotic step in guard cells.     

A novel method of natural cryoprotection : intracellular glass formation in deeply frozen populus

Correlating measurements from differential scanning calorimetry, freeze-fracture freeze-etch electron microscopy, and survival of twigs after two-step cooling experiments, we provide strong evidence that winter-hardened Populus balsamifera v. virginiana (Sarg.) resists the stresses of freezing below −28°C by amorphous solidification (glass formation) of most of its intracellular contents during slow cooling (≤5°C per hour). It is shown that other components of the intracellular medium go through glass transitions during slow cooling at about −45°C and below −70°C. This `three glass' model was then used to predict the results of differential scanning calorimetry, freeze-fracture freeze-etch electron microscopy, and biological experiments. This model is the first definitive explanation for the resistance of a woody plant to liquid N₂ temperatures even if quench cooling (1200°C per minute) begins at temperatures as high as −20°C and warming is very slow (≤5°C per hour). It is also the first time high temperature natural intracellular glass formation has been demonstrated.     

Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation

The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below −30°C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below −25°C. There was a close association between the per cent unfrozen water at −40°C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at −40°C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.     

Survival of Plant Tissue at Super-Low Temperature VI. Effects of Cooling and Rewarming Rates on Survival

The survival rates of the cortical parenchymal cells of mulberry tree were determined as a function of cooling and rewarming rates. When cooling was carried out slowly at 1° to 15° per minute, all of the cells still remained viable even when rewarmed either rapidly or slowly. Survival rates gradually decreased to zero as the cooling rate increased from about 15° to 2000° per minute. In the intermediate cooling rates, when the cells were cooled at the rates lower than 14° per minute, from −2.2° to about −10°, these cells could survive subsequent rapid cooling and rewarming.

However, at cooling rates above 1000° per minute and with rapid rewarming, the effect of cooling rate reversed and survival increased, reaching a maximum at about 200,000° per minute. As the cooling rate increased above 15° per minute, survival rates became increasingly dependent on the rewarming rate, with rapid rewarming becoming less deleterious than slow rewarming.

The temperature range at which damage occurred during rewarming following removal from liquid nitrogen and in which growth rate of ice crystallization was greatest, was −30° to −40°. The survival rates even in the prefrozen cells at −30° decreased considerably by keeping them at −30° for 10 minutes after removal from liquid nitrogen. This fact indicates that intracellular freezable water remains to some degree even in the prefrozen cells at −30°. After removal from liquid nitrogen, all cells retained their viability, when they were passed rapidly through a temperature range between −50° and −2.5° within about 2 seconds, namely at the rates greater than 1000° per minute.

These observations are explained in terms of the size of the crystals formed within the cortical cells.

     

Stomatal opening quantitatively related to potassium transport: evidence from electron probe analysis

When stomata of Vicia faba opened (from a stomatal aperture of about 2 micrometers to one of 12 micrometers) the solute content of the guard cells increased by 4.8 × 10⁻¹² osmoles per stoma. During the same time an average of 4.0 × 10⁻¹² gram equivalents of K⁺ were transported into each pair of guard cells. This amount of K⁺, if associated with dibasic anions, is sufficient to produce the changes in guard cell volume and osmotic pressure associated with stomatal opening. Analysis of Cl, P, and S showed that these elements were not transported in significant amounts during stomatal opening. This finding suggests that the anions balancing K⁺ were predominantly organic. K⁺ was specifically required because no other elements, likely to be present as cations, were found to accumulate in appreciable quantities in guard cells of open stomata.     

Changes in Osmotic Pressure and Mucilage during Low-Temperature Acclimation of Opuntia ficus-indica

Opuntia ficus-indica, a Crassulacean acid metabolism plant cultivated for its fruits and cladodes, was used to examine chemical and physiological events accompanying low-temperature acclimation. Changes in osmotic pressure, water content, low molecular weight solutes, and extracellular mucilage were monitored in the photosynthetic chlorenchyma and the water-storage parenchyma when plants maintained at day/night air temperatures of 30/20°C were shifted to 10/0°C. An increase in osmotic pressure of 0.13 megapascal occurred after 13 days at 10/0°C. Synthesis of glucose, fructose, and glycerol accounted for most of the observed increase in osmotic pressure during the low-temperature acclimation. Extracellular mucilage and the relative apoplastic water content increased by 24 and 10%, respectively, during exposure to low temperatures. These increases apparently favor the extracellular nucleation of ice closer to the equilibrium freezing temperature for plants at 10/0°C, which could make the cellular dehydration more gradual and less damaging. Nuclear magnetic resonance studies helped elucidate the cellular processes during ice formation, such as those revealed by changes in the relaxation times of two water fractions in the chlorenchyma. The latter results suggested a restricted mobility of intracellular water and an increased mobility of extracellular water for plants at 10/0°C compared with those at 30/20°C. Increased mobility of extracellular water could facilitate extracellular ice growth and thus delay the potentially lethal intracellular freezing during low-temperature acclimation.     

Freezing injury and root development in winter cereals

Upon exposure to 2°C, the leaves and crowns of rye (Secale cereale L. cv `Puma') and wheat (Triticum aestivum L. cv `Norstar' and `Cappelle') increased in cold hardiness, whereas little change in root cold hardiness was observed. Both root and shoot growth were severely reduced in cold-hardened Norstar wheat plants frozen to −11°C or lower and transplanted to soil. In contrast, shoot growth of plants grown in a nutrient agar medium and subjected to the same hardening and freezing conditions was not affected by freezing temperatures of −20°C while root growth was reduced at −15°C. Thus, it was apparent that lack of root development limited the ability of plants to survive freezing under natural conditions.

Generally, the temperatures at which 50% of the plants were killed as determined by the conductivity method were lower than those obtained by regrowth. A simple explanation for this difference is that the majority of cells in the crown are still alive while a small portion of the cells which are critical for regrowth are injured or killed.

Suspension cultures of Norstar wheat grown in B-5 liquid medium supplemented with 3 milligrams per liter of 2,4-dichlorophenoxyacetic acid could be cold hardened to the same levels as soil growth plants. These cultures produce roots when transferred to the same growth medium supplemented with a low rate of 2,4-dichlorophenoxyacetic acid (<1 milligram per liter). When frozen to −15°C regrowth of cultures was 50% of the control, whereas the percentage of calli with root development was reduced 50% in cultures frozen to −11°C. These results suggest that freezing affects root morphogenesis rather than just killing the cells responsible for root regeneration.

     

Cation exchange in mammalian erythrocytes. III. The prolytic effect of x-rays on human cells

Freshly drawn heparinized human whole blood is exposed to x-rays in amounts up to 54,000 r in vitro and then equilibrated under a controlled atmosphere at 24 or 38°C. For as long as 26 hours following exposure, potassium is progressively lost from the cells and quantitatively replaced by sodium with little, if any, osmotic disturbance. The mean rate of loss at 20,000 r and 24°C. is about 0.4 per cent of the initial cell potassium per hour and approximately doubles for a 20,000 r increase. It is accentuated if blood is stored at low temperature (5°C.) following radiation exposure. Isotope experiments show that the rate of entrance of potassium into the cells is practically unaltered, the principal effect being an acceleration of the rate from cells to plasma. This suggests that radiation may have interfered with a mechanism of selective potassium accumulation based on preferential retention of the element. The sodium which enters the cells following irradiation contributes to the rapidly exchanging portion of the cellular sodium, suggesting that this fraction is ionic sodium.     

Role of Abscisic Acid in the Induction of Freezing Tolerance in Brassica napus Suspension-Cultured Cells

Brassica napus suspension-cultured cells could be hardened in 6 days at 25°C by the addition of mefluidide or ABA to the culture medium. Cells treated with mefluidide (10 milligrams per liter) or ABA (50 micromolar) attained an LT₅₀ of −17.5°C or −18°C, respectively, while the LT₅₀ for the comparable nonhardened control (sucrose) was −10°C. The increased freezing tolerance of mefluidide-treated cells was paralleled by a 4- to 23-fold increase in ABA, as measured by gas-liquid chromatography using electron capture detection. Application of 1 milligram per liter of fluridone, an inhibitor of abscisic acid biosynthesis, prevented the mefluidide-induced increase in freezing tolerance and the accumulation of ABA. Both these inhibitory effects of fluridone were overridden by 50 micromolar ABA in the culture medium. On the basis of these results, we concluded that increased ABA levels are important for the induction of freezing tolerance in suspension-cultured cells.     

STUDIES ON OSMOTIC EQUILIBRIUM AND ON THE KINETICS OF OSMOSIS IN LIVING CELLS BY A DIFFRACTION METHOD

1. Osmotic equilibrium and kinetics of osmosis of living cells (unfertilized eggs of Arbacia punctulata) have been studied by a diffraction method. This method consists of illuminating a suspension of cells by parallel monochromatic light and measuring, by means of telescope and scale, the angular dimensions of the resulting diffraction pattern from which the average volume of the cells may be computed. The method is far less laborious and possesses several advantages over direct measurement of individual cells. The average size of a large number of cells is obtained from a single measurement of the diffraction pattern and thus individual variability is averaged out. The observations can be made at intervals of a few seconds, permitting changes in volume to be followed satisfactorily. During the measurements the cells are in suspension and are constantly stirred. 2. Volumes of cells in equilibrium with solutions of different osmotic pressure have been determined. In agreement with our previous experiments, based upon direct microscope measurements, we have confirmed the applicability of the law of Boyle-van''t Hoff to these cells; that is to say, the product of volume and pressure has been found to be approximately constant if allowance be made for the volume of osmotically inactive material of the cell contents. The volume of osmotically inactive material was found to be, on the average, 12 per cent of the initial cell volume; in eggs from different animals this value ranged from 6 to 20 per cent. 3. Permeability to water of the Arbacia egg has been found to average, at 22°C., 0.106 cubic micra of water per square micron of cell surface, per minute, per atmosphere of difference in osmotic pressure. 4. Permeability to ethylene glycol has been found to average, at 24°C., 4.0 x 10^–15 mols, per square micron of cell surface, per minute, for a concentration difference of 1 mol per liter. This is in agreement with the values reported by Stewart and Jacobs.     

Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves

The freezing tolerance and incidence of two forms of freezing injury (expansion-induced lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT₅₀ (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from −5°C to −25°C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT₅₀. However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts that were frozen to temperatures over the range of -5 to -10°C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane.