Relative influence of unfrozen fraction and salt concentration on the survival of slowly frozen eight-cell mouse embryos

This laboratory has previously reported that the survival of frozen-thawed human erythrocytes is determined more by the fraction of the extracellular solution that remains unfrozen than by the salt concentration in that fraction, especially when the cells are frozen at low hematocrit. To determine the extent to which these findings are applicable to nucleated mammalian cells, we have studied the survival of some 3300 mouse embryos as a function of the unfrozen fraction and the concentration of salt in that unfrozen fraction. Also varied in the study was the weight percentage ratio of glycerol to salt. The concentration of embryos in these experiments (i.e., the cytocrit) was so low that embryo-embryo contacts should have been rare during the freezing. As in the case of the red cells at low hematocrit, we find that the survival of slowly frozen eight-cell embryos is not affected by the high concentrations of salt produced by freezing, at least up to 3.3 molal NaCl, and therefore is not affected by the extent to which the cells shrink below their isotonic volume, nor in general is survival influenced by the temperature at which given salt concentrations and unfrozen fractions are attained or by the glycerol concentration at those temperatures. On the other hand, the attainment of low values of the unfrozen fraction (U) is damaging, but the damage appears in part to be due to the fact that low values of U had to be achieved by placing embryos in solutions hypotonic with respect to NaCl, which caused their volume to be greater than isotonic prior to freezing.     

Permeability Properties of Rickettsia mooseri

The passive permeability properties of Rickettsia mooseri to both inorganic and organic solutes have been examined. Visual observations by phase-contrast microscopy of rickettsiae in macerated yolk sacs taken directly from heavily infected eggs revealed plasmolysis with hypertonic NaCl and KCl as well as with sucrose solutions. In contrast, similar visual studies of rickettsiae which had been subjected to freezing or to a purification process, or both, were plasmolyzed by hypertonic sucrose but not by hypertonic NaCl and KCl. These primary observations were extended to a variety of solutes and were placed on a quantitative basis by use of optical density and radioisotope dilution methods. Intracellular Na(+) and K(+) concentrations in processed rickettsiae, measured by flame photometry, closely paralleled the concentration of these ions in the suspending medium. It was concluded that R. mooseri appears to possess an osmotically active, functional, and structural membrane distinct from the cell wall, located at the surface of a structure analogous to the bacterial protoplast. In the intact organism, this membrane is passively impermeable to sucrose, NaCl, and KCl. However, altered permeability properties, especially to inorganic electrolytes, may be expected in rickettsiae which have been stored in the frozen state and subjected to a lengthy purification process.     

Analysis of “solution effects” injury: Rabbit renal cortex frozen in the presence of dimethyl sulfoxide

Slices of rabbit renal cortex were frozen in 0.64 or 1.92 M dimethyl sulfoxide (Me₂SO) to various subzero temperatures, thawed, and assayed for viability. Salt and Me₂SO concentrations were calculated and correlated with the injury taking place during freezing. In separate experiments, slices were treated with NaCl or Me₂SO in concentrations sufficient to simulate the exposure brought about as a result of freezing. The effects of these treatments on cortical viability were compared with the results of freezing to equivalent concentrations of either NaCl or Me₂SO. The results show that whereas slices will tolerate exposure to at least six times the isotonic concentration of NaCl at 0 °C, they are unable to tolerate even three times the isotonic salt concentration when frozen in 1.92 M Me₂SO. They can, however, tolerate 3 × NaCl when frozen in 0.64 M Me₂SO. Freezing damage did not depend upon the amount of ice formed per se, since slices frozen in the low concentration of Me₂SO tolerated removal of about 75% of the initial fluid content of the system, whereas slices frozen in 1.92 M Me₂SO did not tolerate an identical removal of unfrozen solution. It was found that treatment of slices with high concentrations of Me₂SO at subzero temperatures in accordance with Elford's application (14) of Farrant's method (20) produced damage which correlated approximately with the damage observed when the same concentrations of Me₂SO were produced by freezing. It is concluded that most of the damage caused by freezing in 1.92 M Me₂SO is produced either directly or indirectly by Me₂SO. Possible mechanisms for this injury are discussed.     

The effect of initial tonicity on freeze/thaw injury to human red cells suspended in solutions of sodium chloride

Human red blood cells, suspended in solutions of sodium chloride, have been frozen to temperatures between -2 and -14 degrees C and thawed, and the extent of hemolysis was measured. In parallel experiments, red cells were exposed to similar cycles of change in the composition of the suspending solution, but by dialysis at 21 degrees C. The tonicity of the saline in which the cells were initially suspended was varied between 0.6x isotonic and 4x isotonic; some samples from each experimental treatment were returned to isotonic saline before hemolysis was measured. It was found that the tonicity of the saline used to suspend the cells for the main body of the experiment affected the amount of hemolysis measured: raising the tonicity from 0.6x to 1x to 2x reduced hemolysis, both in the freezing and in the dialysis experiments, whereas raising the tonicity further to 4x reversed that trend. There was little difference between the freeze/thaw and the dialysis treatments for the cells suspended in 1x or 2x saline, whether or not the cells were returned to isotonic conditions. However, the cells suspended in 0.6x saline showed greater damage from freezing and thawing than from the comparable change in the composition of the solution, whether or not they were returned to isotonic conditions. Cells that were suspended in 4x saline and exposed to changes in salt concentration by dialysis showed less hemolysis when they were assayed in the 4x solution than cells that had received the comparable freezing/thaw treatment, but when the experiment included a return to isotonicity, the two treatments gave similar results. Returning the cells to isotonic saline had a negligible affect on the cells in 0.6x and 1x saline, but caused considerable hemolysis in the 2x and 4x samples, more so after dialysis than after freezing and thawing. We conclude that cells suspended in 0.6x and 4x saline behave differently from cells suspended in 1x and 2x saline and hence that cells suspended in a range of solutions of differing initial tonicity should not be treated as a homogeneous population. We argue that an effect of the unfrozen fraction of water (U) cannot be distinguished, within the framework of these freeze/thaw experiments alone, from an effect of initial tonicity, and that the biphasic nature of the correlation between haemolysis and U makes a causal connection improbable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Ultrastructure-function correlative studies for cardiac cryopreservation. V. Absence of a correlation between electrolyte toxicity and cryoinjury in the slowly frozen,cryoprotected rat heart

Hearts were frozen to ?17 °C in the initial presence of 2.1 m DMSO. Attempts were made to prevent or minimize the consequences of an osmotic shock based on Lovelock's classical hypothesis of freezing injury. Substitution of mannitol or potassium for NaCl before freezing did not improve the results, nor did perfusion of thawed hearts with hyperosmotic perfusate. It was found that freezing and thawing resulted in a significant attenuation of coronary flow and that, as a result of this, DMSO was apparently retained within the heart after thawing. DMSO was also difficult to remove at 30 °C in the absence of prior freezing and caused a significant drop in coronary flow upon institution of DMSO washout with balanced salt solution. The blanching of freezing and thawing was also seen, in milder form, in nonfrozen hearts. For both frozen-thawed and nonfrozen hearts, the blanching was associated with DMSO washout with balanced salt solution. Flow was improved by perfusion with hyperosmotic perfusate in both nonfrozen and in frozen-thawed hearts, but the improvement was largely temporary. Evidence from earlier studies indicates that electrolyte concentrations during freezing cannot be correlated with cardiac cryoinjury, in support of the present findings. It is suggested instead that cryoprotectant toxicity may be the chief agent of injury under the conditions studied.     

The freezing point depression of mammalian tissues in relation to the question of osmotic activity of cell fluid

The freezing point depression of freshly excised frozen tissues, pulverized in a hydraulic press or in a mortar, is greater than that of plasma. Even at 0°C. the freezing point depression of such homogenates increases significantly with time. Dilution data indicate that such freezing point data are valid. The presence of intact cells has been shown in smears of tissues pulverized in a mortar, but not in smears of those crushed in a hydraulic press. The osmolarity of various diluent solutions affects the calculated osmotic activity of tissue homogenates presumably because of delayed diffusion between the diluent and cell fluid. With a hypertonic NaCl diluent, spuriously low values of tissue osmotic activity are found from calculations assuming instantaneous mixing between homogenates and diluents. The limitations of data from cryoscopic experiments and from tissue-swelling experiments are discussed in relation to the basic question of whether or not cell fluid is isotonic to extracellular fluid.     

Roles of unfrozen fraction, salt concentration, and changes in cell volume in the survival of frozen human erythrocytes

The cause of slow freezing injury and the basis of the protection by solutes like glycerol are subjects of debate. During slow freezing, cells are sequestered in unfrozen channels between ice crystals that grow by removing pure water from the channels. As a consequence, the solute concentration in the channels rises and the volume of liquid in the channels progressively decreases. The rise in solute concentration, in turn, causes the cells to progressively shrink osmotically. Until recently cryobiologists have ascribed slow freezing injury to either the rise in solute (electrolyte) concentrations in the channels or to the consequent cell shrinkage, rather than to the decrease in the of the channels. Although ordinarily reciprocally coupled, it is possible to separate the composition of the channels from their size, or more precisely from the magnitude of the unfrozen fraction, by suspending cells in NaCl/cryoprotectant solutions in which the mole ratio of the two is held constant, but the molality of the NaCl is allowed to vary below and above isotonic. When human red cells are frozen in such solutions to temperatures that produce given NaCl concentrations (ms), but varying unfrozen fractions (U), survival at low U is found to be strongly dependent on U but independent of ms. At higher values of U, survival becomes inversely dependent on both ms and U. Although cell volume during freezing is independent of the NaCl tonicity in the solution, the cells in the several solutions differ in volume both prior to the onset of freezing and after the completion of thawing. We have now examined and compared the effect of returning the thawed cells to isotonic solutions and isotonic volume or nearly so, and find that there is little change in survival after exposure to low U, but that survival after exposure to high U values exhibits substantially increased sensitivity to ms, a sensitivity that is probably a manifestation of posthypertonic hemolysis. Low values of U were in general attained by the use of solutions with low tonicities of NaCl, and as a consequence cells frozen to low U values had larger volumes prior to freezing than cells frozen to higher U values. The significance of this confounding is discussed.     

On the mechanism of injury to slowly frozen erythrocytes.

When cells are frozen slowly in aqueous suspensions, the solutes in the suspending solution concentrate as the amount of ice increases; the cells undergo osmotic dehydration and are sequestered in ever-narrowing liquid-filled channels. Cryoprotective solutes, such as glycerol, reduce the amount of ice that forms at any specified subzero temperature, thereby controlling the buildup in concentration of those other solutes present, as well as increasing the volume of the channels that remain to accommodate the cells. It has generally been thought that freezing injury is mediated by the increase in electrolyte concentration in the milieu surrounding the cells, rather than reduction of temperature or any direct action of ice. In this study we have frozen human erythrocytes in isotonic solutions of sodium chloride and glycerol and have demonstrated a correlation between the extent of damage at specific subzero temperatures, and that caused by the action at 0 degrees C of solutions having the same composition as those produced by freezing. The cell lysis observed increased directly with glycerol concentration, both in the freezing experiments and when the cells were exposed to corresponding solutions at 0 degrees C, showing that the concentration of sodium chloride alone is not sufficient to account quantitatively for the damage observed. We then studied the effect of freezing in anisotonic solutions to break the fixed relationship between solute concentration and the volume of the unfrozen fraction, as described by Mazur, P., W. F. Rall, and N. Rigopoulos (1981. Biophys. J. 653-675). We confirmed their experimental findings, but we explain them differently. We ascribe the apparently dominant effect of the unfrozen fraction to the fact that the cells were frozen in, and returned to, anisotonic solutions in which their volume was either less than, or greater than, their physiological volume. When similar cell suspensions were subjected to a similar cycle of increase and then decrease in solution strength, but in the absence of ice (at 20 degrees C), a similar pattern of hemolysis was observed. We conclude that freezing injury to human erythrocytes is due solely to changes that occur in the composition of their surrounding milieu, and is most probably mediated by a temporary leak in the plasma membrane that occurs during the thawing (reexpansion) phase.     

Factors contributing to inactivation of isolated thylakoid membranes during freezing in the presence of variable amounts of glucose and NaCl.

During freezing of isolated spinach thylakoids in sugar/salt solutions, the two solutes affected membrane survival in opposite ways: membrane damage due to increased electrolyte concentration can be prevented by sugar. Calculation of the final concentrations of NaCl or glucose reached in the residual unfrozen portion of the system revealed that the effects of the solutes on membrane activity can be explained in part by colligative action. In addition, the fraction of the residual liquid in the frozen system contributes to membrane injury. During severe freezing in the presence of very low initial solute concentrations, membrane damage drastically increased with a decrease in the volume of the unfrozen solution. Freezing injury under these conditions is likely to be due to mechanical damage by the ice crystals that occupy a very high fraction of the frozen system. At higher starting concentrations of sugar plus salt, membrane damage increased with an increase in the amount of the residual unfrozen liquid. Thylakoid inactivation at these higher initial solute concentrations can be largely attributed to dilution of the membrane fraction, as freezing damage at a given sugar/salt ratio decreased with increasing the thylakoid concentration in the sample. Moreover, membrane survival in the absence of freezing decreased with lowering the temperature, indicating that the temperature affected membrane damage not only via alterations related to the ice formation. From the data it was evident that damage of thylakoid membranes was determined by various individual factors, such as the amount of ice formed, the final concentrations of solutes and membranes in the residual unfrozen solution, the final volume of this fraction, the temperature and the freezing time. The relative contribution of these factors depended on the experimental conditions, mainly the sugar/salt ratio, the initial solute concentrations, and the freezing temperature.     

Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes.

As suspensions of cells freeze, the electrolytes and other solutes in the external solution concentrate progressively, and the cells undergo osmotic dehydration if cooling is slow. The progressive concentration of solute comes about as increasing amounts of pure ice precipitate out of solution and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. The consensus has been that slow freezing injury is related to the composition of the solution in these channels and not to the amount of residual liquid. The purpose of the research reported here was to test this assumption on human erythrocytes. Ordinarily, solute concentration and the amount of liquid in the unfrozen channels are inversely coupled. To vary them independently, one must vary the initial solute concentration. Two solutes were used here: NaCl and the permeating protective additive glycerol. To vary the total initial solute concentration while holding the mass ratio of glycerol to NaCl constant, we had to allow the NaCl tonicity to depart from isotonic. Specifically, human red cells were suspended in solutions with weight ratios of glycerol to NaCl of either 5.42 or 11.26, where the concentrations of NaCl were 0.6, 0.75, 1.0, 2.0, 3.0, or 4.0 times isotonic. Samples were then frozen to various subzero temperatures, which were chosen to produce various molalities of NaCl (0.24-3.30) while holding the fraction of unfrozen water constant, or conversely to produce various unfrozen fractions (0.03-0.5) while holding the molality of salt constant. (Not all combinations of these values were possible). The following general findings emerged: (a) few cells survived the freezing of greater than 90% of the extracellular water regardless of the salt concentration in the residual unfrozen portion. (b) When the fraction of frozen water was less than 75% the majority of the cells survived even when the salt concentration in the unfrozen portion exceeded 2 molal. (c) Salt concentration affected survival significantly only when the frozen fraction lay between 75 and 90%. To find a major effect on survival of the fraction of water that remains unfrozen was unexpected. It may require major modifications in how cryobiologists view solution-effect injury and its prevention.     

Intracellular freezing of glycerolized red cells.

The response of glycerolized human red blood cells to freezing has been evaluated in terms of the thermodynamic state of the frozen intracellular medium. The physiochemical conditions requisite for intracellular freezing, characterized by the cooling rate and the degree of extracellular supercooling, are altered appreciably by the prefreezing addition of glycerol to the cells.Fresh human erythrocytes were suspended in an isotonic glycerol solution yielding a final cryophylactic concentration of either 1.5 or 3.0 m. Subsequently the cell suspension was frozen on a special low temperature stage, mounted on a light microscope, at controlled constant cooling rates with varying degrees of extracellular supercooling (ΔT_sc). The formation of a pure intracellular ice phase was detected by direct observation of the cells.The addition of glycerol produced several significant variations in the freezing characteristics of the blood. As in unmodified cells, the incidence of intracellular freezing increased with the magnitudes of both the cooling rate and the extracellular supercooling. However, the glycerolized cells exhibited a much greater tendency to supercool prior to the initial nucleation of ice. Values of ΔT_sc > ?20 °C were readily obtained. Also, the transition from 0 to 100% occurrence of intracellular ice covered a cooling rate spectrum in excess of 300 to 600 °K/min, as compared with 10 °C/min for unmodified cells. Thus, the incidence of intracellular ice formation was significantly increased in glycerolized cells.     

DNA replication in eucaryotic nuclei. Evidence suggesting a specific model of replication

Preimplantation-stage mouse embryos suspended in dimethyl sulfoxide (DMSO) have been used as a model to study details of the response of a simple multicellular system to freezing and thawing. Rapid freezing to ?196 °C kills the embryos unless they have first been cooled very slowly to at least below ?50 °C. The survival of both 2-cell and 8-cell embryos has been found to depend as critically on the rate at which the frozen embryos were thawed as on the rate at which they were first frozen. The damaging consequences of thawing frozen embryos too rapidly have been shown to occur between ?70 and ?20 °C. Finally, the survival of embryos as a function of the time in DMSO prior to freezing and thawing has been compared with their volume changes as a function of time in DMSO. This comparison leads to the tentative conclusion that dimethyl sulfoxide need not permeate the embryos to protect them against freezing damage. Overall, the embryos' response to freezing and thawing is qualitatively similar to that displayed by many other cell types.     

The freezing point depression of mammalian tissues after sudden heating in boiling distilled water

The calculated freezing point depression of freshly excised boiled mammalian tissue is approximately the same as that of plasma. The boiling procedure was chosen to eliminate the influence of metabolism on the level of the freezing point depression. Problems created by the boiling, such as equilibrium between tissue and diluent, change in activity coefficient by dilution, and loss of CO(2) content, are discussed. A frozen crushed tissue homogenate is hypertonic to plasma. Boiling and dilution of such hypertonic homogenate exposed to room temperature for 5 to 15 minutes did not produce significant or unexplicable decreases in its osmotic activity. Moreover, freezing and crushing of a boiled diluted tissue did not produce any increase of the isoosmotic level of freezing point depression. It is possible to explain these data either with the hypothesis of hypertonic cell fluid or with that of isotonic cell fluid. In the case of an assumed isotonic cell fluid, data can be explained with one assumption, experimentally backed. In the case of an assumed hypertonic theory data can be explained only with the help of at least three ad hoc postulates. The data support the validity of the classical concept which holds that cell fluid is isotonic to extracellular fluid.     

Effect of cooling and rewarming rates on glycerolated human erythrocytes.

Human erythrocytes suspended in a sodium-free buffered salt solution containing glycerol in 1 m concentration (1 part of packed cells to 4 parts buffered salt solution) were frozen by slow, moderately rapid, or very rapid cooling to various subzero C temperatures. The frozen specimens, after a 5-min storage period at a given temperature, were thawed at low, moderately high, or very high rates. The hemolysis in the frozen and thawed samples was measured by a colorimetric determination of the hemoglobin released from the damaged cells. At ?10 °C, the highest freezing temperature employed, nearly 100% recovery of intact erythrocytes was obtained irrespective of the cooling and rewarming conditions. The extent of the hemolysis after exposure to lower freezing temperatures depended upon the cooling and rewarming conditions. Moderately rapid and very rapid freezing to, and thawing from temperatures below ?40 °C permitted significantly higher recoveries of intact cells than the other freezing/ thawing combinations. In the temperature range ?15 to ?30 °C the combination slow cooling and slow rewarming afforded maximum protection. Very rapid freezing/ slow thawing was the most damaging combination throughout the entire freezing range. The results were interpreted in part by a conventional two-factor analysis, lower cooling rates allowing concentrated salts to determine hemolysis, higher cooling rates destroying the cells by intracellular freezing. Apparent anomalies were explained in terms of a generalized “thermal/osmotic” shock according to which the erythrocytes were subject to greater hemolysis the higher the rates of cooling and/or warming.     

Effect of hypertonicity on survival of unprotected human cultured cells following freezing and thawing

An investigation was carried out on the post-thaw survival of unprotected human heteroploid EUE cells, either maintained in isotonic medium (0.137 M NaCl) or adapted to hypertonicity (0.356 M NaCl) and frozen in medium with an increased concentration of NaCl. A fivefold increase in the survival fraction of the adapted cells in comparison with the unadapted ones was observed when cells were frozen in isotonic medium. When cells were frozen in hypertonic medium (0.356 M NaCl), the two cell types exhibit comparable survival values. The results are discussed, with special attention to cell defense mechanisms against freezing injury.     

Some factors contributing to the survival of rapidly cooled plant cells

The stability of innocuous intracellularly frozen cells was found to be influenced remarkably by the surrounding solutions and the degree of prefreezing. In the cells suspended in isotonic or slightly hypertonic glucose solution, innocuous intracellular freezing readily occurred and these rapidly frozen cells remained stable even at −30 °C for 20 min, while cells prefrozen to −10 °C after suspending in water survived standing at −30 °C only for 5 sec at most. These facts suggest that when the innocuous intracellularly frozen cells are surrounded with concentrated sugar solutions, these cells remain far more stable than those surrounded with ice.     

Effects of freezing of bovine preimplantation embryos derived from oocytes fertilized in vitro on survival of their inner cell mass cells

The morphology of the inner cell mass (ICM) cells and the proportion of dead ICM cells in frozen-thawed bovine preimplantation embryos were investigated by differential fluorochrome staining. Embryos at the blastocyst stage of development were frozen and thawed by two different techniques (three-step and one-step) in two different basic salt solutions (PBS and TCM 199) containing 1.36M glycerol. After thawing and glycerol removal, embryos were co-cultured in a cumulus cells monolayer in TCM 199 for 48 hr (morula) or 24 hr (blastocysts). Differential cell counts of the ICM and trophectoderm were then done using differential fluorochrome staining. Overall, there was no significant difference in the viability of embryos frozen in the two basic salt solutions. Low proportions of dead ICM cells were observed in embryos frozen at the morula stage in both PBS (19.1%) or TCM 199 (18.0%). However, blastocyst stage embryos frozen by the three-step technique had a higher (P < 0.05) proportion of dead ICM cells in TCM 199 (37.7%) than in PBS (18.2%). Blastocysts frozen by the one-step technique had a higher (P < 0.05) proportion of dead ICM cells (42.2%) than those frozen by the three-step technique (18.2%), regardless of basic salt solutions. Results indicate that freezing and thawing damages ICM cells in morphologically normal embryos and that the degree of damage depended on the basic salt solution and the freezing method. © 1994 Wiley-Liss, Inc.     

Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Summary Pea (Pisum sativum) root nodule cells infected by the diazotrophRhizobium leguminosarum have been well characterized by chemical fixation techniques. Propane-jet freezing and high pressure freezing were used in this study to compare rapidly frozen and chemically fixed pea root nodule cells. Cells that had been incubated in 2-(N-morpholino)ethanesulfonic acid buffer and frozen with the propane-jet freezer were better preserved than cells that had been chemically fixed or frozen with the high-pressure freezer. Rapidly frozen infected nodule cells showed that the rough endoplasmic reticulum had a high frequency of associations with the peribacteroid membrane and the infection thread. The peribacteroid space also varied in size depending on the method of preservation; however, it was most reduced in size and devoid of inclusions in the propane-jet frozen tissue. The biological significance of these observations is discussed.Abbreviations HPF high-pressure freezing - MES 2-(N-morpholino)ethanesulfonic acid - PBM peribacteroid membrane - PBS peribacteroid space - PJF propane-jet freezing - RER rough endoplasmic reticulum