Abstracts: CryoLetters 23 (3), 2002

CryoLetters is a bimonthly, international journal for low temperature science and technology

CryoLetters 23, 141-150 (2000)
©CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Shen Fanyi1*, Liu Wenji 2, Gao Rongfu 3, Zhang Wenjie1, Zhao Qi4

1College of Basic Sciences and Technique, Beijing Forestry University, Beijing 100083, China.
2Department of Basic Courses, Zhengzhou Institute of Light Industry, Zhengzhou 450002, China.
3College of Biology, Beijing Forestry University, Beijing 100083, China. 
4Department of Biology, Capital Normal University, Beijing 100037, China 


The temperature at which ice grows through narrow, hydrophilic capillary is known to be depressed (9, 26). Further, the nucleation temperature near a hydrophilic surface varies with the size, geometry and the properties of a particle (10, 11, 12, 13). In this paper we show how these two effects are additive for the water that freezes on the wall of a capillary without the presence of pre-existing ice. The combined effect is a substantial lowering of nucleation temperature that could, if this analysis is correct, have important cryobiological consequences.

Keywords: Plant water, Deep supercooling, Melting point shift, Heterogeneous nucleation, Freezing temperature.



CryoLetters 23, 151_156 (2000)
©CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


T. Hoshino1*, M. Tojo2, H. Kanda3, M.L. Herrero4, A.M. Tronsmo4, M. Kiriaki1, Y. Yokota1 and I. Yumoto1

1 Research Institute of Biological Resources, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1, Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan
2 Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1, Gakuen-chou, Sakai, Osaka 599-8531, Japan
3 National Institute of Polar Research, 1-9-10, Kaga, Itabashi-ku, Tokyo 173-8515 Japan
4 The Norwegian Crop Research Institute, Fellesbygget, N-1432 Ås, Norway


Chilling resistances in moss pathogenic fungi, Pythium ultimum var. ultimum, from Longyearbyen, Svalbard (78˚N, 15˚E), located in the Arctic Zone and in the same isolates from Temperate Zone, were determined. Both strains had similar optimum growth temperatures. However, the strains from Svalbard could grow and survive at 0 - 5˚C. In addition, chilling treatment induced irregular mycelial morphology in the Arctic isolates. On the other hand, the isolates from Japan did not grow at temperatures below 5˚C and were destroyed after chilling stress (0˚C for 3 days or at 4˚C for 1 week). The results suggested that isolates from Svalbard highly adapted to the severe spring condition in Polar environments.

Key words: chilling resistance, fungi, Pythium ultimum var. ultimum, Svalbard



CryoLetters 23, 157-166 (2000)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

What is 'unfreezable water', how unfreezable is it and how much is there?

Joe Wolfe1*, Gary Bryant 2 and Karen L. Koster3

1 School of Physics, The University of New South Wales, Sydney.

2 Department of Applied Physics, RMIT University, Melbourne.

3 Department of Biology, The University of South Dakota, Vermillion, SD.


Water that remains unfrozen at temperatures below the equilibrium bulk freezing temperature, in the presence of ice, is sometimes called 'unfreezable' or 'bound'. This paper analyses the phenomenon in terms of quantitative measurements of the hydration interaction among membranes or macromolecules at freezing temperatures. These results are related to analogous measurements in which osmotic stress or mechanical compression is used to equilibrate water of hydration with a bulk phase. The analysis provides formulæ to estimate, at a given sub-freezing temperature, the amount of unfrozen water due to equilibrium hydration effects. Even at tens of degrees below freezing, this hydration effect alone can explain an unfrozen water volume that considerably exceeds that of a single 'hydration shell' surrounding the hydrophilic surfaces. The formulæ provided give a lower bound to the amount of unfrozen water for two reasons. First, the well-known freezing point depression due to small solutes is, to zeroth order, independent of the membrane or macromolecular hydration effect. Further, the unfrozen solution found between membranes or macromolecules at freezing temperatures has high viscosity and small dimensions. This means that dehydration of such systems, especially at freezing temperatures, takes so long that equilibrium is rarely achieved over normal experimental time scales. So, in many cases, the amount of unfrozen water exceeds that expected at equilibrium, which in turn usually exceeds that calculated for a single 'hydration shell'.

Keywords: unfreezable water, water of hydration, bound water, dehydration, freezing, freezing point depression



CryoLetters 23, 167-172 (2000)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


A Medrano*1, F Cabrera2, F González2, M Batista2, A Gracia2

1 Department of Animal Sciences, Faculty of Superior Studies-Cuautitlan, National Autonomous University of Mexico. Cuautitlan Izcalli, Mexico.
2 Unit of Reproduction and Obstetrics, Faculty of Veterinary, University of Las Palmas de Gran Canaria. Arucas, Las Palmas, Spain.


A series of experiments was carried out to validate a –150°C ultra-low temperature freezer for its possible use to properly freeze and store semen. In the first part, crude sample handling was simulated to see whether temperature of stored samples was maintained within a safe range; also, the freezing point and latent heat of fusion plateau of a semen extender were monitored. In the second part, buck semen was (i) frozen in liquid nitrogen and stored in the ultra-low freezer, (ii) frozen and stored in the ultra-low freezer, and (iii) frozen and stored in liquid nitrogen, to compare sperm cryosurvival between freezing methods. Both, frequent removal of samples and long opening of the freezer door did not negatively affect stored sample temperature; latent heat of fusion plateau was 5 minutes long. Semen stored either at –150°C or at –196°C cryosurvived similarly after 2 days and after 2 months of cryopreservation.

Keywords: semen, goat, freezing, ultra-low temperature freezer.



CryoLetters 23, 173-182 (2000)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cold hardiness and supercooling capacity in the pea leafminer Liriomyza huidobrensis

Bing Chen and Le Kang*

Institute of Zoology, the Chinese Academy of Sciences, Beijing, 100080, China. email:


Pupal SCP (supercooling point) of Liriomyza huidobrensis showed no variation with age, with an average of –20.9C. Low temperature survival of different ages of pupae showed no correlation with their SCP. Nonlinear regression analysis found that the response of L. huidobrensis pupae to exposure time under different low temperature regimes above –5C was best fitted by a logistic equation. Both low temperature and exposure time had significant effects on pupal mortality. Temperatures above 5C do not prevent pupae from emergence. L. huidobrensis was shown to be a freeze susceptible, and at the same time, a chill tolerant insect. It can tolerate subzero temperatures by supercooling. Compared with L. sativae, another dominant leafminer in China (31), L. huidobrensis is more cold tolerant. Our results explain differences between the species in geographic distribution and phenology.

Keywords: cold hardiness, Liriomyza huidobrensis, leafminer, supercooling, Liriomyza sativae



CryoLetters 23, 183-190 (2000)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Thomas F. Sørensen and Hans Ramløv*

Department of Life Sciences and Chemistry, Roskilde University, P.O.Box 260, 18.1, DK-4000 Roskilde, Denmark. email:


The viviparous teleost Zoarces viviparus inhabits the coastal areas of the eastern North Atlantic and Baltic region. Antifreeze activity was measured in serum from pregnant females and embryos during their embryological development inside the ovary and found to be up to 0.95C and 0.85C respectively. Lower levels of antifreeze activity (up to 0.34C) were discovered in the ovarian fluid. The result is higher hysteresis freezing points and thus less protection to freezing of the ovarian fluid compared to the serum in embryos and the maternal organism. The pertinence of this to the cold tolerance of Z. viviparus is presently unknown. SDS-PAGE of ovarian fluid shows only few protein bands and a difference in proportional density of proteins compared to maternal and embryo serum. This could indicate that another mechanism than rupture of blood vessels in the ovarian wall is of importance for the occurrence of small proteins in the ovarian fluid.

Keywords: Zoarces viviparus, Zoarcidae, Antifreeze proteins, Embryos, Viviparity



CryoLetters 23, 191-196 (2000)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Studies on Cryopreservation of luc gene transfected bluegill sunfish fibroblast cell line

Tiantian Zhang* and David M. Rawson.

Luton Institute of Research in the Applied Natural Sciences, University of Luton, 2 Adelaide Street, Luton, Bedfordshire, LU1 5DU, United Kingdom


The effect of cryopreservation on the survival of luc gene transfected bluegill sunfish fibroblast (BF-2) cells was investigated. Propane-1,2-diol was found to be the least toxic cryoprotectant when compared with DMSO and methanol. Both propane-1,2-diol and DMSO are effective in protecting cells from freezing damage. Whilst there were no significant differences in cell survival between cryoprotectant concentration (10 or 15%) and culture age used in this study, 7-day old culture appeared to be more resistant to freezing without cryoprotectant when compared with 3- and 14-day old culture. The highest cell survival values obtained with propane-1,2-diol (10%) and DMSO (10%) protection were 96.2 ± 1.2% and 94.0 ± 3.1% respectively. Initial subsequent cell growth after cryopreservation was slower than their non-frozen controls. The survival of transfected BF-2 cells (BF-2/luc1) after cryopreservation were very similar to those obtained with wild type cells being: 94.0 ± 3.1% and 95.2 ± 1.5% respectively with 10% DMSO protection. These results suggested that genetically modified fish cell lines may be equally amenable to cryopreservation as the wild type.

Keywords: bluegill sunfish fibroblast (BF-2), luc gene, transfection, cryoprotectant toxicity, cryopreservation.



CryoLetters 23, 197-208 (2000)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


J. Burch* and T. Wilkinson

Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK.


If an ex situ programme for the conservation of rare and threatened bryophytes is to succeed, a method of long-term, basal storage is required. The aim of this study was to determine the effectiveness of sucrose, ABA and sucrose/ABA combined, as cryoprotectant pretreatments, for the long-term storage of protonemata of the endangered Cornish path moss, Ditrichum cornubicum. This work is a prelude to the in vitro conservation of rare and endangered UK bryophytes. The addition of sucrose and/or ABA in the pre-culture medium, reduced protonemal growth rate. However, pretreatment afforded a high level of protection against tissue damage during dehydration and freezing, with 100 % regeneration of pretreated plants after thawing, compared to 53 % regeneration of controls. Sucrose combined with ABA in the pre-culture medium gave the most effective cryoprotection, with protonemata having fewer necrotic tissues and rapid recovery of growth.

Keywords: cryopreservation, bryophyte, ex situ conservation., sucrose, ABA


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