Tokio Nei

Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures. I. Microscopic observation of hemolyzing erythrocytes during the freezing and thawing process.

Summary Observations with a special cryomicroscope have shown that a number of crythrocytes are damaged during freezing over a temperature range from −1.5°C to −10°C. The erythrocytes which remain intact after freezing hemolyze when the frozen material is thawed.

Growth of ice crystals in frozen specimens

The formation of ice crystals, which might be the possible artefact in cryo‐techniques for electron microscopy, was examined during the rewarming process of rapidly frozen erythrocytes.

Freezing injury to erythrocytes. I. Freezing patterns and post-thaw hemolysis

The extent of hemolysis of human red blood cells suspended in different concentrations of glycerol and frozen at various cooling rates was investigated on the basis of morphological observation in the frozen state. Hemolysis of the cells in the absence of glycerol showed a V-shaped curve in terms of cooling rates. There was 70% hemolysis at an optimal cooling rate of approximately 103 °C/min and 100% hemolysis at all other rates tested. Morphologically, a lower than optimal cooling rate resulted in cellular shrinkage, while a higher than optimal rate resulted in the formation of intracellular ice. The cryoprotective effect of glycerol was dependent upon its concentration and on the cooling rate. Samples frozen at 103 and 104 °C/min showed freezing patterns which differed from cell to cell. The size of intraand extracellular ice particles became smaller, and there was less shrinkage or deformation of cells as the rate of cooling and concentration of glycerol were increased. There was some correlation between the morphology of frozen cells and the extent of post-thaw hemolysis, but the minimum size of intracellular ice crystals which might cause hemolysis could not be estimated. As a cryotechnique for electron microscopy, the addition of 30% glycerol and ultrarapid freezing at 105 °C/min are minimum requirements for the inhibition of ice formation and the prevention of the corresponding artifacts in erythrocytes.

Mechanism of freezing injury to erythrocytes: effect of initial cell concentration on the post-thaw hemolysis.

Abstract It has been previously reported that the post-thaw hemolysis of erythrocytes, frozen under various conditions, depends upon the initial cell concentration; increasing the cell concentration decreases the proportion of intact cells after freeze-thawing. In the present study, the effect of cell concentration upon post-thaw hemolysis, examined mainly by the morphological observation of freezing patterns in specimens with or without cryoprotectant glycerol, was most marked in concentrated cell suspensions in which the cells had become shrunken as a result of extracellular freezing. The addition of glycerol lessened the packing effect progressively as the concentration was increased. The results thus obtained may be explained by assuming that cells, deformed in the freezing process, and rigid at low temperatures, might undergo mechanical damage when subjected to compression and abnormal contact.

Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures: II. Investigations of factors affecting hemolysis by freezing

Summary The mechanism of hemolysis during freezing and thawing at temperatures ranging from the freezing point to −10°C was investigated under various experimental conditions. The cell and solute concentrations of the specimen have a direct effect on hemolysis during freezing. Investigations of freezing injury to erythrocytes in the frozen state showed that the high hemolysis observed can hardly be explained by increases in salt concentration alone. Morphological observations agree with the quantitative measurements of hemolysis in almost all cases. The results of the present experiments suggest that, in the freezing process at near-zero temperatures, mechanical damage is one of the most important causes of hemolysis of erythrocytes.

Effect of residual moisture content on the survival of freeze-dried bacteria during storage under various conditions

Summary Aqueous suspensions of Escherichia coli were dried under various conditions of freeze-drying to obtain residual moisture contents ranging from approximately 0 to 20%. The effect of the residual moisture content on the survival rate of these cells during relatively short storage periods was investigated. The results showed that a high residual moisture content produced a rapidly lowered survival rate in cells stored under vacuum or nitrogen, and that the reverse was true for cells stored in air. Higher storage temperatures also produced lowered survival rates.

Freezing injury to erythrocytes. II. Morphological alterations of cell membranes.

Abstract Morphological alterations of human red blood cell membranes were examined with the cells containing different concentrations of glycerol being subjected to rapid rates of cooling, approximately 104 and 105 °C/min, and subsequent rewarming. Small membrane defects, similar to holes, were observed in specimens frozen with and without 10% glycerol. Various degrees of roughness were found on the surface of the cells at all freezing rates tested. The membrane alterations were reduced with increasing glycerol concentration, although roughness also appeared on the surface of the cells in 30% glycerol suspensions, frozen rapidly, and rewarmed to −80 or −60 °C. The cell membrane surface texture correlated with the growth of intra- and extracellular ice particles. There was also a positive correlation between these alterations and post-thaw hemolysis. It is concluded, therefore, that morphological alterations appearing on the erythrocyte membranes may be a manifestation of freezing damage.

Studies of the effect of drying conditions on residual moisture content and cell viability in the freeze-drying of microorganisms

Summary A series of studies were made to investigate the effect of the dehydration of cellular water upon cell viability during the drying process in freeze-drying of microorganisms. Water suspensions of Escherichia coli cells were used in these experiments, and the drying conditions were controlled in order to determine how to obtain dried cells that retained a high percentage of residual moisture. The influence of the temperature and the drying rate upon the relationship between the residual moisture content and cell survival was investigated. From the results obtained, it was ascertained that the temperature and the drying rate in secondary drying did not greatly influence the survival rate of the dried cells. In general, it was assumed that survival of the dried cells is primarily affected by dehydration of unfreezable cellular water and therefore depends upon the residual moisture content.

Freezing and freeze-drying of microorganisms☆

Some papers reporting the effects of intense cold on microorganisms had already appeared in the 19th century and since then there have been a great many investigations of the freezing of microorganisms.3* 53 ‘I, 12 These experiments demonstrate that a wide variety of microorganisms can survive cooling to and storage at very low temperatures. In the past ten years, basic research on. freezing of living cells has advanced spectacularly, particularly investigations of the mechanism of cellular injury produced by freezing and thawing of microorganisms. However, time does :not permit me to give detailed descriptions of all of these investigations. I would like to mention the reasons why microorganisms are frozen. The primary reason is the preservation of their viability at low temperatures. Repeated subculturing, a common procedure in culture collections, can be eliminated by freezing.’ Another reason is to maintain cellular morphology rather than viability. In t’his case, freezing is just the first step in the procedure of freeze-substitution or freeze-drying. The third reason is just the opposite of the first t,wo. In this case freezing is used as a method of disintegrating microorganisms. Such freezing results in mechanical destruction of the cell structure and release of the cellular constituents with little or no effect upon the enzymatic activity. Thus, there are many reasons for freezing microorganisms. In practical use, the freezing conditions, particularly the cooling rates, must be carefully controlled in every instance, since the freezing pattern depends primarily upon the rate of cooling. The materials must be cooled at the most effect’ive rate to obtain the desired results : for ins-tance, preservation of viability requires slow cooling, preservation of morphology requires ultrarapid cooling and disintegration requires repeated, rapid freezing.

Freeze-drying process of biological specimens observed with a scanning electron microscope

A pit membrane was observed with a cryo‐SEM during the course of dehydration at low temperatures. The freeze‐drying process of sea‐urchin eggs and parenchyma cells of higher plants was also examined with this microscope. Conditions for observation of frozen specimens in the native state were discussed on the basis of morphological studies of alterations such as shrinkage or deformation which appeared during the freeze‐drying process.