There is a special reason for reviewing this book at this time: it is the 50th edition of a compendium that is known and used frequently in most chemical and physical laboratories in many parts of the world. Surely, a publication that has been published for 56 years, withstanding the vagaries of science in this century, must have had something to offer. There is another reason: while the book is a standard fixture in most chemical and physical laboratories, including those in medical centers, it is not as frequently seen in the laboratories of physician's offices (those either in solo or group practice). I believe that the Handbook can be useful in those laboratories. One of the reasons, among others, is that the various basic items of information it offers may be helpful in new tests, either physical or chemical, which are continuously being published. The basic information may relate

Handbook of Chemistry and Physics

Water is the most abundant component of biological material, but it is systematically excluded from conventional electron microscopy. This is because water evaporates rapidly under the vacuum conditions of an electron microscope. Cryoelectron microscopy has long been seen as a possible avenue to overcome this limitation, but until recently the direct observation of frozen-hydrated specimens was relatively unsuccessful because of a number of serious difficulties. These were, in particular, due to the absence of a good cryospecimen holder, the inherently low contrast of hydrated specimens and the structural damage due to ice crystals formed during freezing. As a consequence, the cryomethods which have flourished in electron microscopy during the last 20 years were not aimed at preserving the hydration of the specimen in the electron microscope. Freezing was only used as an aid to preparation. The objects ultimately observed in the electron microscope were dry and at room temperature. Such cryomethods have recently been reviewed in detail (Robards and Sleytr 1985).

/pdf/cryo-electron-microscopy-of-vitrified-specimens-46t0snibk6.pdf

Cryo-electron microscopy of vitrified specimens.

Cells can endure storage at low temperatures such as--196 degrees C for centuries. The challenge is to determine how they can survive both the cooling to such temperatures and the subsequent return to physiological conditions. A major factor is whether they freeze intracellularly. They do so if cooling is too rapid, because with rapid cooling insufficient cell water is removed osmotically to eliminate supercooling. Equations have been developed that describe the kinetics of this water loss and permit one to predict the likelihood of intracellular freezing as a function of cooling rate. Such predictions agree well with observations. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration. The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage. Recent experiments, however, indicate that the damage is due more to the decrease in the size of the unfrozen channels. This new view of the mechanism of slow freezing injury ought to facilitate the development of procedures for the preservation of complex assemblages of cells of biological, medical, and agricultural significance.

Freezing of living cells: mechanisms and implications

The failure of complex mammalian organs, such as the kidney, to function following freezing to low temperatures is thought to be due largely to mechanical disruption of the intercellular architecture by the formation of extracellular ice. Classical approaches to the avoidance of ice formation through the imposition of ultra-rapid cooling and warming rates or by gradual depression of the equilibrium freezing point during cooling to -80 degrees C have not been adequate. An alternative approach relies on the ability of highly concentrated aqueous solutions of cryoprotective agents to supercool to very low temperatures. At sufficiently low temperatures, these solutions become so viscous that they solidify without the formation of ice, a process termed vitrification. When embryo suspensions are cryopreserved using conventional procedures, this supercooling behaviour allows intracellular vitrification, even in the presence of extracellular ice. We have therefore used mouse embryos to examine the feasibility of obtaining high survival following vitrification of both the intra- and extracellular solutions and report here that in properly controlled conditions embryos seem to survive in high proportions after cryopreservation in the absence of ice.

Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification.

http://science.sciencemag.org/content/sci/168/3934/939.full.pdf

Cryobiology: The Freezing of Biological Systems

IN 1959, Lovelock and Bishop1 reported the protective action of dimethylsulphoxide (DMSO) in preventing freezing injury to living calls. The “cryoprotective” action of this substance, now in wide use2 for the frozen preservation of various types of living cell, presumably resides in part in its ability to reduce the extent to which salts are concentrated when water is converted to ice. On the basis of this assumption Farrant3,4 proposed and developed two procedures by which DMSO might be used to prevent altogether the concentration of the soluble electrolytes in tissues cooled to temperatures in the range −78° to −196° C. In the course of his studies, Farrant obtained preliminary data describing the phase equilibrium behaviour of the system water–DMSO, both in the absence and in the presence of small quantities of salts. In view of the current interest in the use of DMSO we undertook a more extensive determination of the phase equilibrium and non-equilibrium behaviour of solutions of this compound in water. These measurements form the subject of this communication. They indicate (1) that the system water–DMSO can, in all proportions, be crystallized completely, and (2) that, in certain conditions, a stable hydrate (DMSO.3H2O) is formed.

Phase Diagram for the System Water–Dimethylsulphoxide

Study by differential thermal analysis of the temperatures of instability of rapidly cooled solutions of glycerol, ethylene glycol, sucrose and glucose.

The specific heats and densities of supercooled water and heavy water have been measured from the respective melting points to the respective homogeneous nucleation temperatures. The calorimetric measurements indicate structural contributions to the differences between the specific heats of the liquids and the corresponding crystals. These ``structural specific heats'' are exponential functions of the supercooling. The results are interpreted in terms of Frenkel's heterophase fluctuation theory. It is concluded that, during supercooling, water molecules tend to form icelike clusters incorporating six or more monomers.

Clustering in supercooled water

A new experimental technique for studying the dynamics of bubble growth in thermoplastics using scanning electron microscopy is developed. The influence of temperature, saturation pressure, molecular weight, and the nature of physical blowing agent are investigated. The experimental results show that, the above, process variables control the growth of foams during processing. The existing Newtonian model for the growth of a single bubble in an infinite amount of polymer has been modified to account for the non-Newtonian effects by modeling the polymer as a power law fluid. The experimental data has been compared with the appropriate viscoelastic cell model which considers the growth of closely spaced spherical bubbles during the foaming process. The simulation results indicate that the predictions of the cell model are in qualitative agreement with the trends of the experimental data and the quantitative agreement is reasonable. The cell model also gives an equilibrium radius which agrees with the experimental data. Other viscous models do not predict the equilibrium radius of the bubble and underpredict the experimental data.

Don H. Rasmussen

Papers

Phase Diagram for the System Water–Dimethylsulphoxide

Study by differential thermal analysis of the temperatures of instability of rapidly cooled solutions of glycerol, ethylene glycol, sucrose and glucose.

Clustering in supercooled water

Numerical and experimental studies of bubble growth during the microcellular foaming process

The glass transition in amorphous water. Application of the measurements to problems arising in cryobiology.