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PubMed1990

Computerized slow-staged freezing of semen from men with testicular tumors or Hodgkin's disease preserves sperm better than standard vapor freezing.

Two methods of freezing semen taken from patients with testicular tumors or Hodgkin's disease before treatment were compared. Many patients already have semen abnormalities, so an optimal method is extremely important. Ejaculates from 8 patients with testicular tumors and 20 with Hodgkin's disease were frozen by fast-freezing or by slow-staged freezing. Effects of motility, viability, and swelling after thawing were significantly impaired with both methods. However, cryosurvival was better after slow- than fast-freezing: motility 24% +/- 12.4% versus 15% +/- 11.2%; viability 24.1% +/- 11.4% versus 17.3% +/- 10.4%, swelling 33.3% +/- 11% versus 27.6% +/- 12.8%. The effects were equal for normal and abnormal sperm. Sperm from tumor patients should be frozen by slow-staged freezing method in spite of the higher cost and longer time.

Computers↗

PubMed1994

A Comparison of Freezing Injury in Oat and Rye: Two Cereals at the Extremes of Freezing Tolerance.

A detailed analysis of cold acclimation of a winter rye (Secale cereale L. cv Puma), a winter oat (Avena sativa L. cv Kanota), and a spring oat cultivar (Ogle) revealed that freezing injury of leaves of nonacclimated seedlings occurred at -2[deg]C in both the winter and spring cultivars of oat but did not occur in winter rye leaves until after freezing at -4[deg]C. The maximum freezing tolerance was attained in all cultivars after 4 weeks of cold acclimation, and the temperature at which 50% electrolyte leakage occurred decreased to -8[deg]C for spring oat, -10[deg]C for winter oat, and -21[deg]C for winter rye. In protoplasts isolated from leaves of nonacclimated spring oat, expansion-induced lysis was the predominant form of injury over the range of -2 to -4[deg]C. At temperatures lower than -4[deg]C, loss of osmotic responsiveness, which was associated with the formation of the hexagonal II phase in the plasma membrane and subtending lamellae, was the predominant form of injury. In protoplasts isolated from leaves of cold-acclimated oat, loss of osmotic responsiveness was the predominant form of injury at all injurious temperatures; however, the hexagonal II phase was not observed. Rather, injury was associated with the occurrence of localized deviations of the plasma membrane fracture plane to closely appressed lamellae, which we refer to as the "fracture-jump lesion." Although the freeze-induced lesions in the plasma membrane of protoplasts of spring oat were identical with those reported previously for protoplasts of winter rye, they occurred at significantly higher temperatures that correspond to the lethal freezing temperature.

Journal Article↗

PubMed1982

Spin-Probe Studies during Freezing of Cells Isolated from Cold-Hardened and Nonhardened Winter Rye : MOLECULAR MECHANISM OF MEMBRANE FREEZING INJURY.

Mesophyll cells isolated from cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) were spin-labeled with the fatty-acid spin probe N-oxyl-4,4-dimethyloxazolidine 5-ketostearic acid. The probe was intercalated within the cellular membranes and changes in probe motion were followed during extracellular freezing of the cells. A correlation was observed between the lethal freezing temperatures (LT(50)) of the cells and the maximum hyperfine splitting value achieved by the incorporated probe. Rigid limit spectra indicated that a more ordered average packing was attained by membranes of hardened cells which survived freezing to lower temperatures.Nonhardened cells fixed with osmic acid at lethal freezing temperatures, in the frozen state, showed both conversion of normal bilayered cellular membrane ultrastructure to an amorphous state and condensation of cellular membranes to form densely packed multibilayered vesicles. Taken together with the spin-labeling data, these results suggest that at least one molecular mechanism of extracellular freezing injury involves the irreversible conversion of planar membrane bilayers to structures having less ordered packing and increased surface curvatures.

Journal Article↗

PubMed1984

Freezing behavior of water in small pores and the possible role in the freezing of plant tissues.

Two model systems were used to study the freezing of water in small diameter pores. Water in pores having a diameter of less than 100 nanometers froze at lower temperatures than bulk water. Data obtained with a range of pore sizes were consistent with predicted values based on equations developed by Mazur (1965 Ann NY Acad Sci 125: 658-676), and Homshaw (1980 J Soil Sci 31: 399-414). The addition of solutes lowered the freezing point of water in small pores. We propose that the freezing behavior of water in small pores may account for some of the freezing patterns observed in plant tissues. In tissues where cells are tightly packed, share common walls, and lack intercellular spaces, the presence of water in cell wall microcapillaries would alter the freezing temperature of tissue water, impede the spread of ice, and facilitate supercooling.

Journal Article↗

PubMed1986

Studies on Freezing Injury in Plant Cells : II. Protein and Lipid Changes in the Plasma Membranes of Jerusalem Artichoke Tubers during a Lethal Freezing in Vivo.

Plasma membranes were isolated from both unfrozen and frozen tissues of Jerusalem artichoke tubers (Helianthus tuberosus L.) in high purity utilizing an aqueous two-polymer phase partition system. Although the recovery of the plasma membranes was decreased significantly by freezing of tissues even at the nonlethal temperature (-5 degrees C), the isolated plasma membrane samples were considered to be representative of the plasma membranes in situ. Freezing of the tissues at sublethal temperatures resulted in marked changes in the chemical composition of the plasma membrane. Those are losses of sterols and phosphatidylethanolamine from the plasma membranes, and a change of specific proteins with relatively high molecular weights into low molecular weight peptides. These specific proteins were designated as frost susceptible proteins. The properties of the plasma membrane ATPase seem to be not affected so much by the in vivo freezing of cells. However, inhibition of the plasma membrane ATPase by N,N'-dicyclohexylcarbodiimide (DCCD) was relatively low before and after freezing in vivo at the nonlethal temperature at -5 degrees C, but was markedly enhanced by freezing in vivo at sublethal temperatures below -10 degrees C. From the results, it is assumed either that the enzyme molecule was partially modified, especially at the presumed DCCD binding sites or that the DCCD had become more accessible to the enzyme as a result of increased permeability of the plasma membranes. These observed changes are discussed in connection with the mechanism of cell injury.

Journal Article↗

PubMed1984

Pulmonary type II cell lamellar body ultrastructure preserved by rapid freezing and freeze drying.

Lamellar body ultrastructure was examined in cultured type II alveolar epithelial cells processed by a method of rapid freezing and freeze drying in the absence of both chemical fixation and solvent dehydration. This method of specimen preparation was chosen to optimize the retention of soluble substances within the type II cell. The use of cultured cell aggregates in which type II cells line the free surface facilitated the effectiveness of rapid freezing for the preservation of lamellar body fine structure. Lamellar bodies of frozen/frozen dried type II cells showed none of the often profound lipid extraction artifact produced by conventional processing. Instead they exhibited a substructure with noteworthy characteristics in common with lamellar bodies processed by resin dehydration lipid retention methods (Stratton, 1976). Importantly, the lamellae of frozen/frozen dried lamellar bodies were contiguous, with no interlamellar space, as is commonly observed in solvent-processed (extracted) specimens. The dimensions of lamellar components in frozen/frozen dried lamellar bodies were, however, different from published values for resin-dehydrated lipid-retained specimens. Lamellar width and the widths of component phospholipid head and fatty acid tail regions in frozen/frozen dried lamellar bodies were approximately 35% smaller than values reported for resin-dehydrated lamellar bodies. This difference was attributed to shrinkage of lamellar components as water was removed from the unfixed tissue during the freeze-drying process. Lamellar bodies preserved by rapid freezing/freeze drying to optimize the in situ retention of intracellular components possess closely adherent concentric membranous lamellae. This supports the contention that the widely appreciated lamellar pattern of the pulmonary lamellar body represents the in vivo molecular organization of intracellular surfactant phospholipids.

Animals↗

PubMed1988

Maturation changes of the plasma membrane of rat spermatozoa observed by surface replica, rapid-freeze and deep-etch, and freeze-fracture methods.

Rat spermatozoa from the epididymis and ductus deferens were observed by surface replica, rapid-freeze and deep-etch, and conventional freeze-fracture methods. By the surface replica method, parallel periodical ridges were observed in the acrosomal region of the spermatozoa from the distal part of the cauda epididymis (zone 6) and from the ductus deferens. The periodicity of the ridges forming a domain was about 35 nm. A quantitative analysis of the spermatozoa along the reproductive tract indicated that 39.4% and 73.5% of the population in zone 6 of the epididymis and in the ductus deferens, respectively, had the domain. None of the spermatozoa from zone 1 through zone 5 had the domain. The results of the rapid-freeze and deep-etch procedure showed that the ridges observed by the surface replica method consisted of linear arrangements of elliptical particles on the ES face of the plasma membrane. The particles were about 30 nm in length and 15 nm in width. On the corresponding PF face of the plasma membrane, linear arrangements of the intramembrane particles (IMPs) of about 8 nm in diameter were observed by both the deep-etch and freeze-fracture methods. The IMPs tended to run in paired parallel lines. A close relationship was observed between the lines of the elliptical particles on the ES and of the IMPs on the PF faces. The elliptical particle may be a protruded part of the IMP(s) or other protein(s) bound to the IMP(s).

Animals↗

PubMed1993

Cryofixation of basement membranes followed by freeze substitution or freeze drying demonstrates that they are composed of a tridimensional network of irregular cords.

Since conventional chemical fixation may extract tissue components and thus alter structural organization, cryofixation was used to reexamine the ultrastructure of three thick basement membranes: lens capsule, Reichert's membrane, and Engelbreth-Holm-Swarm (EHS) tumor matrix, and two thin basement membranes, those of epididymis and semi-niferous tubules. Cryofixation was achieved by slam freezing followed by either freeze substitution in dry acetone containing 1% osmium tetroxide and 0.05% uranyl acetate or freeze drying in a molecular distillation dryer. The results by both procedures demonstrate that thick basement membranes and the lamina densa of thin basement membranes are composed of a network of anastomosing strands referred to as cords. The cords vary in density and distinctiveness, but their thickness averages 3 to 5 nm in every tissue examined. The spaces separating the cords vary within wide limits, but their mean diameter is approximately 15 nm in every case. Two other common features are 1) the presence within the network of a few 1.5-3.0-nm-thick filaments and 2) 4.5-nm-wide sets of parallel lines referred to as double tracks. When these results are compared with those previously described after conventional fixation, no significant difference is observed in either the cord network or the associated filaments and "double tracks." However, in the thin basement membranes processed by cryofixation, the lamina densa is in direct contact with epithelial cells, whereas, after conventional fixation, the lamina densa is separated from the epithelial cells by a pale layer referred to as lamina lucida or lamina rara. Immunogold labeling of three basement membranes after cryofixation and freeze substitution in acetone containing 0.3% glutaraldehyde yields strong reactions for laminin, type IV collagen, and heparan sulfate proteoglycan. Comparison with previous results indicates that conventional formaldehyde fixation adequately preserves laminin and type IV collagen but causes the loss of some proteoglycan. It is concluded that, except for this loss and the absence of lamina lucida in cryofixed thin basement membranes, the morphological and antigenic features obtained after cryofixation are similar to those observed in the past after conventional fixation.

Animals↗

PubMed1997

Deformation of loaded articular cartilage prepared for scanning electron microscopy with rapid freezing and freeze-substitution fixation.

To investigate the effect of joint loading on collagen fibers in articular cartilage, 45 knees of adult rabbits were examined by scanning electron microscopy. The knees were loaded at the patella with a simulated "quadriceps force" of 0.5-4 times body weight for 0.5 or 25 minutes, plunge-frozen, and fixed by freeze-substitution with aldehydes. Six knees were loaded for 3 hours and then fixed conventionally. Fixed tibial plateaus were examined and then freeze-fractured through the area of tibiofemoral contact, dried, coated, and examined by scanning electron microscopy to assess the overall deformation of the tibial articular surface and matrix collagen fibers. With tissue prepared by conventional fixation used as a standard, the quality of fixation was graded by light and transmission electron microscopy of patellar cartilage taken from half of the freeze-fixed knees. In loaded specimens, an indentation was present where the femur contacted the tibial plateau. The diameter and apparent depth of the dent were proportional to the magnitude and duration of the load; no dent was seen in the controls. The thickness of the cartilage at the center of the indentation was reduced 15-80%. Meniscectomy always produced larger deformations in otherwise equivalent conditions. Icecrystal damage to cells was evident by transmission electron microscopy and scanning electron microscopy, but at magnifications as high as x 30,000 the collagen fibrils prepared by freeze-substitution and conventional aqueous methods were identical. In loaded regions, the collagen matrix of the tibial cartilage was deformed in two ways: (a) radial collagen fibers exhibited a periodic crimp, and (b) in regions where an indentation was created by the femoral condyle, the radial fibers were bent, in effect creating a tangential zone where none had existed before. The radial fibers apparently are loaded axially and buckle under normal loads.

Animals↗

PubMed1987

Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution.

The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4-6 nm in diameter. Fine filaments 8-10 nm in diameter and 30-50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3-5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.

Animals↗

PubMed1977

The influence of freezing and freeze-drying of tissue specimens on enzyme activity.

In the presented study the influence of freezing and freeze-drying on enzyme activity is described. Attention is paid to 16 enzymes which can be used for quantitative enzyme histochemical techniques. With the exception of succinate dehydrogenase only, no significant inactivation during freezing and freeze-drying procedures could be demonstrated with lactate dehydrogenase, malate dehydrogenase (NAD+), malate dehydrogenase (decarboxylating) (NADP+), isocitrate dehydrogenase (NADP+), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADH-oxydoreductase, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome c oxidase, phosphoglucomutase, glucosephosphate isomerase, glucose-6-phosphatase, acid phosphatase, beta-glucuronidase and non specific aryl esterase. Therefore, the results supply a sound foundation for those quantitative enzyme histochemical techniques in which tissue specimens are frozen or frozen-dried before enzyme estimations are performed.

Animals↗

PubMed2006

Ultrastructure of Helicobacter pylori in human gastric mucosa and H. pylori-infected human gastric mucosa using transmission electron microscopy and the high-pressure freezing-freeze substitution technique.

BACKGROUND: Conventional ultrastructural analyses of Helicobacter pylori and H. pylori-infected gastric mucosa by transmission electron microscopy (TEM) have limitations because of structural artifacts introduced during fixation. METHODS: We used high-pressure freezing (HPF) followed by freeze substitution for TEM to investigate the ultrastructure of H. pylori and H. pylori-infected gastric mucosa. For HPF-freeze substitution, human gastric biopsy specimens were placed in the HPF instrument at a pressure of approximately 2,000 atm at the temperature of liquid nitrogen. Specimens were then transferred to an instrument for freeze substitution. Specimens were first placed in acetone containing 2% OsO4 at -85 degrees C. The temperature was increased to -3 degrees C, followed by embedding in Quetol 812. Ultrathin sections were double-stained by uranium acetate/lead nitrate. HPF and conventionally prepared samples were examined by TEM. RESULTS: The H. pylori envelope was clearly seen to consist of an outer membrane, periplasmic space, and plasma membrane. The periplasmic space was filled with electron-dense materials. A peptidoglycan layer was only occasionally visible. A thick, very fine filamentous or reticular fringe corresponding to the bacterial glycocalyx was seen surrounding the H. pylori cells. At the adhesion loci of H. pylori to gastric epithelial cells, H. pylori was connected to the epithelial cells by very fine, thickly arranged filaments or more closely, with a contact zone. The epithelial cells showed indentations or pedestals. CONCLUSIONS: The well-developed, thick bacterial glycocalyx of H. pylori appears to strongly interact with external cellular components and may play an important role in the adhesion of H. pylori to epithelial cells.

Adult↗

PubMed1983

Viability of micrococci and lactobacilli upon freezing and freeze-drying in the presence of different cryoprotectants.

Studies were conducted on the viability of Micrococcus varians strain M95 and Lactobacillus plantarum strain L4 upon freezing and freeze-drying using five cryoprotectants (sucrose, lactose, sodium glutamate, peptone, dry nonfat milk) singly or in combinations with gelatin, glutamic acid, and sodium acetate. The number of survivals was determined immediately after treatment and after storage at room temperature or refrigeration temperatures, under vacuum or in air. Dry nonfat milk and peptone introduced at the levels of 8 and 5%, respectively, to broth culture, were found to be the best cryoprotectants providing a 100% viability determined immediately after the treatment of the strains under investigation. Immediately after freezing and freeze-drying, the numbers of viable micrococci remain high, the percentage viability in the presence of almost all the protectants used being 100%. During storage, those numbers decrease rapidly, reaching zero in 3 months upon storage at room temperature in air. The storage ability of lactobacilli is considerably better and, regardless of the fact that the percentage viability decreases, sufficient numbers of viable cells remain after 6 months of storage at both test temperatures. The best results are obtained on storing the microorganisms under vacuum in ampoules under reduced temperatures (+5 degrees C).

Cryoprotective Agents↗

PubMed1980

Cryofixation without cryoprotectants. Freeze substitution and freeze etching of an insect olfactory receptor.

Antennae of the silk moth, Bombyx mori, were frozen by immersion into propane at -180 degrees C, and further processed by (a) freeze substitution (FS) or (b) freeze etching (FE). Although no cryoprotectant was used, freezing damage was observed in deeper tissue regions only. Data from FS specimens closely resemble those from FE replicas. Therefore, FS usually does not induce noticeable secondary artefacts by the preparation steps subsequent to freezing. When compared with chemically fixed antennae, the superior quality of cryofixation in this tissue is evident, particularly where cell surfaces and processes border the receptor lymph cavity; membranes are smooth following a steady course; dendrites and axons are round in cross-section with evenly distributed microtubules. The value of cryofixation is discussed with special reference to structures of presumed functional significance (e.g. stimulus conducting pore tubules, intramembrane particles of the receptor membrane, the ciliary segment of the dendrites, intercellular dilations, membrane junctions).

Animals↗

PubMed1983

Artifacts associated with quick-freezing and freeze-drying.

We have studied the structures produced when nonbiological samples were subjected to quick-freezing and freeze-drying with a liquid helium cooled freeze-slamming device. Samples examined in this way included sodium chloride, sucrose, and Tris buffer. A variety of filamentlike and trabeculumlike structures were formed in these preparations. These structures may represent eutectic mixtures formed during the growth of small ice crystals during the freezing process, and exposed during the rapid sublimation of pure ice during the etching process. Samples of biological membranes (isolated chloroplast membranes) were prepared in various buffers by means of this technique. In distilled water, excellent replicas of membrane surfaces were obtained. In salt solutions, however, the membranes appeared to be embedded in a network of thin filaments appearing very much like a cytoskeletal lattice. It is concluded that extreme caution must be used when employing this preparation technique for studies of cell architecture, and that extensive washing of cell components in distilled water may be necessary to obtain faithful representations of cell structure.

Buffers↗

PubMed1981

Quick-freeze fixation and freeze-drying of isolated rat pancreatic islets: application to the ultrastructural localization of inorganic phosphate in the pancreatic beta cell.

A bounce-free mechanical quick-freeze assembly and a Coulter-Terracio freeze-dry apparatus were successfully coupled to obtain high quality ultrastructural preservation of pancreatic beta cells in a simple and dependable manner. Except for obvious shrinkage spaces, morphological relationships at the tissue, cellular, and subcellular levels were all intact. Beta cell secretory granules demonstrated a dense core surrounded by an electron lucent halo as typically described in specimens after aqueous fixation. Cell membranes and intracellular membranes demonstrated a trilaminar appearance. Golgi apparatus were well preserved. Two clearly defined populations of mitochondria were found. One group of very dark mitochondria had extremely dense matrices in which cristae were barely visible. A second group of mitochondria had light matrices with prominent cristae. The combined quick-freeze fixation and freeze-drying was applied to reevaluate the ultrastructural localization of inorganic phosphate that had been precipitated with lead in the beta cells of pancreatic islets. Accumulation of inorganic phosphate adjacent to the plasma membrane and over the nucleolus of the beta cell in nonstimulated islets was documented with better detail than heretofore possible.

Animals↗

PubMed1985

Ultrastructure and energy-dispersive x-ray microanalysis of cartilage after rapid freezing, low temperature freeze drying, and embedding in Spurr's resin.

In order to undertake meaningful high-resolution x-ray microanalysis of tissues, methods should be used that minimize the introduction of artefacts produced by loss or translocation of ions. The most ideal method is rapid freezing but the subsequent sectioning of frozen tissues is technically difficult. An alternative method is to freeze dry the tissues at a low temperature, and then embed them in resin. This facilitates the rapid production of reproducible thin sections. With freeze-dried, embedded hypertrophic cartilage, the morphology was similar to that seen using aqueous fixatives even when no additional electron density is introduced by the use of osmium vapor. Energy-dispersive analysis of specific areas show that little or no loss or migration of ions occurs from structures such as mitochondria. Mitochondrial granules consisting of calcium and phosphorus precipitates were not observed except where the cells were damaged as a result of the freezing process. This may suggest that these granules only appear when tissue is damaged because of inadequate preservation.

Animals↗

PubMed1993

Freeze-drying of cephalothin sodium: granularly agglomerated crystallization during freezing. II.

The mechanism of granularly agglomerated crystallization during freezing of cephalothin sodium (CET-Na) in aqueous solution has been discussed. Our previous report noted that strict control of the following is important in order to obtain crystalline granular agglomerates of CET-Na: (1) thermal history of the solution before receiving freezing, (2) cooling rate in freezing, and (3) aging temperature level in the crystal growth step. In order to clarify the physico/chemical meanings of the individual controls, further investigations have been made: 1) with varying thermal history, aqueous 30-% CET-Na solutions were prepared for storage, first at 0 degrees C with its supersaturation and secondly at 25 degrees C, in an unsaturated state for observing any structural changes by viscosimetry, refractometry, and surface tensiometry; 2) morphological changes in ice crystals during freezing at varying cooling rates, as well as those during the crystal growth step, were observed by polarized-light cryomicroscopy and scanning electron microscopy; 3) melting, as well as crystal growth, at several aging temperature levels were observed by electrical conductometry and also by the above-mentioned techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Cephalothin↗