The effect of undissolved air on isochoric freezing

This study evaluates the effect of undissolved air on isochoric freezing of aqueous solutions. Isochoric freezing is concerned with freezing in a constant volume thermodynamic system. A possible advantage of the process is that it substantially reduces the percentage of ice in the system at every subzero temperature, relative to atmospheric freezing. At the pressures generated by isochoric freezing, or high pressure isobaric freezing, air cannot be considered an incompressible substance and the presence of undissolved air substantially increases the amount of ice that forms at any subfreezing temperature. This effect is measurable at air volumes as low as 1%. Therefore eliminating the undissolved air, or any separate gaseous phase, from the system is essential for retaining the properties of isochoric freezing.     

Effects of low-temperature acclimation on the survival and cold tolerance of an antarctic mite

The effects of long-term exposures to constant temperatures (+4, 0, −5, −10, −15 and −20°C) on the survival of a cryptostigmatid mite, Alaskozetes antarcticus, were studied during 1983–1984 on Signy Island, South Orkney Islands, in the maritime Antarctic. Field-fresh samples collected during the austral summer showed very large (e.g. about 60 percentage points) variations in survival when placed at constant temperatures, as a result of collection-date effects. Pretreatment acclimations (10-day) at +4 and 0°C (especially) reduced this variation. Short-term modulations in cold-hardiness levels were related to ambient temperature fluctuations. However, samples collected on the same occasion, from microhabitats 20 m apart, also showed significant cold-hardiness variation. For twelve summer samples, survival after 24 h at −15°C was highly correlated with supercooling capacity. Winter samples showed little variation in survival, interms of collection-date. Percentage survival remained greater than 85% at −5, −10 and −15°C, for exposures up to 100 days. Samples with median supercooling points of about −30°C, showed 52% survival after 250 days at −15°C, and 73% survival after 100 days at −20°C. At −15°C, supercooling capacity was used up at an estimated rate of 0.06 deg day⁻¹, as a result of a time-temperature interaction effect on the probability of heterogeneous nucleation. Adult mites showed 78% survival after 21 days encasement in distilled water ice, at −15°C. Survival differences between post-larval stages were not detected. In conclusion, survival ability under controlled laboratory conditions appeared to exceed the requirements of average winter-habitat temperatures, but the effects of fluctuating and extreme temperatures require investigation. Supercooling points are considered to be accurate indicators of low-temperature survival capability in this species.     

Sterilization by Cooling in Isochoric Conditions: The Case of Escherichia coli

High hydrostatic pressure (HHP) affects the structure, metabolism and survival of micro-organisms including bacteria. For this reason HHP is a promising treatment in the food industry. The aim of this work is to evaluate the effect of high pressure, under isochoric cooling conditions, on Escherichia coli, where such high pressure develops due to the fact water cannot expand. We combine survival curves obtained by spectrophotometry and images of atomic force microscopy in this study. Our results show that cooling at -20 and -30°C leads to a partial destruction of a Escherichia coli population. However, cooling at -15°C causes a total extermination of bacteria. This intriguing result is explained by the phase diagram of water. In the first case, the simultaneous formation of ice III and ice Ih crystals provides a safe environment for bacteria. In the second case (-15°C) Escherichia coli remains in a metastable and amorphous free-of-crystals liquid subjected to high pressure. Our work is the first experimental study carried out to inactivate Escherichia coli under isochoric cooling conditions. Unlike HHP, which is based on the application of an external load to augment the pressure, this technique only requires cooling. The method could be used for annihilation of other Escherichia coli strains and perhaps other micro-organisms.     

Freeze avoidance: a dehydrating moss gathers no ice

Using cryo‐SEM with EDX fundamental structural and mechanical properties of the moss Ceratodon purpureus (Hedw.) Brid. were studied in relation to tolerance of freezing temperatures. In contrast to more complex plants, no ice accumulated within the moss during the freezing event. External ice induced desiccation with the response being a function of cell type; water‐filled hydroid cells cavitated and were embolized at ?4 °C while parenchyma cells of the inner cortex exhibited cytorrhysis, decreasing to ～20% of their original volume at a nadir temperature of ?20 °C. Chlorophyll fluorescence showed that these winter acclimated mosses displayed no evidence of damage after thawing from ?20 °C while GCMS showed that sugar concentrations were not sufficient to confer this level of freezing tolerance. In addition, differential scanning calorimetry showed internal ice nucleation occurred in hydrated moss at ～?12 °C while desiccated moss showed no evidence of freezing with lowering of nadir temperature to ?20 °C. Therefore the rapid dehydration of the moss provides an elegantly simple solution to the problem of freezing; remove that which freezes.     

Fate of Escherichia coli O157:H7 in ground beef following high‐pressure processing and freezing

Aims: The purposes of this study were to evaluate the efficacy of high pressure to inactivate Escherichia coli O157:H7 in ground beef at ambient and subzero treatment temperatures and to study the fate of surviving bacteria postprocess and during frozen storage. Methods and Results: Fresh ground beef was inoculated with a five‐strain cocktail of E. coli O157:H7 vacuum‐packaged, pressure‐treated at 400 MPa for 10 min at ?5 or 20°C and stored at ?20 or 4°C for 5–30 days. A 3‐log CFU g^?1 reduction of E. coli O157:H7 in the initial inoculum of 1 × 10⁶ CFU g^?1 was observed immediately after pressure treatment at 20°C. During frozen storage, levels of E. coli O157:H7 declined to <1 × 10² CFU g^?1 after 5 days. The physiological status of the surviving E. coli was affected by high pressure, sensitizing the cells to pH levels 3 and 4, bile salts at 5% and 10% and mild cooking temperatures of 55–65°C. Conclusions: High‐pressure processing (HPP) reduced E. coli O157:H7 in ground beef by 3 log CFU g^?1 and caused substantial sublethal injury resulting in further log reductions of bacteria during frozen storage. Significance and Impact of the Study: HPP treatment of packaged ground beef has potential in the meat industry for postprocess control of pathogens such as E. coli O157:H7 with enhanced safety of the product.     

Increased cold hardiness in eggs of Arcynopteryx compacta (Plecoptera) by dehydration

Eggs of the stonefly, Arcynopteryx compacta, that overwinter in the alpine region of Norwegian mountains, increase their cold-hardiness by dehydration. Eggs enclosed in ice at −22°C survive the loss of about two-thirds of their total water content by shrinkage due to passive diffusion of body water along the concentration gradient. Fully hydrated eggs are killed by freezing at their supercooling point of −26°C, and by direct cooling to −30°C. Dehydrated eggs have a mean supercooling point of −31°C, and survive exposure at −27 and −29°C in ice. Judged from their melting points the eggs do not accumulate low-molecular-weight cryoprotective substances. The difference between freezing and melting points corresponds to a thermal hysteresis of up to 1.8°C. The presence of thermal hysteresis antifreezes may stabilize their supercooled state when enclosed by ice during overwintering. The eggs enter diapause in the autumn, and diapause completion is enhanced both by temperature and time during enclosure in ice.     

Role of thiol redox systems in <Emphasis Type="Italic">Escherichia coli</Emphasis> response to thermal and antibiotic stresses

Isogenic knockout mutants of Escherichia coli deficient in components of the glutathione and thioredoxin redox systems and growing at various temperatures (20–46°C) exhibited considerable differences in growth rate and survival, as well as in expression of the antioxidant genes. In the parental strain E. coli BW25113 (wt) treated with ciprofloxacin, ampicillin, or streptomycin, dependence of survival on growth temperature was a V-shaped curve with the maximum sensitivity within the range corresponding to high growth rates (40–44°C). Significant inverse correlation was observed between log CFU at different temperatures and specific growth rate prior to antibiotic addition. This applied to most of the mutants, which exhibited higher resistance to the three antibiotics tested at nonoptimal temperatures (20 and 46°C) than at 37 and 40°C. No correlation was found between resistance to stress and antibiotics and expression of the antioxidant genes. The role of global regulators ppGpp and σ^s in E. coli resistance to antibiotics and nonoptimal temperatures was shown.     

Resilience to Freezing in the Vegetative Cells of the Microalga Lobosphaera incisa (Trebouxiophyceae,Chlorophyta)

The chlorophyte microalga Lobosphaera incisa was isolated from the snowy slopes of Mt. Tateyama in Japan. This microalga stores exceptionally high amounts of the omega-6 LC-PUFA arachidonic acid in triacylglycerols, and therefore represents a potent photosynthetic source for this essential LC-PUFA. Assuming that freezing tolerance may play a role in adaptation of L. incisa to specific ecological niches, we examined the capability of L. incisa to tolerate extreme sub-zero temperatures. We report here, that the vegetative cells of L. incisa survived freezing at −20°C and −80°C (over 1 month), without cryoprotective agents or prior treatments. Cells successfully recovered upon thawing and proliferated under optimal growth conditions (25°C). However, cells frozen at −80°C showed better recovery and lower cellular ROS generation upon thawing, compared to those preserved at −20°C. Photosynthetic yield of PSII, estimated by F_v/F_m, temporarily decreased at day 1 post freezing and resumed to the original level at day 3. Interestingly, the thawed algal cultures produced a higher level of chlorophylls, exceeding the control culture. The polar metabolome of the vegetative cells comprised a range of compatible solutes, dominated by glutamate, sucrose, and proline. We posit that the presence of endogenous cryoprotectants, a rigid multilayer cell wall, the high LC-PUFA content in membrane lipids, and putative cold-responsive proteins may contribute to the retention of functionality upon recovery from the frozen state, and therefore for the survival under cryospheric conditions. From the applied perspective, this beneficial property holds promise for the cryopreservation of starter cultures for research and commercial purposes.     

Physiological constraints on the life cycle and distribution of the Antarctic fairy shrimp <Emphasis Type="Italic">Branchinecta gaini</Emphasis>

Maturation to adulthood and successful reproduction in the Antarctic fairy shrimp, Branchinecta gaini, must be completed within a physiologically challenging temporal window of ca. 2.5 months in the southern Antarctic Peninsula. Although adults show considerable metabolic opportunism at positive temperatures, little is known of their tolerance of two physiological insults potentially typical to pool life in the maritime Antarctic: sub-zero temperatures and salinity. B. gaini are freeze-avoiding crustaceans with temperatures of crystallisation (T _cs) of −5°C. No antifreeze proteins were detected in the haemolymph. Adults osmoregulate in relation to temperature, but rapid mortality in saline solutions of even low concentration, indicate they cannot osmoregulate in relation to salinity. Survival of ice encasement at temperatures above their T _c was found to be pressure but not time dependent: at severe inoculative ice pressures, there was little immediate survival and none survived after 48 h below −2°C; at mild inoculative ice pressures, immediate survival was ca. 100% at −3°C, but <20% after 48 h. There was no significant difference in survival after 1 and 6 h encasement at −3°C. Observations of ventilation suggest that it is not low temperature per se, but ice that represents the primary cryo-stress, with ventilatory appendages physically handcuffed below the freezing point of pool water. Both sub-zero temperatures and salinity represent real physiological constraints on adult fairy shrimp.     

Effects of extended and repeated exposures to low temperature on mortality of the peach-potato aphid Myzus persicae

Abstract.

• 1 The survival of adult and first-instar Myzus persicae reared at 20°C and 10°C was investigated after brief (1 min) exposure in the absence of plant material to temperatures between −5°C and −25°C, and extended exposures on plants of 1–10 days at a constant 5°C, 3°C and −5°C and a 24 h cycling regime between 5°C (18 h) and −5°C (6 h).
• 2 Life stage, rearing temperature, period of exposure and temperature regime all had a significant effect on the ability of aphids to survive cold. The effects of life stage and rearing temperature were most noticeable following exposure to cycling temperatures and extended exposures at −5°C, and least apparent after 1 min exposures at lower sub-zero temperatures.
• 3 Mortality following exposure to temperatures cycling between −5°C and 5°C was greater than that at 3°C (the mean of the cycling temperatures) and less than at a constant −5°C, suggesting that when temperatures fluctuate by a few degrees around 0°C the minimum temperature may affect survival to a greater extent than the mean.
• 4 These results suggest that an overwintering population of acclimated M.persicae would persist without significant mortality after a period of 7–10 days with −5°C frosts each night.

     

Low-temperature conditioning of the ice nucleation active bacterium,Erwinia herbicola

Rapid “low-temperature conditioning” and “solute conditioning” of the ice nucleation active bacterium Erwinia herbicola No. 26 are described. Conditioning is the process by which the ability to initiate ice at high temperatures is gained in these bacteria. The cumulative ice nucleator concentration, N[T], was used to measure the number of ice nucleators present in the bacterial systems. N[T] was determined at temperatures from −2 ° to −10 °C and was measured under varying conditioning temperature, time, and solute regimes. Values of N[T] increased rapidly on cooling samples from 30 to 5 °C. The optimum low temperature for conditioning was 5 °C. The conditioning process followed first-order reaction kinetics and time constants (1/rate constant) were between 43 and 62 min at 5 °C. Individual ice nucleators were isolated in droplets and were stable for at least 2 hr. Low-temperature conditioning did not occur when protein synthesis was inhibited by eliminating amino acids in the low-temperature conditioning media or by using the protein synthesis inhibitors chloramphenicol and streptomycin. Analysis of low-temperature conditioning, using heterogeneous ice nucleation theory predicted that ice nucleators are large and have diameters ranging from 80 Å (active at −8 °C) to 300 Å (active at −3 °C). In conclusion, it was predicted that conditioning resulted from growth of the nucleator from about 80 to 300 Å, from a change in the surface properties of 300 Å nucleator making it more similar to ice, or from a combination of these.     

Behavioral responses of hatchling painted turtles (Chrysemys picta) and snapping turtles (Chelydra serpentina) at subzero temperatures

We monitored behavioral responses of cold-acclimated hatchling painted turtles (Chrysemys picta) indigenous to Nebraska and hatchling snapping turtles (Chelydra serpentina) indigenous to Nebraska and Arkansas during cooling (0.1°C/min) to temperatures as low as −19°C. All turtles made exploratory movements during cooling and locomotion occurred at temperatures as low as −2 to −4°C, but C. picta maintained relatively higher levels of locomotor activity than C. serpentina, and no differences in motility occurred between northern and southern groups of C. serpentina. Slow movements of the head and limbs were observed in supercooled hatchling C. picta at temperatures as low as −10°C, whereas at about −5°C, C. serpentina exhibited an increase in spontaneous motor activity followed by muscle contracture, immobility, and spontaneous freezing. C. picta spontaneously froze at about −16°C without exhibiting cold contracture, suggesting that they are better adapted to survive exposure to extreme cold.     

Deep supercooling enabled by surface impregnation with lipophilic substances explains the survival of overwintering buds at extreme freezing

The frost survival mechanism of vegetative buds of angiosperms was suggested to be extracellular freezing causing dehydration, elevated osmotic potential to prevent freezing. However, extreme dehydration would be needed to avoid freezing at the temperatures down to ?45°C encountered by many trees. Buds of Alnus alnobetula, in common with other frost hardy angiosperms, excrete a lipophilic substance, whose functional role remains unclear. Freezing of buds was studied by infrared thermography, psychrometry, and cryomicroscopy. Buds of A. alnobetula did not survive by extracellular ice tolerance but by deep supercooling, down to ?45°C. An internal ice barrier prevented ice penetration from the frozen stem into the bud. Cryomicroscopy revealed a new freezing mechanism. Until now, supercooled buds lost water towards ice masses that form in the subtending stem and/or bud scales. In A. alnobetula, ice forms harmlessly inside the bud between the supercooled leaves. This would immediately trigger intracellular freezing and kill the supercooled bud in other species. In A. alnobetula, lipophilic substances (triterpenoids and flavonoid aglycones) impregnate the surface of bud leaves. These prevent extrinsic ice nucleation so allowing supercooling. This suggests a means to protect forestry and agricultural crops from extrinsic ice nucleation allowing transient supercooling during night frosts.     

Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.     

Freezing tolerance and avoidance in high-elevation Hawaiian plants

Freezing resistance mechanisms were studied in five endemic Hawaiian species growing at high elevations on Haleakala volcano, Hawaii, where nocturnal subzero (°C) air temperatures frequently occur. Extracellular freezing occurred at around -5°C in leaves of Argyroxiphium sandwicense and Sophora chrysophylla, but these leaves can tolerate extracellular ice accumulation to -15°C and -12°C, respectively. Mucilage, which apparently acted as an ice nucleator, comprised 9 to 11% of the dry weight of leaf tissue in these two species. Leaves of Vaccinium reticulatum and Styphelia tameiameiae were also found to tolerate substantial extracellular freezing. Dubautia menziesii, on the other hand, exhibited the characteristics of permanent supercooling; a very rapid decline in liquid water content associated with simultaneous intracellular and extracellular freezing. However, in those species that tolerate extracellular freezing, the decline in liquid water content during freezing is relatively slow. Osmotic potential was lower at pre-dawn than at midday in four of the species studied. Nocturnal production of osmotically active solutes may have helped to prevent intracellular freeze dehydration as well as to provide non-colligative protection of cell membranes. Styphelia tameiameiae supercooled to -9·3°C and tolerated tissue freezing to below -15°C, a unique combination of physiological characteristics related to freezing. Tolerance of extracellular ice formation after considerable supercooling may have resulted from low tissue water content and a high degree of intracellular water binding in this species, as determined by nuclear magnetic resonance studies. The climate at high elevations in Hawaii is relatively unpredictable in terms of the duration of subzero temperatures and the lowest subzero temperature reached during the night. It appears that plants growing in this tropical alpine habitat have been under selective pressures for the evolution of freezing tolerance mechanisms.     

Short- and long-term freezing effects in a coastal (Lobaria virens) versus a widespread lichen (L. pulmonaria)

Lichens are considered freezing tolerant, although few species have been tested. Growth, a robust measure of fitness integrating processes in all partners of a lichen thallus, has not yet been used as a viability measure after freezing. We compared relative growth rates (RGR) after freezing with short-term viability measures of photo- and mycobiont functions in the coastal Lobaria virens and the widespread L. pulmonaria to test the hypothesis that low temperature shapes the coastal distribution of L. virens. Hydrated thalli from sympatric populations were subjected to freezing at −10, −20 and −40 °C for 5 h. The rate of cooling and subsequent warming was 5 °C h⁻¹. Short-term viability measures of photobiont (maximal photosystem II efficiency, effective PSII yield) and mycobiont viability (conductivity index), as well as subsequent RGR, were assessed. The exotherms showed that L. virens froze at −3 °C; L. pulmonaria, at −4 °C. Freezing significantly impaired short-term viability measures of both photo- and mycobiont, particularly in the coastal species. Lobaria pulmonaria grew 2.1 times faster than L. virens, but the short-term damage after one freezing event did not affect the long-term RGR in any species. Thereby, short-term responses were impaired by freezing, long-term responses were not. While the lacking RGR-responses to freezing suggest that freezing tolerance does not shape the coastal distribution of L. virens, the significant reported adverse short-term effects in L. virens may be aggravated by repeated freezing-thawing cycles in cold winters. In such a perspective, repeated freezing may eventually lead to reduced long-term fitness in L. virens.     

Cold tolerance of steinernematid and heterorhabditid nematodes

• 1.1. Nematodes survive subzero temperatures using either a freeze-avoiding or freezing-tolerant strategy. Steinernema anomali, S. feltiae, and Heterorhabditis bacteriophora were all found to be freezing tolerant.
• 2.2. The lower lethal temperatures were −22, −19 and −14°C for S. feltiae, H. bacteriophora and S. anomali, respectively.
• 3.3. Survival after prolonged freezing at −4°C was 6, 5 and 3 days for S. feltiae, H. bacteriophora and S. anomali, respectively.
• 4.4. Acclimation to lower temperatures increased freezing tolerance. The freezing tolerance of Heterorhabditis bacteriophora increased under a stepwise acclimation regime; S. feltiae acclimated better under a direct acclimation regime.

     

High subzero cryofixation: A technique for observing ice within tissues

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above −80 °C without sample temperature alteration. Rat liver sections (1 cm²) were frozen at a rate of −1 °C/min to −20 °C, stored for 1 h at −20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to −80 °C methanol. After 1, 3, or 5 days of −80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at −20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at −20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.     

Cold tolerance of the maize orange leafhopper,Cicadulina bipunctata

Cicadulina bipunctata was originally distributed in tropical and subtropical regions of the Old World. This leafhopper recently expanded its distribution area to southern parts of temperate Japan. In this study, factors affecting the overwintering ability of C. bipunctata were examined. A series of laboratory experiments revealed that cold acclimation at 15 °C for 7 days enhanced the cold tolerance of C. bipunctata to the same level as an overwintering population, adult females were more tolerant of cold temperature than adult males, and survival of acclimated adult females was highly dependent on temperature from −5 to 5 °C and exposure duration to the temperature. The temperature of crystallization of adult females was approximately −19 °C but temperatures in southern temperate Japan rarely dropped below −10 °C in the winter, indicating that overwintering C. bipunctata adults in temperate Japan are not killed by freezing injury but by indirect chilling injury caused by long-term exposure to moderately low temperatures. An overwintering generation of C. bipunctata had extremely low overwinter survival (<1%) in temperate Japan; however, based on winter temperature ranges, there are additional areas amenable to expansion of C. bipunctata in temperate Japan.