A comparative study of the effects of freezing and frozen storage on intact and demembranated bull spermatozoa

The damage caused to bull sperm by freezing and thawing them without cryoprotectants was assessed in both intact and membrane-extracted cells. Preparations of membrane-extracted cells were produced by treating the sperm with 0.1% Triton X-100 and motility was restored with exogenously applied ATP and Mg²⁺. Motile demembranated sperm showed no detectable reduction in motility after freezing and thawing. In contrast, when intact cells where subjected to freezing and thawing they lost all motility. These damaged cells were also restored to motility when exogenous ATP and Mg²⁺ were added to the sperm mixture. Apparently freezing and thawing sperm cells causes damage to the plasma membrane which permits ATP and Mg²⁺ to freely enter or leave the cells, but does not damage the components of the sperm cell which generate motility.The effects of storage temperature on frozen demembranated sperm were also explored. Sperm held at ?20 °C showed marked structural changes and progressively decreased motility after prolonged storage. When sperm were frozen at ?20 °C the mitochondrial structures were completely lost after 48 to 72 hr and ATP caused the disintegration of the flagellum rather than initiating motility. Sperm which were frozen at ?76 °C retained motility after short periods of storage, but showed a significant decline in motility when thawed after 8 days. Demembranated sperm which were kept frozen at ?196 °C showed no significant loss of motility when thawed after 1 year of storage.     

Effect of caffeine on the post-thaw motility of buffalo spermatozoa

Buffalo semen was diluted (1:2) with lactose diluent containing caffeine (2, 4 and 6 mM). Diluted semen samples were frozen in a pellet form (0.15 ml), thawed 24 h after freezing in 2.9% sodium citrate for 30 sec and incubated at 37 degrees C for 3 h. Addition of caffeine to diluted buffalo semen before freezing resulted in a significant increase in the post-thaw motility of spermatozoa over the 3-h incubation period. When caffeine was added to the thawing medium, the post-thaw motility was further improved. Thus, the increase in motility due to caffiene treatment was even more pronounced than in samples treated with caffiene before freezing.     

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.     

The effect of straw size, freezing rate and thawing rate upon post-thaw quality of dog semen

Optimal freeze-thaw processes for dog semen will yield a maximal number of insemination doses from an ejaculate. The objectives of this study were to compare the effects of two straw sizes (0.25- and 0.5-mL French), two freezing rates (straws suspended 3.5 and 8 cm above liquid nitrogen) and two thawing rates (in water at 37 and 70 degrees C) upon post-thaw quality of dog semen, and to determine the best treatment combination. Quality was expressed in terms of the percentage progressively motile sperm 5 and 60 min after thawing and the percentage of abnormal acrosomes 5 min after thawing. One ejaculate from each of eight dogs was frozen. Two straws from each ejaculate were exposed to each of the eight treatment combinations. Data were analyzed by means of a repeated measures factorial analysis of variance and means compared using Bonferroni's test. Dog affected each response variable (P < 0.01). Neither straw size, nor freezing rate, nor thawing rate affected motility 5 min after thawing (P > 0.05). Half-milliliter straws resulted in 5.7% more progressively motile sperm 60 min after thawing and 6.5% fewer abnormal acrosomes than 0.25-mL straws (P < 0.05, n = 64). The percentage progressively motile sperm 60 min after thawing tended to be higher for semen thawed at 70 degrees C compared to 37 degrees C (P < 0.06, n = 64). Semen thawed in water at 70 degrees C had 6.6% fewer abnormal acrosomes than semen thawed in water at 37 degrees C (P < 0.05, n = 64). Freezing rate interacted with thawing rate (P < 0.05) in their effects upon acrosomal morphology and freezing 8 cm above liquid nitrogen and thawing in water at 70 degrees C was best. Dog semen should be frozen in 0.5-mL straws, 8 cm above liquid nitrogen and thawed in water at 70 degrees C.     

Unfractionated human marrow cell cryopreservation using dimethylsulfoxide and hydroxyethyl starch

Unfractionated bone marrow (BM) cells were cryopreserved in 1- to 2-ml aliquots using a mixture containing both 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) in an attempt to increase the viable cell yield and reduce the clumping after thawing, observed when 10% DMSO is used alone. Samples thawed after storage for 6 months in the vapor phase of liquid nitrogen, were assayed. Compared to prefreeze values, there was both a greater number of cells that excluded Trypan Blue (50 +/- 12 vs 28 +/- 12%, P less than .01) and a greater CFU-C Recovery (110 +/- 20 vs 89 +/- 35%, P less than .02) for cells in the DMSO/HES mixture, compared to those in 10% DMSO alone. No macroscopic clumping of the thawed cells was observed for those cryopreserved in the mixture in contrast to those in DMSO alone. Freezing was done without a rate-controlled freezing apparatus by simply placing the samples initially into a -80 degrees C freezer, and then later into a liquid nitrogen freezer. Additional samples stored in the DMSO/HES mixture were kept at only -80 degrees C, and when thawed 12 to 16 months later also gave an excellent CFU-C recovery (105 +/- 39% of prefreeze). The DMSO/HES mixture allows for a simplified BM cryopreservation technique that not only assures excellent recovery of CFU-Cs and eliminates clumping upon thawing, but also does not require either the use of a rate-controlled freezer or liquid nitrogen temperatures for storage up to a year.     

Ultrastructural injury to human spermatozoa after freezing and thawing

The ultrastructure of human spermatozoa at various stages of the freezing and thawing process was studied. In addition to conventional fixations, a freeze-substitution method was used to examine spermatozoa before they were thawed. Dilution in a glycerol-egg yolk-citrate medium caused slight swelling of the acrosome. During slow freezing, when large ice crystals grow in the diluent, the sperm plasmalemma became tighter, the mitochondria had more angular profiles and there was a reduction in electron density of the acrosomal contents. After thawing, the apical segment of the acrosome usually became swollen and the mitochondria appeared rounded. We deduce that these ultrastructural changes occur either during or after the thawing procedure.     

The effects of cooling to 5 degrees C and freezing and thawing on the ultrastructure of bull spermatozoa.

Semen from 6 bulls was examined under the transmission electron microscope immediately after collection, after dilution and cooling to 5 degrees C and after freezing and thawing. Conception rates were determined following artificial insemination of the frozen and thawed semen. Dilution and cooling to 5 degrees C caused acrosomal swelling in about 50% of the spermatozoa. Subsequent freezing and thawing caused considerable ultrastructural changes to the acrosomes (disruption of the plasma and outer acrosomal membranes and dispersion of the acrosomal contents) and middle pieces (breakage of the plasma membrane and a reduction in the electron density of the mitochondrial matrix) of a high proportion of spermatozoa. The average non-return rate following insemination of semen from 5 of the bulls was 61.6% and higher (P greater than 0.001) than for the sixth bull (15%). Although this difference in semen viability was also demonstrated in the structural studies (acrosome, P greater than 0.05: middle piece, P greater than 0.001), more work is required to assess the relationship between structure and function of spermatozoa.     

An approach to successful freezing of demi-embryos derived from day-7 bovine embryos

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.     

Cryopreservation of functionally active blood nuclear cell membranes at −80°C

Structural and functional changes in cytoplasmic membranes and cell organelles play a crucial role in cell damage at low temperatures. These changes are reversible if adequate measures are taken to protect biological membranes against cold-induced injuries. In this study, the possibility of cryopreservation of membrane integrity by long-term storage of samples at low temperatures (?80°C) is demonstrated using differentiated nuclear blood cells as an example. In addition to classical methods, freezing of human leukocyte suspensions was carried out in a novel nontoxic cryoprotecting solution under a newly developed exponential freezing program based on the use of cryoprotectors and repair additives. This program ensures the same cryopreservation effect as the linear program, but is economically more efficient and less labor-consuming. After exponential freezing in a cryoprotecting solution and storage at ?80°C, blood leukocytes retain their eosin resistance (91 ± 5% of thawed leukocytes) and phagocytic activity (76.7 ± 14.7% of thawed neutrophils) for 180 days. The novel technology of cell cryopreservation employing a nontoxic cryoprotecting solution, exponential freezing program, and fast thawing is economically efficient, easy to perform and applicable to storage of any animal cells.     

An improved method for preparing refrozen rethawed human lymphocytes on plates for microcytotoxicity studies.

This report describes simplified methods for the initial freezing and thawing of human lymphocytes and the subsequent use of these cells after refreezing on cytotoxicity plates, storage, and a second thaw. The proposed initial freeze method eliminates some technical inconveniences required previously such as chilling of cells prior to addition of DMSO, preparing cryoprotective mixtures just prior to freezing, controlled rate of freezing and thawing and the washing of cells after thawing. However, pH of the media, blood freshness, type of storage tube used, and constant storage temperature were found to be very important to maintain good cell viability. Most lymphocytes maintain an average viability of 85 to 95% for at least a year when prepared according to the present freezing and thawing technique.When panels of lymphocytes are prepared for refrozen rethawed cytotoxicity test plates, the thaw time between freezes must be brief. Production of test plates on ice, however, was not found to be necessary. As the period of storage of refrozen cells on plates increases, viability of the cells after a second thaw decreases and treatment with DNase to enzymatically remove the dead cells is useful. With this procedure, refrozen rethawed lymphocytes up to a year old can be prepared on microcytotoxicity test plates with average viabilities of 90 ± 1%.     

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.     

Impact of time between repeated sperm freezing cycles on sperm quality

Refreezing of sperm samples would provide the possibility of performing more cycles of fertility treatments. Although the effect of repeated cycles of freezing on sperm quality was studied, the effect of the length of the time interval between each freeze-thaw cycle has not been reported. Hence, we assessed the effect of incubation time on the sperm quality of thawed sperm after repeated freezing.One-hundred samples of potential sperm donations with normal sperm quality were evaluated. The fresh semen samples were analyzed and cryopreserved in liquid nitrogen until use. After thawing, the samples were divided randomly to two groups and reanalyzed for motility, vitality, and DNA fragmentation. They were incubated at room temperature and reanalyzed after either 90 min (group A) or 180 min (group B) of incubation, and once again after a repeated cycle of freezing and thawing.Our results showed that the sperm parameters of fresh samples of both groups were similar. After one freeze-thaw cycle, both groups still had comparable values. At the end of their respective incubation time periods, however, there was a significant difference in the mean values of the assessed parameters between the two groups (p < 0.01). An additional freeze-thaw cycle further exacerbated those differences, with group B undergoing an even more substantial decline (p < 0.001).Our data suggest that thawed human spermatozoa sustain a significant decline in sperm parameters in association with longer incubation time, which is further exacerbated by an additional freeze-thaw cycle.     

Quality improvements to mackerel (Scomber japonicus) muscle tissue frozen using a rapid freezer with the weak oscillating magnetic fields

Although freezing is the most popular long-term food preservation method, the formation of ice crystals during the freezing process often degrades the quality of the product. Recently, several reports have argued that oscillating magnetic fields (OMFs) may affect ice crystallization. In this paper, we investigated the effects of OMFs on fresh mackerel using the Cell Alive System® (CAS®) developed as an additional OMF generator for a rapid freezer. Mackerel fillets were frozen with home freezing (HF), air blast freezing without (ABF) or with CAS (ABF-CAS) (ABI Co. Ltd., Chiba, Japan), and stored them for 2 weeks in the frozen storage between −30 °C and −35 °C. We analyzed the tissue damages of thawed samples histologically. The OMFs has been shown to significantly inhibit tissue damage in mackerel tissue after freezing and thawing (especially, thawing in ice water). And it seems that OMFs suppressed the ice hole counts (p < 0.05), the mean size (p = 0.061), and the increase of interstitial area% (p < 0.05) after freezing/thawing. We also found that it is necessary to avoid re-crystallization during thawing to maintain the quality of the frozen product. The use of OMFs with rapid thawing has the potential to improve cryopreservation in the food industry as well as in the bioscience industry.     

Cryopreservation of seabream (Sparus aurata) spermatozoa

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.     

Optimal conditions for freezing CHO-S and HEK293-EBNA cell lines: influence of Me2SO, freeze density, and PEI-mediated transfection on revitalization and growth of cells, and expression of recombinant protein

To avoid the time consuming, labor intensive seed-train expansion and to improve production reliability and consistency, portions of bulk cryopreserved cells from the same cultivation can be utilized as inocula or alternatively may be used to undertake transient transfections for large-scale bioreactor production. In this study, the conditions for large-scale freezing in cryobags were optimized utilizing a design of experiment approach. We showed that relatively high density of 30-40 x 10(6) cells/mL and relatively low Me(2)SO concentrations of 5-6% in the freezing media are optimal to freeze HEK293-EBNA and CHO-S cells in a controlled manner in order to achieve high viable cell recovery and growth post-thawing. The immediate transfer of freshly thawed cells into culture medium resulted in better cell growth compared to cells that were centrifuged in order to remove Me(2)SO. This was the case as long as the residual Me(2)SO did not exceed 0.2-0.3%. The best time to perform transient 25 kDa polyethylenimine-mediated transfection of pCEP4-EGFP plasmid into freshly thawed, one-step inoculated cells is after 72-96 h in culture. At this time point, the numbers of EGFP-positive cells in the freshly thawed culture mimic perfectly that of cells grown continuously. Finally, our data showed that it is possible to freeze transiently polyethyleneimine-transfected HEK293-EBNA cells and maintain growth rate and expression of recombinant protein following thawing. The optimal time point for freezing cells was 4 h after transfection.     

Thawing dog spermatozoa in just-boiled water: submersion time and effect on sperm quality compared to thawing in water at 70 degrees C

Dog spermatozoa have better quality after thawing in water at 70-75 degrees C instead of 35-38 degrees C. The aim of Experiment 1 was to determine the time needed to thaw 0.5 mL straws in just-boiled (98 degrees C) water and that of Experiment 2 to determine whether thawing frozen dog spermatozoa in just-boiled water will result in better quality than thawing in water at 70 degrees C. Prior to freezing the straws of Experiment 1, a Type J thermocouple with wire diameters of 0.08 mm (Osiris Technical Systems, Centurion, South Africa) was placed in the center of each of ninety-three 0.5 mL straws (IMV Technologies, L'Aigle, France) filled with extender (Biladyl* with 0.5%, v/v of Equex STM paste**) and 54 filled with extender plus 200 x 10(6)spermatozoa/mL (Minitüb, Germany (*) and Nova Chemical Sales, MA (**)). Thirty straws with extender were thawed in water at 70 degrees C and the others in just-boiled water. Temperatures inside straws were recorded 10 times/s during warming. Two ejaculates were then collected from each of eight dogs and one from each of three others. Extended ejaculates from the same dog were pooled, frozen 8 cm above liquid nitrogen, and 2 straws from each of the 11 batches thawed in water at 70 degrees C for 8s and 2 in just-boiled water for 6.5s. Sperm morphology and viability were assessed on eosin-nigrosin smears made after thawing and the percentage progressively motile spermatozoa was estimated immediately, 1, 2 and 3h after thawing. The optimal submersion time in just-boiled water was 6.5s for both sperm concentrations, resulting in average temperatures of 23.6+/-1.5 degrees C (+/-S.E.M.) and 24.9+/-1.6 degrees C inside straws with extender or extender plus spermatozoa (P=0.6). The temperature inside straws thawed in water at 70 degrees C was 13.6+/-1.7 degrees C after 8s. Apart from a 1.5% higher (P<0.05) mean percentage motile sperm 2h after thawing, thawing dog spermatozoa in just-boiled (98 degrees C) water holds no benefit over thawing in water at 70 degrees C, which is easier to do.     

Evaluation of dog semen quality after slow (biological freezer) or rapid (nitrogen vapours) freezing

Three ejaculates were collected from each of five dogs. After initial evaluation, the sperm-rich fractions were diluted to 100 x 10(6) spermatozoa x mL(-1) in two steps with an egg yolk-TRIS extender containing a final concentration of 5% glycerol and 0.5% Equex STM paste. Half of the 0.5 mL straws obtained from each ejaculate were frozen on nitrogen vapours (4 cm above the liquid surface) ("rapid freezing"), while the other half was frozen in a biological freezer at a rate of 0.5 degrees C x min(-1) between 5 degrees C and -10 degrees C and of 8 degrees C x min(-1) between -10 degrees C and -60 degrees C, followed by immersion in liquid nitrogen ("slow freezing"). After an average storage of 30 days, the straws were thawed in a water-bath at 37 degrees C for 1 min. Progressive motility was subjectively estimated hourly for 8 h on semen incubated at 38 degrees C. Immediately after thawing and after 2 h of incubation, motility parameters were also measured by a motility analyser. Sperm membrane function and chromatin stability were assessed immediately post-thaw, using the hypo-osmotic swelling test and acridine orange staining, respectively. Slow freezing significantly improved total post-thaw motility, which showed a slower decline over time, although spermatozoal average path and straight line velocity were lower compared to the fast rate. Also the number of intact membrane spermatozoa was significantly higher in slow-frozen samples while the proportion of spermatozoa with single-stranded DNA was minimal after both freezing procedures.     

Influence of cryoprotective diluent on post-thaw viability and acrosomal integrity of spermatozoa of the African elephant (Loxodonta africana)

Electroejaculates from free-ranging, African elephants were frozen to test various seminal diluents, freezing methods and thawing media on post-thaw sperm viability and structural integrity. In Study I, each ejaculate was tested with each of 7 cryoprotective diluents. After cooling to 5 degrees C and equilibration on ice (4 degrees C) for 120 min, each aliquant was pellet frozen on solid CO2, stored in liquid nitrogen and thawed (37 degrees C) in saline or tissue culture solution. Amongst all diluents, post-thaw sperm motility, motility duration in vitro (37 degrees C) and acrosomal integrity were greatest (P less than 0.05) when diluent BF5F was used. Thawing medium had no effect on results. In Study II, the optimal diluent from Study I (BF5F) was compared with the diluent SGI. Results were not affected by a 90- or a 150-min cooling-equilibration interval in an electronic cooler (5 degrees C); however, post-thaw sperm motility rating and duration of motility in vitro were greater (P less than 0.01) with the pellet than the straw container freezing method. When the pelleting method was used, diluents BF5F and SGI provided comparable cryoprotection. Duration of post-thaw motility was enhanced 2-fold and up to 12 h by maintaining thawed semen at 21 rather than 37 degrees C (P less than 0.05). All diluents provided some protection on acrosomal integrity, but the overall proportion of intact acrosomes after thawing was markedly less in Study II, apparently as a result of the slower initial cooling rate (approximately 1.5 degrees C/min) compared to that of Study I (approximately 6.5 degrees C/min). This study demonstrates the feasibility of cryopreserving semen from free-ranging African elephants and indicates that spermatozoa must effectively survive freezing when the BF5F or SGI diluent is used in conjunction with the pelleting method.     

Microbiological aspects of determination of trichloroacetic acid in soil

Soils have been shown to possess a strong microbial trichloroacetic acid (TCA)-degrading activity. High TCA-degradation rate was also observed during soil extraction with water. For correct measurements of TCA levels in soil all TCA-degrading activities have to be inhibited immediately after sampling before analysis. We used rapid freezing of soil samples (optimally in liquid nitrogen) with subsequent storage and slow thawing before analysis as an efficient technique for suppressing the degradation. Frozen soil samples stored overnight at -20 degrees C and then thawed slowly exhibited very low residual TCA-degrading activity for several hours. Omitting the above procedure could lead to the confusing differences between the TCA levels previously reported in the literature.     

Survival ofEscherichia coli after storage in various frozen menstrua

The effect of storage at –9 C onEscherichia coli was examined. In buffer or water, survival after three days was less than 40%. Dimethylsulfoxide (DMSO) (10%) and glycerol (10%) were very protective with over 90% survivors. Variability of replicate samples was greater with frozen than with non-frozen suspensions.With a slide culture technique, it was found that the time required for the thawed cells to complete their first division was increased up to a time equivalent to over two divisions, dependent upon the protective storage menstrua.Injury as shown by inability to grow on a minimal medium after thawing was negligible when the cells were frozen in DMSO or glycerol. Cells stored in frozen buffer were sensitive to a 20 min treatment with actinomycin D following thawing but cells frozen in glycerol or DMSO showed little death or injury. The results suggest that an alteration of the cell envelope is initially responsible for death by freezing.This work was supported in part by U.S. Public Health Service Research Grant EF-428 from the Division of Environmental Engineering and Food Protection.