Influence of cold acclimation on membrane injury in frozen plant tissue

Cold-acclimated twigs of Amelanchier alnifolia Nutt. released less HCN at −4.5 C than nonacclimated twigs following slow freezing to −25 C or rapid freezing to −78 C. Cold-acclimated twigs frozen slowly to −25 C released more HCN than cold-acclimated twigs frozen only to −4.5 C. Cold-acclimated twigs frozen slowly to −25 C and then rapidly to −78 C released less HCN at −4.5 C than cold-acclimated twigs frozen rapidly to −78 C. In general, K⁺ efflux and the inability to reduce triphenyl tetrazolium chloride following freezing and thawing paralleled HCN release at −4.5 C. Because low K⁺ efflux and high triphenyl tetrazolium chloride reduction are known to depend upon membrane integrity, the increased K⁺ efflux and the decreased triphenyl tetrazolium chloride reduction following freezing and thawing provide indirect evidence that HCN release at −4.5 C is a measure of membrane damage in frozen cells.     

Freezing stress and membrane injury of Norway spruce (Picea abies) tissues

Effects of sub-zero temperatures (−5 to −35°C) on the tissues of needles, buds and shoots of Norway spruce [ Picea abies (L.) Karst.] were studied. The freezing caused increased efflux of cellular electrolytes. Freezing injury of the primordial shoots and 1-year-old shoots was the result of the spontaneous freezing of a deep supercooled cellular water. The crystallization injures the cellular membranes leading to the loss of semipermeability and to the drastic efflux of K⁺. In the needles there was no deep supercooling of water and two patterns of changes in the membranes, depending upon the range of the applied temperatures, could be distinguished. At 0 to – 25°C, which do not kill the cells, we observed a disturbance in the membrane semipermeability as monitored by electrolytes efflux within a few hours after thawing of the needles. At lower temperatures (−35°C) we observed irreversible loss of the membrane semipermeability, and death of the tissue. Those changes occurred 10 h after thawing and were probably caused by the released lytic enzymes and some toxic compounds, which acted on the cellular membranes.     

The mechanism of freezing injury in xylem of winter apple twigs

In acclimated winter twigs of Haralson apple (Pyrus Malus L.), a lag in temperature during cooling at a constant rate was observed at about −41 C by differential thermal analysis. The temperature at which this low temperature exotherm occurred was essentially unaffected by the cooling rate. During thawing there was no lag in temperature (endotherm) near the temperature at which the low temperature exotherm occurred, but upon subsequent refreezing the exotherm reappeared at a somewhat higher temperature when twigs were rewarmed to at least −5 C before refreezing. These observations indicate that a small fraction of water may remain unfrozen to as low as −42 C after freezing of the bulk water in stems. The low temperature exotherm was not present in twigs freeze-dried to a water content below 8.5% (per unit fresh weight), but it reappeared when twigs were rehydrated to 20% water. When freeze-dried twigs were ground to a fine powder prior to rehydration, no exotherm was observed. Previous work has shown that the low temperature exotherm arises from xylem and pith tissues, and that injury to living cells in these tissues invariably occurs only when twigs are cooled below, but not above the temperature of the low temperature exotherm. This study revealed that the low temperature exotherm resulted from the freezing of a water fraction, that the freezing of this water was independent of the freezing of the bulk water, that the exotherm was associated with some gross structural feature but not the viability of the tissue, and that injury to living cells in the xylem and pith was closely and perhaps causally related to the initial freezing of this water.     

Manifestations of cell damage after freezing and thawing

The nature of the primary lesions suffered by cells during freezing and thawing is unclear, although the plasma membrane is often considered the primary site for freezing injury. This study was designed to investigate the nature of damage immediately after thawing, by monitoring several functional tests of the cell and the plasma membrane. Hamster fibroblasts, human lymphocytes, and human granulocytes were subjected to a graded freeze-thaw stress in the absence of cryoprotective compound by cooling at -1 degree C/min to a temperature between -10 and -40 degrees C, and then were either warmed directly in water at 37 degrees C or cooled rapidly to -196 degrees C before rapid warming. Mitochondrial function in the cells was then assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), fluorescein diacetate (FDA), colony growth, and osmometric response in a hypertonic solution. Cells behaved as osmometers after cooling at -1 degree C/min to low temperatures at which there were no responses measured by other assays, indicating that the plasma membrane is not a primary site for injury sustained during slow cooling. These results also indicate that the FDA test does not measure membrane integrity, but reflects the permeability of the channels through which fluorescein leaves the cells. Fewer cells could respond osmotically after cooling under conditions where intracellular freezing was likely, implying that the plasma membrane is directly damaged by the conditions leading to intracellular freezing. A general model of freezing injury to nucleated mammalian cells is proposed in which disruption of the lysosomes constitutes the primary lesion in cells cooled under conditions where the cells are dehydrated at low temperatures.     

The destruction of Salmonella typhimurium in chicken exudate by different freeze-thaw treatments

Antibacterial treatments for frozen poultry, including holding at -5°C and slow thawing at 4°C to which exponential phase cells of Salmonella typhimurium were susceptible, were found to be relatively ineffective against stationary phase cells. Exposure of the latter, however, to a pre-freezing triple stress treatment of cold-shock exposure at 5°C to a solution containing 5 mg/l of free available chlorine in 1% succinic acid (pH 2.5) for 20 min substantially lowered the resistance of the cells to subsequent freezing, storage and thawing in poultry flesh exudate. Cell survival was further decreased by storage of exudate at - 18°C for 28 d and this reduced the proportion of stationary phase cells to less than 1% of initial numbers, with a concomitant increase in sensitivity to deoxycholate. Such a combined pre-treatment may have practical potential for salmonella decontamination in the production of frozen poultry.     

Cryopreservation of the milt of the northern pike

Seven published extenders, three thawing media and two thawing temperatures were tested in order to determine their suitability for cryopreservation of northern pike ( Esox lucius L.) sperm. Sperm motility during successive steps of cryopreservation was evaluated. Erdahl and Graham's extender with the addition of egg yolk proved to be the most efficient (maximum hatching rate of 74.5%) when semen was thawed in 120 m m NaCl solution warmed up to 30° C. No correlations between motility of sperm (diluted in extenders or diluted in extenders and then activated with thawing solution) and the subsequent hatching success were observed. The relationship between motility of thawed sperm and its fertilization ability was considerable, but correlation was not significant. Spermatozoa frozen in some extenders were frequently motile after thawing but they were not able to fertilize the eggs, this resulted in a poor hatching rate. Depending on the extender, the addition of yolk induced either positive or detrimental effects on fertilization success.     

Identification of sites of injury in Lactobacillus bulgaricus during heat stress

Heat resistance of Lactobacillus bulgaricus in skimmed milk at 62°, 64°, 65° and 66°C was studied. The response to increasing temperatures in this range was not linear, with temperatures at 65°C and above giving a lower survival rate than would be predicted from experiments at lower temperatures. To identify sites of injury at these temperatures, chemical markers were used. Heating at 64°C and below resulted in damage to the cytoplasmic membrane. At temperatures of 65°C and above chemical markers also indicated damage in the cell wall and proteins. Using differential scanning calorimetry analysis of whole cells of Lact. bulgaricus seven main peaks were observed (l–51, m₁–61, m₂–73, n–80, p–89, q–100,r–112°C). Three of these peaks (l_r, m_r and p_r) were the result of reversible reactions. Analysis of cell fractions identified the cell structure involved in giving rise to each of the three reversible peaks; l_r, cell membrane lipids, m_r, ribosomes, and p_r, DNA. The evidence presented in this paper shows that irreversible reactions in the cell ribosomes are a critical site of damage in Lact. bulgaricus during heat stress in liquid media at 65°C and above.     

Effects of temperature and drying rate during dehydration of celery seeds on germination, leakage and response to gibberellin and cytokinin

A high temperature treatment of 32°C which prevents dehydration injury in celery seeds imbibed for 3 days at 17°C and then dried at 20°C, reduced leakage during rehydration, compared with seeds not given the high temperature treatment. Treatments which would normally release celery seeds from dormancy, such as low temperature imbibition or gibberellin (GA_4/7) and benzyladenine (BA) applications had little effect on the germination of seeds exhibiting desiccation injury. However, GA_4/7 did induce splitting of the seed coat and swelling of the endosperm, and this effect was enhanced by BA. It is suggested that in celery seeds high temperature prevents irreversible embryo damage, including membrane damage, caused by drying.     

Mechanisms of inactivation of HSV-2 during storage in frozen and lyophilized forms

The structural integrity of herpes simplex virus 2 (HSV-2) during freezing, thawing, and lyophilization has been studied using scanning and transmission electron microscopy. Viral particles should be thawed quickly from -80 to 37 degrees C to avoid artifacts of thawing. To avoid freezing damage, the virus should be rapidly frozen (>20 K s(-1)) rather than slowly frozen as occurs on the shelf of a lyophilizer (<1 K s(-1)). Fast freezing and thawing allows six cycles of freeze thaw with no loss of viral titer TCID50. Viral particles were characterized using immunogold labeling methods. Freshly thawed virus had 19 +/- 4 polyclonal immunogold particles virus(-1); virus stored at -80 degrees C for at least 4 months had 17 +/- 3 particles virus(-1); virus stored for 1 week at 4 degrees C had 8 +/- 4 particles virus(-1). By bulk lyophilization the number of particles was 4 +/- 4, but by fast freezing and lyophilization the number of gold particles improved to 12 +/- 5. The loss of viral membrane was directly observed, and the in vitro loss was demonstrated to occur through three possible pathways, including (i) simultaneous release of tegument and membrane, (ii) sequential release of membrane and then tegument, and (iii) release like by in vivo infection. The capsids were not further degraded as indicated by the lack of free DNA, which was only released by boiling the viral samples with 1% SDS, followed by a dilution to 0.001% w/v SDS for the real-time PCR reaction.     

Effect of hypertonic stress on mammalian cell lines and its relevance to freeze-thaw injury

The effect of subjecting the mammalian cell lines MRC-5 and CHO to hypertonic salt concentrations (0.16 to 2.4 m) and returning them to isotonic conditions was investigated. Parameters for measuring cell size, viability and release of radiochemical markers were used to determine the relative susceptibilities of the two cell lines to hypertonic stress and the relative effects of increasing and decreasing hypertonicity. The aim of this study was to determine how great a role hypertonic stress plays in freeze-thaw damage of mamalian cells. This type of study has been extensively used for erythrocytes but not for nucleated mamamlian cell lines.The findings were that considerable cell shrinkage occurred, with a minimum size at 0.6 m NaCl, but that this caused no cell injury or death. Injury, measured by cation leakage and release of membrane and cytoplasmic labels occurred whilst the cell was swelling after reaching its minimum volume. MRC-5 cells succumbed at relatively low salt concentrations and became denatured. CHO cells withstood far high salt concentrations but were then damaged during dilution back to isotonic conditions. Comparison of the data obtained from hypertonic stress experiments and freeze-thaw experiments showed many similarities for CHO cells and indicated that the cell membrane could withstand high salt concentrations both at constant and changing temperatures but were prone to injury on dilution back to isotonic conditions. MRC-5 cells were shown to be very prone to cold shock and the results indicated that they probably succumb to damage and death during the hypertonic phase of cooling rather than thawing thus explaining their much lower survival from freeze-thaw experiments than CHO cells. The influence of DMSO in delaying cell damage to higher salt concentrations and lessening disruptive swelling during dilution were also demonstrated.     

Relation between ultrastructure and viability of frozen-thawed Chinese hamster tissue-culture cells

Electron microscopic examination of Chinese hamster tissue-culture cells showed that freezing and thawing result in structural alterations, the type and magnitude of which depend on the cooling and warming velocities used. Cells suspended in Hanks balanced salt solution and cooled to −196 °C at rates exceeding the survival optimum exhibit different patterns and extents of ultrastructural alterations than do cells cooled or warmed at rates lower than optimum. Even though the addition of 0.4 M dimethyl sulfoxide confers some protection, in terms of survival, it does not prevent structural alterations. The types of alterations, however, differ from those found in cells frozen in Hanks alone. Freeze-thaw treatments producing similar percentages of cell survival do not necessarily cause similar structural alterations, nor is there a simple correlation between structural alterations and survival.     

Heat treatment to decrease chilling injury in tomato fruit. Effects on lipids, pericarp lesions and fungal growth

Tomato fruits are sensitive to low temperature and develop chilling injury, while at nonchilling temperatures they ripen rapidly. Previously, a hot-air treatment was found to reduce the sensitivity of the fruit to low temperatures. In the present study hot air was compared to hot water and their effects on reducing chilling injury and fungal decay were investigated. Tomatoes ( Lycopersicon esculentum cv. Daniella) at the breaker stage were subjected to hot air, 48 h at 38°C, or various hot water dips, 30 min at 40°C or 2 min at 46, 48 or 50°C, before holding at 2°C. The unheated tomatoes developed chilling injury and fungal infections at 2°C, but not at 12°C. All the heat treatments reduced chilling injury and decay in tomatoes held for 3 weeks at 2°C. The outer pericarp tissue of heated tomatoes had higher phospholipid and lower sterol contents than unheated tomatoes. Heated tomatoes also had less saturated fatty acids than unheated tomatoes held at 2°C, but not at 12°C. Scanning electron micrograph observations showed that all the fruits had microcracks in their surface, but the unheated chilled tomatoes had also fungal growth in the cracks, while those of the heated tomato fruit did not. In the areas of chilling injury collapsed cells were present under the peel and could also support pathogen development. It is suggested that the heat treatment institutes a response to high temperature stress in the fruit tissue that leads to strengthened membranes. This prevents the loss of function and cell collapse which was found in the chilling-injured areas of affected fruit.     

The role of direct chilling injury and inoculative freezing in cold tolerance of Amblyomma americanum, Dermacentor variabilis and Ixodes scapularis

Abstract. Supercooling points and chill tolerance were compared among nymphs and adults of the ixodid ticks Dermacentor variabilis, Amblyomma americanum and Ixodes scapularis (Acari: Ixodidae).Supercooling points in the range of <-22 to -18°C were observed for nymphs, and -22 to -8°C for adults.The lower lethal temperatures observed under dry conditions, -14 to -10°C, were warmer than the supercooling points, but still much colder than -4.8°C, the lowest temperature recorded from a likely tick habitat in southwestern Ohio.Based on our experiments, spontaneous freezing and direct chilling injury are not significant mortality factors in these species in the field.Mortality was observed between -5 and -3°C for A.americanum and D.variabilis nymphs chilled for 2 h while in direct contact with ice.This mortality is probably due to inoculative freezing.Given the requirement for a rather humid microhabitat for off-host survival, these findings suggest that inoculative freezing is an important cause of overwintering mortality in these medically important species.     

Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice

Successful cryopreservation demands there be little or no intracellular ice. One procedure is classical slow equilibrium freezing, and it has been successful in many cases. However, for some important cell types, including some mammalian oocytes, it has not. For the latter, there are increasing attempts to cryopreserve them by vitrification. However, even if intracellular ice formation (IIF) is prevented during cooling, it can still occur during the warming of a vitrified sample. Here, we examine two aspects of this occurrence in mouse oocytes. One took place in oocytes that were partly dehydrated by an initial hold for 12 min at -25 degrees C. They were then cooled rapidly to -70 degrees C and warmed slowly, or they were warmed rapidly to intermediate temperatures and held. These oocytes underwent no IIF during cooling but blackened from IIF during warming. The blackening rate increased about 5-fold for each five-degree rise in temperature. Upon thawing, they were dead. The second aspect involved oocytes that had been vitrified by cooling to -196 degrees C while suspended in a concentrated solution of cryoprotectants and warmed at rates ranging from 140 degrees C/min to 3300 degrees C/min. Survivals after warming at 140 degrees C/min and 250 degrees C/min were low (<30%). Survivals after warming at > or =2200 degrees C/min were high (80%). When warmed slowly, they were killed, apparently by the recrystallization of previously formed small internal ice crystals. The similarities and differences in the consequences of the two types of freezing are discussed.     

Modelling the time–temperature relationship in cold injury and effect of high-temperature interruptions on survival in a chill-sensitive collembolan

1. Temperature- and time-dependent mortalities were studied and modelled in insects exposed in regimes with constant and alternating temperatures. In these experiments, freezing was not a cause of death.
2. Survival rates at a range of constant low temperatures (– 5 to + 1 °C) and for different exposure periods (1–14 days) were measured in the summer acclimated springtail Orchesella cincta .
3. Daily interruptions of the cold exposure with short intervals at high temperature reduced mortality or slowed the increase of mortality. This effect was stronger at higher temperature (19 vs 5 and 12 °C) and increased with the duration of the interruption (0·25–2 h).
4. The injury was reversible when the cold exposure was limited to 2 days.
5. Survival in desiccated animals (14% water loss) was reduced.
6. It is suggested that the mortality of summer acclimated springtails is caused by a complex metabolic disorder and membrane changes at low temperatures.     

The Recovery of Sublethally Injured Escherichia coli from Frozen Meat

Sublethal injury to Escherichia coli , measured as the inability of surviving cells to grow on media containing bile salts, was monitored during frozen storage on meat at —5, —10 and —20° C. More rapid increases in injury occurred at the higher subzero temperatures and log phase cells were more susceptible than those in the stationary phase of growth. Repair of injury in non-selective liquid media took between 2 and 6 h at 25° and was often accompanied by an increase in total viable count. Incubation for a fixed period in broth was, therefore, unsuitable for the quantitative recovery of freeze-injured Esch. coli. Resuscitation on membrane filters avoided confusing repair of injury with multiplication of uninjured or repaired cells. The mean recovery of injured cells following incubation on membranes for 4 h at 35°C on tryptone soya agar, was 94%.     

Freeze-thaw injury to isolated spinach protoplasts and its simulation at above freezing temperatures

Possibilities to account for the mechanism of freeze-thaw injury to isolated protoplasts of Spinacia oleracea L. cv. Winter Bloomsdale were investigated. A freeze-thaw cycle to −3.9 C resulted in 80% lysis of the protoplasts. At −3.9 C, protoplasts are exposed to the equivalent of a 2.1 osmolal solution. Isolated protoplasts behave as ideal osmometers in the range of concentrations tested (0.35 to 2.75 osmolal), arguing against a minimum critical volume as a mechanism of injury. Average protoplast volume after a freeze-thaw cycle was not greatly different than the volume before freezing, arguing against an irreversible influx of solutes while frozen. A wide variety of sugars and sugar alcohols, none of which was freely permeant, were capable of protecting against injury which occurred when protoplasts were frozen in salt solutions. The extent of injury was also dependent upon the type of monovalent ions present, with Li = Na > K = Rb = Cs and Cl ≥ Br > I, in order of decreasing protoplast survival. Osmotic conditions encountered during a freeze-thaw cycle were established at room temperature by exposing protoplasts to high salt concentrations and then diluting the osmoticum. Injury occurred only after dilution of the osmoticum and was correlated with the expansion of the plasma membrane. Injury observed in frozen-thawed protoplasts was correlated with the increase in surface area the plasma membrane should have undergone during thawing, supporting the contention that contraction of the plasma membrane during freezing and its expansion during thawing are two interacting lesions which cause protoplast lysis during a freezethaw cycle.     

Fatty acid desaturation in monogalactosyldiacylglycerol of Brassica napus leaves during low temperature acclimation

Brassica napus plants grown at 5°C have the potential to desaturate fatty acids in the major membrane diacylglycerols of leaves at rates much higher than those of plants grown at 30°C. This low temperature-induced desaturation (LTD) is rapidly deactivated if plants grown at 5°C are transferred to 30°C for several hours. By exposure to ¹⁴CO₂ and a computer simulation program, we estimated the rate of desaturation of monogalactosyldiacylglycerol by (ω9-, ω6- and ω3-desaturases of plants grown at 5, 10, 20 and 30°C. Data show that LTD can be induced in mature leaves of plants grown at 20 and 30°C after transfer to 5°C. Full activation of LTD takes several weeks and occurs more rapidly in plants grown at 20°C than 30°C. This activation is largely due to the increased activity of ω9- and ω6-desaturases on C16 fatty acids and ω6-desaturase on C18 fatty acids.     

Effect of the cryopreservation conditions on the viability of the rumen ciliate Diploplastron (Metadinium) affine

AIMS: To study the viability of Diploplastron (Metadinium) affine after its cryopreservation at two cooling rates, and the effect of procedure conditions on viability. METHODS AND RESULTS: There were differences in viability between cooling rates (1 and 4 degrees C min(-1)) at 15 or 5 degrees C, but not after thawing. When the equilibrium temperature (25 or 5 degrees C), the cryopreservant (glycerol or dimethyl sulfoxide [DMSO]) and the use of membrane protector were tested, there were no differences caused by the cryopreservant or the membrane protector. However, the equilibrium at 25 degrees C increased the viability (P = 0.005) compared with 5 degrees C. CONCLUSIONS: Viability after thawing was 0.10-0.19. Adding the cryopreservant (either glycerol or DMSO) at 25 degrees C instead of 5 degrees C improves viability of D. affine after thawing. SIGNIFICANCE AND IMPACT OF THE STUDY: Conditions of cryopreservation are largely dependent on the species of rumen protozoa. Number of viable cells after thawing would indicate the possibility of culture recovery for D. affine.     

Effects of hardening and freezing stress on membrane lipids and CO2 fixation of Ceratodon purpureus protonemata

Membrane lipids and steady-state CO₂ fixation rates were studied in moss protonemata in order to evaluate separately the effects of growth temperature, freezing stress and the achievement of frost hardiness. Protonemata of Ceratodon purpureus (Hedw.) Brid, were grown at 20 and 4°C and parts of both materials were then hardened. The low growth temperature increased the content and unsaturation level of membrane lipids significantly. This did not, however, cause a noticeable increase in the frost hardiness. Nor was the achievement of frost hardiness in this material accompanied by further changes in the amount or unsaturtion level of any membrane lipid class. Cytoplasmic membranes were abundant in both unhardened and hardened materials grown at 4°C, which agreed with the high phospholipid content of these protonemata. The only significant difference in membrane lipids between unhardened and hardened materials was a 50% lower level of trans 16:1 fatty acid in the phosphatidylglycerol fraction of hardened protonemata.
In hardened protonemata monogalactosyldiacylglycerol (MGDG) was the membrane lipid most liable to decrease during the freeze-thaw cycle. The loss of MGDG was accompanied by partial inhibition of CO₂ fixation. Provided the content of phospholipids remained unchanged (freeze-thaw cycle with – 10°C in hardened protonemata), this inhibition was mostly reversible. If loss of the phospholipids also had occurred during the freeze-thaw cycle, as was the case in unhardened material at or below -10°C, CO₂ fixation was severely and nearly irreversibly inhibited after thawing.