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PubMed2005

Detection of viral, bacterial, and parasitological RNA or DNA of nine intestinal pathogens in fecal samples archived as part of the english infectious intestinal disease study: assessment of the stability of target nucleic acid.

Fecal samples were collected from cases and controls as part of the Infectious Intestinal Disease (IID) study in England and were stored as frozen suspensions for 8 to 12 years. The purpose of this study was to apply PCR-based procedures to assess the stability of pathogen-specific nucleic acid sequences present in this archive. Samples from which Cryptosporidium, Giardia, Salmonella, Campylobacter, enteroaggregative Escherichia coli (EAggEC), enterotoxigenic Clostridium perfringens, rotaviruses, noroviruses, or sapoviruses had been previously detected during the IID study using conventional methods were selected from the archive. A generic nucleic acid extraction method to recover RNA or DNA was used. Complementary DNA was generated from RNA by reverse transcription with random priming. Block-based and real-time PCR assays were used to amplify and detect gene fragments from each of these pathogens. The percentage reconfirmation of target was as follows: Giardia duodenalis 68%, Cryptosporidium 96%, Campylobacter 98%, Salmonella 98%, enterotoxigenic C perfringens 34%, EAggEC 93.3%, rotavirus 95%, norovirus 73%, and sapovirus 85%. This study has shown that nucleic acid can be extracted and specific sequences amplified and detected from archived fecal samples. The IID archive therefore represents a valuable resource for further studies, especially the investigation of the samples from which no pathogens had previously been detected.

Cryopreservation↗
PubMed2006

Application of in vivo cryotechnique to the examination of cells and tissues in living animal organs.

When all biological materials in cells and tissues of living animal organs are quickly and promptly frozen, immunolocalization of their components and structural features in situ is necessary to understand their in vivo functioning states. However, these direct morphological analyses were difficult to achieve by conventional chemical fixation methods during the last century. A new cryofixation method, named the "in vivo cryotechnique", in which the normal blood circulation in living animals is always retained at the moment of freezing, has become a powerful tool to visualize the real native morphology of cells and tissues with functional meaning. The "in vivo cryotechnique" can usually be combined with a wide range of subsequent preparation techniques, and can thereby enable us to perform various direct analyses on biological samples, reflecting the physiological functions of living animal organs.

Animals↗
PubMed1992

Reduction of mortality rates of Bacillus thuringiensis var. israelensis aqueous suspensions due to freezing and thawing.

When studying the behavior (carry, dispersion, persistence) of Bacillus thuringiensis var. israelensis (B.t.i.) formulations used in the treatment of rivers or streams for black fly control, a large number of samples containing small quantities of B.t.i. are required for proper analysis. Freezing is a useful procedure to prevent enzymatic alteration or bacterial growth in samples before bioassays are to be performed. Using Aedes atropalpus neonate larvae, we studied the effect of freezing and thawing of B.t.i. aqueous suspensions by looking at mortality response parameters such as the slope and the LC50 of the probit regression. Initial concentration values of 1, 5, 10 and 20 mg/liter at the moment of freezing of the B.t.i. suspensions did not significantly affect toxicity. The number of freeze-thaw cycles greatly increased the LC50 values without much change to the slope of the log-probit regressions. We derived an equation that allowed us to compensate for the loss of toxicity of a given B.t.i. sample, knowing the number of freeze-thaw cycles.

Aedes↗
PubMed1995

Use of pharmacologically induced ejaculation to obtain semen from a stallion with a fractured radius.

Ejaculation was pharmacologically induced in a 13-year-old Quarter Horse stallion with a spiral fracture of the radius. The owners desired to have semen from the stallion frozen prior to euthanatizing the horse, but because of the debilitating injury, standard methods of semen collection could not be used. With the stallion standing quietly in a stall, a plastic collection bag was positioned over the stallion's penis, and clomipramine hydrochloride (2.2 mg/kg of body weight, IV) was administered. Fifty-five minutes later, xylazine hydrochloride (0.5 mg/kg, IV) was administered. The stallion ejaculated 2 minutes after xylazine administration. The semen sample was of low volume and had a high concentration of spermatozoa; however, motility of spermatazoa was poor and the semen was unacceptable for freezing. The stallion was euthanatized. Semen aspirated from the epididymis after euthanasia was of similar quality to that of the ejaculated sample. Pharmacologic induction of ejaculation can be useful for obtaining semen from stallions when standard techniques cannot be applied.

Animals↗
PubMedDate not supplied

Effect of cryoprotectants on freezing, lyophilization, and storage of lyophilized recombinant alpha 1-antitrypsin formulations.

Stabilizing effect of several commonly used cryoprotectants, namely lactose, sucrose, and polyvinyl-pyrrolidone (PVP), on recombinant alpha 1-antitrypsin (rAAT) were studied. A solution of rAAT in a phosphate/citrate buffer (pH 7.0) was made with and without a given cryoprotectant and filled into small vials. The vials were frozen on shelf, at -40 degrees C in a lyophilizer. At the end of the freezing cycle, half of the batch was thawed at ambient temperature and the other half was continued to the completion of lyophilization. Sample vials taken before freezing, after thawing, and after lyophilization were analyzed for total rAAT protein, monomeric content, and elastase-inhibitory activity. Results indicated that neither freeze-and-thaw nor lyophilization caused any damage to rAAT, contrary to what was generally believed. The control formulation (i.e., without a cryoprotectant) performed as good as those containing cryoprotectants. Freezing rates and protein concentration in formulation did not influence the stability of rAAT either. Lyophilized rAAT samples retained the activity and purity during the 12 month stability period, at room temperature and 2-8 degrees C.

Buffers↗
PubMedDate not supplied

Analyte stability study of N-methylcarbamate pesticides in beef and poultry liver tissues by liquid chromatography.

To optimize conditions for sample collection, preparation, storage, and analysis and to assure the validity of our previously published liquid chromatographic (LC) method for carbamate analysis in tissue, stabilities of 16 N-methylcarbamates in beef, duck, and chicken liver tissues were studied by using 2 sampling protocols. Tissue samples were fortified at room temperature to a concentration 5 to 10 times greater than either the Environmental Protection Agency tolerance level for each compound (if established) or the concentration used in the previously published method. Thereafter, samples were continuously frozen at -4 degrees C for varying time intervals. In the first study, samples were analyzed one day (initial) and 0.5, 1, 1.5, 2, 3, 4, 5, and 6 months after fortification. In the second study, samples were analyzed one day (initial) and 0.5, 1, 2, 3, and 6 months after fortification. For each residue and species, a minimum of 4 samples were analyzed by LC at each point in time, and the mean represented analyte concentration at the end of each time interval. Rates of residue depletion varied among analytes and among species. Depletion rates were greater in duck livers than in beef livers. Methomyl and oxamyl were depleted completely within 2 weeks. Between 2 and 6 months after sample fortification, residue depletions to levels below detection limits were observed for aldicarb, aldicarb sulfoxide, aldicarb sulfone, dioxacarb, promecarb, propoxur, and bendiocarb. The initial loss of certain carbamates during sample preparation in tissues exposed to room temperature for up to 8 h was greater than the subsequent rate of loss. Results indicate that cryogenic conditions are required for sample preparation and storage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Cryopreservation of rat and human liver slices by rapid freezing.

The cryopreservation of human liver slices is a promising way to enhance the ability to test the metabolism of drug candidates. This study demonstrates the use of a novel technique for the cryopreservation of both rat and human liver slices. In this technique the slices are treated with Me2SO and sandwiched between aluminum plates separated by a thin gasket. The device is then submerged in liquid nitrogen to freeze the slices, which can then be stored until use. To thaw the slices, the apparatus is submerged in a water bath at 37 degrees C. Slices frozen and thawed in this manner were compared to those frozen in conventional cryovials. The viability of the slices was determined by incubating them in 12-well plates and measuring urea synthesis, ethoxycoumarin metabolism, and cytosolic enzyme leakage (LDH and ALT). The viability of rat slices frozen between plates approached that of fresh slices and was consistently higher than slices frozen in cryovials. Slices from two human samples gave similar results. The technique was found to work over a wide range of Me2SO concentrations (4.5 to 22% was tested) with an optimal concentration between 10 and 15%.

Aged↗
PubMedDate not supplied

Effect of centrifuge speed, refrigeration medium, and sperm washing medium on cryopreserved sperm quality after thawing.

Cryopreservation and subsequent thawing of semen for assisted reproductive procedures decreases sperm motility; motility further reduces when the cryoprotectant medium is removed because of the osmotic shock and centrifugation done to prepare the sperm. To compare motility and thus pregnancy rates, this study examined the effects of adding an additional refrigeration medium and three different centrifugation speeds for sperm preparation. Semen samples from 16 healthy normal volunteers were obtained by masturbation after 48 h of abstinence. Sperm motility and other motion characteristics were analyzed with a computer-assisted semen analyzer before freezing, after thawing, and after centrifugation. Each sample was divided into 6 aliquots and frozen using the liquid nitrogen vapor method. After thawing, human tubal fluid (HTF) with 5% human serum albumin was added to 3 aliquots, and refrigeration medium (identical to freezing medium without glycerol) was added to the remaining 3 tubes for each specimen. The tubes containing the two media were then washed by centrifugation at 100 g, 300 g, and 500 g for 10 min. Aliquots with refrigeration medium did not significantly differ from those with HTF for percent motility, curvilinear velocity, straight-line velocity, and amplitude of lateral head displacement at any centrifugation speed. Motile sperm count was significantly greater only at 100 g and 300 g for refrigeration medium (p = .02 and .01) and HTF (p = 0.001); at 300 g, average path velocity in refrigeration medium aliquots (p = .01) and linearity in HTF (p = .01) were greater. The results indicated that the reduction in motility and other motion characteristics probably cannot be overcome by changing factors such as the sperm preparation medium or centrifugation speed. More effective cryopreservation techniques or preparation methods that eliminate centrifugation need to be developed.

Centrifugation↗
PubMedDate not supplied

Recovery of potato apices after several years of storage in liquid nitrogen.

A method for the systematic cryopreservation of potato apices was developed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and the Institute for Crop and Grassland Science of the Federal Agricultural Research Centre (FAL, Braunschweig). Designed specifically for routine use in genebanks, this method uses a very simple ultra-rapid freezing approach and was applied to a wide range of varieties within the Federal Centre for Breeding Research on Cultivated Plants (BAZ, Quedlinburg) Potato Collection. After several years of storage in liquid nitrogen, shoot tips from a random sample of 51 varieties were thawed and the survival and shoot regeneration percentages compared to those measured immediately after freezing. There were no major changes in either survival or recovery of frozen apices. Data presented are not the outcome of a systematic experiment but from that accumulated during our work from 1992 to 1999.

Cryopreservation↗
PubMed1999

A novel approach to sperm cryopreservation.

Human spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been achieved by the standard approaches of cryobiology. Conventional approaches to cryopreservation impose a linear change of temperature with time; however, the stresses that cells encounter during cryopreservation are all non-linear with time. In this paper it is shown that improved methods of cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes instead of imposing a linear temperature reduction. Several treatments were compared: control of solidification to achieve constant ice formation with time was more damaging than the standard linear reduction in temperature. However, treatments which followed a chosen non-linear concentration profile, referred to as 'controlled concentration' allowed recovery of almost all the cells which were motile before freezing. The biophysical basis of these different responses was examined using the cryostage of a scanning electron microscope and freeze substitution and it was found that, surprisingly, all samples of spermatozoa in the frozen state were neither osmotically dehydrated nor had any visible intracellular ice. Viability on thawing did not appear to correlate with conventional theories of cellular freezing injury, which suggests that for human spermatozoa other factors determine viability following freezing and thawing.

Cryoelectron Microscopy↗
PubMed2001

Tissue factor activity in human monocytes is regulated by plasma: implications for the high and low responder phenomenon.

The 'high and low responder phenomenon' of monocyte tissue factor (MTF) activity has been attributed to effects on monocytes by granulocytes, platelets and lipopolysaccharide (LPS). To study the possible contribution of plasma to the high and low responder phenomenon, we measured the MTF activity in isolated cryopreserved human monocytes from two donors (monocytes A and monocytes B) after incubation in a plasma environment depleted of granulocytes, platelets and LPS. In buffer only, MTF activity was 643 and 679 fM (fM = final concentration of tissue factor), in normal pooled plasma, it was 1478 and 1615 fM (P = 0.001), respectively, in monocytes A and in monocytes B. Incubation with individual plasma samples from healthy controls (n = 43) gave a median MTF of 1355 fM (range 1044-1976 fM) and 1329 fM (range 858-1951 fM) respectively. A plasma consistently induced a higher or lower level of MTF activity in both monocytes: r = 0.82 (P < 0.00001). Coumarin use did not influence the high and low responder phenomenon. In the absence of granulocytes, platelets and LPS, plasma determines the high and low responder phenomenon. This phenomenon is not influenced by coumarin treatment.

Aged↗
PubMed2001

Generation of live rat offspring by intrauterine insemination with epididymal spermatozoa cryopreserved at -196 degrees C.

This study reports the development of a reliable method for cryopreservation of rat epididymal spermatozoa and the production of live young by artificial insemination using these cryopreserved spermatozoa. The motility and membrane integrity of rat spermatozoa were investigated after spermatozoa had been subjected to physical stress and frozen with various concentrations of glycerol (0, 3 and 6%) either in the presence or absence of Equex Stem as cryoprotective agents. The ability of cryopreserved spermatozoa to generate normal offspring by intrauterine insemination was also evaluated. Rat spermatozoa that had been centrifuged at 700 g for 5 min showed a significant decrease in motility compared with non-centrifuged spermatozoa. In addition, after centrifugation three times the percentage of membrane-intact spermatozoa decreased to approximately 0%. The percentage of membrane-intact spermatozoa was significantly higher (P < 0.01) in semen samples that had been frozen in medium without glycerol than in samples frozen in medium with 3% glycerol. Although the addition of 0.7% Equex Stem to medium without glycerol or with 3% glycerol did not influence rates of sperm motility after freezing and thawing, the percentage of membrane-intact spermatozoa was improved by the presence of 0.7% Equex (P < 0.05). Therefore, rat spermatozoa were handled gently to avoid physical stress and were frozen in medium containing 23% egg yolk, 8% lactose monohydrate and 0.7% Equex Stem, at pH 7.4 adjusted with 10% Tris(hydroxymethyl)aminomethane solution. Thirteen female rats were inseminated into the oviductal end of both uterine horns with frozen-thawed spermatozoa. Forty-one normal live offspring were obtained from nine of the inseminated females. These results indicate that frozen-thawed rat spermatozoa can generate normal offspring. To our knowledge, this procedure is the first successful production of offspring using spermatozoa cryopreserved in liquid nitrogen.

Animals↗
PubMed1995

Freeze-fracture study of Bodo sp. (Kinetoplastida: Bodonidae).

Freeze-fracture technique was used to analyse the structure of conventionally fixed and quickly frozen Bodo sp., a free-living kinetoplastid. In the former method, chemically fixed and cryopreserved cells presented a corrugated membrane pattern in the flagella and cell body surfaces. In the latter, however, replicas from quickly frozen unfixed flagellates showed membranes with a smoother aspect, allowing the observation of intramembranous particles (IMPs) on the fracture faces, hardly detectable in previously fixed samples. The IMPs were randomly distributed throughout the cell surface, except in the sparsely seen short IMP rows.

Animals↗
PubMed1988

Fluorometric evaluation of cryopreserved bovine spermatozoa extended in egg yolk and milk.

A comparison of fluorogenically quantifiable parameters of cryopreserved, bovine spermatozoa that had been processed in homogenized milk and egg yolk citrate-based extenders was made using flow cytometry. Semen from four bulls was processed in egg yolk-citrate or homogenized milk extenders, packaged in straws and frozen at -196 degrees C. Samples were thawed at 37 degrees C, subdivided into three portions and stained after 0, 1.5 and 3 h of incubation at 37 degrees C. Spermatozoa were stained using a combination of carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) and analyzed by dual parameter flow cytometry. The sperm cells were quantified fluorometrically at each time interval for both green and red fluorescence. The proportion of spermatozoa retaining the fluorescent CFDA derivative was larger at each time interval for samples in egg yolk citrate than those in milk. Differences in the retention of spermatozoal viability were detected between identical samples of bovine spermatozoa extended in milk or egg yolk based media.

Journal Article↗
PubMed2005

A new method to measure air-borne pyrogens based on human whole blood cytokine response.

Air-borne microorganisms, as well as their fragments and components, are increasingly recognized to be associated with pulmonary diseases, e.g. organic dust toxic syndrome, humidifier lung, building-related illness, "Monday sickness." We have previously described and validated a new method for the detection of pyrogenic (fever-inducing) microbial contaminations in injectable drugs, based on the inflammatory reaction of human blood to pyrogens. We have now adapted this test to evaluate the total inflammatory capacity of air samples. Air was drawn onto PTFE membrane filters, which were incubated with human whole blood from healthy volunteers inside the collection device. Cytokine release was measured by ELISA. The test detects endotoxins and non-endotoxins, such as fungal spores, Gram-positive bacteria and their lipoteichoic acid moiety and pyrogenic dust particles with high sensitivity, thus reflecting the total inflammatory capacity of a sample. When air from different surroundings such as working environments and animal housing was assayed, the method yielded reproducible data which correlated with other parameters of microbial burden tested. We further developed a standard material for quantification and showed that this assay can be performed with cryopreserved as well as fresh blood. The method offers a test to measure the integral inflammatory capacity of air-borne microbial contaminations relevant to humans. It could thus be employed to assess air quality in different living and work environments.

Air Microbiology↗
PubMed1991

Determination of 6-thioguanine resistant lymphocytes in human blood by immunohistochemical antibromodeoxyuridine staining.

An immunohistochemical method for the determination of 6-thioguanine resistant (TGr) lymphocytes in human blood samples has been developed. The new technique is a modification of the autoradiographic assay and uses labelling of DNA synthesizing cells by bromodeoxyuridine (BrdU) instead of tritiated thymidine. The label is detected immunohistochemically by using a monoclonal antibody against BrdU in single-stranded DNA. Thawed cryopreserved isolated mononuclear cells are cultured with (TG cultures) and without (control cultures) TG for 40 h, the last 16 h with BrdU. The cells are harvested, fixed and put on microscopic slides; for the TG cultures, all material is used, while slides from the control culture are prepared at a dilution of 1:300. The immunohistochemical staining of the slides involves denaturation of DNA in formamide, incubation with anti-BrdU-DNA antibody, followed by a secondary antibody conjugated with peroxidase and staining with diaminobenzidine. Nuclei that have incorporated BrdU appear brown while all other material remains unstained. The number of all labelled nuclei is scored similarly in control and TG slides and the frequency of TGr lymphocytes is obtained by dividing the number of labelled nuclei in TG slides with that (multiplied by the dilution factor) in control slides. The anti-BrdU method will much simplify and speed up the microscopical evaluation of the TG variants and is very suitable for automation.

Bromodeoxyuridine↗
PubMedDate not supplied

Effects of cryopreservation on testicular sperm nuclear DNA fragmentation and its relationship with assisted conception outcome following ICSI with testicular spermatozoa.

The objective of the study was to investigate the effects of freeze-thawing on testicular sperm DNA fragmentation, fertilization rates and pregnancy rates following intracytoplasmic sperm injection with testicular spermatozoa (TESE). This ongoing prospective study included 88 couples attending for infertility treatment where the man presented with obstructive azoospermia at the Regional Fertility Centre, Belfast, UK. Patients were allocated to receive TESE treatment with fresh or freeze-thawed spermatozoa. Sperm aliquots were stored in liquid nitrogen at -196 degrees C following static phase vapour cooling or cooling at controlled rates using a programmable freezer. Samples were thawed at either room temperature or 37 degrees C. Sperm nuclear DNA; assessed by the alkaline Comet assay, was significantly damaged by slow freezing followed by fast thawing. Pregnancies were more likely to be achieved with spermatozoa displaying markedly less DNA damage. However, no differences were observed in the fertilization rates, the number of blastomeres or the cumulative embryo score between TESE cycles using either fresh or frozen thawed testicular spermatozoa. The pregnancy rates tended to be higher following fresh TESE cycles (30%) compared with TESE cycles using frozen-thawed testicular spermatozoa (26%), although this difference did not reach statistical significance. It is concluded that cryopreservation of testicular spermatozoa may reduce pregnancy rates, although this will only be confirmed by a much larger multi-centre trial.

Biopsy↗
PubMed1993

Cryopreservation of human spermatozoa. IV. The effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity, and mitochondrial function.

OBJECTIVE: To test the hypotheses that there is a two-factor aspect of cellular damage during cryopreservation that occurs in human sperm (osmotic effects versus intracellular ice formation) and that there is a cooling rate by warming rate interaction related to this damage. DESIGN: Ejaculates from healthy men were cooled at 0.1, 1.0, 10, 175, or 800 degrees C/min to -80 degrees C in a solution of 0.85 M glycerol and plunged into liquid nitrogen. Samples were warmed at 400 degrees C/min (experiment 1) or either 1 degrees C or 400 degrees C/min (experiment 2). After warming, sperm were assessed for survival using motility as the endpoint in experiment 1 and motility, plasma membrane integrity, and mitochondrial function in experiment 2. RESULTS: In experiment 1, over the various cooling rates with a standard 400 degrees C/min warming rate, a plot of motility versus cooling rate produced a classical inverted U-shaped curve (n = 6) with maximum motility at the 10 degrees C/min cooling rate. In experiment 2, over the various cooling rates, both 1 and 400 degrees C/min warming rates produced similar but shifted plots of motility, plasma membrane integrity, and mitochondrial function versus cooling rate, which also produced inverted U-shaped patterns (n = 11). Maximal survival for each of the three endpoints occurred at 10 degrees C/min cooling rate for the rapidly warmed sperm and at 1 degree C/min for the slowly warmed sperm. CONCLUSIONS: These data support the hypotheses that a two-factor hypothesis of cryodamage applies to human spermatozoa and that an interaction exists between cooling rate and warming rate. These data also suggest that motility, plasma membrane integrity, and mitochondrial function are not differently affected by cooling and warming during cryopreservation.

Adult↗