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.     

Influence of cell concentration,temperature, and press performance on flow characteristics and disintegration in the freeze-pressing of Saccharomyces cerevisiae with the X-press

The pressure required for initiation of flow when freeze-pressing with the X-press is related to the phase boundaries of water, particularly those between ice I and liquid even at temperatures around ?25°C and lower. Widening the orifice of the pressure chamber to diameters larger than 2.5 mm leads to lower pressures and less extensive cell disintegration. Pressing Saccharomyces cerevisiae slowly with the aid of a manual hydraulic jack at ?25°C produces a disintegration of 60–75% irrespective of cell concentration. Pressing at ?35°C shows no clear differences. Pressing more rapidly with the aid of a motor-driven hydraulic press produces a similar extent of disruption of diluted cell suspensions (5.4 mg/g) as slow pressing. However, freeze-pressing a paste of baker's yeast (270 mg/g) increases the degree of disintegration. Under these conditions the disintegration is further enhanced by a lower temperature, ?35°C, and by a high velocity of flow through the orifice, such that more than 95% of the S. cerevisiae is disrupted by one pressing at less than 2 × 10⁸ Pa. Mechanisms for flow through the X-press are suggested and discussed in relation to the phase diagram of water.     

Effects of temperature on the growth and microstructure formation of cuttlebone from cuttlefish Sepia pharaonis

ABSTRACT

This study investigated the effects of three temperatures (20°C, 27°C, and 31°C) on the physiological performance (survival and growth) and cuttlebone micromorphological features (chamber number, chamber height and lamellae number) of cuttlefish Sepia pharaonis. We examined gross morphological characteristics of the internal calcareous cuttlebone to determine whether lamellar matrix was impacted by temperature. Juvenile survival was significantly affected by temperature (χ²?=?54.580, df?=?2, P?<?0.001). Cuttlebone weight, length and width were also positively correlated with temperature. Scanning electron microscopy revealed that a single chamber structure consists of septa, lamellae and pillars. At 20°C the chamber number of 89.0?±?10.8 was significantly higher than at 27°C with 44.8?±?3.6 or 31°C with 47.5?±?4.3 (Kruskal–Wallis [KW], χ^2?=?26.391, df?=?2, P?=?0.0001), whilst chamber height was lower at 20°C (KW χ^2?=?27.842, df?=?2, P?=?0.0001). Moreover, lamellae number varied among the treatments (KW χ^2?=?22.411, df?=?2, P?=?0.0002). Lamellae numbers at 20°C, 27°C and 31°C were 3–6, 6–9 and 5–8, respectively. The results indicated that the intrinsic lamellar matrix structure was significantly affected by temperature and that this effect may be used in cuttlebone growth studies.     

The state of water on hydrated collagen as studied by pulsed NMR

The spin-lattice relaxation time (T₁) of water adsorbed on collagen fibers was determined at six frequencies and temperatures varying from 25° to ?80°C. Care was taken to eliminate the contributions to the signal of protons other than those in the adsorbed water. Quantitative calculations were made on T₁ and the results were compared with the experimental data. It is suggested that a maximum of about 0.50–0.55 g water per g collagen forms a hydration layer, which cannot be frozen down to ?90°C and exhibits a distribution of motional correlation times. For collagen samples containing a larger quantity of adsorbed water, the additional water molecules behave like ordinary isotropic water, having a single correlation time and a freezing temperature of about ?10°C.     

Formulation Screening and Freeze-Drying Process Optimization of Ginkgolide B Lyophilized Powder for Injection

The purpose of this study was to prepare ginkgolide B (GB) lyophilized powder for injection with excellent appearance and stable quality through a formulation screening and by optimizing the freeze-drying process. Cremophor EL as a solubilizer, PEG 400 as a latent solvent, and mannitol as an excipient were mixed to increase the solubility of GB in water to more than 18 times (about from 2.5 × 10^?4 mol/L (0.106 mg/mL) to 1.914 mg/mL). Formulation screening was conducted by orthogonal design where the content of GB in the solution before lyophilization (using external standard method of HPLC) and reconstitution time after lyophilization were the two evaluation indexes. The optimized formulations were GB in an amount of 2 mg/mL, Cremophor EL in an amount of 16% (v/v), PEG 400 in an amount of 9% (v/v), mannitol in an amount of 8% (w/v), and the solution pH of 6.5. Through four single-factor experiments (GB adding order, preparation temperature of GB solution, adding amount, and adsorption time of activated carbon), the preparation process of GB solution was confirmed. The glass transition temperature of maximally GB freeze-concentrated solution was ? 17.6°C through the electric resistance method. GB lyophilized powder began to collapse at ? 14.0°C, and the fully collapsed temperature was ? 13.0°C, which were determined by freeze-drying microscope. When the collapse temperature was determined, the primary drying temperature was obtained. Thereby, the freeze-drying curve of GB lyophilized powder was initially identified. The freeze-drying process was optimized by orthogonal design, the qualified product appearance and residual moisture content were the two evaluation indexes. The optimized process parameters and process were (1) shelf temperature, decreased from room temperature to ? 45.0°C, at 0.5°C/min in 2 h; (2) shelf temperature increased from ? 45.0 to ? 25.0°C, at 0.1°C/min, maintained for 3 h, and the chamber pressure was held at 10 Pa; (3) shelf temperature was increased from ? 25.0 to ? 15.0°C at 0.1 °C/min, maintained for 4 h, and the chamber pressure was held at 10 Pa; and (4) shelf temperature was increased from ? 15.0 to 20.0°C at 1.0 °C/min, maintained for 4 h, and the chamber pressure was raised up to 80 Pa. In these lyophilization process conditions, the products complied with relevant provisions of the lyophilized powders for injection. Meanwhile, the reproducibility was satisfactory. Post-freezing annealing had no significantly beneficial effects on shortening the freeze-drying cycle and improving the quality of GB lyophilized powder.     

Thermodynamics and rheology of the Hughes press

Among the methods used for the rupture of cells by means of solid shear, the Hughes press is best known. It operates by forcing an aqueous suspension of cells previously frozen at ?27°C under pressure through a narrow circumferential gap. The mode of action requires a knowledge of the isentropic compression of the solid phase and its possible involvement with a phase change from ice I to ice III. It is shown that the mechanical properties of the solid mixture are relevant. A model is proposed of the plastic flow for the geometry of the press and it is shown that cell breakage is proportional to the velocity of the plunger and inversely proportional to gap width.     

Adsorption of molecular oxygen by polymer covalently bonded co(II) porhyrin complex in toluene

Reaction betwenn molecular oxygen and polystyrene covalently bonded Co(II) protoporphyrin IX complex, which was prepaired by the incorporation of a cobaltous ion into the metal-free porphyrin polymer, was studied in the presence of N-ethylimidazole by measuring visible absorption and electron spin resonance spectra. It was found that the complex forms a monomeric oxygen adduct reversibly at low temperature dependent on oxygen pressure. In the presence of molecular oxygen, a new electron spin resonance signal due to the oxygen complex at g_iso=2.02 shows no superhyperfine splitting structure in fluid toluene solution even at ?80 °C, but it was observed in frozen toulene glass solution at ?120°C, The oxygen adducts of the complexes between C0(II) protoporphyrin IX dimethyl ester and N-ethylimidazole and copoly(styrene-N-vinylimidazole) showed eight resolved superhyperfine splitting at ?40 and ?60°C, respectively. The polymer covalently bonded Co(II) complex with N-ethylimidazole was oxidized at room temperature under oxygen atmosphere. It was suggested that a Co(II) porphyrin–oxygen adduct with an axial ligand may be oxidised monomolecularly at high temperature.     

Fertilizability of cryopreserved zona-free hamster ova

Mature unfertilized ova from superovulated hamsters were freed from all investments and frozen at ?50°C. They were cooled at about 1°C/min to 0°C then at 0.8° to 0.6°C/min to ?50°C. At 0°C, dimethyl sulfoxide was added to a final concentration of 1.25 M. The ova were stored at ?50°C for up to four months. Thawing was performed at 2–4°C/min and followed by several washes with insemination medium. Approximately 90% of the ova were normal in appearance after thawing. The frozen and thawed ova with normal appearance could be penetrated by hamster or human spermatozoa at a rate comparable to unfrozen controls. The ability of hamster ova to tolerate storage at a relatively convenient temperature (?50°C) for long periods (tested for up to four months) makes possible their shipment at low cost to institutions lacking this resource. There they can be used for basic biological studies of sperm–egg interaction or in the clinical assessment of human sperm quality.     

Critical temperature for skin necrosis in experimental cryosurgery

To investigate the minimal lethal freezing temperature required to produce skin necrosis in dogs, multiple skin sites were frozen with cryosurgical equipment. Tissue temperatures were recorded from thermocouple sites placed at diverse distances, usually 5 mm from the edge of the freezing probe. In single freezing cycles of about 3 min duration, tissue temperatures in the range of 0 to ?60 °C were produced. Punch biopsies of the skin at the thermocouple sites 3 days after freezing injury provided tissues for estimation of viability by histologic examination.The histologic findings permitted classification of the biopsy tissue into three groups, that is, viable, borderline, or necrotic. When classified as borderline, the division between the necrotic and viable tissue was evident on the histologic slide. The viable specimens were scattered through the 0 to ?35 °C range. All specimens frozen to ?10 °C or warmer were viable. In biopsies classified as borderline, the range of viability extended from ?11 ° to ?50 °C. The necrotic biopsies covered a range of ?14 ° to ?50 °C. Cell death was certain at temperatures colder than ?50 °C. The data showed cryosurgical freezing conditions produced a range of temperatures in which viability or death of tissue may occur and that the ranges of viability and necrosis overlapped to a great extent.The wide range of temperatures at which cells were viable shows the need to achieve tissue temperatures in the range of ?50 °C in the cryosurgical treatment of cancer.     

Quorum sensing: an emerging link between temperature and membrane biofouling in membrane bioreactors

Lab-scale membrane bioreactors (MBRs) were investigated at 12, 18, and 25?°C to identify the correlation between quorum sensing (QS) and biofouling at different temperatures. The lower the reactor temperature, the more severe the membrane biofouling measured in terms of the transmembrane pressure (TMP) during filtration. More extracellular polymeric substances (EPSs) that cause biofouling were produced at 18?°C than at 25?°C, particularly polysaccharides, closely associated with QS via the production of N-acyl homoserine lactone (AHL). However, at 12?°C, AHL production decreased, but the release of EPSs due to deflocculation increased the soluble EPS concentration. To confirm the temperature effect related to QS, bacteria producing AHL were isolated from MBR sludge and identified as Aeromonas sp., Leclercia sp., and Enterobacter sp. through a 16S rDNA sequencing analysis. Batch assays at 18 and 25?°C showed that there was a positive correlation between QS through AHL and biofilm formation in that temperature range.     

Denaturation of Soybean Protein by Freezing

When a solution of soybean acid-precipitated or 11S protein was frozen and stored at ?1 to ?5°C, the protein became partially insoluble after thawing. Ultracentrifugation and disc-electrophoresis of freeze-stored 11S protein solution after removing insoluble components revealed that new components which may be aggregates or associates of the 11S component were formed. When concentrated and stored at 5°C, disc-electrophoresis of 11S component showed that associates were formed. Mercaptoethanol could dissolve the insoluble protein and also convert the associates to the original 11S component. NEM–11S was not insolubilized by frozen storage at ?5°C or storage at 5°C after being concentrated. From these facts it can be concluded that denaturation of soybean protein by freezing may be caused by intermolecular reactions through S-S bonds as a result of concentration by freezing. This may suggest a mechanism of the formation of sponge-like texture in kori-tofu which is made by frozen storage of soybean curd for 15 to 20 days at ?1 to ?3°C.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of γ-glutamyltranspeptidase in the liver. On exposure to ?2·5°C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4°C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at ?2·5°C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at ?2·5°C on cooling; a 10·5-fold increase after 12 h at ?2·5°C on the initiation of freezing; and a 22-fold increase after 24 h at ?2·5°C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1·8-fold in the thawed state, while plasma ketone levels were increased 1·8-fold in the frozen state and 1·5-fold in the thawed state. Plasma total T₃ levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of γ-glutamyltranspeptidase was found to be elevated at all stages of the freeze–thaw process. After 1, 12 and 24 h at ?2·5°C, activities were increased 2·5-, 2·3-, 2·4-fold respectively in the homogenates and 2·5-, 2·2-, 2·4-fold respectively in the plasma membranes. After thawing, activities were still increased 1·9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of γ-glutamyltranspeptidase was increased 1·4-fold after 1 h at ?2·5°C after which it returned to normal. The role of thyroid hormone in producing the increase in γ-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.     

野生鸡枞菌种长期保存方法比较

野生鸡枞菌种质资源的有效保存是对野生鸡枞加以保护和利用的前提。以自行分离的5个野生鸡枞菌株作为研究对象,采用蒸馏水保藏法和-80°C冻结保藏法对野生鸡枞菌种长期保存的方法进行了实验研究,蒸馏水法分别保存于室温和4°C,-80°C冻结保藏同时采用程控降温法和泡沫盒降温法,保存20个月后对4种不同方法保存的5个菌株的保存效果进行比较。实验结果表明:蒸馏水室温保存法菌种存活率为100%,萌发期较短,为4-10 d,是一种简便、实用、有效而成本低廉的长期保存方法;-80°C冻结保藏法的存活率为56%-76%,萌发期7-16 d,泡沫盒降温法可以很好地控制降温速度,是一种简便有效的控温方法。     

Experiments with an apparatus for lyophile desiccation of micro-organisms

Summary Lyophyle desiccation is an extremely valuable method of preserving micro-organisms. The apparatus described above proves satisfactory. The material should be frozen as rapidly as possible at a temperature of −35°C. or lower, and the apparatus should be constructed in such a way that the pressure remains approximatively constant. During the sealing of the ampoules care should be taken that no dry material passes from the latter to the apparatus. A time limit for the desiccation process is hard to fix. When water has condensed on the outside surface of the ampoules, the apparatus is left untouched for about an hour. This brings the total duration of the desiccation as a rule up to 1 1/2 to 2 hours. A volume of 0.25 ml skimmed milk suffices for the preservation of a strain. It is advisable to start from a fresh culture that has been grown on a solid medium and to suspend the required amount in skimmed milk at a pH of 7.4–7.8. The suspension should directly be frozen in a thin layer. Bofore the frozen ampoules are submitted to the desiccation process, they may be kept for some time at a temperature of −35°C. For this as well as for many valuable indications we are greatly indebted to the Staff of the Kamerlingh Onnes Laboratory.     

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.     

Visualization of intracellular ice crystals formed in very rapidly frozen cells at −27 °C

Tumor cells of an ascites sarcoma of rat were primarily frozen very rapidly with the original host ascitic fluid at ?27 °C by the spraying method. Frozen specimens were fractured and replicated at about ?100 °C under vacuum by a special spray-sandwich method for freeze-etching, and the morphological appearance of ice crystals formed in and around the frozen cells were observed by electron microscopy.The cells cooled very rapidly at ?27 °C actually froze intracellularly, and intracellular ice crystals ranged from 0.03 to 0.5 μm in grain size due to the initial freezing rate of the specimens. In the cells having granulous intracellular ice crystals less than 0.05 μm in grain size, cytoplasmic organelles seemed to maintain their original structures.We suggested in our previous report that these tumor cells, frozen very rapidly at temperatures above ?30 °C, survived intracellular freezing as long as they remained translucent, and optically no ice crystals appeared within them, as seen in intact unfrozen cells. It may therefore be concluded that the tumor cells frozen very rapidly at temperatures near ?30 °C actually freeze intracellularly and probably maintain their viability as long as the size of individual intracellular ice-crystals is kept smaller than 0.05 μm, although the exact critical size of innocuous intracellular ice crystals is uncertain.     

Preservation of Phakopsora pachyrhizi Uredospores

This study compared different temperatures and dormancy‐reversion procedures for preservation of Phakopsora pachyrhizi uredospores. The storage temperatures tested were room temperature, 5°C, ?20°C and ?80°C. Dehydrated and non‐dehydrated uredospores were used, and evaluations for germination (%) and infectivity (no. of lesions/cm²) were made with fresh harvested spores and after 15, 29, 76, 154 and 231 days of storage. The dormancy‐reversion procedures evaluated were thermal shock (40°C/5 min) followed or not by hydration (moist chamber/24 h). Uredospores stored at room temperature were viable only up to a month of storage, regardless of their hydration condition. Survival of uredospores increased with storage at lower temperatures. Dehydration of uredospores prior to storage increased their viability, mainly for uredospores stored at 5°C, ?20°C and ?80°C. At 5°C and ?20°C, dehydrated uredospores showed increases in viability of at least 47 and 127 days, respectively, compared to non‐dehydrated spores. Uredospore germination and infectivity after storage for 231 days (7.7 months), could only be observed at ?80°C, for both hydration conditions. At this storage temperature, dehydrated and non‐dehydrated uredospores exhibited 56 and 28% of germination at the end of the experiment, respectively. Storage at ?80°C also maintained uredospore infectivity, based upon levels of infection frequency, for both hydration conditions. Among the dormancy‐reversion treatments applied to spores stored at ?80°C, those involving hydration allowed recoveries of 85 to 92% of the initial germination.     

Pressure-temperature stability of DNA in neutral salt solutions

The effect of pressure on the melting temperature of DNA (Cl. perfringens) has been examined in several concentrations of neutral salt by measurement of uv absorbance. The results indicate that the apparent transition volume increases as the salt concentration, and hence the melting temperature, is raised, and suggest that the transition occurs without a change in volume at a T_m of 59°C. Additional experiments were conducted in an effort to determine whether transition behaviour can be found in other regions of pressure and temperature; no additional transition behaviour was observed in experiments conducted with temperatures as low as ?21.85°C at 2000 atm and pressures in excess of 9000 atm at 58°C.     

Cryopreservation of monogonont rotifers

Development of techniques to maintain viable rotifer clones in a frozen state would preserve the genotype and reduce routine maintenance for those clones not being actively studied. To this end we have frozen Brachionus plicatilis in dimethyl sulfoxide at concentrations ranging from 6% to 18%. Survival rates decreased as the endpoint temperature was reduced from ?20 °C to ?45 °C, but did not decrease when the temperature was further reduced to ?196 °C (liquid nitrogen). Only 2% of the individuals survived freezing in liquid nitrogen.     

Supercooling ability and cold hardiness of the pollen beetle Meligethes aeneus

Supercooling point (SCP) and cold‐hardiness of the pollen beetle Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae) were investigated. Mature eggs from the oviduct were supercooled on average to ?28.0 °C and from oilseed rape buds to ?24.4 °C; first instars were supercooled to ?21.0 °C and second instars to ?16.8 °C. Despite their high supercooling ability, none of the eggs survived 24 h exposure to ?2.5 °C. The supercooling ability of adults varied significantly among feeding and non‐feeding beetles: high SCPs prevailed during the whole warm period, being about ?12 °C; low values of SCP of ?20 °C dominated in non‐feeding beetles. In spring and autumn, beetles displayed the same acclimation efficiency: after 1 week of exposure at 2.0 °C with no access to food their SCPs were depressed equally by about 3 °C. Meligethes aeneus beetles have a different response to low temperatures depending on the season. The lowest tolerance was found in reproductively active beetles after emergence from overwintering sites; the time needed to kill 50% of individuals (Ltime₅₀) was 56.2 h at ?7 °C and the lower lethal temperature needed to kill 50% (Ltemp₅₀) after 24 h exposure was ?8.6 °C. Cold hardiness increased from midsummer to midwinter; Ltime₅₀ was 80 h in August, 182.8 h in September, and 418.1 h in January. Lethal temperature after 24 h exposure was ?9.1 °C in August and ?9.8 °C in September. In February, after diapause, the beetles started to loose their cold tolerance, and Ltemp₅₀ was slightly increased to ?9.5 °C. Hibernating beetles tolerated long exposure at ?7 °C well, but mortality was high after short exposure if the temperature dropped below ?9 °C for 24 h. Despite the season, the beetles died at temperatures well above their mean SCP; consequently, SCP is not a suitable index for cold hardiness of M. aeneus.