Effect of low temperature and culture media on the growth and freeze-thawing tolerance of Exiguobacterium strains

Bacteria of the genus Exiguobacterium have been repeatedly isolated from ancient permafrost sediments of the Kolyma lowland of Northeast Eurasia. Here we report that the Siberian permafrost isolates Exiguobacterium sibiricum 255-15, E. sibiricum 7-3, Exiguobacterium undae 190-11 and E. sp. 5138, as well as Exiguobacterium antarcticum DSM 14480, isolated from a microbial mat sample of Lake Fryxell (McMurdo Dry Valleys, Antarctica), were able to grow at temperatures ranging from -6 to 40 degrees C. In comparison to cells grown at 24 degrees C, the cold-grown cells of these strains tended to be longer and wider. We also investigated the effect of growth conditions (broth or surface growth, and temperature) on cryotolerance of the Exiguobacterium strains. Bacteria grown in broth at 4 degrees C showed markedly greater survival following freeze-thawing treatments (20 repeated cycles) than bacteria grown in broth at 24 degrees C. Surprisingly, significant protection to repeated freeze-thawing was also observed when bacteria were grown on agar at either 4 or 24 degrees C.     

Putative transposases conserved in Exiguobacterium isolates from ancient Siberian permafrost and from contemporary surface habitats

Gram-positive bacteria of the genus Exiguobacterium have been repeatedly isolated from Siberian permafrost ranging in age from 20,000 to 2 to 3 million years and have been sporadically recovered from markedly diverse habitats, including microbial mats in Lake Fryxell (Antarctic), surface water, and food-processing environments. However, there is currently no information on genomic diversity of this microorganism or on the physiological strategies that have allowed its survival under prolonged freezing in the permafrost. Analysis of the genome sequence of the most ancient available Exiguobacterium isolate (Exiguobacterium sp. strain 255-15, from 2 to 3 million-year-old Siberian permafrost) revealed numerous putative transposase sequences, primarily of the IS200/IS605, IS30, and IS3 families, with four transposase families identified. Several of the transposase genes appeared to be part of insertion sequences. Southern blots with different transposase probes yielded high-resolution genomic fingerprints which differentiated the different permafrost isolates from each other and from the Exiguobacterium spp. type strains which have been derived from diverse surface habitats. Each of the Exiguobacterium sp. strain 255-15 transposases that were used as probes had highly conserved homologs in the genome of other Exiguobacterium strains, both from permafrost and from modern sites. These findings suggest that, prior to their entrapment in permafrost, Exiguobacterium isolates had acquired transposases and that conserved transposases are present in Exiguobacterium spp., which now can be isolated from various modern surface habitats.     

Reproduction and metabolism at - 10 degrees C of bacteria isolated from Siberian permafrost

We report the isolation and properties of several species of bacteria from Siberian permafrost. Half of the isolates were spore-forming bacteria unable to grow or metabolize at subzero temperatures. Other Gram-positive isolates metabolized, but never exhibited any growth at - 10 degrees C. One Gram-negative isolate metabolized and grew at - 10 degrees C, with a measured doubling time of 39 days. Metabolic studies of several isolates suggested that as temperature decreased below + 4 degrees C, the partitioning of energy changes with much more energy being used for cell maintenance as the temperature decreases. In addition, cells grown at - 10 degrees C exhibited major morphological changes at the ultrastructural level.     

Metabolic activity of Siberian permafrost isolates, <Emphasis Type="Italic">Psychrobacter arcticus</Emphasis> and <Emphasis Type="Italic">Exiguobacterium sibiricum</Emphasis>, at low water activities

The Siberian permafrost is an extreme, yet stable environment due to its continuously frozen state. Microbes maintain membrane potential and respiratory activity at average temperatures of -10 to -12 degrees C that concentrate solutes to an a (w) = 0.90 (5 osm), The isolation of viable Psychrobacter arcticus sp. 273-4 and Exiguobacterium sibiricum sp. 255-15 from ancient permafrost suggests that these bacteria have maintained some level of metabolic activity for thousands of years. Permafrost water activity was simulated using (1/2) TSB + 2.79 m NaCl (5 osm) at and cells were held at 22 and 4 degrees C. Many cells reduced cyano-tetrazolium chloride (CTC) indicating functioning electron transport systems. Increased membrane permeability was not responsible for this lack of electron transport, as more cells were determined to be intact by LIVE/DEAD staining than were reducing CTC. Low rates of aerobic respiration were determined by the slope of the reduced resazurin line for P. arcticus, and E. sibiricum. Tritiated leucine was incorporated into new proteins at rates indicating basal level metabolism. The continued membrane potential, electron transport and aerobic respiration, coupled with incorporation of radio-labeled leucine into cell material when incubated in high osmolarity media, show that some of the population is metabolically active under simulated in situ conditions.     

Intracellular freezing and survival in the freeze tolerant alpine cockroach Celatoblatta quinquemaculata

The alpine cockroach Celatoblatta quinquemaculata is common at altitudes of around 1500 m on the Rock and Pillar range of Central Otago, New Zealand where it experiences freezing conditions in the winter. The cockroach is freeze tolerant, but only to c. -9 degrees C. The cause of death at temperatures below this is unknown but likely to be due to osmotic damage to cells (shrinkage). This study compared the effect of different ice nucleation temperatures (-2 and -4 degrees C) on the viability of three types of cockroach tissue (midgut, Malpighian tubules and fat body cells) and cooling to three different temperatures (-5, -8, -12 degrees C). Two types of observations were made (i) cryomicroscope observations of ice formation and cell shrinkage (ii) cell integrity (viability) using vital stains. Cell viability decreased with lower treatment temperatures but ice nucleation temperature had no significant effect. Cryomicroscope observations showed that ice spread through tissue faster at -4 than -2 degrees C and that intracellular freezing only occurred when nucleated at -4 degrees C. From temperature records during cooling, it was observed that when freezing occurred, latent heat immediately increased the insect's body temperature close to its melting point (c. -0.3 degrees C). This "rebound" temperature was independent of nucleation temperature. Some tissues were more vulnerable to damage than others. As the gut is thought to be the site of freezing, it is significant that this tissue was the most robust. The ecological importance of the effect of nucleation temperature on survival of whole animals under field conditions is discussed.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

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.     

Characterization of Exiguobacterium isolates from the Siberian permafrost. Description of Exiguobacterium sibiricum sp. nov.

Three Gram-positive bacterial strains, 7-3, 255-15 and 190-11, previously isolated from Siberian permafrost, were characterized and taxonomically classified. These microorganisms are rod-shaped, facultative aerobic, motile with peritrichous flagella and their growth ranges are from -2.5 to 40 degrees C. The chemotaxonomic markers indicated that the three strains belong to the genus Exiguobacterium. Their peptidoglycan type was A3alpha L-Lys-Gly. The predominant menaquinone detected in all three strains was MK7. The polar lipids present were phosphatidyl-glycerol, diphosphatidyl-glycerol and phosphatidyl-ethanolamine. The major fatty acids were iso-C13:0, anteiso-C13:0, iso-C15:0, C16:0 and iso-C17:0. Phylogenetic analysis based on 16S rRNA and six diverse genes, gyrB (gyrase subunit B), rpoB (DNA-directed RNA polymerase beta subunit), recA (homologous recombination), csp (cold shock protein), hsp70 (ClassI-heat shock protein-chaperonin) and citC (isocitrate dehydrogenase), indicated that the strains were closely related to Exiguobacterium undae (DSM 14481(T)) and Exiguobacterium antarcticum (DSM 14480(T)). On the basis of the phenotypic characteristics, phylogenetic data and DNA-DNA reassociation data, strain 190-11 was classified as E. undae, while the other two isolates, 7-3 and 255-15, comprise a novel species, for which the name Exiguobacterium sibiricum sp. nov. is proposed.     

Avoidance of intracellular freezing by the freezing-tolerant New Zealand Alpine weta Hemideina maori (Orthoptera: Stenopelmatidae)

Calorimetric analysis indicates that 82% of the body water of Hemideina maori is converted into ice at 10 degrees C. This is a high proportion and led us to investigate whether intracellular freezing occurs in H. maori tissue. Malpighian tubules and fat bodies were frozen in haemolymph on a microscope cold stage. No fat body cells, and 2% of Malpighian tubule cells froze during cooling to -8 degrees C. Unfrozen cells appeared shrunken after ice formed in the extracellular medium. There was no difference between the survival of control tissues and those frozen to -8 degrees C. At temperatures below -15 degrees C (lethal temperatures for weta), there was a decline in survival, which was strongly correlated with temperature, but no change in the appearance of tissue. It is concluded that intracellular freezing is avoided by Hemideina maori through osmotic dehydration and freeze concentration effects, but the reasons for low temperature mortality remain unclear. The freezing process in H. maori appears to rely on extracellular ice nucleation, possibly with the aid of an ice nucleating protein, to osmotically dehydrate the cells and avoid intracellular freezing. The lower lethal temperature of H. maori (-10 degrees C) is high compared to organisms that survive intracellular freezing. This suggests that the category of 'freezing tolerance' is an oversimplification, and that it may encompass at least two strategies: intracellular freezing tolerance and avoidance.     

Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures.     

Antifreeze proteins modify the freezing process in planta

During cold acclimation, winter rye (Secale cereale L. cv Musketeer) plants accumulate antifreeze proteins (AFPs) in the apoplast of leaves and crowns. The goal of this study was to determine whether these AFPs influence survival at subzero temperatures by modifying the freezing process or by acting as cryoprotectants. In order to inhibit the growth of ice, AFPs must be mobile so that they can bind to specific sites on the ice crystal lattice. Guttate obtained from cold-acclimated winter rye leaves exhibited antifreeze activity, indicating that the AFPs are free in solution. Infrared video thermography was used to observe freezing in winter rye leaves. In the absence of an ice nucleator, AFPs had no effect on the supercooling temperature of the leaves. However, in the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by 0.3 degrees C to 1.2 degrees C. In vitro studies showed that apoplastic proteins extracted from cold-acclimated winter rye leaves inhibited the recrystallization of ice and also slowed the rate of migration of ice through solution-saturated filter paper. When we examined the possible role of winter rye AFPs in cryoprotection, we found that lactate dehydrogenase activity was higher after freezing in the presence of AFPs compared with buffer, but the same effect was obtained by adding bovine serum albumin. AFPs had no effect on unstacked thylakoid volume after freezing, but did inhibit stacking of the thylakoids, thus indicating a loss of thylakoid function. We conclude that rye AFPs have no specific cryoprotective activity; rather, they interact directly with ice in planta and reduce freezing injury by slowing the growth and recrystallization of ice.     

Depression of the ice-nucleation temperature of rapidly cooled mouse embryos by glycerol and dimethyl sulfoxide.

The temperature at which ice formation occurs in supercooled cytoplasm is an important element in predicting the likelihood of intracellular freezing of cells cooled by various procedures to subzero temperatures. We have confirmed and extended prior indications that permeating cryoprotective additives decrease the ice nucleation temperature of cells, and have determined some possible mechanisms for the decrease. Our experiments were carried out on eight-cell mouse embryos equilibrated with various concentrations (0-2.0 M) of dimethyl sulfoxide or glycerol and then cooled rapidly. Two methods were used to assess the nucleation temperature. The first, indirect, method was to determine the in vitro survival of the rapidly cooled embryos as a function of temperature. The temperatures over which an abrupt drop in survival occurs are generally diagnostic of the temperature range for intracellular freezing. The second, direct, method was to observe the microscopic appearance during rapid cooling and note the temperature at which nucleation occurred. Both methods showed that the nucleation temperature decreased from - 10 to - 15 degrees C in saline alone to between - 38 degrees and - 44 degrees C in 1.0-2.0 M glycerol and dimethyl sulfoxide. The latter two temperatures are close to the homogeneous nucleation temperatures of the solutions in the embryo cytoplasm, and suggest that embryos equilibrated in these solutions do not contain heterogeneous nucleating agents and are not accessible to any extracellular nucleating agents, such as extracellular ice. The much higher freezing temperatures of cells in saline or in low concentrations of additive indicate that they are being nucleated by heterogeneous agents or, more likely, by extracellular ice.     

Microbial metabolism in ice and brine at -5°C

Metabolic activity, but not growth, has been observed in ice at temperatures from -5°C to -32°C. To improve understanding of metabolism in ice, we simultaneously examined various aspects of metabolism ((14) C-acetate utilization, macromolecule syntheses and viability via reduction of CTC) of the glacial isolates Sporosarcina sp. B5 and Chryseobacterium sp. V3519-10 during incubation in nutrient-rich ice and brine at -5°C for 50 days. Measured rates of acetate utilization and macromolecule syntheses were high in the first 20 days suggesting adjustment to the lower temperatures and higher salt concentrations of both the liquid vein network in the ice and the brine. Following this adjustment, reproductive growth of both organisms was evident in brine, and suggested for Sporosarcina sp. B5 in ice by increases in cell numbers and biomass. Chryseobacterium sp. V3519-10 cells incubated in ice remained active. These data indicate that neither low temperature nor high salt concentrations prohibit growth in ice, but some other aspect of living within ice slows growth to within the detection limits of current methodologies. These results imply that microbial growth is plausible in natural ice systems with comparable temperatures and sufficient nutrients, such as debris-rich basal ices of glaciers and ice masses.     

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.     

A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing

The changes in morphology of Penicillium expansum Link and Phytophthora nicotianae Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage. Viability of 0.5-1.0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 degrees C in the presence or absence of glycerol. In P. expansum shrinkage occurred in all hyphae at rates of cooling of less than 15 degrees C min-1; at faster rates intracellular ice nucleation occurred. The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18 degrees C min-1 to 55 degrees C min-1. This species was particularly resistant to freezing injury and recovery was greater than 60% at all rates of cooling examined. At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated. In contrast, during the cooling of Ph. nicotianae in the growth medium, shrinkage occurred and no samples survived on thawing from -196 degrees C. However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 degrees C min-1 the loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.     

Inhibition of bacterial ice nucleation by polyglycerol polymers

The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nucleating activity of proteins from the ice nucleating bacterium Pseudomonas syringae. PGL of molecular mass 750 Da was added to a solution consisting of 1 ppm freeze-dried P. syringae 31A in water. Differential ice nucleator spectra were determined by measuring the distribution of freezing temperatures in a population of 98 drops of 1 microL volume. The mean freezing temperature was lowered from -6.8 degrees C (control) to -8.0,-9.4,-12.5, and -13.4 degrees C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2 degrees C). PGL was found to be an ineffective inhibitor of seven defined organic ice nucleating agents, whereas the general ice nucleation inhibitor polyvinyl alcohol (PVA) was found to be effective against five of the seven. The activity of PGL therefore seems to be specific against bacterial ice nucleating protein. PGL alone was an ineffective inhibitor of ice nucleation in small volumes of environmental or laboratory water samples, suggesting that the numerical majority of ice nucleating contaminants in nature may be of nonbacterial origin. However, PGL was more effective than PVA at suppressing initial ice nucleation events in large volumes, suggesting a ubiquitous sparse background of bacterial ice nucleating proteins with high nucleation efficiency. The combination of PGL and PVA was particularly effective for reducing ice formation in solutions used for cryopreservation by vitrification.     

Expression of a beetle, Dendroides canadensis, antifreeze protein in Drosophila melanogaster

Antifreeze protein 1 (DAFP-1), from the beetle Dendroides canadensis, was expressed in Drosophila melanogaster. Mean thermal hysteresis values (the difference between freezing and melting points), indicative of antifreeze protein activity, in the hemolymph of transgenic flies were found to be as high as 6.23+/-0.10 degrees C (using the nanoliter osmometer). Direct comparisons of the capillary and nanoliter osmometer techniques for measuring THA were made, illustrating the much higher values obtained by the latter. Transgenic Drosophila had supercooling points, both in contact with ice and not, that were slightly, but significantly, lower than wild-type controls (1.5-2.0 degrees C and 2.0-4.0 degrees C, respectively). The results indicate functionality of DAFP-1 in Drosophila melanogaster (the ability of DAFP-1 to inhibit both inoculative freezing across the cuticle and freezing initiated by endogenous ice nucleators). The much larger effects of DAFPs in inhibiting inoculative freezing and ice nucleation in Dendroides canadensis relative to the transgenic Drosophila may partially result from the lower DAFP concentrations and activities in Drosophila, however the absence of multiple types of DAFPs and absence of tissue specific expression may also contribute. Transgenic Drosophila were also able to live significantly longer than controls at 0 degrees C and 4 degrees C, indicating that DAFP-1 is able to increase cold tolerance at above freezing temperatures.     

Influence of simulated acid snow stress on leaf tissue of wintering herbaceous plants

Acid snow might be an environmental stress factor for wintering plants since acid precipitates are locally concentrated in snow and the period in which ice crystals are in contact with shoots might be longer than that of acid precipitates in rain. In this study, 'equilibrium' and 'prolonged' freezing tests with sulfuric acid, which simulate situations of temperature depression and chronic freezing at a subzero temperature with acid precipitate as acid snow stress, respectively, were carried out using leaf segments of cold-acclimated winter wheat. When leaf segments were frozen in the presence of sulfuric acid solution (pH 4.0, 3.0 or 2.0) by equilibrium freezing with ice seeding, the survival rate of leaf samples treated with sulfuric acid solution of pH 2.0 decreased markedly. Leaf samples after supercooling to -4 and -8 degrees C in the presence of sulfuric acid solution (pH 2.0) without ice seeding were less damaged. When leaf samples were subjected to prolonged freezing at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0), the survival rates of leaf samples exposed to sulfuric acid decreased more than those of leaf samples treated with water. On the other hand, leaf samples were less damaged by prolonged supercooling at -4 and -8 degrees C for 7 d with sulfuric acid (pH 2.0). The results suggest that an acid condition (pH 2.0) in the process of extracellular freezing and/or thawing promotes freezing injury of wheat leaves.     

Cryomicroscopic analysis of intracellular ice formation during freezing of mouse oocytes without cryoadditives

Kinetics of intracellular ice formation (IIF) under various freezing conditions was investigated for mouse oocytes at metaphase II obtained from B6D2F1 mice. A new cryostage with improved optical performance and "isothermal" temperature field was used for nucleation experiments. The maximum thermal gradient across the window was less than 0.1 degrees C/10 mm at sample temperatures near 0 degrees C. The dependence of IIF on the initial concentration of the suspending medium was found to be pronounced. The mean IIF temperatures were found to be -9.56, -12.49, -17.63, -22.20 degrees C for freezing at 120 degrees C/min in 200, 285, 510, and 735 mosm phosphate-buffered saline, respectively. For concentrations higher than 735 mosm, the kinetics of IIF showed a break point at approximately -31 degrees C. Below -31 degrees C, all the remaining unfrozen oocytes underwent IIF almost immediately over a temperature range of less than 3 degrees C. This dramatic shift in the kinetics of IIF suggests that there were two distinct mechanisms responsible for IIF during freezing. The effect of the cooling rate on the kinetics of IIF was also investigated in isotonic PBS. At 1 degrees C/min none of the oocytes contained ice, whereas, at 5 degrees C/min all the oocytes contained ice. The mean IIF temperatures for cooling rates between 1 and 120 degrees C/min were almost constant with an average of -12.82 +/- 0.6 degrees C (SEM). In addition, constant temperature experiments were conducted in isotonic PBS. The percentages of oocytes with IIF were 0, 50, 60, and 95% for -3.8, -6.4, -7.72, and -8.85 degrees C. In undercooling experiments, IIF was not observed until approximately -20 degrees C (at which temperature the whole suspension was frozen spontaneously), suggesting the involvement of the external ice in the initiation of IIF between approximately -5 and -31 degrees C during freezing of oocytes.     

Preservation of viable cells in the undercooled state

Previous studies into the mechanisms governing the freezing of cells in the absence of extracellular ice have been extended to develop a method for the preservation of viable cells in the undercooled state. Deep undercooling of cells is achieved by suspending fine droplets of the cells in oil to make an emulsion, thus minimizing initiation of extracellular ice nucleation. Attempts to preserve yeast cells, cultured sainfoin cells, and dissected shoot-tips (pea and potato) in this way are described. The main findings are that yeast cells can be preserved undercooled at -20 degrees C for at least 16 weeks with no detectable loss of viability, showing that -20 degrees C is a low enough temperature for inhibition of significant biochemical deterioration and that the emulsions are stable over long periods. In preliminary experiments, sainfoin cells survived 24 hr at -10 degrees C, and shoot-tips survived 48 hr at -10 degrees C. Sainfoin cells, conditioned by growth in medium supplemented with sorbitol, showed enhanced survival after exposure to low temperatures and a lower intracellular freezing point than control cells. Possible reasons for this are discussed.