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PubMed1998

Cold inhibition of cell volume regulation during the freezing of insect malpighian tubules.

Cells in freeze-tolerant tissues must survive substantial shrinkage during exposure to the hyperosmolarity that results as solutes are excluded from extracellular ice. We investigated the possibility that this hyperosmotic shock elicits an acute regulatory volume increase (RVI) by monitoring the response of epithelial cell volume in the Malpighian tubules of the New Zealand alpine weta (Hemideina maori) during exposure to low temperature/hyperosmolarity (mimicking freezing conditions) or during an actual freeze/thaw cycle. The cross-sectional area of cells in isolated Malpighian tubules was measured using differential interference contrast microscopy. At 20 degrees C, cells held in saline containing 400 mmol1-1 glucose exhibit an RVI in response to hyperosmotic shock. Cross-sectional area decreased by 30% immediately after a change from iso-osmotic (0.7 osmol1-1) to hyper-osmotic saline (2.1 osmol1-1, equal to the osmotic shock encountered during freezing to -4 degrees C) and then returned to 21% below the control value 30 min after the exposure. Although substantial cellular function of Malpighian tubules was retained at low temperature (the rate of fluid secretion by isolated tubules at 4 degrees C was 72% of that measured at 20 degrees C), no RVI was observed at 0% degrees C; cross-sectional area was 39% below the control value immediately after the hyperosmotic exposure and 36% below the control value 30 min after hyperosmotic exposure. Dibutyryl cyclic AMP potentiated the RVI observed at 20 degrees C, but failed to elicit an RVI at 0 degrees C. A substantial RVI was also absent when the saline contained trehalose rather than glucose, regardless of whether the tubules were held at 20 degrees C or 0 degrees C. The cross-sectional area of cells in saline containing glucose remained at approximately 30% below the control value during an entire 30 min period of actual freezing to -4 degrees C, suggesting that an acute volume regulatory response was in fact inhibited during mild freezing. The inhibition of an acute RVI during mild freezing may serve to avoid the energetic expenditure associated with volume regulation at a time when the normal defence of cell volume appears to be unnecessary.

Animals↗
PubMed1989

In vitro stability of insulin-loaded erythrocytes after freezing storage.

In order to clarify the utility of resealed erythrocytes as a cell carrier of insulin the freezing stabilities of loaded cells encapsulating two different amounts of insulin (about 0.1 and 7.0 IU/ml of packed cells) were investigated in an anti-freezing reagent containing a high concentration of glycerol at -80 degrees C. After storing for 1 week under the above condition the preparation was thawed at 37 degrees C. Although the osmotic resistance of the loaded cells was lowered more than that of intact cells, the freezing and thawing treatment did not change the resistance of the loaded cells to osmotic shock. Recoveries of hemoglobin from loaded cells with a low insulin content (0.1 IU/ml of cells) and the cell volume after freezing and thawing were similar to those from intact cells and the cells resealed without insulin. However, the recovery of insulin from the cells with a high insulin content (7.0 IU/ml of cells) was the same as that from one with a low insulin content (about 16%). In the in vitro stability experiment at 37 degrees C the insulin content in the loaded cells decreased according to a biexponential function. The considerable fraction of the loaded cells were lysed by freezing and thawing. In the in vitro stability experiment the decline curve of insulin in the cells showed apparently monoexponential kinetics. From these results, it was established that insulin-loaded erythrocytes may be stored in a high concentration of glycerol under freezing at -80 degrees C, although a partial decrease in the stability of cells was observed after thawing.

Animals↗
PubMed2001

Improving the freeze tolerance of bakers' yeast by loading with trehalose.

We examined the freeze tolerance of bakers' yeast loaded with exogenous trehalose. Freeze-tolerant and freeze-sensitive compressed bakers' yeast samples were soaked at several temperatures in 0.5 M and 1 M trehalose and analyzed. The intracellular trehalose contents in both types of bakers' yeast increased with increasing soaking period. The initial trehalose-accumulation rate increased with increasing exogenous trehalose concentration and soaking temperature. The maximum trehalose content was almost identical (200-250 mg/g of dry cells) irrespective of the soaking temperature and the type of bakers' yeast, but depended on the exogenous trehalose concentration. The leavening ability of both types of bakers' yeast loaded with trehalose was almost identical to that of the respective original cells, irrespective of the soaking conditions. The freeze-tolerant ratio (FTR) of both types of bakers' yeast increased with increasing intracellular trehalose content. However, FTR decreased during over-soaking after the maximum amount of trehalose had accumulated. FTR of the freeze-sensitive bakers' yeast was more efficiently improved than that of the freeze-tolerant type.

Freezing↗
PubMed2002

Low-temperature brewing by freeze-dried immobilized cells.

We propose a novel biocatalyst in brewing. A cryotolerant strain of Saccharomyces cerevisiae was immobilized on delignified cellulosic material followed by freeze-drying of the immobilized cells without the use of any cryoprotectant. The freeze-dried immobilized biocatalyst was used in repeated-batch fermentation of wort and showed reduced fermentation time and increased productivities as compared with free freeze-dried cells (FFDCs). It also demonstrated suitability for low-temperature brewing (5 and 0 degrees C). The fermentation time in repeated-batch fermentations at 15 degrees C was 1.5-2 d for a period of 13 mo, showing a high operational stability of the system. At 0 degrees C the freeze-dried immobilized biocatalyst showed a 2- to 3.5-fold decrease in fermentation time in comparison with FFDCs. Polyphenol contents, bitterness, and diacetyl concentration were lower in beers produced by freeze-dried immobilized cells as compared with FFDCs. At 0 degrees C polyphenols were 40% lower than at 15 degrees C. Higher alcohols were reduced and ethyl acetate increased in comparison with FFDCs. Amyl alcohols at 0 degrees C were lower than half of their content at 15 degrees C, while ethyl acetate was 31 mg/L at 0 degrees C and 18 mg/L at 15 degrees C. These data justify the improved aroma and taste of beers produced by freeze-dried immobilized biocatalyst mainly at low temperatures.

Beer↗
PubMed2000

Freeze-fracture cytochemistry: a new fracture-labeling method for topological analysis of biomembrane molecules.

Freeze-fracture cytochemistry allows visualization of cellular and molecular characteristics of biomembranes in situ. In this review, we discuss freeze-fracture cytochemistry with special reference to a new cytochemical labeling of replicas, the detergent-digestion fracture-labeling technique. In this procedure, unfixed cells are rapidly-frozen, freeze-fractured, and physically stabilized by evaporated platinum/carbon. The frozen cells are then removed from the freeze-fracture apparatus to thaw and are subsequently treated with detergents. After detergent-digestion, replicas are labeled with cytochemical markers. We demonstrate that the technique is a versatile tool for direct analysis of the macromolecular architecture of biomembranes and allows identification of particular intracellular membrane organelles. In addition, we demonstrate the application of ultrasmall gold to freeze-fracture immunocytochemistry. Freeze-fracture cytochemistry is a valuable technique for investigating topology and dynamics of membrane molecules.

Cell Membrane↗
PubMed2005

Freeze-thaw effects on phosphorus loss in runoff from manured and catch-cropped soils.

Concern over nonpoint source P losses from agricultural lands to surface waters in frigid climates has focused attention on the role of freezing and thawing on P loss from catch crops (cover crops). This study evaluated the effect of freezing and thawing on the fate of P in bare soils, soils mixed with dairy manure, and soils with an established catch crop of annual ryegrass (Lolium multiflorum L.). Experiments were conducted to evaluate changes in P runoff from packed soil boxes (100 by 20 by 5 cm) and P leaching from intact soil columns (30 cm deep). Before freezing and thawing, total P (TP) in runoff from catch-cropped soils was lower than from manured and bare soils due to lower erosion. Repeated freezing and thawing significantly increased water-extractable P (WEP) from catch crop biomass and resulted in significantly elevated concentrations of dissolved P in runoff (9.7 mg L(-1)) compared with manured (0.18 mg L(-1)) and bare soils (0.14 mg L(-1)). Catch crop WEP was strongly correlated with the number of freeze-thaw cycles. Freezing and thawing did not change the WEP of soils mixed with manures, nor were differences observed in subsurface losses of P between catch-cropped and bare soils before or after manure application. This study illustrates the trade-offs of establishing catch crops in frigid climates, which can enhance P uptake by biomass and reduce erosion potential but increase dissolved P runoff.

Agriculture↗
PubMed1998

Effect of prerigor freezing and postrigor calcium chloride injection on the tenderness of callipyge longissimus.

The effect of rapid prerigor freezing and postrigor freezing and postrigor calcium chloride injection on the tenderness of callipyge longissimus was studied. Ewe and wether lambs (n = 49; 1/2 Dorset x 1/2 Romanov) were grain-fed and slaughtered at approximately 250 d of age. Based on leg conformation scores, 23 of the carcasses had the callipyge phenotype. Within each phenotype, approximately one-half of the carcasses were chilled conventionally (24 h at -2 degrees C). At approximately 17 min postmortem, the remaining carcasses were submersed in liquid nitrogen for 15 min and then held at -2 degrees C for 4 d. At 1 d postmortem for carcasses chilled conventionally and at 4 d postmortem for carcasses frozen in liquid nitrogen, the longissimus muscles from both sides were removed. The longissimus from one side of each carcass was vacuum-packaged and aged (1 degrees C) conventionally for 7 or 14 d. The remaining muscles were injected with a 2.22% solution of food-grade calcium chloride at 5% by weight, vacuum-packaged, and aged as above. Liquid nitrogen freezing was effective in limiting sarcomere shortening (1.99 vs 1.63 microm; P < .05). Warner-Bratzler shear force values of callipyge longissimus were 222 and 232% of that of normal longissimus after 7 and 14 d postmortem, respectively (P < .001). Also, trained panel tenderness rating was decreased by 49.4% in untreated callipyge longissimus after 14 d postmortem. Liquid nitrogen, calcium chloride injection and their combination did not affect d-14 longissimus shear force and sensory tenderness for normal lambs because untreated muscles were already tender. Liquid nitrogen freezing improved the shear force and sensory tenderness rating of callipyge longissimus by 30 and 86.2% after 14 d postmortem, respectively. Calcium chloride injection improved the shear force and sensory tenderness of callipyge longissimus by 36.7 and 86.2% after 14 d postmortem, respectively (P < .001). The most effective treatment for mitigating the callipyge effect on tenderness was the combination (freezing and calcium chloride injection) treatment, which improved the shear force and sensory tenderness by 51.2 and 124.2% after 14 d postmortem, respectively (P < .001). We conclude that either treatment can effectively mitigate the negative effect of callipyge phenotype on longissimus tenderness. Callipyge lamb carcasses subjected to the combination of prerigor liquid nitrogen freezing, postrigor calcium chloride injection, and 14 d postmortem storage had tenderness similar (P > .05) to that of normal, untreated carcasses after 14 d of postmortem storage.

Animals↗
PubMed2003

Effect of freezing on Fossomatic cell counting in ewe milk.

Using the Fossomatic method, a total of 10,072 analytical somatic cell count (SCC) observations were carried out on 4760 aliquots taken from 70 individual ewe milk samples with the objective of studying whether freezing showed significant differences of SCC in comparison with refrigeration, according to different analytical conditions. These conditions were four preservation procedures (without preservation, potassium dichromate, azidiol, and bronopol), two storage temperatures (refrigeration and freezing), five milk ages within storage (24 h postcollection in refrigeration, and 24 h, 15, 30, and 60 d postcollection in freezing), two thawing types (rapid and slow), and two analytical temperatures (40 and 60 degrees C). Preservation, storage, and analytical temperature, type of thawing and milk age within storage, and most of the interactions showed a significant effect on the SCC variation. On average, the SCC was lower after freezing than in refrigeration. This effect depended specifically on type of preservation and analytical temperature of milk. The SCC of milk unpreserved or preserved with bronopol or potassium dichromate, and analyzed at 40 degrees C, was not affected by freezing; however, use of azidiol as a preservative before freezing, and heating the milk to 60 degrees C following thawing resulted in significantly decreased SCC. Milk age had little quantitative influence on SCC of thawed milk. The type of thawing (rapid and slow) did not significantly influence SCC of milk analyzed at 40 degrees C. As a result, when using properly handled samples, the Fossomatic method could be used to enumerate SCC in samples frozen over the 60 d.

Animals↗
PubMed2026

Physiological sub-typing of cold and freezing injury in Triticum turgidum subspecies with bioinformatic and expression characterization of glutathione reductase.

BACKGROUND: This study examined how different subspecies of Triticum turgidum (T. durum, T. polonicum, T. turanicum) respond to cold and freezing, assessing their water status, stress responses, and antioxidant system, with particular focus on the structure and function of glutathione reductase (TtGR). METHODS: TtGR genes were first identified from the T. turgidum genome using publicly available genomic resources such as Ensembl Plants. Promoter regions (~2 kb upstream) were analyzed to identify cis-regulatory elements using PlantCARE. Gene classification was performed based on predicted subcellular localization and conserved domain features. Plants were subjected to cold acclimation and freezing treatments, and physiological, biochemical, and enzymatic parameters were measured. RESULTS: Bioinformatics analyses identified four TtGR genes in the T. turgidum genome. The genes in two groups: cytosolic (Class I) and chloroplastic (Class II). Gene structure analysis showed a conserved exon-intron organization, while motif analysis confirmed the presence of Nicotinamide Adenine Dinucleotide Phosphate (NADPH)-binding and redox-active domains across all TtGR proteins. Several regulatory sequences in the promoters are involved in cold (DRE), abscisic acid (ABRE), and stress (STRE) responses, indicating that TtGR genes are dynamically regulated in response to environmental changes. Physiological analyses showed that freezing treatment reduces leaf water content in all genotypes, leading to turgor loss, hydrogen peroxide (H2O2) accumulation, and increased malondealdehyte (MDA) levels. However, tolerance mechanisms addressing water stress and membrane damage differ among genotypes. At the biochemical level, activation of the antioxidant defense system occurs in all genotypes. T. turanicum displays strong defense by significantly increasing enzyme activities, ensuring that the ascorbate-glutathione cycle continues under stress. By contrast, T. polonicum, although showing increased overall enzyme activities, experiences a dramatic drop in glutathione reductase (GR) activity at freezing temperatures, which restricts reduced glutathione (GSH) regeneration and creates a functional bottleneck in the antioxidant cycle. T. durum fails to sustain enzyme activities over the stress period, leading to an intermediate-sensitive response. Thus, whereas T. turanicum effectively maintains antioxidant function during freezing, T. polonicum and T. durum exhibit less efficient stress responses, either through enzymatic bottlenecks or a lack of sustained defense. CONCLUSIONS: One of the most striking findings of this study is the observed dissociation between TtGR gene expression levels and enzyme activities. Low temperature limits the link between transcription and enzyme function. The primary determinant of low-temperature tolerance in T. turgidum subspecies is the sustainability of GR enzyme activity and GSH regeneration under freezing conditions.

Triticum↗
PubMed1999

Effect of freeze-dried soybean curd (tofu) on various bodily functions.

The present study was designed to examine the effect of freeze-dried soybean curd (tofu) on various bodily functions. The dietary experiment consisted of 4 days of a non-prescribed ordinary diet, 10 days of an experimental diet that contained 190 g of meat contributing about 38 g of protein (hereinafter referred to as the "meat period"), and 39 days of an experimental diet that contributed vegetable protein, i.e., freeze-dried tofu, corresponding to the protein that 190 g of meat would provide (hereinafter referred to as the "freeze-dried tofu period"). Three eggs (about 150 g) and 180 ml of cow's milk were prescribed for the daily diet, and staple and other foods were dispensed freely during both the meat and the freeze-dried tofu periods. The results showed that serum cholesterol levels and diastolic blood pressure significantly decreased in the freeze-dried tofu period as compared with the meat period. Freeze-dried tofu was found to be a valuable food for preventing lifestyle-related chronic diseases.

Adult↗
PubMed1998

[Experimental study of homograft of repeated freezing-thawing treated and ultra-low-temperature treated tendon].

In order to compare the immunogenecity and biological properties of homologous tendon grafts after treatment from different methods of freezing, tendons from chickens received repeated freezing-thawing treatment or ultra-low-temperature treatment, and then, the post-treatment tendons were preserved in liquid nitrogen for 3 months before transplantation. The autogenous tendon transplantation was served as the control. It was found that in the group of repeated freezing-thawing treated tendons, the tendon cells all died and while in the ultra-low temperature treated tendons the active rate of tendon cells was 92.5% +/- 3.4%, and the histological observation showed that transplantation of frozen tendons would result in extensive infiltration of inflammatory cells in the grafted tendons and the peritendinous adhesion was serious than that of the autografts. The active flexion function, hydroxyproline levels and the biomechanical analysis showed no significant differences between the repeated freezing-thawing treated homografts and the ultra-low-temperature treated homografts, and that the autografts was definitely superior to the homografts. The conclusions were: (1) Transplantation of the homologous tendons from the two different methods of freezing could receive considerable success and there was no significant difference between them; (2) Transplantation of frozen homologous tendon graft might give successful result which was probably due to the preservation of the cellular activity of the tendon cells following freezing treatment and elimination of the antigen presenting cells in the tendon as well, and (3) Although the cellular components of the tendon were damaged and the antigenicity of the tendon was lowered, it did not necessarily mean that homologous tendon graft would always be successful in transplantation.

Animals↗
PubMed2002

Effect of extracellular polymers on freeze-thaw conditioning of activated sludge.

In this study, the effects of extracellular polymers on freeze-thaw conditioning of activated sludge are investigated. Various physical and chemical extraction methods including centrifugation, blending, heat, EDTA, EGTA, and NaOH extraction were used to remove extracellular material from sludge matrix. The improvements in freeze-thaw conditioning were evaluated by commonly used measures of sludge dewaterability. The results of this study indicate that removal of extracellular polymers using relatively gentle extraction methods before freeze-thaw conditioning improves the sludge dewaterability after the freeze-thaw. In addition to extracellular polymers, cations also play an important role in determining the freeze-thaw effectiveness on activated sludge. Best dewaterability is achieved when both extracellular polymers and cations are removed from activated sludge before freeze-thaw conditioning.

Bioreactors↗
PubMed2003

[Experimental study of repairing segmental bone defect with reconstituted freeze-dried bone allograft].

OBJECTIVE: To study the effect of basic fibroblast growth factor (bFGF) and hyaluronic acid gel (HAG) combined with freeze-dried bone allograft in repairing segmental bone defect and to explore their mechanism. METHODS: The 15 mm segmental bone/periosteum defects were created on bilateral radius in 50 New Zealand rabbits and were treated with four different kinds of implants on 25 radius respectively (group A: bFGF and HAG combined with freeze-dried bone; group B: bFGF combined with freeze-dried bone; group C: HAG combined with freeze-dried bone; group D: simple freeze-dried bone as a control). The repair of defect was observed radiologically and histologically and were analyzed by radionuclide bone imaging and measurement of calcium contents at different periods. RESULTS: The new bone formation, bone metabolic activity and calcium contents of defects were higher in group A than in group B (P < 0.05), and were higher in group B than in groups C and D (P < 0.05). There were no significant difference between groups C and D. The bone defects healed in the 8th week in group A, in the 10th week in group B, but did not healed in the 10th week in groups C and D. CONCLUSION: As an osteogenetic factor, bFGF promotes the new bone formation; as a slow-release carrier, HAG enhances the effectiveness of bFGF. The combination of bFGF, HAG and freeze-dried bone allograft can repair the segmental bone defect more effectively.

Animals↗
PubMed1964

EFFECTS OF GASTRIC FREEZING ON THE ISOLATED DENERVATED GASTRIC POUCH IN DOGS.

The purpose of this study was to investigate the effects of gastric freezing on the chief and parietal cells of the stomach. To carry this out six Heidenhain pouch dogs were prepared. The secretions of these pouches were studied, both for HCl levels and pepsin activity, for 30 days before and 30 days after the freezing of the pouch. Freezing was carried out for one hour at -17 to -20 degrees C. The results show that no essential depression of either the HCl levels or the pepsin activity of the juice was effected by freezing. Therefore, it is deduced that freezing does not affect the functional activity of either the chief or the parietal cells. Complications such as ulceration and fistula formation into adjacent organs occurred as a result of freezing in certain pouch dogs.

Animals↗
PubMed1992

Scanning electron microscopic and light microscopic observations on morphological changes of freeze-dried bone implantation in rats: comparison with fresh autogenous bone transplantation.

Bone remodelling after the implantation of freeze-dried autogenous bone in rat parietal bone was compared with fresh autogenous bone transplantation, using a scanning electron and light microscope revealed the time intervals after transplantation/implantation. The light microscope revealed the time delay of the bone remodelling in the implantation, compared with the transplantations. The scanning electron microscope showed that the differences between the two groups were in the states of bone union and bone resorption. In the fresh bone group, the newly-formed bone filled the spaces between host and the transplanted bones at 2 to 3 weeks after the transplantation: the newly-formed bone fused and melted into the transplanted bone. New bone formation was more dominant on the bone surface in the dura mater side than in the skin side. The union was almost completed at 5 weeks. In freeze-dried bone implantation, the bone union in the contact space was very poor and the implanted bone was mainly covered by the new bone, which developed from the host bone surface in the dura mater side at 2 to 3 weeks after the implantation. What is noteworthy is that bone resorbed areas showing numerous Howship's lacunae were mainly observed on the host bone surface in the vicinity of newly-formed bone. However in freeze-dried bone implantation, the bone resorption was greater on the host and implanted bone surface than that of fresh bone transplantation: the resorption of host bone was considerably larger at certain periods after freeze-dried bone implantation. The present results show that the healing process of freeze-dried bone implantation, even though autogenous bone was used, differed from that of fresh autogenous bone transplantation, and the differences are concerned not only with time sequences but also with qualitative changes. This suggests that the host would have some different responses to the freeze-dried autogenous bone from fresh materials.

Animals↗
PubMedDate not supplied

Long-term stabilization of a new freeze-dried and albumin-free formulation of recombinant human interferon alpha 2b.

In this work, we evaluate the stability of a new freeze-dried and albumin-free formulation of recombinant human IFN alpha 2b (rhIFN-alpha2b) to be used in humans. The freeze-dried, albumin-free formulation was stored at the recommended temperature of 4 degrees C, and under accelerated storage conditions (28 degrees C). The stability of this product was also compared with the stability of a liquid albumin-free formulation of this cytokine. Finally, the stability of the freeze-dried albumin-free formulation was examined after reconstitution and storage at 4 degrees C and room temperature (28 degrees C) for 30 days. Samples were periodically subjected to biological activity assay (antiviral titration), reversed phase high-performance liquid chromatography (RP-HPLC), pyrogens, sterility and enzyme-linked immunosorbent assay (ELISA) testing, abnormal toxicity screening, organoleptic evaluation, and measurement of residual moisture and pH. Accelerated storage (28 degrees C) data for the freeze-dried albumin-free formulation showed biochemical stability of the active ingredient throughout the 6-month study, showing activity between 85 and 125% of its nominal value. RP-HPLC-determined purity showed that rhIFN-alpha2b remained above 95%. Additionally, the formulation was non-pyrogenic, non-toxic, sterile, and organoleptically acceptable. The real-time storage data confirmed the good biochemical long-term (30 months) stability of the freeze-dried formulation of this cytokine. Comparison with the liquid rhIFN-alpha2b albumin-free preparation showed that the freeze-dried albumin-free formulation maintained the stability of the active ingredient better than the liquid preparation. The formulation was also stable after reconstitution and storage at 4 degrees C and 28 degrees C, for 30 days.

Albumins↗
PubMed1991

A correlative study of the freezing patterns in rat myocardium using scanning and transmission electron microscopes.

Rat myocardial tissue, both fresh and chemically fixed, was quench frozen in melting Freon 22 at rates that resulted in in the formation of large ice-crystals. The material was studied with both scanning and transmission electron microscopy. Particular attention was paid to the characteristic freezing patterns within the myofibrils and mitochondria, and to features exposed by such freezing that might otherwise remain obscured. The freezing patterns found in tissue subjected to different processes were compared with the ultrastructure of unfrozen, chemically fixed counterparts. Ice-crystal cavities in the contractile material varied considerably along the length of a given myofiber in which the freezing front had progressed in parallel with the long axis of the myofibers. It is suggested that the intercalated disc functioned as a barrier to the freezing process. Ice generally compressed and distorted the contractile material beyond recognition, although the positions of the Z-bands remained evident and in register. Most single-fixed mitochondria were devoid of visible ice-crystal cavities, and the cristae generally remained intact. On the other hand, ice-crystal cavities were commonly seen between cristae of double-fixed mitochondria. No signs of perforations were seen in lipid bilayer membranes. Numerous strand-like connections between mitochondria as well as between mitochondria and the myofibrillar surface, which were believed to be sarcotubules, were made visible by the slow freezing process. Other strands, transverse to the myofibrils, connecting adjacent Z-bands and joining Z-bands to the sarcolemma, were interpreted as cytoskeletal elements. These strands, together with what apparently were SR tubules, exhibited high resistance to the stresses associated with the formation of ice-crystals.

Actin Cytoskeleton↗
PubMed1990

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing.

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.

Animals↗