Visualization of freezing damage. II. Structural alterations during warming

There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from ?20 ° to ?50 °C and then rapidly cooled to ?196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above ?40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as ?45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as ?20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.     

Heat-induced stimulation of 3-O-methylglucose transport in rat thymocytes.

Cells incubated at 41–46 °C show a gradual increase in the initial rate of 3-O-methylglucose uptake when subsequently assayed at 37 °C. Cellular ATP levels remain constant throughout this temperature range, but at temperatures higher than 46 °C, ATP levels decline as does the extent of transport stimulation. Cells incubated at 45 °C for 5 min continue to show a gradual increase in transport activity throughout a subsequent 25-min incubation period at 37 °C. The increase in transport activity is characterized by an increase in the proportion of the rapid phase of 3-O-methylglucose uptake, with little or no change in the half-time of either the rapid phase or the slow phase. Transport stimulation at high temperatures is blocked by inhibitors of oxidative phosphorylation. Cells depleted of intracellular exchangeable Ca²⁺ by treatment with the ionophore A23187 in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid show nearly the same degree of stimulation at high temperatures as untreated cells, suggesting that exchangeable Ca²⁺ ions do not play an obligatory role in the mechanism of transport stimulation. It is suggested that structural changes occur at 41–46 °C in the membrane proteins controlling glucose transport activity.     

Gas chromatography: elution temperature, speed of analysis, and separation, efficiency as influenced by rate of temperature programming and carrier gas velocity in open tubular glass capillary columns.

The elution temperatures and degree of separation of a model system series of hydrocarbons were followed as functions of the rate of temperature programming and carrier gas velocity. Graphs are presented that permit individual assessment of these variables. Lower elution temperatures can be achieved by raising the carrier gas velocity or lowering the rate of temperature programming. In glass capillary columns of normal dimensions (0.25-mm i.d. × 50 m), the former is much less effective and results in lower column efficiency. The elution temperature of n-pentadecane varied from 139 to 309°C and the separation efficiency of the column decreased by 50% as the program rate varied from 1 to 32°C/min at constant pressure drop. A simplified glass inlet splitter and constant pressure-drop device is also described.     

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.     

Turning up the heat on subzero fish: thermal dependence of sustained swimming in an Antarctic notothenioid

We determined the maximum sustained swimming speed (U_crit), and resting and maximum ventilation rates of the Antarctic fish Pagothenia borchgrevinki at five temperatures between −1°C and 8°C. We also determined resting metabolic rate (VO₂) at −1°C, 2°C, and 4°C. U_crit of P. borchgrevinki was highest at −1°C (2.7±0.1 BL s⁻¹) and rapidly decreased with temperature, representing a thermal performance breadth of only 5°C. This narrow thermal performance supports our prediction that specialisation to the subzero Antarctic marine environment is associated with a physiological trade-off in performance at high temperatures. Resting oxygen consumption and ventilation rate increased by more than 200% across the temperature range, which most likely contribute to the decrease in aerobic swimming capabilities at higher temperatures.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

Short-term effects of temperature upon the growth of intertidal fucales

The growth rate of five species of intertidal Fucales (Pelvetia canaliculata (L.) Dec. et Thur., Fucus spiralis L., Fucus vesiculosus L., Fucus serratus L., Ascophyllum nodosum (L.) Le Jolis) was measured at temperatures from 2.5 to 35 °C. An increase in temperature immediately causes a high growth rate, and during the first hour it increases linearly with temperature; at 35 °C it is 20 times the control at 7 °C. This acceleration of growth is based mainly on stored photosynthate. After the first few hours the growth rate decreases rapidly, particularly at the highest temperatures. After 2–3 weeks a temperature optimum below 17.5 °C is indicated. High temperatures, 30–35 °C, were lethal to all species, with a survival time corresponding to their vertical zonation in the natural habitat.     

Growth of Kluyveromyces marxianus and formation of ethyl acetate depending on temperature

Conversion of lactose into ethyl acetate by Kluyveromyces marxianus allows economic reuse of whey-borne sugar. The high volatility of ethyl acetate enables its process-integrated recovery by stripping. This stripping is governed by both the aeration rate and the partition coefficient, K _EA,L/G. Cultivation at elevated temperatures should decrease the K _EA,L/G value and thus favor stripping. K. marxianus DSM 5422 as a potent producer of ethyl acetate was cultivated aerobically in whey-borne media for studying temperature-dependent growth and ester formation. Shake flask cultivation proved thermal tolerance of this yeast growing from 7 to 47 °C with a maximum rate of 0.75 h^?1 at 40 °C. The biomass yield was 0.41 g/g at moderate temperatures while low and high temperatures caused distinct drops. The observed μ-T and Y _X/S-T dependencies were described by mathematical models. Further cultivations were done in an 1-L stirred reactor for exploring the effect of temperature on ester synthesis. Cultivation at 32 °C caused significant ester formation (Y _EA/S?=?0.197 g/g) while cultivation at 42 °C suppressed ester synthesis (Y _EA/S?=?0.002 g/g). The high temperature affected metal dissolution from the bioreactor delivering iron for yeast growth and preventing ester synthesis. Cultivation at 32 °C with a switch to 42 °C at the onset of ester synthesis allowed quick and efficient ester production (Y _EA/S?=?0.289 g/g). The high temperature lowered the K _EA,L/G value from 78 to 44 L/L which heightened the gas-phase ester concentration (favoring ester recovery) without increasing the liquid-phase concentration (avoiding product inhibition).     

The effect of insulin on the heart rate and temperature of the ground squirrel <Emphasis Type="Italic">Spermofilus undulatus</Emphasis> during arousal from hibernation

The effect of insulin on the heart rate and body temperature, measured per rectum, of ground squirrels (Spermophilus undulatus) during triggered arousal from winter hibernation was studied. We found that the outcomes of insulin injection to hibernating ground squirrels varied in the course of arousal. During the first stage, while body temperatures were less than 10°C, the heart rates and rectal temperatures in both control and insulin-treated groups changed in the same manner. During the next stage of arousal, when the body temperature rose above 12°C, elevation of the heart rate and rectal temperature in the insulin-treated animals was significantly retarded and lasted 110 min compared to 80 min in the control group. Conversely, in the final stage of arousal at body temperatures above 20°C, the heart rate and body temperature increased more rapidly in the insulin-treated animals that reached normal body temperature within 40 min compared to 60 min in the control group. Suggested mechanisms of bidirectional effects of insulin on the heart rates and body temperatures in ground squirrels at the particular stages of arousal, with regard to the progression of endogenous insulin and glucose levels in the blood serum, are discussed.     

Coupling of insulin receptors to glucose transport: a temperature-dependent time lag in activation of glucose transport.

We have studied the ability of occupied insulin receptors to activate (or couple to) the glucose transport system in isolated rat adipocytes. Maximal insulin action is seen when only a small proportion (<10%) of the receptors is occupied, and this fraction can be rapidly filled (<5 s) at an insulin concentration of 100 ng/ml. Additionally, control studies show that when the extracellular glucose concentration is tripled, the rate of transport triples within 10 s, indicating that changes in transport activity can be observed nearly instantaneously. Therefore, when cells are exposed to a high insulin concentration (100 ng/ml), any delay in the onset of insulin action beyond this time must be due to the time required for coupling of occupied insulin receptors to the glucose transport system. At 24 °C there is a lag of at least 200 s after insulin addition before a significant stimulation of 2-deoxyglucose transport is seen. The length of this lag phase is temperature dependent, decreasing to 45 s at 37 °C. An Arrhenius plot of the coupling lag is linear, with an activation energy of 25 kcal/mol. After the delay in the onset of initial transport activation the full response appears in a gradual manner, requiring 20 min at 24 °C to attain maximal stimulation. The time required for the full insulin response to appear is also temperature dependent, decreasing to 5 min at 37 °C. Similar results were obtained for the kinetics of insulin activation of 3-O-methyl glucose transport. Thus, the coupling of insulin receptors to the glucose transport system can be divided into two components: an initial absolute time lag followed by a gradual incremental process before the maximal, or full, effect of insulin is achieved. In conclusion, (1) there is an absolute delay in the onset of the insulin's initial action on glucose transport, (2) after an initial delay, activation of transport proceeds in a gradual manner, and (3) the coupling process between insulin receptors and the glucose transport system is temperature dependent and can be described by a linear Arrhenius plot. This suggests that the rate of activation is not limited by membrane fluidity.     

Influence of heat shock on chlorophyll fluorescence of white oak (<Emphasis Type="Italic">Quercus pubescens</Emphasis> Willd.) leaves

Chlorophyll fluorescence parameters of Quercus pubescens Willd. as response to heat shock (HS) by immersing leaves for 5 and 15 min in water of temperatures between 38 and 59 °C were examined. Fluorescence was measured after different periods of recovery (15, 30, 90, 210, and 1 440 min at 24/26 °C night/day temperature and 100 % humidity). The effective quantum yield of photosystem 2 (Y) in control and HS-treated leaves was always measured after previous 15 min irradiation. Under a 5 min HS, Y did not change after using temperatures below 44 °C, was rapidly restored after HS of moderate temperatures (44–48 °C), and progressively decreased and recovered eventually to the initial value after HS of high temperatures (48–52 °C). Y did not recover after HS with temperatures higher than 52 °C. Increase in the duration of HS from 5 to 15 min lead to change of the initial Y at each HS temperature, but the recovery processes were similar to those characteristic after 5 min incubation. The processes of recovery may depend mainly on the specificity of injuries caused by different heat shock temperatures. Thus Q. pubescens is able to preserve and recover the functional potential of its photosynthetic apparatus in response to HS up to 52 °C.     

Strategies of freeze avoidance in larvae of the goldenrod gall moth,Epiblema scudderiana: Laboratory investigations of temperature cues in the regulation of cold hardiness

Laboratory manipulations of ambient temperature were used to investigate the role of temperature in triggering or modulating cold-hardiness adaptations, supercooling-point depression and cryoprotectant accumulation, in larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens), a freeze-intolerant species. Low temperature strongly facilitated cryoprotectant synthesis; larvae subjected to a 1°C per day decrease in temperature showed a major increase in the rate of glycerol synthesis when temperature fell below 5°C with highest rates of synthesis, greater than 90 μmol g⁻¹ d⁻¹, at temperatures between 0 and −10°C. Conversely, abrupt rewarming of larvae from −18 to 23°C in mid-November stimulated a rapid loss of glycerol (from a starting level of 1763 ± 278 μmol/g wet weight) with a half time of only 1.5 days. Supercooling-point depression was not keyed to ambient temperature but appeared to be an endogenous event occurring over the same time interval in laboratory animals held at warm or cold temperatures, as well as in outdoor animals. Rewarming of cold-adapted larvae in November resulted in only a small rise in supercooling point (and did not break diapause) but rewarming in February resulted in a 19°C increase in supercooling point in 4 days, followed rapidly by pupation.     

The effect of elevated temperature and reactor shutdown on the benthic marine flora of the millstone thermal quarry,Connecticut

Forty-nine species and one variety of benthic blue-green, red, brown and green algae were found over a 1.5 year period in a thermal sea water dump where temperatures average 10°C above ambient Long Island Sound waters. Of these, 58% can survive temperatures exceeding 30°C, but only six show survival after prolonged excessive temperature. At temperatures less than 27°C, the number of taxa is independent of temperature, but at greater temperatures there is a significant negative correlation of temperature to taxa count, reaching a minimum of 3 species. Rapid temperature drops cause concomitant drops in taxa counts, 14% of this variation being attributed to drastic temperature change which affects the algae.     

Temperatures in Pigs During 3 T MRI Temperatures,Heart Rates,and Breathing Rates of Pigs During RF Power Deposition in a 3 T (128 MHz) Body Coil

Exposure to radiofrequency (RF) power deposition during magnetic resonance imaging (MRI) induces elevated body‐tissue temperatures and may cause changes in heart and breathing rates, disturbing thermoregulation. Eleven temperature sensors were placed in muscle tissue and one sensor in the rectum (measured in 10 cm depth) of 20 free‐breathing anesthetized pigs to verify temperature curves during RF exposure. Tissue temperatures and heart and breathing rates were measured before, during, and after RF exposure. Pigs were placed into a 60‐cm diameter whole‐body resonator of a 3 T MRI system. Nineteen anesthetized pigs were divided into four RF exposure groups: sham (0 W/kg), low‐exposure (2.7 W/kg, mean exposure time 56 min), moderate‐exposure (4.8 W/kg, mean exposure time 31 min), and high‐exposure (4.4 W/kg, mean exposure time 61 min). One pig was exposed to a whole‐body specific absorption rate (wbSAR) of 11.4 W/kg (extreme‐exposure). Hotspot temperatures, measured by sensor 2, increased by mean 5.0 ± 0.9°C, min 3.9; max 6.3 (low), 7.0 ± 2.3°C, min 4.6; max 9.9 (moderate), and 9.2 ± 4.4°C, min 6.1, max 17.9 (high) compared with 0.3 ± 0.3°C in the sham‐exposure group (min 0.1, max 0.6). Four time‐temperature curves were identified: sinusoidal, parabolic, plateau, and linear. These curve shapes did not correlate with RF intensity, rectal temperature, breathing rate, or heart rate. In all pigs, rectal temperatures increased (2.1 ± 0.9°C) during and even after RF exposure, while hotspot temperatures decreased after exposure. When rectal temperature increased by 1°C, hotspot temperature increased up to 42.8°C within 37 min (low‐exposure) or up to 43.8°C within 24 min (high‐exposure). Global wbSAR did not correlate with maximum hotspot. Bioelectromagnetics. 2021;42:37–50. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society     

Effect of constant and fluctuating temperature on the circadian foraging rhythm of the red imported fire ant,Solenopsis invicta Buren (Hymenoptera: Formicidae)

Understanding circadian foraging rhythms activity of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae) foragers at different temperatures is an important step towards developing control measures in Integrated Pest Management (IPM) programs. In this study, the circadian foraging rhythm activities of S. invicta foragersat different temperature were investigated under laboratory and field conditions. Results indicated that the foraging activity increased after sunrise, and maximum foraging occurred at 14:00 (foraging rate was 69.22 ± 0.57 and 72.58 ± 1.15 foragers/min in the first and second year, respectively) in the tea fields of Guangzhou during autumn. Furthermore, foragers demonstrated circadian rhythms and exhibited a unimodal after 24 h. A significant correlation was found between foraging activity and temperature. S. invicta colonies were active at moderate soil temperatures (approximately 26.65 °C to 29.24 °C). The preferred temperature of the colonies was 26 °C, followed by 22 °C and 18 °C in the laboratory. The individual S. invicta activity was maximum at 17:00 (18.67 ± 1.66 times /10 min) and minimum at 5:00 (8.33 ± 2.51 times/10 min) at 26 °C. The fluctuating temperature had a significant impact on individual locomotor activity (r = 0.8979, P < 0.01) but did not alter the rhythm activity. Our results demonstrated that temperature might play an important role in circadian foraging rhythms activity of S. invicta. These results may have implications for the development of more effective fire ant management strategies.     

Freezing tolerance in relation to cooling rate in an adult insect

In the adult tenebrionid beetle Upis ceramboides unusually low cooling rates are required to demonstrate maximum freezing tolerance, and a very slight change in rate can reduce survival from 100 to 0%. Freezing to ?50 °C results in 100% mortality at rates above 0.35 °C/min, but no injury is apparent if the cooling rate is 0.28 °C/min. The lower lethal temperature, determined with a cooling rate of 0.17 °C/min, is about ?60 °C. The maximum cooling rate which allows full survival is nearly identical to optimal cooling rates previously found for mouse embryos and some lymphocytes, but the striking sensitivity to very slight changes in rate is unique to Upis. Most studies dealing with insect freezing tolerance have utilized rates of 1 °C/min or faster, and the failure of some of these laboratory studies to observe freezing survival may be due to the use of lethal cooling rates.     

High sensitivity differential scanning calorimetry of the bilayer to hexagonal phase transitions of diacylphosphatidylethanolamines

The bilayer to hexagonal phase transition of dioleoylphosphatidylethanolamine has been detected for the first time by differential scanning calorimetry. The observed transition is dependent on scan rate. This dependence can be explained by assuming that at rapid scan rates, the rate of conversion of bilayer to hexagonal phase is too slow at low temperatures for equilibration to take place. At higher temperatures the rate of interconversion becomes more rapid. The transition is observed to occur at 14°C using a scan rate of 0.74 K/min while it is centered at 8°C using a scan rate of 0.19 K/min. The enthalpy of the transition is 290 ± 40 cal/mol lipid and the transition is characterized by a ΔCp of −9 ± 1 mcal K⁻¹ (g lipid)⁻¹. The bilayer to hexagonal phase transition of dielaidoylphosphatidylethanolamine and of 1-palmitoy1-2-oleoylphosphatidylethanolamine occurs at 65.6°C and 71.4°C, respecitvely, with a corresponding transition enthalpy of 450 ± 20 and 400 ± 30 cal/mol lipid. The transitions of these phosphatidylethanolamines, occuring at higher temperatures, are independent of scan rate and show a higher degree of cooperativity than that of dioleoylphosphatidylethanolamine. Compared with the gel to liquid-crystalline transition of bilayer phospholipids the transition to hexagonal phase has a much lower enthalpy.     

Zur fortpflanzungsbiologie von mesostoma ehrenbergii (Focke, 1836) (Turbellaria)

1. At temperature levels from 10 to 25°C animals from resting eggs produce subitaneous eggs independent on temperature. In contrast animals from subitaneous eggs produce subitaneous eggs dependent on temperature. At a high rate subitaneous eggs are only formed at temperature levels above 20°C.
2. Below 10°C no development occurs in the juveniles. At temperatures of 30/22°C (24.7°C) the first subitaneous eggs are formed after 6–9 days, at 14/9°C (10.7°C) they are formed after 34 days. At different temperature levels the developmental rate of the young is from 10.5 to 42 days. One generation extends over 16.5 (30/22°C) to 75 days (14/9°C). The average egg production is 10–20 subitaneous eggs or 30–60 resting eggs. The maximum egg production of one individual is 50 subitaneous eggs or 84 resting eggs. 50% of the animals have just formed resting eggs, before the juveniles are hatched. Resting eggs in the first egg-batch are formed 6–20 days later than subitaneous eggs. The duration of life is between 65 (30/22°C) and 140 days (19/13°C).
3. Young worms in resting eggs have a dormance period of at least 15–30 days.

At room temperatures (20°C) no juvenile in resting eggs hatches from water. By combining room and refrigerator (3.5°C) temperatures the hatching rate increases to a maximum of 85%. To reach a hatching rate of 50–65% the influence of low temperatures must be at least 30 days. At room temperatures 60% of the young in resting eggs hatch from mud covered with water. Combining high and low temperatures the hatching success is between 67 and 81%, where the highest percentage of the young may hatch at room temperature. Up to 90 days low temperatures cause a maximum hatching rate of 79%. It decreases to approximately 30% after 180 days. At high temperatures resting eggs preserved in 100% moist mud, survive for two months. By adding a period of low temperatures the hatching rate increases to a maximum of 52%. Low temperatures are survived for more than 6 months. Up to 30 days preservation at 3.5°C causes a maximum hatching rate of 61%, up to 12o days it decreases to 30%. At room temperature the young in resting eggs are not resistant against air-dried mud (30–40% rel. air moisture). Combining high and low temperatures air-dried mud is endured 1 month (hatching rate 5–14%). Preservation of 30–120 days at 3.5°C and 70% rel. air moisture result in a hatching rate of 43–61%. li]4. In the open air in Middle-Europe there occur 5–6 generations of M. ehrenbergii per life-cycle. The first generation hatches from resting eggs in May, where the production of subitaneous eggs is independent on temperature. All other generations up to October hatch from subitaneous eggs. The egg-production of those worms is dependent on environmental factors. In summer subitaneous egg production prevails, in autumn resting egg production. The abundance during the life-cycle is dependent on the number of animals which produce subitaneous eggs. Resting eggs are predestinated to endure periods of dryness and cold. The life-cycles of the species M. lingua and M. productum are different from those of M. ehrenbergii in length and in the number of generations. In both species 7 generations occur over 8 to 8.5 respectively 5.5 months. M. nigrirostrum only forms resting eggs. The life-cycle consists of one generation from February/March to May/June.     

The contuation of the phase-boundary between ice I and liquid into the region of ice III and II and its relation to freeze-pressing of biological material.

The trajectory of the phase-boundary between ice I and liquid has been continuously followed by compression of deionized water, 0.10 m KCl, 0.10 m NaCl, and deionized water with suspended yeast cells (Saccharomyces cerevisiae, 180 mg/g) in a close-ended pressure chamber at temperatures below 0 °. Upon increasing pressure on deionized H₂O at ?8.6 °C the temperature first increases, until the transition line between ice I and liquid is reached. Then the sample cools on further compression, which is concomitant with an increase in electrical conductivity, indicating the gradual formation of liquid. At ?34.8 °C the pressure drops spontaneously from 3 × 10⁸ to 2.4 × 10⁸ Pa, the conductivity decreases, and the volume of the samples becomes further reduced to ?3.1 cm³/mole of H₂O, making the formation of ice III probable. On increase of pressure on 0.10 m KCl and 0.10 m NaCl the sample is gradually cooled, as the fusion line of the respective eutectic solid is reached. 0.10 m KCl is then super-cooled into the region of ice III and II, whereas 0.10 m NaCl is desalinated with a final conductivity of the suspension of 3–10 nmho/cm. In the sample with S. cerevisiae 180 mg/g the ice I-liquid phase-boundary was followed to ?36.0 °C into the region and ice III and II.These results are of great importance to the understanding of the freeze-pressing process, since they indicate that a transition from ice I to liquid may occur even at temperatures between ?22 °C and ?35 °C, thus facilitating flow of material through the press. This way they shed light on the pressures needed to initiate flow at different temperatures and compositions of the sample to be freeze-pressed.