Freezing of Cells and Bacteria

In 1968, Meryman [1.20] presented his ideas about the minimum cell volume and hypothesized that during feezing cell are damaged in two steps. Initially water diffuses from the cell to the surrounding, the freezing solution concentrating the solu- 

De Antoni et al. [1.23] demonstrated that the addition of trehalose during freezing and thawing of two strains of Lactobacillus bulgaricus improved the survival rate differentially, but in both cases considerably. The samples (1 mL) were frozen at 18 °C/min to -60 °C and thawed to 37 °C at 15 °C/min. The solution consisted of distilled water, culture medium and 10% milk with or without trehalose. It was shown that after three freeze-thaw cycles, milk alone resulted in a survival rate of 24 or 65%, whereas with trehalose this was could be improved to 32 and 100%, respectively. The efficacy in the case of both strains was clearly different. De Antoni et al. suggested that the efficiency of milk was related to its Ca2+ content, whereas the trehalose could replace water molecules in the phospholipids of the membranes. However, no mention was made of whether other sugar molecules in milk showed any effect. 

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Preservation of cell structure, food taste, and avoidance of thermal degradation are reasons for the removal of moisture from such materials by sublimation. The process is preceded by quick freezing which forms small crystals and thus minimum damage to cell walls, and is likely to destroy bacteria. Some of the materials that are being freeze dried commercially are listed in Table 19.9(b). 

In terms of shape, the first of these are rod-shaped and are called bacilli (singular, bacillus). The bacilli often have small, whip-like structures known as flagella, with which they are able to move about. Some bacilli have oval, egg-shaped, or spherical bodies in their cells, known as spores. Under adverse conditions, such as dehydration, and in the presence of disinfectants, the bacteria may die, but the spores may be able to live on. The spores germinate when the conditions become favorable, and form new bacterial cells. Some are so resistant that they can withstand boiling and freezing temperatures and prolonged desiccation. See Fig. 1. 

The field of cryobiology is also rather diffuse, for it includes some who use low temperatures as a practical tool, others who are concerned with how they affect plants and animals in nature, and still others who are interested in the physical and chemical phenomena induced in cells by freezing. To date, it is the practical aspects that have received the major share of attention [ ]. There have been, for example, numerous attempts to preserve the viability of living cells and tissues by freezing and low-temperature storage. Many of these have been successful, but others have not. Cells and tissues that have been successfully frozen include sperm of various animals, red blood cells, a variety of human tissues, bacteria, molds, viruses, and cancer tissues [-]. 

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