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Abstracts: Cryoletters 22 (2), 2001

CryoLetters 22, 77-90 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

1-2 September 2000,St. Martin's College, Ambleside, Cumbria

Glyn N. Stacey1* and John G. Day2

1 National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Herts., EN6 3QG, UK.
2 Centre for Ecology and Hydrology, Far Sawrey, Ambleside, Cumbria, LA22 0LP, UK

Symposium: Time Travel for Genes

Dr Stanley Leibo (ACRES, Louisiana) reviewed a broad range of issues in the long term viability of preserved cells with particular reference to his experience in the preservation of sperm and oocytes. He reviewed the key developments and factors in successful cryopreservation of a range of cell types. Dr Leibo went on to highlight a significant and largely ignored problem of the variability of success in long term storage between different groups of workers. Theoretically it is evident that it is highly unlikely that the survival of cells cryopreserved by similar methods should show significant differences in between laboratories. Dr Leibo suggested that it is more likely that the differences arose from the quality of the storage process. He related a number of examples of catastrophic failure in maintenance of storage vessels resulting in the loss of precious biological material.

Dr Peter Glenister (FESA, Harwell UK) described successful approaches in the preservation of mouse embryos and new developments that have enabled the preservation of mouse sperm. He identified the variable response of some strains of mouse to embryo cryopreservation. Dr Glenister also raised the issue of viral contamination and methods to remove contaminating virus from embryos by washing. In general discussion it was evident that the procedures required to achieve virus removal depended on species specific characteristics of the outer surface of the embryo.

The role of Biological Resource Centres (BRCs) in the provision of reference strains of microorganisms for industry, agriculture and research was presented by Dr David Smith (CABI Bioscience, Egham, UK). He described the dependence of such centres on the existence of effective cryopreservation methods and emphasised the need for BRCs to maintain close involvement in the development of cryopreservation technologies. This was seen as vital to improve current preservation methods and also address the substantial groups of microorganism for which successful preservation techniques do not as yet exist.

In the fields of agriculture and biodiversity conservation cryopreservation is playing an increasingly important role. Dr Barbara Reed (USDA, Corvallis, Oregon) described the major issues in preserving and maintaining frozen stocks of plant tissue from a diverse species range. It appears that plant tissue preservation presents some particularly complex challenges since even closely related varieties and species show very different responses to preservation procedures. 

The symposium discussion revisited a number of issues including: the importance of cellular nutrition prior to cryopreservation, avoidance of chilling injury at 0oC by rapid cooling in the early phases of the cooling profile and most importantly the requirement for adequate desiccation.

The symposium was jointly organised by the SLTB and the UK Federation for Culture Collections (UKFCC). Dr Smith's and Dr Reed's full papers are included in this issue of CryoLetters.


Scientific Meeting of the Society for low temperature biology

The state-of-the-art in the preservation of human tissue was reviewed by Professor David Pegg (University of York, UK) who described the challenges encountered in the preservation, the techniques employed to measure cryoprotection and the use of in vitro cell culture systems to model tissue responses. His paper appears in this issue of CryoLetters.

New developments in cryobiology were presented by speakers from a wide range of scientific disciplines. Mr Q Huang (University of York, UK) presented data on the penetration of skin specimens by cryoprotectant for tissue banking procedures. From the field of plant biology the mode of action of plant fructans as a novel class of cryoprotectants was described (Dr Dirk Hincha, Max Plank Institute, Germany) and new achievements in the preservation of garlic and blackberry species were reported by Graham Souch (University of Derby, UK) and Graham Sherlock (University of Abertay, Dundee, UK) respectively. The development of preservation protocols for rare species of plant is made difficult by the lack of experimental material. Musuthami Jayanthi (Forest Research Institute, Kuala Lumpur, Malaysia) presented data on the Taguchi experimental design as an alternative approach to traditional factorial experimental designs in an attempt to reduce the quantity of germplasm material consumed in the development of cryopreservation techniques for tropical rain forest tree species. Preservation of cryopreservation recalcitrant fungi belonging to the genus Serpula was described by Dr Nia White (University of Abertay, Dundee, UK). She described successful preservation of these organisms in desiccated alginate beads. This method is becoming increasingly popular for preservation of plant and fungal species for which standard preservation protocols have proven unsuccessful. Mathew Ryan (CABI Bioscience, Egham, UK) described the use of molecular techniques to measure the influence cryopreservation on the stability of gene function. This presentation was awarded the George Planer Prize for the best oral presentation by a young scientist at the meeting, and the full paper appears in this issue of CryoLetters. The presentations also included some novel observations on cryopreservation using non-linear cooling rates (Jane Morris, Asymptote Ltd., Cambridge, UK) and the process by which water is excluded from erythrocytes during freezing (John Morris, Asymptote Ltd., Cambridge, UK). These provoked stimulating in depth discussion, which indicate that there are significant areas of cryobiology that would benefit from further fundamental research work.

In addition to the oral presentations, there were 19 poster papers covering diverse topics including; the application of specialist techniques in cryobiology (Differential Scanning Calorimetry, Electron Microscopy and 1H NMR); the development of gene banks and database development and electronic knowledge sharing.

Undoubtedly, one of the highlights of the meeting was the annual SLTB Lecture, given by Dr Christopher Polge FRS who reviewed the early development of cryopreservation. He described the events leading to his seminal discovery of the cryoprotective effects of glycerol which facilitated, for the first time, the cryopreservation of animal cells. He emphasised that even with the dramatic progress made in this field in the last 50 years the critical factor in successful cryopreservation remained the desiccation step prior to cooling and freezing.

The Ambleside meeting acted as a focus for the collation and discussions on a huge amount of cryobiological research undertaken over the last 50 years. No single approach or cell type dominated the meeting and valuable interactions between those involved with the preservation of micro-organisms, gametes and embryos, plant tissues and human cells and tissues formed the most important and stimulating sections of the meeting. Furthermore, the detailed discussions on long-term storage identified some common issues and novel solutions to assure the security, reliability and safety of low temperature storage systems.


Acknowledgement: The authors would like to thank the Wellcome Trust and the UK Federation for Culture Collections who provided sponsorship for the symposium "Time Travel for Genes".



CryoLetters 22, 91-96 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Provision and maintenance of micro-organisms for industry and international research networks

David Smith

CABI Bioscience UK Centre, Egham, Surrey, TW20 9TY. UK


Biological Resource Centres of preserved micro-organisms provide important biological resources for industry, agriculture and research. The development of cryopreservation methods has enabled the stocks of organisms to be maintained in a stable form free from the hazards of contamination, genetic variation and laboratory accidents. Resource centres can therefore offer reliable and reproducible sources of particular micro-organisms and groups working on biodiversity have methods available to carry out in vitro biological conservation. Nevertheless, some micro-organisms remain recalcitrant to standard cryopreservation methods and new developments in cryopreservation techniques are needed to provide the necessary methodologies for reliable preservation of some important micro-organisms.

Keywords: cryopreservation, cryomicroscopy, fungi, nematodes, culture collections, biodiversity



CryoLetters 22, 97-104 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Barbara M. Reed

National Clonal Germplasm Repository, U.S. Dept. of Agriculture, Agricultural Research Service, 33447 Peoria Rd, Corvallis, OR 97333-2521, USA. Email:


Methodology for plant germplasm cryopreservation was created in the 1970s, expanded in the 1980s, and implemented at the end of the 20th century.  Translating experimental techniques into routine cryostorage of a clonal collection requires attention to details beyond those normally required for methods development.  Early decisions include the choice of accessions to be stored, number of each accession per storage unit, number of replicates, location of storage, viability testing, record keeping and proper control groups.  Emphasis should be placed on selecting a secure storage site and compiling complete records needed for the recovery of plant material. Secure remote storage, duplicate locations, and secure, accurate records are all important in ensuring the safety and usefulness of base collections.  Evaluation of cryostored collections should be initiated to determine the longevity of plants and stability of storage conditions. Collections of several clonal genera are now stored in liquid nitrogen and more are in progress worldwide. These base-storage collections of clonal germplasm provide security for safeguarding long-term access to genetic diversity that is vital to food security and to continued improvement of many clonally-propagated agricultural crops.

Keywords: germplasm, cryopreservation



CryoLetters 22, 105-114 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


David E Pegg

Medical Cryobiology Unit, Dept of Biology, University of York, York YO10 5YW,UK.


Cryopreservation plays an important role in tissue banking and will assume even greater importance when tissue engineering becomes an everyday reality. For some tissue grafts, living cells are unnecessary and adequate preservation methods are usually available. For other tissues living and functioning cells are needed and preservation methods are much less advanced. The basic requirements for cell recovery can usually be defined if a few basic biophysical properties of the cell are known and some standard measurements of the effect of cryobiological variables are carried out. The problems in tissue cryopreservation are not usually due to difficulties in preserving the living cells per se, but arise from the properties of the intregrated cell/matrix systems upon which tissue function almost always depends. Some examples of such difficulties are described. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most likely way forward. However, two major problems still to be overcome are cryoprotectant toxicity and recrystallization during rewarming. Less obvious, and certainly less well understood is chilling injury  – damage caused by reduction in temperature per se; this may yet turn out to be of fundamental importance.

Keywords:  Tissue cryopreservation; dimethyl sulphoxide; artery; cartilage; chilling injury; dielectric heating; antifreeze protein.



CryoLetters 22, 115-124 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Matthew J. Ryan*1, Peter Jeffries2, Paul D. Bridge3& David Smith1

1CABI Bioscience, Bakeham Lane, Egham, Surrey. TW20 9TY,  2Department of Biosciences, University of Kent, Canterbury, Kent. CT2 7NJ,  3Department of Biological and Chemical Sciences, Birkbeck, University of London, Malet Street, WC1E 7HX, UK
*Corresponding author


Protocols that involve a freezing process are frequently used for the preservation of fungi, but there have been few studies to assess the physiological and genetic stability of isolates after preservation. In this study, the effects of cryopreservation and lyophilisation on the viability, physiology and genetic stability of isolates of Metarhizium anisopliae, Fusarium oxysporum and Serpula lacrymans were examined. It was found that preservation regime influenced the characters of some of the test isolates. Secondary metabolite profiles and extracellular enzyme production can be affected by preservation. Polymorphism's were detected after PCR fingerprinting in replicates of 2 isolates of Metarhizium that had been preserved by cryopreservation and lyophilisation. The results indicated a need to improve existing preservation protocols. Research is being undertaken to develop novel cryopreservation regimes for recalcitrant micro-organisms. Biochemical and molecular techniques will be used to assess the effectiveness of preservation.

Keywords: Cryopreservation, Lyophilisation, Stability, PCR fingerprinting



CryoLetters 22, 125-134 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Biologically Relevant Environmental Data: Macros to make the most of microclimate recordings

Brent J. Sinclair

Department of Zoology, University of Otago, PO Box 56, Dunedin, New Zealand.


The increased availability and use of automatic data loggers has made collection of microclimatic data cheap and easy. I present macros for the quantitative analysis and comparison of microclimatic (particularly temperature) data in the spreadsheet package Microsoft Excel. The macros presented will (1) collate maximum and minimum values of temperature (or other) cycles, (2) provide a count of the number of times a temperature recording crosses a given threshold, (3) measure the period of time spent above or below a given threshold, and (4) measure the rate of change of a data set as it approaches a threshold. A case study of the freeze tolerant grasshopper Sigaus australis (Orthoptera: Acrididae) and winter temperatures from its alpine habitat is used to demonstrate the macros. 

Keywords: Microclimate, freeze tolerance, cold hardiness, Orthoptera, data logger, Microsoft Excel



CryoLetters 22, 135-140 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Cryopreservation of lipid-rich seeds: effect of moisture content and cooling rate on germination

M. Elena González-Benito* and Félix Pérez-García

Departamento de Biología Vegetal, Escuela Universitaria de Ingeniería Técnica Agrícola, Universidad Politécnica de Madrid, Ciudad Universitaria, 28040 Madrid, Spain.


The effect of fast and slow cooling on germination of seeds from two Brassicaceae species (Eruca vesicaria (L.) Cav., Brassica napus L. var. oleifera (Moench) DC cv. Bingo) and cypselas from three Compositae species (Onopordum nervosum Boiss., Onopordum acanthium L., Helianthus annuus L. cv. Viky) at different moisture contents was studied. Seed lipid content (dry weight basis) ranged from 15% (O. nervosum) to 41% (H. annuus). For each species, seeds with four moisture contents were cryopreserved either by direct immersion in liquid nitrogen or by previous cooling at 10ºC/min from room temperature to –50ºC. In three species (E. vesicaria, B. napus, and H. annuus) germination of air-dried (6.2-8.9% moisture content) seeds cooled by direct immersion in liquid nitrogen was not significantly different from germination of control seeds (air-dried, non-cooled). In the two Onorpordum species the best response among cooling treatments was observed when air-dried seeds were slowly cooled.

Abbreviations: mc= moisture content; RH= relative humidity

Keywords: Brassicaceae, Compositae, cooling rate, cryopreservation, lipid content, liquid nitrogen, moisture content, oilseed rape, seed germination, sunflower.

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