Freezing Chamber Research Articles

Study of Preparation of Prescribed Pore Configuration in Zirconium Dioxide Ceramic Due to Carbamide Directional Solidification

The possibility is demonstrated of preparing porous ceramic based on zirconium dioxide micro- and nanopowder using crystallization of carbamide in a suspension. Different suspension cooling regimes are studied for oxide powders (in air, in a freezing chamber, in snow). It is shown that sintered ceramic porosity and pore size depend directly on suspension composition and cooling method. As a result of this ceramic is prepared with porosity of 30 – 60% with pore diameter of 0.2 – 200 μm.

고수압 해저터널에 급속 인공동결공법 적용시 간극수의 염분 농도가 동결속도에 미치는 영향 평가: 실내 동결챔버시험 위주로

고수압 조건의 해저터널 공사에 기존의 그라우팅 공법은 제한을 받게 되어 굴착작업이 어려울 수 있으나, 인공동결공법을 적용할 경우 신속한 차수 및 지반보강 효과를 얻을 수 있다. 인공동결공법을 적용할 경우, 동결 조건에 따라 동결토의 거동이 크게 변화하므로 기존의 연구 사례를 바탕으로 해저지반 등 특수한 조건에서 동결토의 역학적 거동을 예측하는 것에는 한계가 있다. 또한, 인공동결공법의 설계를 위해서는 동결체 형성에 필요한 소요시간 및 동결범위를 산정해야 하며, 해저지반의 경우 간극수 내 염분의 영향을 반영해야 한다. 본 논문에서는 동결토의 열적 특성을 파악하기 위해 인조규사 시료에 대한 실내 열전도도 측정시험과 동결챔버시험을 수행하였다. 동결토와 비동결 포화토 간극수의 염도를 조절하여 동결 과정과 염분에 따른 유효 열전도도를 비정상 열선법을 적용하여 측정하였다. 인조규사 간극수의 염도에 의한 동결특성 변화를 파악하기 위해 동결챔버를 설계 및 제작하였고 이를 통하여 동결 조건을 변화시키며 동결챔버시험을 수행하였다. 동결 조건에 따른 시료내 온도 변화를 분석하고 이를 통해 동결 조건이 사질토의 열전달 특성에 미치는 영향을 평가하였다. 비동결 포화토의 경우는 간극수가 담수(염도 0%)인 조건과 염수(염도 3.5%)인 조건 모두 유효 열전도도가 유사하게 평가되었으나, 동결토의 경우는 간극수가 염수인 조건이 보다 큰 유효 열전도도를 보였다. 이는 동결챔버시험에서 간극수가 염수인 조건이 담수인 조건보다 동결속도가 더 빠른 결과와 일치한다. It is extremely difficult to apply conventional grouting methods to subsea tunnelling construction in the high water pressure condition. In such a condition, the rapid artificial freezing method can be an alternative to grouting to form a watertight zone around freezing pipes. For a proper design of the artificial freezing method, the influence of salinity on the freezing process has to be considered. However, there are few domestic tunnel construction that adopted the artificial freezing method, and influential factors on the freezing of the soil are not clearly identified. In this paper, a series of laboratory experiments were performed to identify the physical characteristics of frozen soil. Thermal conductivity of the frozen and unfrozen soil samples was measured through the thermal sensor adopting transient hot-wire method. Moreover, a lab-scale freezing chamber was devised to simulate freezing process of silica sand with consideration of the salinity of pore-water. The temperature in the silica sand sample was measured during the freezing process to evaluate the effect of pore-water salinity on the frozen rate that is one of the key parameters in designing the artificial freezing method in subsea tunnelling. In case of unfrozen soil, the soil samples saturated with fresh water (salinity of 0%) and brine water (salinity of 3.5%) showed a similar value of thermal conductivity. However, the frozen soil sample saturated with brine water led to the thermal conductivity notably higher than that of fresh water, which corresponds to the fact that the freezing rate of brine water was greater than that of fresh water in the freezing chamber test.

Direct visualization of solute locations in laboratory ice samples

Abstract. Many important chemical reactions occur in polar snow, where solutes may be present in several reservoirs, including at the air–ice interface and in liquid-like regions within the ice matrix. Some recent laboratory studies suggest chemical reaction rates may differ in these two reservoirs. While investigations have examined where solutes are found in natural snow and ice, few studies have examined either solute locations in laboratory samples or the possible factors controlling solute segregation. To address this, we used micro-computed tomography (microCT) to examine solute locations in ice samples prepared from either aqueous cesium chloride (CsCl) or rose bengal solutions that were frozen using several different methods. Samples frozen in a laboratory freezer had the largest liquid-like inclusions and air bubbles, while samples frozen in a custom freeze chamber had somewhat smaller air bubbles and inclusions; in contrast, samples frozen in liquid nitrogen showed much smaller concentrated inclusions and air bubbles, only slightly larger than the resolution limit of our images (∼ 2 µm). Freezing solutions in plastic vs. glass vials had significant impacts on the sample structure, perhaps because the poor heat conductivity of plastic vials changes how heat is removed from the sample as it cools. Similarly, the choice of solute had a significant impact on sample structure, with rose bengal solutions yielding smaller inclusions and air bubbles compared to CsCl solutions frozen using the same method. Additional experiments using higher-resolution imaging of an ice sample show that CsCl moves in a thermal gradient, supporting the idea that the solutes in ice are present in mobile liquid-like regions. Our work shows that the structure of laboratory ice samples, including the location of solutes, is sensitive to the freezing method, sample container, and solute characteristics, requiring careful experimental design and interpretation of results.

투명 온도제어형 삼축셀을 이용한 흙의 동상 실내실험

최근 들어 우리나라에 이상한파가 자주 발생하고 있으며, 이로 인해 지반에서는 동상에 따른 도로, 철도, 사면과 같은 구조물을 대상으로 지반변형에 따른 문제가 야기되고 있다. 동상현상은 가장 대표적인 동토지반의 공학적 특성으로, 구조물 설계에 있어서 동착 및 크리프 현상과 함께 주요한 설계정수로서 고려된다. 그러므로 극한지 지반구조물 설계에 관한 연구를 위해서는 동상현상에 대한 기초적 연구가 선행되어야 한다. 이미 다양한 동상 관련 실험장비가 개발되어 연구에 활용되고 있지만, 기존의 동상 연구를 위한 실험장비들은 기초의 설계정수로서 동상현상을 분석하기에는 한계점이 나타난다고 판단된다. 국내에는 동상을 실험적 모사하기에는 필요한 장치와 냉동챔버의 고비용으로 시험방법 및 기준이 전무한 실정이다. 이에 본 논문에서는 다양한 방법으로 동결-융해를 모사할 수 있는 투명 온도제어형 삼축셀 을 소개하고자 한다. 새롭게 개발된 동결-융해 실험장치를 활용해 동상 실내실험을 수행하였으며, 본 장치를 활용한 실내실험법을 제안하고자 한다. Nowadays abnormal coldness happens frequently in Korea and frost heaving causes unexpected ground deformation which results in severe problems for structures such as roadway, railroad and cutoff slope. 'Frost heave' as one of the primary phenomenon is considered to be an important factor together with 'adfreeze bond-strength' and 'creep deformation' for structural design process in permafrost area. Therefore, the fundamental study for frost heave has to be preceded for design of geo-structures in cold region. While various experimental apparatuses have been developed, there still exist a certain level of limitation to evaluate the frost-heave characteristics as design parameters. There are no standard testing method and criteria for analyzing frost heaving in Korea because temperature controlled testing apparatuses including a freezing chamber are expensive. In this paper, a new standard freezing and thawing testing apparatus is introduced, which simulates various freezing and thawing conditions in a soil specimen by using a temperature controlled triaxial cell. Frost heaving tests were performed to assess the new testing apparatus and experimental procedure to evaluate frost heaving for soils is proposed.

Effects of glazing and chitosan-based coating application on frozen salmon preservation during six-month storage in industrial freezing chambers

Freezing and glazing are techniques commonly used to reduce the incidence of fish deterioration processes. In order to find an alternative to complement freezing and replace water glazing, the present work aimed at evaluating the effect of water glazing and edible coatings of 0.5% w/v and 1.5% w/v chitosan on quality parameters of frozen fish. Both types of coatings – water glazing and chitosan coatings – were applied directly on frozen Atlantic salmon (Salmo salar) and stored for 9 months at −22 °C. Several parameters such as coating/glazing loss, weight loss, drip loss, Total Viable Counts (TVC), Total Volatile Basic-Nitrogen (TVB-N), K-value, pH and color coordinates L*a*b* were periodically evaluated in order to compare glazing with the chitosan-based coatings and uncoated control samples. Samples coated with 1.5% w/v chitosan performed better in maintaining the color of the salmon and controlling microbial contamination of frozen and thawed samples.

CONTROLLED FREEZING AS A LOW-TEMPERATURE TOLERANCE TEST FOR APPLE ROOTSTOCKS

In the years 2009-2010, frost resistance of artificially frozen plants of nine Polish apple rootstocks (P 2, P 14, P 16, P 22, P 59, P 60, P 66, P 67 and P 68) bred at the Research Institute of Pomology and Floriculture (recently renamed Research Institute of Horticulture), and of M.7, M.9, M.26, M.106 rootstocks and Antonovka seedlings was tested. Controlled freezing was performed in a freezing chamber manufactured by Binder GmbH, Germany. Prior to exposing the rootstocks to freezing temperatures, they had been kept in sealed plastic bags in a cold store (0°C). During freezing, 10 rootstocks of each genotype were inside the same plastic bag to protect them from drying. Freezing was performed on 3-5 March 2009 and on 15-17 February 2010. On each freezing date, the plants were subjected to the temperatures of -8°C, -10°C and -12°C for 3 hours. During freezing, the temperature was lowered at a rate of 2°C per hour, and after freezing it was raised at the same rate until it reached 0°C. Rootstocks of the listed clones treated in the same way but not frozen served as the control. After freezing, the plastic bags with rootstocks were again placed in a cold store and subsequently planted in a field at the beginning of April. After planting, the rootstocks were cut back to a height of 5 cm above the soil level and their regeneration was evaluated during the growing season. The best regeneration (average for two years of evaluation) showed rootstocks P 60, P 67 and P 68; rootstocks P 59, P 66 as well as M.7, M.9, Antonovka seedlings and M.106 also regenerated well.

동결된 화강풍화토와 알루미늄판 접촉면에서 발현되는 동착강도 측정 연구

Bearing capacity of pile is governed by only skin friction in frozen ground condition, while it is generally governed both by skin friction and end bearing capacity in typically unfrozen ground condition. Skin friction force, which arises from the interaction between pile and frozen soils, is defined as adfreeze bond strength, and adfreeze bond strength is one of the most important key parameters for design of pile in frozen soils. Many studies have been carried out in order to analyze adfreeze bond strength characteristics over the last fifty years. However, many studies for adfreeze bond strength have been conducted with limited circumstances, since adfreeze bond strength is sensitively affected by various influence factors such as intrinsic material properties, pile surface roughness, and externally imposed testing conditions. In this study, direct shear test is carried out inside of large-scaled freezing chamber in order to analyze the adfreeze bond strength characteristics with varying freezing temperature and normal stress. Also, the relationship between adfreeze bond strength and shear strength of the frozen soil obtained from previous study was analyzed. The coefficient of adfreeze bond strength was evaluated in order to predict adfreeze bond strength based on shear strength, and coefficients suggested from this and previous studies were compared.

078 Improving standarised freezing protocols

In applications like cell and tissue banking, the method of freezing is used for long term storage of biological specimens at low temperatures. Lowering the temperatures reduces the metabolism of cells and therefore can prohibit their quality loss over time. During the freezing process different kinds of factors have been identified as cell damaging mechanisms [1]. However, a correct setting of process parameters including the cooling rate, warming rate, nucleation temperature, (Tn) and type and concentration of cryoprotective agents can reduce cellular damage and result in high quality output after freezing. For comparison of the qualities of different biological specimens, freezing protocols and viability analyses need to be standardised. Most of the earlier studies did not account for the control of all process parameters, especially the nucleation temperature [1]. Therefore, the aim of this study was to control all possible process parameters during freezing of human endothelial cells using different experimental methods. A standardised freezing protocol was developed which is based on our earlier experimental results [2]. The protocol includes a cooling rate of 5 K/min, heating rate of 100 K/min, 5% (v/v) dimethyl sulfoxide and a nucleation temperature of −8 °C. The first method used was a biological freezer (CM2000, Carburos Metalicos) where 1 mL cell suspensions were frozen in cryovials. For the control of the nucleation temperature a new setup was developed which is based on nucleation via Peltier elements [3]. A second method used a cryomicroscopic system including a freezing chamber (FDCS, Linkam) and a digital camera (Retiga, QImaging). Therefore, another new setup was constructed to control Tn. In this setup nucleation is induced by a liquid nitrogen cooled copper rod which is positioned towards the specimen by a step motor system. Both constructions allowed the control of the nucleation temperature and contribute to a further standardisation of freezing protocols. After freezing of the endothelial cells, the quality of the protocol was determined using staining methods based on trypan blue, calcein AM and ethidium homodimer. As a general quality test the absolute viability was determined which is defined as the ratio of viable cell number after freezing to the total cell number before the freezing protocol. Using this method, the viability of endothelial cells after freezing was 90%. To achieve a standardisation of freezing protocols we propose the necessity to control all process parameters including the nucleation temperature and the use of the absolute viability as a general method for quality comparison of different cellular specimens after freezing. [1] P. Mazur, S.P. Leibo, E.H.Y. Chu: A two-factor hypothesis of freezing injury, Experimental Cell Research 71 (1972) 2, 345–35 [2] I. Bernemann, N. Hofmann, R. Spindler, A. Szentivanyi, B. Glasmacher: Strategy to improve cryopreservation protocols, CryoLetters 29 (2008) 1, 73–88 [3] R. Spindler, B. Rosenhahn, N. Hofmann, B. Glasmacher: Video analysis of osmotic cell response during cryopreservation, Cryobiology 64 (2012) 3, 250–260 Source of funding: This work is supported by funding from the Deutsche Forschungsgemeinschaft for the Cluster of Excellence REBIRTH (EXC 62/1). Conflict of interest: None declared. glasmacher@imp.uni-hannover.de

동결토의 일축압축강도와 전단강도 상관관계 분석에 관한 연구

동결토는 물과 얼음의 상변화에 따라 일반적인 흙과는 다른 거동을 보이며, 동결토의 강도는 온도, 함수비, 시간, 토질 특성 등 다양한 인자에 의해 영향을 받는다. 특히 지반이 동결되는 경우, 얼음과 흙 입자의 결합력에 의해 높은 강도 특성을 보이며, 이때의 전단강도는 흙의 구조적 결합에 영향을 미치는 얼음과 세립분의 함량에 따라 달라지며, 이로 인해 점착력이 동결토의 전단강도에 미치는 영향은 증가한다. 본 논문에서는 동결조건에서 직접전단시험과 일축압축강도시험을 실시하여 온도에 따른 흙의 강도 특성을 확인하였으며, 점착력과 일축압축강도와의 상관관계를 확인하였다. The mechanical behavior of frozen soils is different from that of unfrozen soils due to the phase change between water and ice. The strength characteristics of frozen soils are governed by the intrinsic material properties such as grain size, ice and water content, air bubbles, and by externally imposed testing conditions such as temperature, freezing time, and strain rate. Especially, the strength of the frozen soils is generally higher than that of unfrozen soils due to ice binding capacity with soil particles, and is strongly affected by a highly complex interaction between the solid soil skeleton and the pore matrix, composed of ice and unfrozen water. In this study, the direct shear test and unconfined compression test are carried out inside of a large-scaled freezing chamber, and the relationships between cohesion and unconfined compression strength under various freezing temperature conditions are discussed.

Quality and Safety of Hematopoietic Progenitor Cell (HPC) Products Cryopreserved by Simple Passive Gradual Freezing Method

Abstract 4410To provide quality HPC products for transplantation, harvests of bone marrow (BM), Peripheral Blood Stem Cells (PBSCs) and Cord Blood (CB) are routinely cryopreserved by gradual cooling from ambient temperature to −90°C using Controlled Rate Freezing (CRF) systems before placement in liquid/vapor nitrogen storage. CRF systems generally utilize a freezing chamber and computer-controlled freezing cycle with an established sequence of steps where timing, target temperatures and cooling rates are defined. System failures that endanger product quality may occur due to impurities in liquid nitrogen, mechanical/software problems and probe malfunction. A more convenient and economical freezing may be performed by simple “passive freezing” (PF) where products are placed directly into an ultra-low freezer (−80°C). Continuous temperature monitoring during the freeze process is achieved using individual data loggers. In the PF process, products remain undisturbed during the freeze process, and are removed for placement in liquid/vapor nitrogen storage when the target product temperature is reached (−80°C). Probe specific freezing curves are generated from downloaded data logger information.In an internal validation study, the St. Louis Cord Blood Bank (SLCBB) investigated quality and safety of HPC products cryopreserved using this PF method. Evaluation was based on the analysis of freezing kinetics, post-thaw cell recoveries, and available transplant outcome data in reference to those obtained by the conventional CRF method. A total of twelve (12) red blood cell and plasma reduced CB products cryopreserved in 10% DMSO/Dextran by PF were thawed according to a validated albumin/dextran reconstitution thaw method. Compared to the programmed CRF system, PF product cooling rate to a temperature of −60°C was faster (1.2– 2.0°C/min compared to 1°C/min), ultimately followed by slower cooling rate to a product target temperature of −80°C (approximately 0.4°C/min compared to 10°C/min). The kinetics of PF curves resulted in longer freezing cycles with an average freeze time of 180 minutes compared to 80 minutes by CRF method. Post-thaw cell recoveries of the PF data set were comparable to those obtained from an established thaw control group of RBC and plasma reduced CB cryopreserved by CRF system (n = 25). Average percent recoveries for CB products cryopreserved by PF methodology compared to CRF system were reported as follows, respectively: viable nucleated cells (NC) by trypan blue = 84% +/− 5% compared to 71% +/− 6%, viable (7AAD) CD34+ cells = 67% +/− 7% compared to 63% +/− 15%, and colony forming unit (CFU) = 73% +/− 7% compared to 72% +/− 14%.Subsequently, the SLCBB reviewed post-thaw cell recoveries of ten (10) PBSC harvests cryopreserved by PF and thawed at the SLCBB for clinical transplantation. Similar to the CB evaluation, results indicated a high quality HPC product post-thaw with average percent recoveries reported as follows: viable nucleated cells (NC) by trypan blue = 79% +/− 10%, viable (7AAD) CD34+ cells = 81.2% +/− 20.2, and CFU = 73.6% +/− 21.9%. All PBSC products were thawed and infused with no reports of infusion-related adverse events. These PBSC products engrafted shortly within predicted time; absolute neutrophil count (ANC) > 500/uL was reported within 12 days, while platelets > 20,000/uL was achieved within 22 days.Retrospective review of more than 1,800 CB products distributed by the SLCBB for transplant showed that five (5) PF products were transplanted as singlet products to treat patients with myeloid malignancies. Transplant outcome data received by CIBMTR for those cases indicated 100% success of engraftment and 100% survival at 100 days post-infusion. Engraftment data reports indicated ANC > 500 was achieved within 6–30 days, and platelet count > 20,000 was achieved within 25–163 days. The transplant outcome of PF product is comparable to that reported historically for CRF CB products. Conclusions:In vitro results and clinical findings support the quality and safety of HPC products cryopreserved using PF methodology and would recommend PF as valid alternative to the use of CRF systems. Disclosures:No relevant conflicts of interest to declare.

Current and emerging screening methods to identify post-head-emergence frost adaptation in wheat and barley

Cereal crops can suffer substantial damage if frosts occur at heading. Identification of post-head-emergence frost (PHEF) resistance in cereals poses a number of unique and difficult challenges. Many decades of research have failed to identify genotypes with PHEF resistance that could offer economically significant benefit to growers. Research and breeding gains have been limited by the available screening systems. Using traditional frost screening systems, genotypes that escape frost injury in trials due to spatial temperature differences and/or small differences in phenology can be misidentified as resistant. We believe that by improving techniques to minimize frost escapes, such 'false-positive' results can be confidently identified and eliminated. Artificial freezing chambers or manipulated natural frost treatments offer many potential advantages but are not yet at the stage where they can be reliably used for frost screening in breeding programmes. Here we describe the development of a novel photoperiod gradient method (PGM) that facilitates screening of genotypes of different phenology under natural field frosts at matched developmental stages. By identifying frost escapes and increasing the efficiency of field screening, the PGM ensures that research effort can be focused on finding genotypes with improved PHEF resistance. To maximize the likelihood of identifying PHEF resistance, we propose that the PGM form part of an integrated strategy to (i) source germplasm;(ii) facilitate high throughput screening; and (iii) permit detailed validation. PGM may also be useful in other studies where either a range of developmental stages and/or synchronized development are desired.

Numerical Modeling of the Freezing of a Porous Humid Food inside a Cavity due to Natural Convection

The conjugate heat transfer problem of food freezing inside a cavity was numerically investigated. A vegetable sponge has been considered as a food block freezing inside a square freezing chamber due to natural convection. The need for specifying the block surface convective heat transfer coefficients was eliminated by solving the surrounding cooling fluid and, therefore, a comprehensive understanding on heat transfer and airflow during freezing can be achieved. The 2-D unsteady Navier-Stokes and energy equations were solved using a finite volume method with the semi-implicit SIMPLE algorithm. Thermophysical properties of food block components were considered to be dependent on temperature, as well as moisture and ice content, which has been rarely considered in food freezing studies. The specific heat capacity method was employed to model the freezing process. The Krischer model was adopted for predicting the thermal conductivity of the food, which indicates that the model works with reasonable accuracy for humid porous foods. The mechanism of natural convection in the cavity was carefully studied and the effects of different parameters on the freezing time were examined. The food freezing curves were investigated for various Rayleigh numbers in the range of 104 ≤ Ra ≤ 106, with different area ratios of A = 1/16, 1/9, and 1/4, and food initial water contents of X tw = 0.48, 0.58, and 0.68. It was concluded that increasing the Rayleigh number reduces the freezing time. On the other hand, area ratio and initial water content of the food were proved to extend the freezing time.

Freezing pattern and frost killing temperature of apple (Malus domestica) wood under controlled conditions and in nature

The freezing pattern and frost killing temperatures of apple (Malus domestica Borkh.) xylem were determined by differential thermal analysis and infrared differential thermal analysis (IDTA). Results from detached or attached twigs in controlled freezing experiments and during natural field freezing of trees were compared. Non-lethal freezing of apoplastic water in apple xylem as monitored during natural winter frosts in the field occurred at -1.9 ± 0.4 °C and did not change seasonally. The pattern of whole tree freezing was variable and specific to the environmental conditions. On detached twigs high-temperature freezing exotherms (HTEs) occurred 2.8 K below the temperature observed under natural frosts in the field with a seasonal mean of -4.7 ± 0.5 °C. Microporous apple xylem showed freezing without a specific pattern within a few seconds in IDTA images during HTEs, which is in contrast to macroporous xylem where a 2D freezing pattern mirrors anatomical structures. The pith tissue always remained unfrozen. Increasing twig length increased ice nucleation temperature; for increased twig diameter the effect was not significant. In attached twigs frozen in field portable freezing chambers, HTEs were recorded at a similar mean temperature (-4.6 ± 1.0 °C) to those for detached twigs. Upon lethal intracellular freezing of apple xylem parenchyma cells (XPCs) low-temperature freezing exotherms (LTEs) can be recorded. Low-temperature freezing exotherms determined on detached twigs varied significantly between a winter minimum of -36.9 °C and a summer maximum -12.7 °C. Within the temperature range wherein LTEs were recorded by IDTA in summer (-12.7 ± 0.5 to -20.3 ± 1.1 °C) various tiny clearly separated discontinuous freezing events could be detected similar to that in other species with contrasting XPC anatomy. These freezing events appeared to be initially located in the primary and only later in the secondary xylem. During the LTE no freezing events in the bark and central pith tissue were recorded. Attached twigs were exposed to various freezing temperatures at which LTEs occur. Even if 60% of XPCs were frost-damaged twigs were able to recuperate and showed full re-growth indicating a high regeneration capacity even after severe frost damage to XPCs.

Freeze tolerance of forage bermudagrasses

AbstractThe ability to survive harsh winters is one of the primary factors limiting bermudagrass (Cynodon spp.) distribution, especially those used for forage. Consequently, improved stress tolerance has been a goal of programmes for breeding bermudagrasses. While significant progress has been made in developing and evaluating freeze tolerance of turfgrasses, information on forage bermudagrasses is limited. Our objective was to evaluate freeze tolerance of recently released forage bermudagrasses compared with standard cultivars. Plants were established and acclimated in growth chambers and exposed to sub‐freezing temperatures in a programmable freeze chamber. Plant responses to low‐temperature exposures were quantified by regrowth mass. Freeze tolerance of Tifton 44 and Coastal was not significantly different from Midland, the reference cultivar. Coastcross, Tifton 85 and Tifton 68 were more susceptible to low‐temperature injury than Midland, while Hardie, Goodwell, Midland 99 and Ozark were more freeze tolerant than Midland. Ozark, Midland 99, Goodwell and Hardie are less likely to sustain winterkill than Coastcross, Tifton 85 and Tifton 68 in areas that frequently experience low temperatures. Cultivars with one or more parents originating in Kenya had poor freeze tolerance, while germplasm from Yugoslavia and Afghanistan produced plants that were more freeze tolerant.

10.1007/s11983-008-1007-0

For the first time, based on direct observations of carbon dioxide concentration at modern Russian drifting stations, the role of the Arctic sea ice in maximum seasonal changes in carbon dioxide distribution is considered. Carbon dioxide is generated during growth of ice mass at the sea ice undersurface and is absorbed at the surface of melting sea ice. The time of ice growth is three or four times greater than that of its melting in summer months, and, as result, the time of carbon dioxide generation is longer that that of its uptake. Consequently, the Arctic Ocean is a source of carbon dioxide on the mean annual scale (the effect of Arctic “breathing”). Experiments in a freezing chamber confirm these conclusions.

Condensation of acetamide in aqueous solutions in the presence of sulfur(IV) dioxide

We found that reaction of acetamide proceeding in SO2–H3СC(О)NH2–H2O solution affords cyclic oquinoid compound І that formally can be regarded as a product of acetamide condensation along the scheme: SO2 with the rate 10 to 15 ml min until homogenous mixture has formed. The viscous oily yellow solution as placed to a freezing chamber (≈ –15°С). After a few weeks the SO2 excess was neutralized at –5 to –10°С by adding Na2CO3 in small portions until gas evolution stopped. The solution was decanted and again placed into freezing chamber (~ –15°С). After ~14 days the solution obtained was placed to a desiccator over calcium chloride at room temperature and kept until a precipitate has fiormed. We isolated 5.2 g (62 %) of product І, white, easily sublimating at room temperature. The IR spectrum, ν, cm: 3420 m [νas(NH)]; 3342 s [νs(NH)]; 3080 sh [νas(СH2)(=CH2)]; 2930 s [νs(СH2)(=CH2)]; 2857 s [νs(СH2)]; 1637 m [ν(СО), ν(С=С)]; 1562 s [δ(NH2), [ν(С=С)ring]; 1453 s [δ(СH2), δ(СNН)]; 1397 m [δ(СH2)planar.]; 1361 m [δ(СH2), r(NH2)]; 1071 w [νas(СN)(C–NH2)]; 1061 m [δ(ССC)planar,ring ]; 969 w [νring]; 823 m, br [t(NH2)]; 797 m [δ(СH2)]; 733 m [δ(ССC)out-of-plane, ring]; 711 m [r (СH2), t(NH2)]; 611 m [δ(ССC)out-of-plane, ring]; 497 m [δ(ОСС)ring ]. The Н NMR spectrum (CD3OD), δ, ppm (J, Hz): 1.33–1.37 m (4H, C–NH2), 1.44–1.53 m (4Н, CH2–NH2) 2.63 t (4Н, CH2–NH2, JНН 7.2), 4.87 s (4H, C=СH2). The mass spectrum (FAB, solution of I in 3-nitrobenzyl alcohol), m/z (Irel %): 250 (100) [M + 2Н]; (FAB, solution of I in 3-nitrobenzyl alcohol with NaI adding) m/z (Irel, %): 271 (65) [M + Na], 250 (53) [M + 2Н]. In the FAB mass-spectrum occurs a peak of [M + 2Н], characteristic of o-quinones that are reduced to the corresponding catechols, that is not inherent in p-quinones [5]. Found, %: C 57.43; H 6.41; DOI: 10.1134/S107036320906036X

Critical temperature point for storage of vitrified embryos

OBJECTIVE: Objective of this study was to determine the critical temperature for storage of vitrified embryos with minimal cryo-injury and adverse effects on the embryonic development following thawing. DESIGN: Mouse embryos were vitrified and stored under liquid nitrogen (control group) or at −90 °C, −100°C, −110 °C, −122.5°C (experimental groups) for 5 hours. Embryos were then thawed and their developmental potential was evaluated. MATERIALS AND METHODS: 8-cell mouse embryos, previously frozen at 2-cell stage and cultured to the 8-cell stage after thawing were vitrified by treatment with 7.5% Ethylene Glycol + 7.5% DMSO, and 15% Ethylene Glycol + 15% DMASO + 0.5M sucrose and loading into V-Kim vitrification straws, a recently developed closed vitrifcation straw system (Isatis Scientific, California) before plunging into liquid nitrogen. Some of the straws were kept in liquid nitrogen (control group) and the rest were placed in a controlled freezing chamber at temperatures of −90 °C, −100°C, −110 °C, −122.5°C (experimental groups) for a period of 5 hours. Embryos were then removed from their respective temperature of storage and thawed by transferring to 1 and 0.5 M sucrose, and wash medium supplemented with 10% serum. Embryo survival and blastocyst formation rates were evaluated. Results were analyzed by one-way ANOVA. RESULTS: A total of 163 embryos were vitrified and divided into ‘Control’, ‘-90 °C’, ‘-100°C’, ‘-110 °C’, ‘-122.5°C’ groups. After thawing, 92 % of embryos survived in the control group and 82% developed to the blastocyst stage. Survival rates after thawing were 56%, 84%, 87%, and 90%; and blastocyst formation rates were 39%, 78%; 78%, and 79% for the −90 °C, −100°C, −110 °C, −122.5°C groups respectively. There were no significant differences in survival and blastocyst formation rates in all groups (P>0.05) except in the −90 °C group which had lower survival and blastocyst formation rates when compared with all other groups (P<0.05). CONCLUSIONS: The critical temperature for storage of vitrified mouse embryos is around −100 °C’. Storing below this temperature even for a short period of time has adverse effects on thaw rates and blastocyst formation. Experiments are underway to determine if this finding applies to other stages of mouse embryonic development and to human embryos.

On the role of the Arctic sea ice in seasonal variability of carbon dioxide concentration in Northern latitudes

For the first time, based on direct observations of carbon dioxide concentration at modern Russian drifting stations, the role of the Arctic sea ice in maximum seasonal changes in carbon dioxide distribution is considered. Carbon dioxide is generated during growth of ice mass at the sea ice undersurface and is absorbed at the surface of melting sea ice. The time of ice growth is three or four times greater than that of its melting in summer months, and, as result, the time of carbon dioxide generation is longer that that of its uptake. Consequently, the Arctic Ocean is a source of carbon dioxide on the mean annual scale (the effect of Arctic “breathing”). Experiments in a freezing chamber confirm these conclusions.

The Effect of Blanching, Freezing and Freeze-Storage on Changes of Some Chemical Compounds Content in New Zealand Spinach

The Effect of Blanching, Freezing and Freeze-Storage on Changes of Some Chemical Compounds Content in New Zealand Spinach The aim of the work conducted in 2003-2004 was the estimation of the effect of blanching, freezing and freeze-storage on the content of chosen chemical compounds in plants of New Zealand spinach grown from seedlings. The determinations included dry matter, total protein, L-ascorbic acid, titrable acidity, total sugars, crude fibre, total ash, pH, nitrates, nitrites and chlorophyll content in the experimental material (fresh, blanched, frozen and stored shoots with leaves of New Zealand spinach). Used in the experiment procedures had a significant effect on changes of dry matter, total protein, L-ascorbic acid, titrable acidity, total sugars, crude fibre, total ash content and pH. After blanching their losses reached respectively: 34.9, 6.7, 70.2, 45.0, 56.3, 19.3, 52.9%, in relation to the raw material. However, the value of pH increased to 6.75. After freezing the losses, in relation to the blanched material, were: 21.7% for total protein, 21.4% - L-ascorbic acid, 27.3% - titrable acidity, 42.9% - total sugars, 4.5% - crude fibre, 2.5% - total ash, and pH decreased to 6.45. Frozen material was stored for 3, 6 and 9 months in a freezing chamber at -25 ÷ -27°C. The period of storage did not have statistically significant effect on the preservation of determined chemical components.

An investigation into the similarities and differences governing the cryopreservation success of koala ( Phascolarctos cinereus: goldfuss) and common wombat ( Vombatus ursinus: shaw) spermatozoa

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm’s response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 μM), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala ( n = 4) and wombat ( n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast—3 cm above liquid N2 interface; slow—6 °C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris–citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better ( P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410 mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility.