An experimental comparison of intracellular ice formation and freeze-thaw survival of hela S-3 cells

A small-volume fluorescent dye viability assay has been successfully applied to a conduction cryomicroscope freezing-thawing stage as a means of determining post-thaw survival of the nucleated mammalian cell HeLa S-3. The survival signature for HeLa S-3 cells has been determined, revealing an optimum cooling rate of −30 °C/min where the maximum survival is 30%. No cells survive for cooling rates greater than −128 °C/min and the decreased survival at supraoptimal cooling rates coincides with a linear increase in the percentage of cells containing intracellular ice from 0% at −16 °C/min to 100% at −128 °C/min.Although no data were taken to identify increased salt concentration as the mechanism responsible for cell injury at suboptimal cooling rates, the post-thaw leakage of intracellular fluorescent dye at these rates takes approximately 4–10 min as opposed to instantaneous release of dye for cells which contain ice at the high cooling rates. This indicates two modes of damage.Cell number density has been identified as an important parameter in freezing studies since survival can be enhanced at slow rates by packing cells together in groups. Packing also causes a greater fraction of the cells in a sample to have intracellular ice present, thus decreasing survival at the faster rates. These responses can be explained by assuming that the outer cells in a group protect the inner ones from solution damage at slow rates, yet restrict water flux from the inner cells at faster rates, causing an increased likelihood of intracellular ice formation. Both of these results are consistent with the dual-mechanism freezing damage theory proposed by Mazur.     

Effect of nifedipine on core cooling in rats during tail cold water immersion

Male rats (450 g, n=11/group) were heated at an ambient temperature of 42°C until a rectal temperature of 42.8°C was attained. Rats, then received either saline (30°C)+tail ice water immersion (F+I) or saline (30°C)+tail ice water immersion+Nifedipine, a peripheral vasodilator, (F+I+N) to determine cooling rate effectiveness and survivability. The time to reach a rectal temperature of 42.8°C averaged 172 min in both groups resulting in similar heating rates (0.029°C/min). The cooling rates in group F+I and F+I+N were not significantly different from each other. We conclude that since Nifedipine did not improve cooling rates when combined with fluid+tail ice water immersion, its use as a cooling adjunct does not seem warranted.     

Evaluation of different cooling rates, equilibration periods and diluents for effects on deep-freezing, enzyme leakage and fertility of taurine bull spermatozoa

We studied the effects of 2 diluents (Tris and milk), 4 cooling rates (10°C/30°C to 5°C for 1 or 2 h), 2 equilibration periods (0 and 2 h) and their interactions on the freezability, glutamic oxaloacetic transaminase (GOT) leakage and fertility of frozen-thawed semen in 18 ejaculates from 3 Friesian bulls. The means of pre- and post-freezing motility, GOT leakage and fertility rates (52.81% based on follow up of 267 inseminated cows) were significantly (P<0.01) influenced by the bulls, cooling rates & equilibration periods, but not by diluents or the interactions studied. The mean prefreeze motility of spermatozoa following 1 h of cooling from 10°C to 5°C was significantly lower (60.38%) and that after 2 h of cooling from 30°C to 5°C was higher (72.38%) than 2 h of cooling from 10°C to 5°C (66.57%) or 1 h of cooling from 30°C to 5°C (67.96%). The mean post-thaw motility observed following 2 h of prefreeze cooling was, however, significantly greater (45%) than after 1 h of cooling (35%) for both the initial temperatures. Leakage of GOT pre- and post-freezing was significantly less following 2h of cooling from 30°C to 5°C (17.26 and 27.36 μmole/L) than after 1 h of cooling from either 10°C (19.71 and 30.13 μmole/L) or 30°C (18.95 and 29.58 μmole/L) and 2 h of cooling from 10°C to 5°C (21.43 and 34.48 μmole/L). The conception rates for semen frozen at the above cooling rates (66.13, 48.65, 56.67 and 42.25%, respectively) were inverse to GOT leakage. An equilibration period of 2 h over that of 0 h at 5°C adversely affected the prefreeze motility and GOT leakage, but it significantly improved postthaw motility (44.03 vs 35.49%) and fertility rates (57.86 vs 47.24%). These findings suggested that both Trisand milk-based diluents were equally efficacious for cryopreservation of bovine semen, and that slow cooling of semen straws over a period of 2 h from 30°C to 5°C as compared with faster cooling rates or a lower initial temperature (10°C), plus at least 2 h of equilibration time at 5°C were essential for optimal freezability, lower enzyme leakage & higher fertility rates within the tropics.     

Characteristics of galactomannanase for degrading konjac gel

Galactomannanase (Glmnase) is an enzyme product derived from Aspergillus niger. The activity of Glmnase degrading (hydrolyzing) the konjac gel were investigated. Significant loss in the enzyme activity was found when the temperature above 60 °C. Similar observations were obtained when the reaction pH above 5. Further increase in the pH value resulted in entirely loss of enzyme activity at the alkaline pH region (pH 8.0 and above). The optimal hydrolyzing temperature and pH were at 60 °C and 5.0, respectively. For the stability test, the purified Glmnase increased its thermostability up to 70 °C at pH 5.0, but it retained only about 60% activity after 60 min incubation at this temperature and its activity became zero after 20 min incubation at 80 °C. The Glmnase was stable at the pH range from 3.0 to 7.0 at room temperature and retained at least 80% activity for 60 min. For the storage temperature test, the lyophilized Glmnase still conserved about 90% activity during 7 days at 30 °C, and was higher than about 80% at 4 °C. The K_m and V_max, were 0.018 mg/ml konjac powder and 0.20 mg/ml reducing sugar per min, respectively.     

Freeze-Dehydration for Permanent Mounts after Aceto-Orcein Stain

Fresh smears of mammalian material were stained 5 min (only) by La Cour's (1941) aceto-orcein method at 60°C, briefly rinsed and drained, and then dehydrated in a prechilled equal-parts mixture of absolute alcohol and anhydrous acetone held 2 hr or more at — 20°C or lower by means of dry ice. The preparations were differentiated 30 sec at room temperature in 95% alcohol containing 1% HCl, counterstained (optional) in a 0.01% solution of fast green FCF in absolute alcohol for 10 sec, passed through 2 changes of xylene and mounted in HSR or other synthetic resin.     

Cryopreservation of mouse ovarian tissue following prolonged exposure to an Ischemic environment.

In cases in which ovarian tissue is to be cryopreserved for tissue or gene banking it is important to maintain its integrity and viability. This study examined how delays between the death of an animal and the collection/cryopreservation of its ovarian tissue influenced follicle viability. Mouse ovaries were placed in PBS+antibiotic (in vitro) or left within the body (in situ) at room temperature for 0, 3, 6, 12, or 24 h following the death of the donor. These ovaries were cryopreserved at 1 degrees C/min on dry ice or in a -84 degrees C freezer using a passive cooling device or by conventional slow cooling (0.3 degrees C/min). The ovaries were grafted under the kidney capsule of ovariectomized recipient mice and collected 2 weeks later, and the size and number of follicles were determined. Cryopreserved ovarian tissue grafted immediately after the death of the donor contained numerous viable and healthy follicles independent of the cooling procedure (dry ice, 134 +/- 32; -84 degrees C, 165 +/- 54; slow, 214 +/- 55 follicles per half ovary). Tissues stored in vitro before cryopreservation retained viable follicles up to 12 h after death (dry ice, 30 +/- 15; -84 degrees C, 86 +/- 45; slow, 93 +/- 33), whereas tissue left in situ had significantly reduced follicle numbers within 3 h of death (dry ice, 36 +/- 12; -84 degrees C, 19 +/- 6; slow, 28 +/- 7). No significant difference was found between the cooling rates tested, indicating that a passive cooling container which cools at 1 degrees C/min is a suitable alternative to conventional slow cooling. We conclude that ovarian tissues for cryobanking should be cryopreserved as soon as possible after collection or death of the animal to ensure maximal follicular survival.     

Spherulitic crystallization of gelatinized maize starch and its fractions

Binary systems of polymers often display spherulitic morphologies after cooling from the melt, but these phenomena have rarely been reported among food polymers of native-size. Here we report the observation of spherulitic and other morphologies in gelatinized maize starch. The morphology could be manipulated by choosing polymer compositions and kinetic regimes. Spherulites (10 μm diameter) formed from gelatinized high-amylose maize starches and purified amylose at cooling rates of order of magnitude 100 °C/min. They were more numerous and exhibited a higher melting point the greater the ratio of amylose to amylopectin. Rapid cooling rates (150–500 °C/min) resulted in a more even distribution of smaller spherulites. Very rapid (liquid nitrogen quench) or slow (0.1–1 °C/min) cooling rates resulted in mixed morphology, as did addition of 15 or 60% (w/w) sucrose to a 10% (w/w) dispersion of high-amylose starch (HAS). Spherulites were observed in aqueous suspensions of high-amylose maize starch between 5 and 30% (w/w). Lower starch concentrations resulted in a broader size distribution and spherulites of more distinct shape. WAXS patterns of B-type were observed. Negatively birefringent spherulites predominated, but positive spherulites were found. The spherulite melting range overlapped with that for amylose–lipid complex. Evidence indicated that micro-phase separation takes place when a holding period at 95 °C follows gelatinization at 180 °C. Despite the high maximum temperature of treatment (180 °C) there was evidence for a memory effect in samples of 30% HAS. Spherulite morphology closely resembled that of native starch granules in very early stages of development.     

Relative tolerance of cirripede larval stages to acute thermal shock: A laboratory study

(1) Meroplankters drawn into once-through cooling circuits of coastal power plants are subjected to transient thermal stress. The effect of such acute thermal shock on the development of barnacle larvae was studied in the laboratory.

(2) The response of the barnacle larvae (naupliar and cyprid stages) to elevated temperature was dependent on exposure time and their stage of development.

(3) Among the stages tested, N-6 larvae showed maximum tolerance. Exposure to 37°C did not affect larval survival, but delayed development of N-2 larva to cypris by one day.

(4) Exposure at 40°C delayed, hastened or did not affect the development time of N-2 and N-4 larvae through cypris, depending on exposure time.

(5) Ten mins exposure at 43°C proved lethal to all larval stages with mortality ranging from 20 to 86%.

(6) Development success of the surviving larvae, measured in terms of cypris yield, showed no significant difference from controls, at temperatures below 40°C.

(7) Settlement activity was significantly affected in only those cyprid larvae which were exposed to 43°C for 10 min.

(8) Results of the present study indicate that thermal stress experienced in the once-through cooling system does not have significant impact on survival and development of the barnacle larvae at temperatures of 37–40°C.     

Vitrification of human monocytes

Human monocytes purified from peripheral blood by counterflow centrifugal elutriation were cryopreserved in a vitreous state at 1 atm pressure. The vitrification solution was Hanks' balanced salt solution (HBSS) containing (w/v) 20.5% Me2SO, 15.5% acetamide, 10% propylene glycol, and 6% polyethylene glycol. Fifteen milliliters of this solution was added dropwise to 1 ml of a concentrated monocyte suspension at 0 degrees C. Of this, 0.8 ml was drawn into silicone tubing and rapidly cooled to liquid nitrogen temperature, stored for various periods, and rapidly warmed in an ice bath. The vitrification solution was removed by slow addition of HBSS containing 20% fetal calf serum. The numerical cell recovery was about 92% and most of these retained normal phagocytic and chemotactic ability. Differential scanning calorimeter records of the solution show a glass transition at -115 degrees C during cooling and warming, but no evidence of ice formation during cooling. Devitrification occurs at about -70 degrees C during warming at rates as rapid as 80 degrees C/min. The amount of devitrification is dependent upon the warming rate. Freeze-fracture freeze-etch electron microscope observations revealed no ice either intra- or extracellularly in samples rapidly cooled to liquid nitrogen temperatures except for small amounts in some cellular organelles. However, if these cell suspensions were warmed rapidly to -70 degrees C and then held for 5 min, allowing devitrification to occur, the preparation contained significant amounts of both intra- and extracellular ice. Biological data showed that this devitrification was associated with severe loss of cell function.     

Application of the Nauta-Gygax Technic for Degenerating Axons to Mounted Sections

Frozen sections of formalin-fixed brains containing lesions were mounted on slides that had been coated first with albumen-glycerol (1:1) then 4% gelatin and blotted. The slides were placed in formaldehyde vapor at 56° C for 40-60 min, washed, and stored (optional) in 10% formalin-saline. The staining technic was as follows: after washing, soak 30-40 min in 0.5% phosphomolybdic acid, rinse; put in 0.05% potassium permanganate 9-16 min (usually 12 min); decolorize in a 1:1 mixture of 1% hydroquinone and 1% oxalic acid; wash thoroughly; soak in 1.5% AgNO₃ at about 20° C for 25-35 min; rinse; put into an ammino-silver solution (4.5% AgNO₃, 20 ml; pure ethanol, 10 ml; ammonia, sp. gr. 0.880, 2.4 ml; 2.5% NaOH, 1 ml) for 1-2 min; reduce in acidified formalin (distilled water, 400 ml; pure ethanol, 45 ml; 1 % citric acid, 13.5 ml; 10% formalin, 13.5 ml) for 1-3 min; wash; dehydrate through ascending grades of alcohol, including absolute; coat with 0.5% collodion, allow to dry slightly and harden in absolute alcohol-chloroform (2:1); rehydrate and put into 1% Na₂S₂O₃ for 1 min; dehydrate and cover.     

Staining Procedures for Ultrathin Sections of Tissues Embedded in Polyester Resin

Tissues were fixed for 30 min In cold (0-2° C) 1% OsO₄ (Palade) buffered at pH 7.7, to which 0.1% MgCl₂ was added. Dehydration was in a graded ethanol series (containing 0.5% MgCl₂) at 0-2° C, and terminated with 2 changes of absolute ethanol. Tissues were then transferred by a graded series to anhydrous acetone. Infiltration of the tissue with Vestopal-W (a polyester resin), is gradual with the aid of graded solutions of Vestopal-W in acetone. The infiltrated tissue is encapsulated and initial polymerization is done under ultraviolet light at room temperature for 8-16 hr. This is followed by final hardening at 60° C for 36-48 hr. Sections (0.2-1 μ) were cut, dried on slides, placed in acetone for 1 min and then treated by either of the following staining procedures: (1) Thionin-azure-fuchsin staining: Flood the preparation with 0.2% aqueous thionin and heat to 60-80° C for 3 min; if the preparation begins to dry, add stain. Rinse in distilled water. Flood the slide with 0.2% azure B in phosphate buffer at pH 9. Heat to 60-80° C for 3 min; do not permit the preparation to dry. Rinse in distilled water. Dip the slide in MacCallum's variant of Goodpasture's carbol-fuchsin stain for 1-2 sec. Rinse in distilled water. Check the preparation microscopically for intensity of the fuchsin stain. Repeat dips as may be needed to obtain the desired intensity. Rinse in distilled water. Dehydrate quickly in 95% and absolute alcohol; clear in 2 changes of xylene and cover in Permount or similar synthetic resin. (2) Thionin-azure counterstain for the periodic acid-Schiff reaction: Oxidize the tissue in 0.5% periodic acid for 15 min and transfer to Schiff's leucofuchsin solution for 30 min. Counterstain with 0.5% aqueous thionin for 3 min; wash in distilled water; stain in 0.2% azure B in phosphate buffer at pH 5.5; wash in distilled water; dehydrate; clear and cover as in the first method. For temporary preparations let dry after absolute alcohol and apply a drop of immersion oil directly on the section.     

Critical thermal maximum and body water loss in first instar larvae of three Cetoniidae species (Coleoptera)

Critical thermal maximum (CT_max) and body water losses were measured in first instar larvae of Gnorimus nobilis, Osmoderma eremita (Trichiinae) and Cetonischema aeruginosa (Cetoniinae) when air temperature was increased gradually (0.5 °C/min) from 20 °C to the critical thermal maximum (CT_max), in dry air (near 0% R.H.).

The CT_max was significantly lower in O. eremita (45.6±0.7 °C) than in G. nobilis (48.5±0.6) and C. aeruginosa (51.4±0.9 °C).

An increase of 10 °C (30–40 °C) induced a 2-fold increase of the water loss in C. aeruginosa and O. eremita (Q₁₀=2.10±0.12 and 2.13±0.20, respectively). In the range from 40 to 45 °C to CT_max a strong increase of the water loss was observed in O. eremita and C. aeruginosa, respectively. Body water losses were significantly lower in C. aeruginosa than in O. eremita and G. nobilis over the range 20 °C—CT_max; no significant difference occurred between G. nobilis and O. eremita.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus) : II. Effects of pretreatment with a small number of HVJ

Ehrlich ascites tumor cell membranes were completely modified after incubation at 37 °C for 30 min with a small dose of HVJ (about 0.7% of the maximum number of the virus particles that could be adsorbed onto the cells). After this treatment, the cells could adsorb further added HVJ onto their surfaces at 0 °C. But the cell agglutination which was induced by viral adsorption at 0 °C was very weak, and the interaction of the adsorbed virus with the lipid layer of the cell membrane at 37 °C preceding fusion or lysis of the cells was not strong. A discrepancy was observed between acquisition of the modification and liberation of sialic acid (destruction of viral receptors) by viral neuraminidase. The modification proceeded well on incubation at 37 °C but not at lower temperatures. The possibility that the modification is induced by fusion of viral envelopes with cell membranes is discussed.     

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.     

Preparation of enantiomerically pure (S)-flurbiprofen by an esterase from Pseudomonas sp. KCTC 10122BP

Esterase PF1-K from Pseudomonas sp. KTCC 10122BP was overproduced by the fed-batch culture of Escherichia coli. The soluble expression of esterase PF1-K was achieved by shifting the culture temperature from 37 to 25 °C at the time of IPTG induction. The enzyme was partially purified to about 75% purity by a single-step hydrophobic interaction column chromatography. The purified enzyme exhibited a fairly high enantioselectivity towards the hydrolysis of rac-flurbiprofen ethyl ester. The enzymatic chiral resolution was further improved by optimizing the reaction conditions in terms of reaction rate and enantioselectivity. The optimal reaction conditions were found to be 40 °C, pH 10.5 and 600 mM of initial rac-flurbiprofen ethyl ester. After 90 min of batch reaction under the optimal conditions, 50% of the initial rac-flurbiprofen was hydrolyzed with an enantiomeric excess of 99%.     

Characterization of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopy and differential scanning calorimetry (DSC) were used to characterize the incidence of intracellular ice formation (IIF) in 12- to 13-hr-old embryos of Drosophila melanogaster (Oregon-R strain P2) as influenced by the state of the eggcase (untreated, dechorionated, or permeabilized), the composition of the suspending medium (with and without cryoprotectants), and the cooling rate. Untreated eggs underwent IIF over a very narrow temperature range when cooled at 4 or 16 degrees C/min with a median temperature of intracellular ice formation (TIIF50) of -28 degrees C. The freezable water volume of untreated eggs was approximately 5.4 nl as determined by DSC. IIF in dechorionated eggs occurred over a much broader temperature range (-13 to -31 degrees C), but the incidence of IIF increased sharply below -24 degrees C, and the cumulative incidence of IIF at -24 degrees C decreased with cooling rate. In permeabilized eggs without cryoprotectants (CPAs), IIF occurred at much warmer temperatures and over a much wider temperature range than in untreated eggs, and the TIIF50 was cooling rate dependent. At low cooling rates (1 to 2 degrees C/min), TIIF50 increased with cooling rate; at intermediate cooling rates (2 to 16 degrees C/min), TIIF50 decreased with cooling rate. The total incidence of IIF in permeabilized eggs was 54% at 1 degree C/min, and volumetric contraction almost always occurred during cooling. Decreasing the cooling rate to 0.5 degree C/min reduced the incidence of IIF to 43%. At a cooling rate of 4 degrees C/min, ethylene glycol reduced the TIIF50 by about 12 degrees C for each unit increase in molarity of CPA (up to 2.0 M) in the suspending medium. The TIIF50 was cooling rate dependent when embryos were preequilibrated with 1.0 M propylene glycol or ethylene glycol, but was not so in 1.0 M DMSO. For embryos equilibrated in 1.5 M ethylene glycol and then held at -5 degrees C for 1 min before further cooling at 1 degree C/min, the incidence of IIF was decreased to 31%. Increasing the duration of the isothermal hold to 10 min reduced the incidence of IIF to 22% and reduced the volume of freezable water in embryos when intracellular ice formation occurred. If the isothermal hold temperature was -7.5 or -10 degrees C, a 10- to 30-min holding time was required to achieve a comparable reduction in the incidence of IIF.     

Thermoregulatory responses of old men to gradual changes in ambient temperature

1. 1. Thermoregulatory respones to gradual rise and fall in the ambient temperature (T_a) were compared between 8 old (68–78 years) and 8 younger (20–25 years) male subjects.

2. 2. Starting at T_a of 31.5°C (r.h. 40%), T_a was raised to 39.5°C, then lowered to 21.5°C, and raised back to 31.5°C at a constant rate of 0.3°C/min.

3. 3. Noticeable differences in responses between the age groups were as follows: decline of sweating rate and reduction of acral blood flow during room cooling were retarded in the aged group, with wider variations among individuals, compared with those in the younger group; the tympanic and oesophageal temperatures fell considerably during cooling in the elderly group, failing to return to the level at start during the rewarming of the room, in contrast to the younger group.

4. 4. Such sluggish responses may be attributed largely to reduced cutaneous thermal perception with advancing age.

In vitro excystment of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae)

1986. In vitro excystrnent of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae). International Journal for Parasitology 16: 641–645. An optimal hatching success of Plagiorchis species 1 metacercariae (100% excystment, active metacercariae, mean hatching speed 2–10 min, lowest variance of the mean speed) was observed after pretreatment in an HCl-pepsin solution at pH 2.0 and 42°C for 60–70 min, and incubation in a hatching medium at 42 °C and pH 7.3–8.0 with a bile salt (Nacholate), NaHCO₃, and a reductant (cysteine with 100% N₂). The minimum conditions for nearly 100% excystment with lower hatching speeds and higher variances were the presence of NaHCO₃, an oxygen concentration reduced to about 3% in the gas phase, pH> 7.3 and a temperature near 30°C if Na-cholate was absent, or in the presence of the bile salt, a phosphate buffer at pH> 5.0 and room temperature only. Obviously some hatching factors acted interchangeably with compensation for missing stimuli by others. The effect of the bile salt was comparable with that of other surfactants. The metacercariae excysted in nonenzymatic media, which implies an active hatching mechanism.     

Three-step cooling: A preservation method for adult rat heart cells

Adult rat heart cells were exposed to two-step cooling to ?196 °C with different holding periods at different subzero temperatures between both steps. The highest survival based on the percentage of trypan blue-excluding cells was 25% with 10% DMSO and a holding period of 6 min, and 21% with 15% DMSO and a holding period of 30 min. The highest survival based on morphological intactness was about 10%; there was no difference in results after cooling with 10 and 15% DMSO, and after holding between 2 and 30 min. The optimal survival based on the percentage of contracting cells was 52%, with 15% DMSO and a holding period of 2 min.When the holding period was replaced by a programmed cooling stage, the results could be improved. With this threestep cooling method, the optimal values, based on the number of trypan blue-excluding, intact, and contracting cells, were 40, 32, and 60%, respectively. It appeared that in the presence of 10% DMSO, which provided better survival than 5 and 15%, no significantly different results were obtained when the starting temperatures of the second cooling step varied between ?10 and ?20 °C, when the end temperatures varied between ?30 and ?60 °C, or when the cooling rates of the second cooling step varied between 0.1 and 1 °C/min. Three-step cooling provided similar results as linear cooling from 0 to ?100 °C, followed by rapid cooling to ?196 °C.     

Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy

Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at -0.3 degrees C min(-1) to -60 degrees C and osmotic shock invariably resulted in damage to the organism's pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to -130 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C).