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.     

Protective effect of intracellular ice during freezing?

Injury results during freezing when cells are exposed to increasing concentrations of solutes or by the formation of intracellular ice. Methods to protect cells from the damaging effects of freezing have focused on the addition of cryoprotective chemicals and the determination of optimal cooling rates. Based on other studies of innocuous intracellular ice formation, this study investigates the potential for this ice to protect cells from injury during subsequent slow cooling. V-79W Chinese hamster fibroblasts and Madin-Darby Canine Kidney (MDCK) cells were cultured as single attached cells or confluent monolayers. The incidence of intracellular ice formation (IIF) in the cultures at the start of cooling was pre-determined using one of two different extracellular ice nucleation temperatures (-5 or -10 degrees C). Samples were then cooled at 1 degrees C/min to the experimental temperature (-5 to -40 degrees C) where samples were warmed rapidly and cell survival assessed using membrane integrity and metabolic activity. For single attached cells, the lower ice nucleation temperature, corresponding to increased incidence of IIF, resulted in decreased post-thaw cell recovery. In contrast, confluent monolayers in which IIF has been shown to be innocuous, show higher survival after cooling to temperatures as low as -40 degrees C, supporting the concept that intracellular ice confers cryoprotection by preventing cell dehydration during subsequent slow cooling.     

Thermal shock and dilution shock as the causes of freezing injury

We suggest that during slow freezing, cellular membranes are altered by the hypertonic solutions produced. This alteration in itself does not cause membrane leakage of normally impermeant solutes but it renders the cells susceptible to solute leakage on the application of a stress, which is provided during freezing by the reduction in temperature (thermal shock) and during thawing by dilution (dilution shock).During slow freezing the effects of cooling rate changes are due to the different times available for the hypertonic solutions to affect the membrane. At a given cooling rate cryoprotective agents reduce the effect on the cells at each temperature during freezing perhaps by reducing the ionic strength. The thermal shock stress during cooling and the dilution shock during thawing thus damages the cells less. With rapid freezing, there is insufficient time for these effects to take place during cooling, which allows the cells to reach low temperatures without thermal shock damage. However, the presence of extracellular ice and the formation of intracellular ice provide hypertonic conditions that render the cells liable to dilution shock on thawing. The slower the rate of thawing of rapidly cooled cells the greater will be the damage from this dilution shock.     

Ice formation and tissue response in apple twigs

Abstract. The response of apple twig tissue to a freezing stress was examined using a combination of low temperature scanning electron microscopy and freeze substitution techniques. Bark and wood tissues responded differently. In the bark, large extracellular ice crystals were observed in the cortex. The adjacent cortical cells collapsed and a large reduction in cell volume was observed. The extent of cell collapse throughout the bark was not uniform. Cells in the periderm, phloem and cambium exhibited little change in cell volume compared to cortical cells. Large extracellular ice crystals were not observed in the xylem or pith tissues. The xylem ray parenchyma and pith cells did not collapse in response to a freezing stress, but retained their original shape. The pattern of ice formation and cell response was not observed to change with season or the level of cold acclimation. This study supported the concept that bark and xylem tissues exhibit contrasting freezing behaviour. The observations were consistent with the idea that water in bark freezes extracellularly while water in xylem ray parenchyma and pith cells may supercool to temperatures approaching –40 °C prior to freezing intracellularly.     

The mechanism by which fish antifreeze proteins cause thermal hysteresis

Antifreeze proteins are characterised by their ability to prevent ice from growing upon cooling below the bulk melting point. This displacement of the freezing temperature of ice is limited and at a sufficiently low temperature a rapid ice growth takes place. The separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the temperature of ice growth is referred to as the hysteresis freezing point. The hysteresis is supposed to be the result of an adsorption of antifreeze proteins to the crystal surface. This causes the ice to grow as convex surface regions between adjacent adsorbed antifreeze proteins, thus lowering the temperature at which the crystal can visibly expand. The model requires that the antifreeze proteins are irreversibly adsorbed onto the ice surface within the hysteresis gap. This presupposition is apparently in conflict with several characteristic features of the phenomenon; the absence of superheating of ice in the presence of antifreeze proteins, the dependence of the hysteresis activity on the concentration of antifreeze proteins and the different capacities of different types of antifreeze proteins to cause thermal hysteresis at equimolar concentrations. In addition, there are structural obstacles that apparently would preclude irreversible adsorption of the antifreeze proteins to the ice surface; the bond strength necessary for irreversible adsorption and the absence of a clearly defined surface to which the antifreeze proteins may adsorb. This article deals with these apparent conflicts between the prevailing theory and the empirical observations. We first review the mechanism of thermal hysteresis with some modifications: we explain the hysteresis as a result of vapour pressure equilibrium between the ice surface and the ambient fluid fraction within the hysteresis gap due to a pressure build-up within the convex growth zones, and the ice growth as the result of an ice surface nucleation event at the hysteresis freezing point. We then go on to summarise the empirical data to show that the dependence of the hysteresis on the concentration of antifreeze proteins arises from an equilibrium exchange of antifreeze proteins between ice and solution at the melting point. This reversible association between antifreeze proteins and the ice is followed by an irreversible adsorption of the antifreeze proteins onto a newly formed crystal plane when the temperature is lowered below the melting point. The formation of the crystal plane is due to a solidification of the interfacial region, and the necessary bond strength is provided by the protein “freezing” to the surface. In essence: the antifreeze proteins are “melted off” the ice at the bulk melting point and “freeze” to the ice as the temperature is reduced to subfreezing temperatures. We explain the different hysteresis activities caused by different types of antifreeze proteins at equimolar concentrations as a consequence of their solubility features during the phase of reversible association between the proteins and the ice, i.e., at the melting point; a low water solubility results in a large fraction of the proteins being associated with the ice at the melting point. This leads to a greater density of irreversibly adsorbed antifreeze proteins at the ice surface when the temperature drops, and thus to a greater hysteresis activity. Reference is also made to observations on insect antifreeze proteins to emphasise the general validity of this approach.     

The mechanism by which fish antifreeze proteins cause thermal hysteresis

Antifreeze proteins are characterised by their ability to prevent ice from growing upon cooling below the bulk melting point. This displacement of the freezing temperature of ice is limited and at a sufficiently low temperature a rapid ice growth takes place. The separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the temperature of ice growth is referred to as the hysteresis freezing point. The hysteresis is supposed to be the result of an adsorption of antifreeze proteins to the crystal surface. This causes the ice to grow as convex surface regions between adjacent adsorbed antifreeze proteins, thus lowering the temperature at which the crystal can visibly expand. The model requires that the antifreeze proteins are irreversibly adsorbed onto the ice surface within the hysteresis gap. This presupposition is apparently in conflict with several characteristic features of the phenomenon; the absence of superheating of ice in the presence of antifreeze proteins, the dependence of the hysteresis activity on the concentration of antifreeze proteins and the different capacities of different types of antifreeze proteins to cause thermal hysteresis at equimolar concentrations. In addition, there are structural obstacles that apparently would preclude irreversible adsorption of the antifreeze proteins to the ice surface; the bond strength necessary for irreversible adsorption and the absence of a clearly defined surface to which the antifreeze proteins may adsorb. This article deals with these apparent conflicts between the prevailing theory and the empirical observations. We first review the mechanism of thermal hysteresis with some modifications: we explain the hysteresis as a result of vapour pressure equilibrium between the ice surface and the ambient fluid fraction within the hysteresis gap due to a pressure build-up within the convex growth zones, and the ice growth as the result of an ice surface nucleation event at the hysteresis freezing point. We then go on to summarise the empirical data to show that the dependence of the hysteresis on the concentration of antifreeze proteins arises from an equilibrium exchange of antifreeze proteins between ice and solution at the melting point. This reversible association between antifreeze proteins and the ice is followed by an irreversible adsorption of the antifreeze proteins onto a newly formed crystal plane when the temperature is lowered below the melting point. The formation of the crystal plane is due to a solidification of the interfacial region, and the necessary bond strength is provided by the protein "freezing" to the surface. In essence: the antifreeze proteins are "melted off" the ice at the bulk melting point and "freeze" to the ice as the temperature is reduced to subfreezing temperatures. We explain the different hysteresis activities caused by different types of antifreeze proteins at equimolar concentrations as a consequence of their solubility features during the phase of reversible association between the proteins and the ice, i.e., at the melting point; a low water solubility results in a large fraction of the proteins being associated with the ice at the melting point. This leads to a greater density of irreversibly adsorbed antifreeze proteins at the ice surface when the temperature drops, and thus to a greater hysteresis activity. Reference is also made to observations on insect antifreeze proteins to emphasise the general validity of this approach.     

Supercooling in overwintering azalea flower buds: additional freezing parameters

Results of calorimetric, nuclear magnetic resonance, and low temperature light microscopic studies on supercooled azalea (Rhododendron kosterianum, Schneid.) floral primordia are reported. Heat release during freezing of the supercooled floral primordia is in the range predicted for supercooled pure water. Spin-lattice and spin-spin relaxation times measured by pulsed nuclear magnetic resonance spectroscopy decreased after freezing, suggesting that a redistribution of tissue water is associated with injury to the floral primordium. The calorimetric and low temperature microscopy studies showed no detectable ice formation in floral primordia until the major freezing event at low temperature. No resistance to ice growth is found to exist in the primordium tissues, indicating that a freezing barrier or thermodynamic equilibrium exists between the unfrozen primordium and other flower bud parts which contain ice at subfreezing temperatures.     

Thermal stability of collagen fibers in ethylene glycol

The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.     

Changes in denaturation and rheological properties of collagen-hyaluronic acid scaffolds as a result of temperature dependencies

This report describes the effect of temperature on the mechanical viscoelastic properties such as: storage modulus (E′), loss modulus (E″), and loss tangent (tan δ) of the collagen sponges modified with hyaluronic acid (HA). In order to detect collagen–HA copolymer denaturation and to assess its thermal stability, the differential scanning calorimetry (DSC) supplemented by thermogravimetric (TG) measurements was used. The denaturation temperature (T_d) of unmodified collagen samples increased from 69 to 86 °C for cross-linked samples, respectively. These temperature dependencies show remarkable changes in E′ and E″ at selected temperature up to 226 °C for all samples due to the release of loosely and strongly bound water. The influence of HA on the viscoelastic behavior of collagen is manifested by a shift of the tan δ peak associated with the process of decomposition towards higher temperatures resulting in a higher thermo-stability of the modified scaffolds.     

Cold hardiness and deep supercooling in xylem of shagbark hickory

Differential thermal analysis, differential scanning calorimetry, pulsed nuclear magnetic resonance spectroscopy, and low temperature microscopy are utilized to investigate low temperature freezing points or exotherms which occur near −40 C in the xylem of cold-acclimated shagbark hickory (Carya ovata L.). Experiments using these methods demonstrate that the low temperature exotherm results from the freezing of cellular water in a manner predicted for supercooled dilute aqueous solutions. Heat release on freezing, nuclear magnetic resonance relaxation times, and freezing and thawing curves for hickory twigs all point to a supercooled fraction in the xylem at subfreezing temperatures. Calorimetric and low temperature microscopic analyses indicate that freezing occurs intracellularly in the xylem ray parenchyma. The supercooled fraction is found to be extremely stable, even at temperatures only slightly above the homogeneous nucleation temperature for water (−38 C). Xylem water is also observed to be resistant to dehydration when exposed to 80% relative humidity at 20 C. D₂O exchange experiments find that only a weak kinetic barrier to water transport exists in the xylem rays of shagbark hickory.     

Singular efficacy of trimethylamine N-oxide to counter protein destabilization in ice

This study reports the first quantitative estimate of the thermodynamic stability (Delta G degrees ) of a protein in low-temperature partly frozen aqueous solutions in the presence of the protective osmolytes trimethylamine N-oxide (TMAO), glycine betaine, and sarcosine. The method, based on guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, distinguishes between the deleterious effects of subfreezing temperatures from those due specifically to the formation of a solid ice phase. The results point out that in the liquid state molar concentrations of these osmolytes stabilize significantly the native fold and that their effect is maintained on cooling to -15 degrees C. At this temperature, freezing of the solution in the absence of any additive causes a progressive destabilization of the protein, Delta G degrees decreasing up to 3-4 kcal/mol as the fraction of liquid water in equilibrium with ice ( V L) is reduced to less than 1%. The ability of the three osmolytes to prevent the decrease in protein stability at small V L varies significantly among them, ranging from the complete inertness of sarcosine to full protection by TMAO. The singular effectiveness of TMAO among the osmolytes tested until now is maintained high even at concentrations as low as 0.1 M when the additive stabilization of the protein in the liquid state is negligible. In all cases the reduction in Delta G degrees caused by the solidification of water correlates with the decrease in m-value entailing that protein-ice interactions generally conduct to partial unfolding of the native state. It is proposed that the remarkable effectiveness of TMAO to counter the ice perturbation is owed to binding of the osmolyte to ice, thereby inhibiting protein adsorption to the solid phase.     

The Formation and Distribution of Ice within Forsythia Flower Buds

Differential thermal analysis detected two freezing events when dormant forsythia (Forsythia viridissima Lindl.) flower buds were cooled. The first occurred just below 0°C, and was coincident with the freezing of adjacent woody tissues. The second exotherm appeared as a spike between −10 and −25°C and was correlated with the lethal low temperature. Although this pattern of freezing was similar to that observed in other woody species, differences were noted. Both direct observations of frozen buds and examination of buds freeze-fixed at −5°C demonstrated that ice formed within the developing flowers at temperatures above the second exotherm and lethal temperature. Ice crystals had formed within the peduncle and in the lower portions of the developing flower. Ice also formed within the scales. In forsythia buds, the developing floral organ did not freeze as a unit as noted in other species. Instead the low temperature exotherm appeared to correspond to the lethal freezing of supercooled water within the anthers and portions of the pistil.     

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 degrees C and -20 degrees 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 degrees C and -80 degrees 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 degrees 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.     

Direct cell injury associated with eutectic crystallization during freezing

Freezing induced direct cell injury has been explained by a two-factor hypothesis-intracellular ice formation (IIF) at rapid cooling rates, and solution effects at slow cooling rates. Even though IIF is generally believed to be a major injury mechanism at rapid cooling rates, injury by solution effects is not fully understood and several injury mechanisms have been suggested. Solution effects have generally been considered the result of the elevated electrolyte concentration within the intracellular and extracellular space during freezing. In addition to the injury by this elevated electrolyte concentration, freezing injury associated with eutectic crystallization was investigated. To examine the injury associated with eutectic crystallization, two different experiments were designed and performed. In the first experiment, two groups of AT-1 rat prostate tumor cell suspensions were frozen and thawed on a cryomicroscope in the same way except that eutectic crystallization was initiated in only one group. During the second experiment, AT-1 cells were suspended in several different media, which have different eutectic crystallization temperatures, and exposed to a single cooling-warming cycle with varying end temperature of the protocol on a directional solidification stage. After both experiments, post-thaw viability was evaluated and compared. The post-thaw viability drops significantly upon the occurrence of the eutectic crystallization regardless of suspending media, which suggests direct cell injury associated with eutectic crystallization. Based on these observations, two possible injury mechanisms are anticipated: (i) mechanical damage to the cell membrane due to eutectic crystallization, and (ii) intracellular eutectic formation (IEF). The proposed mechanisms provide a more comprehensive physical explanation of freezing induced cell injury and extend the understanding on solution effects.     

Methods and Principles of Fixation by Freeze-Substitution

Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O°C. A 1 to 3 mm. specimen was thrown into 3:1 propane-isopentane cooled by liquid nitrogen to -175°C. (with precautions). The frozen tissue was placed in substituting fluid at -70°C. for 1 week to dissolve ice slowly without distorting tissue structure. Excess substituting agent was washed out, and the specimen was embedded, sectioned, and stained conventionally. For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1 per cent solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, and picric acid in ethanol. Preservation of structure was poorer after substitution in solvent alone. Evidence was obtained that the chemical agent fixes tissue at low temperatures. The chemical mechanisms of fixation are probably similar to those operating at room temperature: new chemical cross-linkages, which contain the fixing agent, join tissue constituents together. This process is distinguished from denaturation by pure solvents. Freeze-substitution has many advantages, particularly the preservation of structure to the limit of resolution with the light microscope, and the accurate localization of many soluble and labile substances.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 degrees C/min while the ice nucleation temperature was varied between -3 and -10 degrees C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below -4 degrees C and cell survival exhibits an optimum at a nucleation temperature of -6 degrees C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at -3 degrees C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at -10 degrees C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in alpha-helical structures and a concomitant increase in beta-sheet structures starting at an onset temperature of approximately 48 degrees C.     

Infective Juveniles of the Entomopathogenic Nematode,Steinernema feltiae Produce Cryoprotectants in Response to Freezing and Cold Acclimation

Steinernema feltiae is a moderately freeze-tolerant entomopathogenic nematode which survives intracellular freezing. We have detected by gas chromatography that infective juveniles of S. feltiae produce cryoprotectants in response to cold acclimation and to freezing. Since the survival of this nematode varies with temperature, we analyzed their cryoprotectant profiles under different acclimation and freezing regimes. The principal cryoprotectants detected were trehalose and glycerol with glucose being the minor component. The amount of cryoprotectants varied with the temperature and duration of exposure. Trehalose was accumulated in higher concentrations when nematodes were acclimated at 5°C for two weeks whereas glycerol level decreased from that of the non-acclimated controls. Nematodes were seeded with a small ice crystal and held at -1°C, a regime that does not produce freezing of the nematodes but their bodies lose water to the surrounding ice (cryoprotective dehydration). This increased the levels of both trehalose and glycerol, with glycerol reaching a higher concentration than trehalose. Nematodes frozen at -3°C, a regime that produces freezing of the nematodes and results in intracellular ice formation, had elevated glycerol levels while trehalose levels did not change. Steinernema feltiae thus has two strategies of cryoprotectant accumulation: one is an acclimation response to low temperature when the body fluids are in a cooled or supercooled state and the infective juveniles produce trehalose before freezing. During this process a portion of the glycerol is converted to trehalose. The second strategy is a rapid response to freezing which induces the production of glycerol but trehalose levels do not change. These low molecular weight compounds are surmised to act as cryoprotectants for this species and to play an important role in its freezing tolerance.     

Differential scanning calorimetry of the thermal denaturation of lactate dehydrogenase

1. Differential scanning calorimetry has been used to study the thermal denaturation of lactate dehydrogenase. At pH 7.0 in 0.1 M potassium phosphate buffer, only one transition was observed. Both the enthalpy of denaturation and the melting temperature are linear function of heating rate. The enthalpy is 430 kcal/mol and the melting temperature 61 degrees C at 0 degrees C/min heating rate. The ratio of the calorimetric heat to the effective enthalpy indicated that the denaturation is highly cooperative. Subunit association does not appear to significantly contribute to the enthalpy of denaturation. 2. Both cofactor and sucrose addition stabilized the protein against thermal denaturation. Pyruvate addition produced no changes. Only a small time-dependent destabilization was observed at low concentrations of urea. Large effects were observed in concentrated NaCl solutions and with sulfhydryl-modified lactate dehydrogenase.     

Does plant height determine the freezing resistance in the páramo plants?

Neotropical ecosystems between treeline and snowline, called páramos, stretch along Andean ranges from Costa Rica to northern Peru. The páramo climate is characterized by regular night frosts occurring throughout the year. Páramo plants use two strategies to deal with freezing temperatures. They either avoid ice formation in the tissues or tolerate extracellular ice formation. We tested the microclimate hypothesis, which suggests that the freezing resistance of the páramo plants is determined by plant height, that is, that taller plants experience a milder microclimate and avoid freezing, whereas smaller plants are exposed to the more extreme thermal conditions near the ground and tolerate them. We measured the temperature at which ice formed inside the plants (the ‘exotherm’), and compared it with the temperature at which 50% damage to the tissue occurred (Lt50); a significant difference between the exotherm and Lt50 would indicate freezing tolerance whereas the absence of a difference would indicate avoidance by supercooling. We analysed the freezing resistance of 38 common Ecuadorian páramo species. We found no correlation between plant height and freezing resistance mechanism or injury temperature and reject the microclimate hypothesis. Tolerant plants reach higher altitudes than avoidant plants, but their altitudinal ranges largely overlap and the Lt50 does not differ between them. These results suggest that there is no qualitative difference between the two strategies to survive the páramo frosts. Shrub leaves were injured at significantly lower temperatures than other life forms, such as herbs, which may reflect leaf anatomical differences among the plants.     

Cold hardiness of the eggs of the plaice,Pleuronectes platessa

Summary The pelagic eggs of the plaice, Pleuronectes platessa, at the stage of first heart beat, can tolerate freezing into solid sea ice at temperatures down to-6°C. A long term freezing period of 5 days, at sublethal temperatures of-3 to-4°C, had only a minor effect on the eggs. When the ice was absent from the medium, the eggs supercooled readily to temperatures around-15° to-20°C. However, no thermal hysteresis agents were present in the eggs at low temperature and the cold hardiness therefore seem to rely on a high tolerance to brine solutions at low temperatures.