Home About us Contact
|
Home About us Contact | ||
|
|
||
Rapid Freezing (rapid + freezing)
Selected AbstractsComparison between computerized slow-stage and static liquid nitrogen vapour freezing methods with respect to the deleterious effect on chromatin and morphology of spermatozoa from fertile and subfertile menINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2001M. E. Hammadeh The purpose of this study was to determine the negative effects (cryodamage) on human spermatozoa after freeze-thawing and to determine whether freeze-thawing of spermatozoa with a programmed slow freezer is better than freezing with liquid nitrogen vapour (rapid freezing) with regard to alterations in sperm chromatin and morphology in semen from fertile (donor) and subfertile, IVF/ICSI, patients. Ninety-five semen samples were obtained either from patients attending our IVF unit for treatment (n=34) or from donors (n=25) with proven fertility and normal sperm quality according to WHO guidelines. Each semen sample was divided into two parts after liquefaction and addition of the cryoprotectant. The first part was frozen using a programmed biological freezer and the second part was frozen by means of liquid nitrogen vapour. Smears were made before the freezing and after the thawing procedure to assess morphology (strict criteria) and chromatin condensation (Acridine Orange test). The mean percentage of chromatin condensed spermatozoa in the samples from donors (control group) was 92.4 ± 8.4% before freezing and this decreased significantly (p < 0.0001) to 88.7 ± 11.2% after freeze-thawing with the computerized slow-stage freezer and to 87.2 ± 12.3% after using static liquid nitrogen vapour (p < 0.001). The corresponding values for semen obtained from patients was 78.9 ± 10.3% before freezing which decreased to 70.7 ± 10.8 and 68.5 ± 14.8%, respectively (p < 0.001). On the other hand, the mean percentage of normal sperm morphology in the control group decreased from 26.3 ± 7.5% before freezing to 22.1 ± 6.4% (p < 0.0001) after thawing with the computerized slow-stage freezer and to 22.2 ± 6.6% (p < 0.0001) after the use of static liquid nitrogen vapour. In the patient group, the mean percentage of normal morphology decreased from 11.7 ± 6.1% after freezing with the biological freezer to 9.3 ± 5.6% and to 8.0 ± 4.9% after freezing with static liquid nitrogen vapour. This study demonstrates that chromatin packaging and morphology of human spermatozoa decrease significantly after the freeze-thawing procedure, not only after the use of static liquid nitrogen vapour but also after the use of a computerized slow-stage freezer. However, the chromatin of semen samples with normal semen parameters (donor sperm) withstand the freeze-thaw injury better than those with low quality semen samples. Therefore, the computerized slow stage freezer could be recommended for freezing of human spermatozoa, especially for subnormal semen samples, for example, ICSI and ICSI/TESE candidates and from patients with testicular tumours or Hodgkin's disease, in order to avoid further damage to the sperm chromatin structure. [source] SURVIVAL OF THREE SALMONELLA SEROTYPES ON BEEF TRIMMINGS DURING SIMULATED COMMERCIAL FREEZING AND FROZEN STORAGEJOURNAL OF FOOD SAFETY, Issue 2 2001G.A DYKES ABSTRACT This study investigated the survival of three Salmonella serotypes (S. Brandenberg, S. Dublin and S. Typhimurium) on beef trimmings during simulated commercial freezing, frozen storage for 9 months and subsequent abusive slow thawing and refreezing conditions. This was achieved by plating samples monthly and after thawing and refreezing on nonselective Tryptic Soy Agar (TSA) and selective Xylose Lysine Desoxycholate Agar (XLD) and incubating both at 37C for 24 h to determine Salmonella counts, aerobic counts and the presence, if any, of sublethal injury of this pathogen. Two freezing temperatures (,18C or ,35C) to simulate slow or rapid freezing respectively, and two inoculation levels (103 cfu g,1 or 105 cfu g,1) were used. Aerobic counts and counts of all the Salmonella serotypes did not change significantly (p > 0.05) during frozen storage or for any of the other treatments applied in this study. This finding was attributed to the insulating nature of the subcutaneous fat layer in this manufacturing cut. These results are important with respect to food safety associated with ground beef processing. [source] Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructureJOURNAL OF MICROSCOPY, Issue 1 2003T. Müller-Reichert Summary Caenorhabditis elegans is one of the most important genetic systems used in current biological research. Increasingly, these genetics-based research projects are including ultrastructural analyses in their attempts to understand the molecular basis for cell function. Here, we present and review state-of-the-art methods for both ultrastructural analysis and immunogold localization in C. elegans. For the initial cryofixation, high-pressure freezing is the method of choice, and in this article we describe two different strategies to prepare these nematode worms for rapid freezing. The first method takes advantage of transparent, porous cellulose capillary tubes to contain the worms, and the second packs the worms in E. coli and/or yeast paste prior to freezing. The latter method facilitates embedding of C. elegans in a thin layer of resin so individual worms can be staged, selected and precisely orientated for serial sectioning followed by immunolabelling or electron tomography. [source] Investigation of freezing- and thawing-induced biological, chemical, and physical changes to enoxaparin solutionJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 3 2009Rahul P. Patel Abstract This study investigated the effect of freezing and thawing on the biological, physical, and chemical properties of enoxaparin solution. Solutions were frozen and thawed under different conditions, in the presence or absence of dimethyl sulfoxide (DMSO) or 1,2-propanediol (1,2-PD), and the antifactor Xa (AFXa) activity was determined. Enoxaparin solution lost more than 60% of its AFXa activity when thawed rapidly after freezing at ,196°C. The loss of AFXa activity was less with higher freezing temperatures and increased with the number of freeze/thaw cycles, but was independent of the duration of freezing. Slow freezing to ,196°C with rapid thawing, or rapid freezing with slow thawing, resulted in negligible loss of AFXa activity. The loss of AFXa activity did not involve the loss of N -sulfate groups, the breakdown of glycosidic bonds or the glassy state transition. Controlling the freezing or thawing conditions, dilution with water or addition of a small percentage of DMSO ameliorated the loss of enoxaparin AFXa activity. The loss in AFXa activity was found by size exclusion chromatography to be primarily due to aggregation and was reversed by sonication in the presence of DMSO. These results may provide insight into solutions for the long-term storage of concentrated or diluted enoxaparin. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:1118,1128, 2009 [source] Freeze-drying of tert- butanol/water cosolvent systems: A case report on formation of a friable freeze-dried powder of tobramycin sulfateJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2002Sakchai Wittaya-Areekul Abstract A case study is presented in which a tert -butanol (TBA)/water cosolvent system was found to be a useful means of producing freeze-dried tobramycin sulfate that readily forms a loose powder upon agitation in a specialized application in which a critical quality attribute is the ability to pour the sterile powder from the vial. Both formulation and processing variables are important in achieving acceptable physical properties of the cake as well as minimizing residual TBA levels. Liquid/liquid phase separation was observed above critical concentrations of both drug and TBA, resulting in a two-layered lyophilized cake with unacceptable appearance, physical properties, and residual TBA levels. However, the choice of tobramycin sulfate and TBA concentrations in the single-phase region of the phase diagram resulted in a lyophilized solid that can readily be poured from vials. Crystallization of TBA before drying is critical to achieving adequately low residual TBA levels, and this is reflected in the effect of thermal history of freezing on residual TBA levels, where rapid freezing results in incomplete crystallization of TBA and relatively high levels of residual solvent. Annealing at a temperature above T,g of the system after an initial freezing step significantly reduces the level of residual TBA. Secondary drying, even at increased temperature and for extended times, is not an effective method of reducing residual TBA levels. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91: 1147,1155, 2002 [source] DNA extraction method for PCR in mycorrhizal fungiLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2001S. Manian Aims: To develop a simple and rapid DNA extraction protocol for PCR in mycorrhizal fungi. Methods and Results: The protocol combines the application of rapid freezing and boiling cycles and passage of the extracts through DNA purification columns. PCR amplifiable DNA was obtained from a number of endo- and ecto-mycorrhizal fungi using minute quantities of spores and mycelium, respectively. Conclusions: DNA extracted following the method, was used to successfully amplify regions of interest from high as well as low copy number genes. The amplicons were suitable for further downstream applications such as sequencing and PCR-RFLPs. Significance and Impact of the Study: The protocol described is simple, short and facilitates rapid isolation of PCR amplifiable genomic DNA from a large number of fungal isolates in a single day. The method requires only minute quantities of starting material and is suitable for mycorrhizal fungi as well as a range of other fungi. [source] An improved protocol for rapid freezing of protein samples for long-term storageACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2004Rapid freezing of protein samples Freezing of purified protein drops directly in liquid nitrogen is a convenient technique for the long-term storage of protein samples. Although this enhances reproducibility in follow-up crystallization experiments, some protein samples are not amenable to this technique. It has been discovered that plunging PCR tubes containing protein samples into liquid nitrogen results in more rapid freezing of the samples and can safely preserve some proteins that are damaged by drop-freezing. The PCR-tube method can also be adapted to a PCR-plate freezing method with applications for high-throughput and structural genomics projects. [source] | |||||||||||||