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The cryopreservation of mammalian embryos has expanded over the past 20 yr by encompassing a range of sophisticated methods to deal with different developmental stages and different sensitivities to low-temperature exposure. We have described a method for slow, controlled-rate freezing of early stage embryos based on exposure to 1,2-propanediol and sucrose, while the method for late-stage (blastocyst) embryos employs mixtures of glycerol and sucrose. Both methods have been used for animal and human embryos. A third rapid cooling or "vitrification" technique is described, which depends on brief but controlled exposure of multicellular embryos to mixtures of glycerol and 1,2-propanediol at high concentrations. This technique is used for successful animal embryo cryopreservation but is not yet widely applied in the clinic. [ABSTRACT FROM AUTHOR]

Two methods for the cryopreservation of mammalian oocytes are described. One method uses a relatively low concentration of the cryoprotectant propanediol plus sucrose and requires controlled-rate cooling equipment to achieve a slow cooling rate. Such a method has produced live births from cryopreserved human oocytes. The second method described employs a high concentration of the cryoprotectant dimethyl sulfoxide plus a low concentration of polyethylene glycol. This method involves cooling by plunging standard straws into liquid nitrogen vapor, hence avoiding the need for specialized equipment, but requires technical ability to manipulate the oocytes quickly in the highly concentrated cryoprotectant solutions. Murine oocytes vitrified, using this technique, have resulted in live births. [ABSTRACT FROM AUTHOR]

Mammalian spermatozoa were among the very first cells to be successfully cryopreserved and over the last five decades the use of frozen-thawed semen for artificial insemination has come to play an important role in domestic livestock production. More recently, semen freezing has increasingly been utilized in the establishment of genetic resource banks for endangered species. Semen is collected, most commonly either by use of an artificial vagina or by electroejaculation of an anaesthetized animal, and basic sperm parameters assessed. Semen is extended using a TEST-egg yolk-glycerol diluent, packaged in 0.25-mL plastic straws and slowly cooled to 5°C over a period of 1-2 h. Cooled straws are frozen by suspending within liquid nitrogen vapor above the liquid nitrogen surface before plunging into the liquid phase. Straws are thawed briefly in air before immersing in a 35°C water bath for 15 s, and often are used directly for insemination without any further processing. [ABSTRACT FROM AUTHOR]

Blood cells can be regarded as a classical field of application of low-temperature biology. Cryopreservation methods have been developed for different categories of blood cells namely red blood cells (RBCs) (erythrocytes), platelets (thrombocytes), mononuclear cells (i.e., lymphocytes, monocytes), and hematopoietic progenitor cells. This chapter outlines the four most commonly applied techniques for RBCs and two for platelets. [ABSTRACT FROM AUTHOR]

Two different approaches have been adopted for the cryopreservation of human embryonic stem cells (hESCs): vitrification and conventional slow cooling/rapid warming. The vitrification method described here is designed for hESCs that grow as discrete colonies on a feeder cell monolayer, and are subcultured by manual subdivision of the colonies into multicellular clumps. hESCs that are subcultured by enzymatic dissociation can more conveniently be cryopreserved by conventional slow cooling/rapid warming methods. Although both methods are suitable for use in a research context, neither is suitable for cryopreservation of embryonic stem cells destined for clinical diagnostic or therapeutic uses without modification. [ABSTRACT FROM AUTHOR]

Cells isolated directly from tissues (primary cultures) have many applications in research and applications in the fields of toxicology, pharmacology, and virology. Where such preparations remain the only scientific option to achieve necessary results it is still possible to refine and reduce the use of animal and human sources of the tissue by cryopreservation of the primary cells for later use, this reduces the need for fresh tissues and enables improvements in standardization through the ability to provide the same cell preparation at different times and to different laboratories. The methods described provide options for adherent cultures as monolayers, harvested cell suspensions, and also lymphocytes isolated from peripheral blood. [ABSTRACT FROM AUTHOR]

To date, more than 25,000 hematopoietic transplants have been carried out across Europe for hematological disorders, the majority being for hematological malignancies. At least 70% of these are autologous transplants, the remaining 30% being allogeneic, which are sourced from related (70% of the allogeneic) or unrelated donors. Peripheral blood mobilized with granulocyte colony stimulating factor is the major source of stem cells for transplantation, being used in approx 95% of autologous transplants and in approx 65% of allogeneic transplants. Other cell sources used for transplantation are bone marrow and umbilical cord blood. One crucial advance in the treatment of these disorders has been the development of the ability to cryopreserve hematopoietic stem cells for future transplantation. For bone marrow and mobilized peripheral blood, the majority of cryopreserved harvests come from autologous collections that are stored prior to a planned infusion following further treatment of the patient or at the time of a subsequent relapse. Other autologous harvests are stored as backup or "rainy day" harvests, the former specifically being intended to rescue patients who develop graft failure following an allogeneic transplant or who may require this transplant at a later date. Allogeneic bone marrow and mobilized peripheral blood are less often cryopreserved than autologous harvests. This is in contrast to umbilical cord blood that may be banked for directed or sibling (related) hematopoietic stem cell transplants, for allogeneic unrelated donations, and for autologous donations. Allogeneic unrelated donations are of particular use for providing a source of hematopoietic stem cells for ethnic minorities, patients with rare human leukocyte antigen types, or where the patient urgently requires a transplant and cannot wait for the weeks to months required to prepare a bone marrow donor. There are currently more than 200,000 banked umbilical cord blood units registered with the Bone Marrow Donors Worldwide registry. In this chapter, we describe several protocols that we have used to cryopreserve these different sources of hematopoietic stem/progenitor cells, keeping in mind that the protocols may vary among transplant processing centers. [ABSTRACT FROM AUTHOR]

The long-term storage of human and animal cell lines is a relatively new branch of the science of cryopreservation of living organisms. The need to maintain the increasing numbers of cell lines, as they started to emerge from research laboratories from the mid-1900s, necessitated a radical approach to the problem. The realization that they could survive cryopreservation, and that a slow rate of cooling is essential for this survival, led to the eventual discovery of cryoprotectants. Subsequent development of mechanical freezers, which can accurately control the rate of cooling, now allows cells to be cryopreserved at their maximum viability. This chapter outlines the essentials steps for the successful preparation, freezing, and storage of cell lines. [ABSTRACT FROM AUTHOR]

This chapter describes a simple system for cryopreservation of avian spermatozoa as pellets, formed by dropping volumes of sperm suspension directly into liquid nitrogen with dimethylacetamide as cryoprotectant. The method originates from the group at the Research Institute of Farm Animal Breeding and Genetics at St. Petersburg-Pushkin and is described here for chicken spermatozoa, but has also been adapted successfully for other species, such as Houbara bustards and pheasants. [ABSTRACT FROM AUTHOR]

This chapter provides detailed descriptions of the protocol used for fish sperm cryopreservation. The principles described can be applied to any species, but details are also given for individual fish species. Successful cryopreservation of fish spermatozoa depends on a range of factors including the collection of high quality sperm, equilibration conditions, choice of cryoprotectant medium, cooling/thawing regimes, and conditions for fertilization. Even though some general rules can be applied to any fish species, optimization of the protocol is needed for each individual species. Furthermore, because sperm derived from marine and freshwater fish differs markedly, the described protocol is mainly focused on the cryopreservation of sperm for freshwater fish because this poses the greater technical challenge. [ABSTRACT FROM AUTHOR]

The cryopreservation of desiccation-tolerant seeds depends on two key steps: specimen dehydration in an environment that ensures the attainment of water contents below the high-moisture freezing limit; and transfer and maintenance at a subzero temperature that may be optimized in relation to the seed-lot moisture content and species. Temperatures about 70°C below the glass transition temperature (Tg-70) or lower are recommended for seed storage. For fully desiccation-tolerant (type I) seeds, cryopreservation success tends to be independent of cooling regime and subzero (°C) temperature treatment when the seeds are dry. However, to maximize recovery of partially desiccation-tolerant (type II) seeds there can be a requirement for slowcooling, avoidance of storage at certain subzero temperatures, and controlled imbibition following cryopreservation, particularly for endospermic seeds that are high in lipid. [ABSTRACT FROM AUTHOR]

Most culturable cyanobacteria and soil microalgae can be cryopreserved with relatively high viability. Furthermore, many freshwater and marine eukaryotic algae can also be cryopreserved, but typically with lower post-thaw viability levels. However, to date, most dinoflagellates, cryptophytes, synurophytes, and raphidophytes cannot be successfully cryopreserved. Marine diatoms can be cryopreserved, and often have high viability, although freshwater diatoms have thus far proven more problematic. Large numbers of strains have been examined, most notably at the four major protistan collections: Culture Collection of Algae and Protozoa (CCAP) (UK), The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) (USA), Sammlung von Algenku Huren Göttingen (SAG) (Germany), and The Culture Collection of Algae at the University of Texas at Austin (UTEX) (USA), and it has been observed that chlorarachniophytes, eustigmatophytes, pelagophytes, phaeothamniophytes, and ulvophytes also have very high success rates, comparable with the other green algae and cyanobacteria. It has been noted that virtually all algae with a large cell size, as well as most filamentous strains, cannot as yet be cryopreserved. There are no known fundamental reasons why large and more complex algae cannot be successfully cryopreserved. Thus, it is anticipated that further research on the basic mechanisms of freezing damage and the empirical development of improved protocols will continue to expand the number and diversity of algal taxa that can be successfully cryopreserved. [ABSTRACT FROM AUTHOR]

Shoot-tip meristem cryopreservation methodologies are reported for the complementary cryoprotective strategies of vitrification and equilibrium freezing using traditional controlled-rate freezing and chemical additive cryoprotection. Pregrowth, pretreatment, and cold acclimation approaches for the improvement of tolerance to liquid nitrogen are also presented. The chapter concludes by reporting an analytical protocol that profiles volatile hydrocarbon stress markers (for ethylene, hydroxyl radicals, and lipid peroxidation products) during cryopreservation. This method uses noninvasive headspace sampling and gas chromatography, and it is widely applicable across cryogenic systems. [ABSTRACT FROM AUTHOR]

The cryopreservation of dedifferentiated cells, grown in suspension culture, is one of the portfolio of techniques employed for the long-term conservation of higher plant germplasm. Suspension cultures are also important in biotechnology, particularly in transformation studies and for the production of specific metabolites, and, here, there is also a pressing need for genetically stable, long-term storage of cell lines. [ABSTRACT FROM AUTHOR]

The aim of preserving a fungus is to maintain it in a viable state without change to its genetic, physiological, or anatomical characters. There are numerous methodologies available to preserve a fungus, but the two methods widely used by culture collections (biological or genetic resource centers) to achieve successful preservation are cryopreservation with liquid nitrogen using controlled-rate freezing and centrifugal freeze-drying. Generic methods are often used, but specific variations of a method may be required in order to achieve optimal stability. No single method can be applied to all fungi. More recently, techniques such as vitrification and encapsulation cryopreservation have been used to preserve recalcitrant fungi. The protocols described within this chapter have been developed over many years at one of the world's largest culture collections of filamentous fungi. [ABSTRACT FROM AUTHOR]

Lyophilization, the removal of water by freezing and then volatilization at low pressure and temperature, has been employed as a standard long-term preservation method for many filamentous fungi. The method outlined involves the use of standard shelf freeze-drying and skimmed milk as a suspending solution/lyoprotectant. This approach has been employed to freeze-dry the majority of the 50,000 fungal strains that have been successfully lyophilized at the Centraal bureau voor Schimmelcultures (CBS) culture collection (http://www.cbs.knaw.nl/). [ABSTRACT FROM AUTHOR]

A method is described that allows a wide range of yeast species to be stored in liquid nitrogen while maintaining a high level of viability. Yeast cultures are sealed in commercially available polypropylene straws after having been mixed with a glycerol-based cryoprotectant. Once placed in a secondary cryotube the temperature of the sealed straws is reduced slowly to -30°C in a methanol bath over a period of up to 3 h. The straws are then transferred directly to the liquid nitrogen and placed in a racking system for long-term storage. [ABSTRACT FROM AUTHOR]

A method is described that allows yeast species to be stored using a variation on the standard freeze-drying method, which employs evaporative cooling in a two-stage process. Yeast cultures are placed in glass ampoules after having been mixed with a lyoprotectant. Primary drying is carried out using a centrifuge head connected to a standard freeze-dryer. Once the centrifuge head is running, air is removed and evaporated liquid is captured in the freeze-dryer. Centrifugation continues for 15 min and primary drying for a further 3 h. The ampoules are constricted using a glass blowing torch. They are then placed on the freeze-dryer manifold for secondary drying under vacuum overnight, using phosphorus pentoxide as a desiccant. The ampoules are sealed and removed from the manifold by melting the constricted section. [ABSTRACT FROM AUTHOR]

Traditionally, the ex situ study of prokaryotes (members of Archaea and Bacteria) has required access to living cultures. Although it may be possible to maintain a small number of strains by serial transfer, such methods are not the best, particularly for long-term storage or for keeping a large collection. One of the major problems is dealing with the vast range of organisms that have been isolated to date. Particularly in the case of methods of cultivating prokaryotes, it is almost impossible to give a simple list of media to use. However, general principles are outlined that should cover most of the currently known prokaryotes in pure culture. The methodologies used for the long-term storage of prokaryotes may be divided into those that involve freezing and those that involve vacuum-drying. The methods described are those that are either used in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (http://www.dsmz.de/index.htm) or in other national culture collections. [ABSTRACT FROM AUTHOR]

Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal and this chapter seeks to analyze some of the mechanisms involved and to show how cooling can be used to produce stable conditions that preserve life. The biological effects of cooling are dominated by the freezing of water, which results in the concentration of the solutes that are dissolved in the remaining liquid phase. Rival theories of freezing injury have envisaged either that ice crystals pierce or tease apart the cells, destroying them by direct mechanical action, or that damage is from secondary effects via changes in the composition of the liquid phase. Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity. Many compounds have such properties, including glycerol, dimethyl sulfoxide, ethanediol, and propanediol. [ABSTRACT FROM AUTHOR]

This chapter describes the methods that can be applied to successfully freeze-dry proteins. Laboratory applications are given at small scale, typified by the purification of a protein intermediate as part of the analytical characterization of a protein, and at intermediate scale, as illustrated by the pilot development of a lyophilized protein reference material such as for use in bioassay or immunoassay. Advice on common problems with freeze-drying of proteins is also given. [ABSTRACT FROM AUTHOR]

This chapter provides an up-to-date overview of freeze-drying (lyophilization) with particulars relevance to stabilizing live cells or viruses for industrial applications as vaccines or seed culture. The chapter discusses the importance of formulation, cycle development, validation, and the need to satisfy pharmaceutical regulatory requirements necessary for the commercial exploitation of freeze-dried products. [ABSTRACT FROM AUTHOR]

The red blood cell (RBC), or erythrocyte, is a flexible biconcave disc 8 µm in diameter. Its main function is to carry oxygen from the lungs to the tissues of the body and to perform this it contains a pigment, hemoglobin. During its 120-d lifespan, it travels about 300 miles around the arteriovenous circulation, repeatedly passing through the capillary bed. As the mean diameter of a capillary is about 3 µm, the red cell has to retain a high degree of flexibility which requires energy. Energy is generated as adenosine triphosphate (ATP) by the anaerobic, glycolytic pathway. [ABSTRACT FROM AUTHOR]

The establishment and maintenance of biological resource centers (BRCs) requires careful attention to implementation of reliable preservation technologies and appropriate quality control to ensure that recovered cultures and other biological materials perform in the same way as the originally isolated culture or material. There are many types of BRC that vary both in the kinds of material they hold and in the purposes for which the materials are provided. All BRCs are expected to provide materials and information of an appropriate quality for their application and work to standards relevant to those applications. There are important industrial, biomedical, and conservation issues that can only be addressed through effective and efficient operation of BRCs in the long term. This requires a high degree of expertise in the maintenance and management of collections of biological materials at ultra-low temperatures, or as freezedried material, to secure their long-term integrity and relevance for future research, development, and conservation. [ABSTRACT FROM AUTHOR]

Spermatozoa were the first mammalian cells to be successfully cryopreserved (1), and the techniques developed by Sherman (2) enabled centers to begin to establish sperm banks for men at risk of losing their fertility as a result of clinical treatments for illness. Banks of cryopreserved donor sperm have become an integral part of assisted reproduction procedures, allowing the treatment of couples with male factor infertility by ensuring that samples are available when required while conferring anonymity. However, more recently it has become essential that human cells for donation are quarantined to reduce the risks of contamination, and semen is required to be stored for several months to ensure the requisite tests for HIV are performed. Although pregnancies from both in vitro fertilization (IVF) and intrauterine insemination (IUI) are common, cryopreservation causes cell lysis, a reduction in motility, an increase in the number of cells undergoing spontaneous acrosome reaction, an alteration in the distribution of acrosin (3, 4), and an overall decrease in fertility (5). Sublethal cryodamage is apparent in postthaw analysis of donor sperm. Spermatozoa may be initially intact and motile, however, viability postthaw is lost rapidly over time. This may be a result of cold shock damage, although it appears that human sperm are less susceptible than many mammalian species, possibly as a result of the high sterol content of the membrane, since high cholesterol levels stabilize membranes during cooling (6). [ABSTRACT FROM AUTHOR]

Although the first observations concerning low temperature preservation of spermatozoa date back to 1776 (when Spallanzani noted that spermatozoa, cooled in snow, became inactive but were revived on warming), successful cryopreservation protocols truly date from only the 1940s and 1950s. Around this time semen preservation was in the forefront of the new science of cryobiology and was important in the formulation of the first theories concerning the causes of cryoinjury. In 1938 Jahnel noted the survival of human spermatozoa stored at the temperature of solid carbon dioxide, an observation taken up by Parkes in 1945 (1), but the single most important development came with the discovery by Polge, Smith, and Parkes in 1949 (2) that glycerol could act as a cryoprotectant for spermatozoa. The initial experiments with glycerol were performed using fowl spermatozoa but were quickly followed by the successful preservation of bull spermatozoa (3) with the first calf from artificial insemination (AI) using frozen/thawed spermatozoa reported in 195l (4). However, early success with the bull rather belied the difficulties that became apparent with other domestic species. The use of glycerol had allowed bull spermatozoa to survive at the comparatively slow freezing rates obtained using methanol cooled with solid CO2, and with semen contained in large glass-freezing ampules. [ABSTRACT FROM AUTHOR]

In contrast to the extensive research and advances in the cryopreservation of animal cell lines and tissues, few methodologies are available for the cryopreservation of invertebrate cells. Several research groups have reported methodologies for marine invertebrate gametes (1-3); these predominantly concentrate on the spermatozoa of the Pacific oyster (Crassostrea gigas). Marine invertebrate gametes, embryos, and larvae have been extensively deployed to monitor marine pollution and to assess the biological quality of coastal and oceanic waters (4-7). This interest stems from the relative ease of their laboratory culture and the sensitivity of the early developmental stages to environmental pollutants, when deployed in biological effects monitoring programs (4,5,7). The use of cryopreserved oyster larvae as material for use in bioassays of water quality has recently been advocated (7). [ABSTRACT FROM AUTHOR]

Storage of seeds is arguably the most effective and efficient method for the ex situ preservation of plant genetic resources. Low storage costs, combined with ease of seed distribution and regeneration of whole plants from genetically diverse material, offer distinct advantages for the storage for conservation of seeds compared with other types of plant tissues, such as meristems and pollen. [ABSTRACT FROM AUTHOR]

Deposition at cryogenic temperatures is generally considered to be the safest method for long-term storage of living materials. (For a general introduction into the storage of plant cell cultures, refer to Chapter 11. [ABSTRACT FROM AUTHOR]

Plant cell and tissue culture is widely used in fundamental research (cell physiology, molecular biology and genetics, developmental biology, and so on) as well as in commercial activities (breeding and vegetative multiplication of food and feed crops and ornamentals). Several factors inherent to these types of work render the long-term storage of the materials urgent. Some of these are: 1. Costs of maintenance: Compared to the culture of many microorganisms, maintenance of plant cells and tissues is costly in terms of labor and equipment. 2. Stability: Plant cells have a remarkable flexibility, with respect to both composition of the genome (endoreduplication to higher ploidy levels is not uncommon in the plant kingdom; somatic mutations, aneuploidy, and amplification frequently occur in tissue cultures) and epigenetics (gene expression patterns are less stable than for most animal cell types). These mechanisms may render material with desired traits valueless. A well-recognized phenomenon is the loss during subculture of cells or tissues of the potential to regenerate into complete plants. 3. Patenting: As a rule, patented material has to be deposited in a publicly accessible culture collection. [ABSTRACT FROM AUTHOR]

Isolated plant protoplasts are most commonly liberated from tissues, [ABSTRACT FROM AUTHOR]

Eukaryotic microalgae and prokaryotic cyanobacteria (blue-green algae) have traditionally been maintained by serial subculture, with the frequency of transfer being largely determined by the growth characteristics of the strain. This technique has the dual disadvantages of being labor intensive and carrying risk of loss in genetic stability of the strain. Compared with other groups of microorganisms, relatively little research has been carried out on the development of long-term preservation methods for microalgae and cyanobacteria. Freeze-drying has not been found to be a successful biostorage method for microalgae, with very low levels of viability (<1% of original population) (1, 2) and a further reduction in viability on prolonged storage (3, 4). The successful lyophilization of the cyanobacterium Nostoc muscorum, using a method similar to that detailed in Chapter 3, has been reported, with no observed reduction in viability after 5 yr storage (5). This technique has been adopted by a small number of researchers to preserve selected cyanobacterial strains. [ABSTRACT FROM AUTHOR]

Chloroplast thylakoids are the site of photosynthetic light reactions, electron transport, adenosine triphosphate (ATP) synthesis and nicotinamide adenine dinucleotide phosphate (NADP) reduction in plant cells. Isolated thylakoids are therefore the necessary prerequisite for many in vitro studies of photosynthetic mechanisms. They are also used to elucidate aspects of photosynthetic responses to freezing, chilling, heat, and high light stress. For many of these studies it is important to work with membranes of closely similar properties over extended periods of time to facilitate direct comparisons of the results. The pigment, protein, and lipid composition of thylakoid membranes, however, vary with plant growth conditions (1-3). Even the solute permeability properties of thylakoids are significantly different between warm-and cold-grown spinach plants (4). [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are unicellular, eukaryotic protozoa found in virtually all soil and aquatic environments (1). All FLA are characterized by a feeding and replicating trophozoite form that, in most genera, can produce a resistant cyst stage in response to adverse conditions (1). FLA claim our interest not only as fundamentally interesting microbes about which there is still much to be learned, but also because of the capacity for certain species to cause serious and often fatal infections in humans. The amoeboflagellate Naegleria fowleri is found in thermally polluted water and causes primary amoebic meningoencephalitis in previously healthy persons (2-4). Members of the genus Acanthamoeba are among the most common FLA and some species cause granulomatous amoebic encephalitis in the immunocompromised host and a destructive eye infection in previously healthy persons termed Acanthamoeba kerdtitis (2, 4) (see Note 1). [ABSTRACT FROM AUTHOR]

Freezing and freeze-drying techniques have become standard methods for the long-term maintenance of bacterial cultures. Both methods of preservation provide varying degrees of success with different species of bacteria, and neither technique results in 100% recovery of preserved cells (1). There is no universally applicable method for the successful preservation of all bacteria, and where it is vitally important that cultures are not lost, it is advisable to use both methods in parallel. [ABSTRACT FROM AUTHOR]

Viruses are noncellular forms of life and are much smaller and less biochemically complex than the simplest unicellular organisms. They consist of either RNA or DNA as a single molecule, or in some cases as a segmented genome, enclosed by one or more proteins. These proteins protect the nucleic acid from degradation; deliver it to the host cells that reproduce the virus; transcribe the nucleic acid (in the case of negative stranded genomes); and assist the virus to expose the nucleic acid to the biochemical machinery inside susceptible host cells. This relative simplicity has in part been the secret of the success of viruses in coexisting with all known life forms. [ABSTRACT FROM AUTHOR]

Nature dictates that biological material will decay and die. The structure and function of organisms will change and be lost with time, as surely in laboratory cultures as in the biologists who study and manipulate them. Attempts to stop the biological clock have been conjured by minds ancient and modern; at the heart of many such schemes have been experiments with temperature and water content. Whereas refrigeration technology provides a means of slowing the rate of deterioration of perishable goods, the use of much lower temperatures has proved a means of storing living organisms in a state of suspended animation for extended periods. The removal of water from viable biological material in the frozen state (freeze-drying) provides another means of arresting the biological clock by withholding water, and commencing again by its addition. [ABSTRACT FROM AUTHOR]