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PubMed2001

Two freezing cycles ensure interface sterilization by cryosurgery during bone tumor resection.

Cryosurgery utilizing an argon-based system allows bone-tumor interface sterilization, while avoiding the risks of conventional cryosurgery. This study was conducted in order to evaluate the number of freezing cycles required for interface sterilization in cases of aggressive human bone tumors. Sixteen tumors were included (six chondrosarcomas, eight metastatic carcinomas, and two giant cell tumors). All occurred within long bones. In all cases a standardized marginal resection was performed. Following thorough curettage, we sampled five different locations within the tumor interface by a cylindrical hollow trephine. The interface viability was assessed using the XTT method. Quantitative histological evaluation was based on the percentage of live cells divided by total lacunae number in five random medium-power fields. One freezing cycle (5 min, -40 degrees C) reduced tumor viability to approximately 5% of prefreezing. However, there were still live specimens. Two or three freezing cycles led to complete interface sterilization. The difference between a single freezing cycle and two freezing cycles was significant (ANOVA, F = 130, P < 0.01). The difference between two freezing cycles and three freezing cycles was not significant (ANOVA, F = 0.14, P < 0.6). The results of the XTT method for the assessment of interface viability correlated well with histological evaluation of the percentage of viable cells (r = 0.89), as well as with cell culture results of frozen vs. prefreezing tumor samples. In conclusion, two freezing cycles are adequate to achieve tumor-bone interface sterilization in aggressive human bone tumors.

Adult↗
PubMed1988

ICV-CRH alters stress-induced freezing behavior without affecting pain sensitivity.

Freezing is an adaptive response often induced by stressful, fear-eliciting stimuli. Three experiments with rats investigated the effects of intracerebroventricular (ICV) administration of corticotropin-releasing hormone (CRH) on freezing behavior and pain sensitivity. Experiments 1 and 3 demonstrated that ICV-CRH (300 ng) enhanced shock-elicited freezing. In Experiment 1, ICV-CRH also enhanced recovery from shock-elicited freezing, suggesting that the peptide has a biphasic effect. Experiments 2 and 3 established that CRH-induced freezing was not caused by heightened pain sensitivity. Interestingly, in Experiment 2, hot-plate exposure produced increased freezing that was attenuated by ICV-CRH. Thus, the direction of the ICV-CRH effect on freezing was found to depend on the nature of the stressor. These results suggest that endogenous CRH systems modulate stress-induced freezing.

Animals↗
PubMed2005

Mammalian freeze-dried sperm can maintain their calcium oscillation-inducing ability when microinjected into mouse eggs.

Mammalian freeze-dried sperm can maintain their genetic integrity and event support full development to term when microinjected into mature oocytes. However, it is unknown whether freeze-dried sperm can still maintain their calcium oscillation-inducing capability. Here, we microinjected mouse and bovine freeze-dried sperm into mouse MII oocytes and examined their calcium oscillation-inducing ability following intracytoplasmic sperm injection (ICSI). Two pieces of information are revealed. First, nearly all oocytes injected with a freeze-dried mouse sperm head or a bovine sperm showed fertilization-like calcium oscillations, indicating that freeze-drying treatment does not affect the activity of the sperm factor responsible for calcium oscillations. Second, freeze-dried sperm exhibited high resistance to external temperature increase. This is shown by the finding that the freeze-dried sperm can maintain their calcium oscillation-inducing capacity even following exposure to 100 degrees C for 3 h. We therefore conclude that mammalian sperm can maintain their calcium oscillation-inducing capability following freeze-drying, rehydration, and ICSI treatments.

Animals↗
PubMed2004

High ice nucleation temperature of zebrafish embryos: slow-freezing is not an option.

Although fish embryos have been used in a number of slow-freezing cryopreservation experiments, they have never been successfully cryopreserved. In part this is because little is known about whether ice forms within the embryo during the slow-freezing dehydration process. Therefore, we examined the temperature of intraembryonic ice formation (T(IIF)) and the temperature of extraembryonic ice formation (T(EIF)), using a cryomicroscope. We used both unmodified zebrafish embryos and those with water channels (aquaporin-3 or AQP3) inserted into their membranes to increase permeability to water and cryoprotectants, examined at 100% epiboly to the 6-somite stage. In these experiments we examined: (1) the spontaneous freezing of (external) solutions; (2) the spontaneous freezing of solutions containing embryos; (3) the effect of preloading the embryos with cryoprotectants on T(IIF); (4) whether preloading the embryos with cryoprotectant helps in survival after nucleating events in the solution; and (5) the damaging effects of extracellular nucleation events versus solution toxicity on the embryos. The solutes alone (embryo medium--EM, sucrose culture medium, 1 M propylene glycol in EM, and 1 M propylene glycol in a sucrose culture medium) froze at -14.9 +/- 1.1, -17.0 +/- 0.3, -17.8 +/- 1.0, and -17.7 +/- 1.4, respectively. There was no difference amongst these means (P > 0.05), thus adding cryoprotectant did not significantly lower the nucleation point. Adding embryos (preloaded with cryoprotectant or not) did not change the basic freezing characteristics of these solutes. In all these experiments, (T(EIF)) equaled (T(IIF)), and there was no difference in the freezing point of the solutions with or without the embryos (P > 0.05). Additionally, there was no difference in the freezing characteristics of embryos with and without aquaporins (P > 0.05). The formation of intraembryonic ice was lethal to the zebrafish embryos in all cases. But this lethal outcome was not related to solution injury effects, because 88-98% of embryos survived when exposed to a higher solute concentration with no ice present. Taken together, these data suggest that slow-freezing is not a suitable option for zebrafish embryos. The mechanism of this high temperature nucleation event in zebrafish embryos is still unknown.

Animals↗
PubMed1993

The use of a liquid-phase controlled rate freezing system for the successful cryopreservation of mouse pre-embryos.

OBJECTIVES: To investigate the ability of a liquid-phase, controlled-rate freezing machine to generate reproducible freezing gradients with a constant rate of change; temperature fluctuations and heat dissipation during seeding; to assess the viability of mouse pre-embryos exposed to the silicone liquid cooling phase and the rates of survival and viability of mouse pre-embryos cryopreserved using this system. DESIGN: Freezing gradients were generated from the bath or a sample and compared for reproducibility and slope. Temperature fluctuations and gradients around the freezing chamber and the temperature rises and dissipation during seeding were measured. The toxicity of the silicone polymer freezing-phase was tested with mouse pronuclear pre-embryos. Different developmental stages of mouse pre-embryos were frozen and thawed and survival recorded in vitro and in vivo. SETTING: Research Laboratories, Sinai Hospital of Baltimore, Baltimore, Maryland. MAIN OUTCOME MEASURES: The reproducibility of freezing gradients; temperature fluctuations during seeding; and the in vitro and in vivo viability of mouse pre-embryos exposed to the silicone polymer or frozen and thawed. RESULTS: The freezing gradients generated were constant and reproducible with a constant rate of change; no temperature differences were recorded around the freezing chamber; temperature changes at seeding are minimal and rapidly dispersed. The silicone polymer was nontoxic to mouse pre-embryos and mouse pre-embryos frozen with the system and subsequently thawed are viable both in vitro and in vivo. CONCLUSIONS: This liquid-phase cryopreservation system is an attractive option for assisted reproductive technologies laboratories because liquid nitrogen is not required for operation, it is reliable, there are small temperature fluctuations during seeding, and it can be kept constantly running.

Animals↗
PubMed2001

The effects of water deprivation on conditioned freezing to contextual cues and to a tone in rats.

In two experiments we used an automated system for quantifying freezing responses in rats to replicate and extend Maren et al. (Maren S, DeCola JP, Fanselow MS. Water deprivation enhances fear conditioning to contextual, but not discrete, conditional stimuli in rats. Behav Neurosci 1994;108:645-9; Maren S, DeCola JP, Swain RA, Fanselow MS, Thompson RF. Parallel augmentation of hippocampal long-term potentiation, theta rhythm and contextual fear conditioning in water deprived rats. Behav Neurosci 1994;108:44-57) who found that water deprivation in rats produced a selective enhancement in conditioning to context, as opposed to conditioning to a tone. In experiment 1 we gave water deprived and non-deprived rats either three or ten pairings of a tone and foot shock. During conditioning water deprivation decreased overall freezing only in rats that received ten pairings. On 2 subsequent days we assessed conditioned freezing (1) to the contextual cues of the conditioning chamber and (2) to the tone when presented in a distinctive, novel environment. We found, in direct contrast to Maren et al. (Maren S, DeCola JP, Fanselow MS. Water deprivation enhances fear conditioning to contextual, but not discrete, conditional stimuli in rats. Behav Neurosci 1994;108:645-9), that (a) water deprived rats did not differ from non-deprived rats in levels of conditioned contextual freezing and that (b) water deprived rats did show reduced levels of freezing to the tone stimulus. In the same experiment we found that the number of tone-shock pairings did not affect levels of conditioned contextual freezing but that rats that had received three pairings did show reduced levels of freezing to the tone stimulus compared with rats that had received ten pairings, thereby demonstrating that the behavioural procedure and analysis system that we used was appropriately sensitive to differences in conditioning. In experiment 2, therefore, we sought to replicate Maren et al. (Maren S, DeCola JP, Fanselow MS. Water deprivation enhances fear conditioning to contextual, but not discrete, conditional stimuli in rats. Behav Neurosci 1994;108:645-9) using, as far as possible, exactly the same procedural parameters. Here we found that water deprivation produced no effects on conditioned freezing to the contextual cues or to the tone. We conclude that there is sufficient reason to doubt the generality of the previously reported findings.

Acoustic Stimulation↗
PubMedDate not supplied

Successful pregnancies in cows following double freezing of a large volume of semen.

The objective of the following paper is to describe a new technology for large volume and double freezing of semen in 12 mL test tubes. Semen from two different bulls was frozen with a new technique using 12 mL test tubes and was refrozen after thawing in mini straws. All freezing was done in a "Multi thermal gradient" (MTG) freezing apparatus, which moves the container at a constant velocity (V) through a thermal gradient (G) producing a controlled cooling rate B = (G) x (V). Each of the two bulls ejaculated were evaluated for post thaw motility in the lab and then in a field trial which was carried out in a split sample mode. We inseminated 105 cows after a double freezing/thawing cycle, and another 123 cows were inseminated with semen frozen in mini-straws and a conventional method. The results showed a 75 +/- 5% post thaw motility after freezing a 12 mL test tube and 50 +/- 5% after a second freezing/thawing in mini-straws, respectively. Controlled vapour freezing showed a 60 +/- 10% post thaw motility. The results of the field trial showed a pregnancy rate of 44% (47/105) for the double freezing group in comparison to 45.5% (56/123) for the controlled group. These results can be beneficial for large volume freezing, and therefore for bull semen cryobanking in a large volume which will be followed by second freezing in a regular insemination volume.

Animals↗
PubMed2003

Ultra-rapid freezing of very low numbers of sperm using cryoloops.

BACKGROUND: With the availability of ICSI, men with severe oligozoospermia (<5x10(6)/ml) are able to reproduce. Current methods for cryopreservation of severe oligozoospermic samples are labour intensive and costly. The objective of this study was to evaluate whether freezing small numbers of motile sperm (approximately 100) was feasible using cryoloops. METHODS: Initial tests assessed the effect of various dilutions of cryoprotectants on pre-freezing sperm motility. Several solutions were further evaluated for their ability to cryoprotect sperm during ultra-rapid freezing. Sperm were placed on cryoloops and held in liquid nitrogen vapour for 5 min prior to freezing (ultra-rapid freezing) or directly submerged into liquid nitrogen. Using the optimal cryoprotectant and technique from these experiments, ultra-rapid and standard slow-rate freezing protocols were compared. RESULTS: Optimal sperm survival was seen when sperm in cryoloops were placed in liquid nitrogen vapour in test yolk buffer with 12% v/v glycerol versus other cryoprotectants. Using this cryoprotectant, post-thaw sperm motility is comparable between ultra-rapid and slow-rate freezing methods. CONCLUSION: Ultra-rapid freezing of very low numbers of sperm is feasible using cryoloops suspended in liquid nitrogen vapour for 5 min.

Cell Survival↗
PubMed1967

Freezing of xylem sap without cavitation.

Freezing of stem sections and entire twigs of hemlock (Tsuga canadensis) has been demonstrated to occur without increasing the resistance to the movement of water through the frozen part after rewarming. This was interpreted to mean that freezing did not produce cavitation in the xylem sap even though A) the sap was unquestionably frozen; B) it contained dissolved gases; and C) it was under tension before freezing and after. Freezing stem sections of some other evergreen gymnosperms during the summer again produced no evidence for cavitation of the xylem sap. On the other hand, freezing stem sections of some angiosperms invariably increased the resistance to sap flow leading to wilting and death in a few hours when the sap tension was at normal daytime values at the time of freezing. These results were interpreted to mean that the bordered pits on the tracheids of gymnosperms function to isolate the freezing sap in each tracheid so that the expansion of water upon freezing not only eliminates any existing tension but also develops positive pressure in the sap. Dissolved gases frozen out of solution may then be redissolved under this positive pressure as melting occurs. As the bubbles are reduced in size by this ice pressure developed in an isolated tracheid, further pressure is applied by the surface tension of the water against air. If the bubbles are redissolved or are reduced to sufficient small size by the time the tension returns to the sap as the last ice crystals melt, then the internal pressure from surface tension in any existing small bubbles may exceed the hydrostatic tension of the melted sap and the bubbles cannot expand and will continue to dissolve.

Journal Article↗
PubMed1983

Maple sap uptake, exudation, and pressure changes correlated with freezing exotherms and thawing endotherms.

Sap flow rates and sap pressure changes were measured in dormant sugar maple trees (Acer saccharum Marsh.). In the forest, sap flow rates and pressure changes were measured from tap holes drilled into tree trunks in mature trees and sap flow rates were measured from the base of excised branches. Excised branches were also brought into the laboratory where air temperature could be carefully controlled in a refrigerated box and sap flow rates and sap pressures were measured from the cut base of the branches.Under both forest and laboratory conditions, sap uptake occurred as the wood temperature declined but much more rapid sap uptake correlated with the onset of the freezing exotherm. When sap pressures were measured under conditions of negligible volume displacement, the sap pressure rapidly fell to -60 to -80 kilopascals at the start of the freezing exotherm. The volume of water uptake and the rate of uptake depended on the rate of freezing. A slow freezing rate correlated with a large volume of water uptake, a fast freezing rate induced a smaller volume of water uptake. The volume of water uptake ranged from 0.02 to 0.055 grams water per gram dry weight of sapwood. The volume of water exuded after thawing was usually less than the volume of uptake so that after several freezing and thawing cycles the sapwood water content increased from 0.7 to 0.8 grams water per gram dry weight.These results are discussed in terms of a physical model of the mechanism of maple sap uptake and exudation first proposed by P. E. R. O'Malley. The proposed mechanism of sap uptake is by vapor distillation in air filled wood fiber lumina during the freezing of minor branches. Gravity and pressurized air bubbles (compressed during freezing) cause sap flow from the canopy down the tree after the thaw.

Journal Article↗
PubMed2006

Influence of the freezing technique (nitrogen liquid vs ultrafreezer of -152 degrees C) and male-to-male variation over the semen quality in Canarian Mastiff breed dogs.

This study was carried out to assess the in vitro quality of canine semen frozen in an ultrafreezer at -152 degrees C and to evaluate the male-to-male variation of frozen semen in five male dogs of the Canarian Mastiff breed. Four ejaculates of each dog were processed individually (5% glycerol and 0.5% Equex) to reach a final concentration of 100 x 10(6) spermatozoa/ml. Then, two freezing techniques were tested to assess the seminal quality (sperm motility, live spermatozoa and abnormal sperm cell percentages) at 1, 30, 60, 120 and 360 days after freezing: (i) semen was frozen and stored in liquid nitrogen; (ii) semen was frozen and stored in the ultrafreezer at -152 degrees C. After freezing-thawing, both freezing protocols showed no significant differences in sperm motility and the percentages of live and abnormal spermatozoa. On the other hand, the microscopic characteristics of spermatozoa in fresh semen were practically similar among males; however, after the semen processing and freezing, significant differences were observed (p < 0.05) among males, especially as regards sperm motility. This inter-individual variability was detected in both freezing protocols, showing that the male-to-male variation in the seminal quality post-freezing was independent of the freezing technique used. The in vitro results obtained in the Canarian Mastiff breed confirmed that the use of ultra-freezers at -152 degrees C is a potential alternative to liquid nitrogen for storing canine semen for long periods of time.

Animals↗
PubMed2003

Modification of standard freezing media to limit capacitation and maximise motility of frozen-thawed equine spermatozoa.

OBJECTIVE: To investigate cryopreservation-induced capacitation-like changes in equine spermatozoa frozen in three different media using chlortetracycline (CTC) fluorescence staining analysis. PROCEDURE: Semen collected from three stallions was diluted in one of three centrifugation media and, after centrifugation and removal of supernatant, extended in corresponding freezing media containing additional egg yolk, glycerol, lactose and Equex paste. The semen was frozen in 5 mL straws and the spermatozoa assessed for motility and membrane quality after thawing. RESULTS: Following centrifugation, spermatozoa diluted with modified Kenney's Centrifugation Medium (MKCM) displayed a higher percentage of (normal) F pattern (94.3%) compared with spermatozoa in Kenney's Centrifugation Medium (KCM) (84.9%) and Glucose-EDTA Centrifugation Medium (GECM) (85.2%). Conversely, the percentage of spermatozoa displaying the (capacitated) B pattern was higher in the KCM (14.1%) and GECM (13.8%) than in the MKCM (5.0%). Following freezing-thawing, there were lower percentages of spermatozoa displaying the AR (acrosome reacted) pattern in modified Kenney's Freezing Medium (MKFM) (45.6%) compared with Kenney's Freezing Medium (KFM) (61.4%) and lactose-EDTA Freezing Medium (LEFM) (61.1%). There was a correspondingly higher percentage of spermatozoa displaying the B pattern in MKFM (52.3%) compared with KFM (37.9%) and LEFM (38.6%). There was no significant difference between the freezing media in the percentage of spermatozoa displaying the F pattern. The percentage of progressively motile spermatozoa was also influenced by the type of freezing medium (P < 0.001). Post-thaw percentages of progressively motile spermatozoa, frozen in MKFM, KFM, and LEFM, were 31.4, 25.8 and 23.3%, respectively. CONCLUSION: MKFM was the preferred medium for cryopreservation of equine spermatozoa due to its superior protection against changes in motility and membrane quality compared with the other freezing media studied.

Animals↗
PubMed1989

Changes in chemical structure and function in Escherichia coli cell membranes caused by freeze-thawing. II. Membrane lipid state and response of cells to dehydration.

Glycerol and spermine prevented the freeze-induced alteration of lipid character in membrane fragments derived from Escherichia coli B logarithmic-phase cells, protecting against loss of membrane function and subsequently maintaining higher cell viability. The membrane specimens derived from the stationary-phase cells exhibited high resistance to freezing-induced alteration of the membrane lipid character, and to freezing injury. Freeze-drying of membrane fragment specimens from both logarithmic- and stationary-phase cells gave rise to alterations in lipid state similar to those shown in freeze-thawing of logarithmic-phase cell membrane specimens. Freeze-drying brought about reduction of cell viability in both growth phase specimens. It is suggested, therefore, that the fundamental cause of the injury induced by freezing of living materials is dehydration of lipid-rich systems such as cellular membranes.

Cell Membrane↗
PubMed1985

Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline.

The relative effectiveness of two natural cryoprotectants, proline and trehalose, in preserving membrane structure and function during freezing was studied. Isolated vesicles of sarcoplasmic reticulum (SR) from lobster muscle (Homarus americanus) were employed to study changes in structure and function during rapid freeze-thaw conditions. Both proline and trehalose were shown to effectively preserve the structure (assessed with freeze fracture) and function (assessed by the ability of the membranes to transport calcium) in the frozen vesicles. As a first step toward determining the mechanism of cryoprotection by these compounds, we have investigated their effectiveness in inhibiting freezing induced fusion between phospholipid vesicles. Pamiltoyloleoyl-phosphatidylcholine: phosphatidylserine (85:15 mole ratio) small unilamellar vesicles (SUVs) were made incorporating one of the following fluorescent probes, and energy donor, cholesteryl anthracene-9-carboxylate, or an energy acceptor, nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine to investigate the amount of membrane mixing during rapid freeze-thaw cycles, and storage at -20 degrees C. Membrane mixing was measured as an energy transfer from donor to acceptor when donor vesicles and acceptor vesicles were mixed before a particular freezing treatment. Membrane mixing was correlated with structural changes in these membranes by freeze-fracture analysis. Both trehalose and proline were found to be more effective in preventing membrane mixing between SUVs than the standard protectants, glycerol and dimethylsulfoxide.

Animals↗
PubMed1984

Olfactory mitral cell integrity after freeze-fixation.

The condition of freeze-fixed mitral cells from the main olfactory bulb of the rat brain was found to depend on the rat at which the tissue is frozen: at very slow rates, mitral cells swell and rupture; at faster rates, cell integrity is preserved. Cooling the tissue to just above its freezing point before rapid freeze-fixation helps to maintain cell integrity at depths well below the surface. When freeze-fixation must be used, as in the 2-deoxyglucose technique, rapid freezing rates should be used to minimize diffusion of isotope and increase autoradiographic resolution. After rapid freezing, better cell integrity was produced by freeze-drying and plastic embedding than by cryostat sectioning and thaw-mounting.

Animals↗
PubMed1996

Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration.

Studies of numerous proteins with infrared spectroscopy have documented that unfolding is a general response of unprotected proteins to freeze-drying. Some proteins that are unfolded in the dried solid aggregate during rehydration, whereas others refold. It has been proposed for the latter case that aggregation is avoided because refolding kinetically outcompetes intermolecular interactions. In contrast, with proteins that normally aggregate after rehydration, minimizing unfolding during freeze-drying with stabilizer has been shown to be needed to favor the recovery of native protein molecules after rehydration. The purpose of the current study was to examine first the opposite situation, in which a denaturant is used to foster additional unfolding in the protein population during freeze-drying. If the protein is not intrinsically resistant to aggregation under the study conditions (e.g., because of intermolecular charge repulsion) and the denaturant does not disrupt intermolecular interactions during rehydration, this treatment should favor aggregation upon rehydration. With infrared spectroscopy we found that at concentrations of the denaturant Na thiocyanate (NaSCN) that only slightly perturbed chymotrypsinogen secondary structure in solution before freeze-drying, there was a large increase in protein unfolding in the dried solid and in protein aggregation measured after rehydration. Bands assigned to intermolecular beta sheet were present in the spectra of samples dried with NaSCN, indicating that aggregation could also arise in the dried solid. By examining the protein structure in the frozen state, we determined that in the absence of NaSCN the protein remains native. NaSCN caused structural perturbations during freezing, without the formation of intermolecular beta sheet, that were intermediate to structural changes noted after freeze-drying. In contrast, samples treated in the presence of NaSCN and sucrose had native-like spectra in the frozen and dried states, and much reduced aggregation after rehydration. These results indicate that during freezing and drying the sugar can counteract and mostly reverse the structural perturbations induced by NaSCN before and during these treatments.

Animals↗
PubMed2001

Phase transitions of glycine in frozen aqueous solutions and during freeze-drying.

PURPOSE: To study the solid-state and phase transitions of glycine, (i) in frozen aqueous solutions, and (ii) during freeze-drying. METHODS: X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to analyze the frozen systems. In situ freeze-drying in the sample chamber of the diffractometer enabled characterization of phase transitions during freeze-drying. RESULTS: Transitions in frozen systems. Rapid (20 degrees C/min) or slow (2 degrees C/min) cooling of aqueous solutions of glycine (15% w/w) to -70 degrees C resulted in crystallization of beta-glycine. Annealing at -10 degrees C led to an increase in the amount of the crystalline phase. When quench-cooled by immersing in liquid nitrogen, glycine formed an amorphous freeze-concentrate. On heating, crystallization of an unidentified phase of glycine occurred at approximately -65 degrees C which disappeared at approximately -55 degrees C, and the peaks of beta-glycine appeared. Annealing caused a transition of beta- to the -gamma- form. The extent of this conversion was a function of the annealing temperature. Slower cooling rates and annealing in frozen solutions increased the crystalline beta-glycine content in the Iyophile. Freeze-drying of quench-cooled solutions led to the formation of gamma-glycine during primary drying resulting in a lyophile consisting of a mixture of beta- and -gamma-glycine. The primary drying temperature as well as the initial solute concentration significantly influenced the solidstate of freeze-dried glycine only in quench-cooled systems. CONCLUSIONS: The cooling rate, annealing conditions and the primary drying temperature influenced the solid-state composition of freeze-dried glycine.

Calorimetry, Differential Scanning↗
PubMed1965

FACTORS AFFECTING THE RATE OF KILLING OF ESCHERICHIA COLI BY REPEATED FREEZING AND THAWING.

Packer, Elliot L. (University of California, Davis), John L. Ingraham, and Stanley Scher. Factors affecting the rate of killing of Escherichia coli by repeated freezing and thawing. J. Bacteriol. 89:718-724. 1965.-Repeated freezing and thawing of cultures of Escherichia coli grown in a minimal medium and frozen in the same medium without carbon source resulted in a linear decrease in the log of the number of surviving cells as a function of the number of freeze-thaw cycles. The slope of this curve, which can be determined accurately, is an index of susceptibility of a culture to death by freezing and thawing. The effect of the physiological state of the culture on the killing rate was determined. Contrary to previous reports, the phase of growth, the state of aerobiosis, and the density of the culture had no effect on the degree of susceptibility to death by freezing and thawing. However, presence of spent growth medium (a filtrate of a stationary culture) in the freezing medium protected cells against death by freezing and thawing. Protection by spent growth medium is effective at high dilutions (1:10(5)), and is lost if spent growth medium is heated in the presence of alkali. It is suggested that the protection afforded by spent growth medium accounts for differences between our results and those reported in the literature.

Cold Temperature↗