Peripheral nerve allograft preservation improves regeneration and decreases systemic cyclosporin A requirements

Peripheral nerve allografting is limited by the need for long-term systemic immunosuppression. The purpose of this study was to determine if nerve allograft preservation reduced the requirements for systemic Cyclosporin A (CsA) immunosuppression. One hundred twenty Lewis rats were randomized to one of seven experimental groups. Group 1 received a 2-cm Lewis posterior tibial nerve autograft. Groups 2-7 received 2-cm ACI posterior tibial nerve allografts. The allograft group was then further subdivided into three groups of two receiving daily subcutaneous injections of 0, 2.5, or 5.0 mg/kg of CsA for 12 weeks. Within each CsA dose, one group received a fresh while the other received a preserved allograft. Preserved grafts were stored in University of Wisconsin solution for 7 days at 5 degrees C prior to implantation. Animals from each group were sacrificed at 6, 12, and 20 weeks postoperatively. Evaluations included histomorphometry, electrophysiology, and serial walking track analysis. Histology revealed varying degrees of nerve regeneration in all groups at 6, 12, and 20 weeks. For a given CsA dose, the group receiving the preserved graft revealed evidence of better nerve regeneration by all histomorphometric parameters including fiber width and density, percentage neural tissue, and total fiber number. There was no statistical difference in walking track analysis between groups at 4 weeks. By 20 weeks, functional recovery statistically poorer than autograft was seen only in the fresh allograft groups receiving 0 or 2.5 mg/kg of CsA. Identical electrophysiologic findings were seen at 20 weeks. These results suggest that nerve graft preservation may decrease systemic immunosuppression requirements while improving functional recovery. As well, storage of nerve grafts is feasible and would facilitate elective surgery and less costly reconstructive repair.     

Degradation of histamine solutions used for bronchoprovocation

STUDY OBJECTIVES: To determine optimal storage conditions for histamine diphosphate (HDP) solutions used for bronchoprovocation. DESIGN: HDP was dissolved in buffered saline solution to concentrations of 0.125 to 16 mg/mL and stored in 3-mL unit dose syringes at different temperatures for varying lengths of time, with and without protection from fluorescent light. SETTING: Dark freezer (-20 degrees C), dark refrigerator (4 degrees C), and laboratory counter top (20 degrees C) illuminated by fluorescent light (375 foot-candles). MEASUREMENTS: HDP concentrations were measured after the solutions were prepared and during storage by a high-performance liquid chromatographic assay that differentiates histamine from its break down products. RESULTS: All dilutions were sterile after preparation and contained 97 to 110% of the labeled amount of HDP. Solutions constantly exposed to fluorescent light (375 foot-candles) and room temperature (20 degrees C) contained only 20 to 37% of the initial concentrations after 7 days. The same dilutions stored at room temperature, but protected from light, contained 83 to 94% of the initial concentrations. Dilutions stored in the dark in a refrigerator (4 degrees C) retained 97% of the initial concentrations after 8 weeks, while dilutions stored in the dark freezer (-20 degrees C) were stable for 12 months. CONCLUSIONS: Exposure to fluorescent light at room temperature results in degradation of histamine solutions used for bronchoprovocation. Dilutions stored in unit dose syringes and protected from light are stable for at least 8 weeks in the refrigerator and up to 12 months frozen. Once removed from the refrigerator or freezer, the solutions should be used within 6 h or discarded.     

Manual circumlaryngeal therapy for functional dysphonia: an evaluation of short- and long-term treatment outcomes

In a liquid environment, at high dilutions, fertility of bull sperm is maintained for 3-5 days when stored at ambient temperatures (10-21 degrees C), after which time it steadily declines at a rate of 3-6% per day. This decline in fertility occurs irrespective of whether the sperm are stored at 5 degrees C or at 15 degrees C, but the rate is greater once storage temperatures exceed 25 degrees C. Sperm motility can be maintained for extended periods in an environment where the extracellular oxidative stress is minimized by reducing the oxygen tension, by addition of antioxidants and chelating agents; however, this will not prevent a significant drop in fertility after five days of storage at ambient temperature. The requirement of energy by the sperm-motility apparatus demands a high level of respiratory activity. This system is very active and the free radicals produced in vivo during this process could lead to chromatin damage. As no internal repair mechanism exists in sperm, an extraneous supply of protectants, or an environment where damage is minimized, is essential to maintain its fertilizing potential. The lack of extended storage potential of sperm, even in the presence of antioxidants, seems to suggest that although oocyte-penetrating ability of the sperm could still be intact, the high rate of intracellular metabolic activity could lead to mitochondrial DNA damage and chromosomal abnormalities that would compromise the viability of the resulting conceptus.     

T cell engraftment in lymphoid tissues of human peripheral blood lymphocyte reconstituted SCID mice with or without prior activation of cells

A controlled study was undertaken to determine the stability of LSD in pooled urine samples. The concentrations of LSD in urine samples were followed over time at various temperatures, in different types of storage containers, at various exposures to different wavelengths of light, and at varying pH values. LSD concentrations were measured quantitatively by the Abuscreen RIA and by HPLC using a fluorescence detection method. Good correlation was observed between the immunoassay and the fluorescent integrity of the LSD molecule. Thermostability studies were conducted in the dark with various containers. These studies demonstrated no significant loss in LSD concentration at 25 degrees C for up to 4 weeks. After 4 weeks of incubation, a 30% loss in LSD concentration at 37 degrees C and up to a 40% at 45 degrees C were observed. Urine fortified with LSD and stored in amber glass or nontransparent polyethylene containers showed no change in concentration under any light conditions. Stability of LSD in transparent containers under light was dependent on the distance between the light source and the samples, the wavelength of light, exposure time, and the intensity of light. After prolonged exposure to heat in alkaline pH conditions, 10 to 15% of the parent LSD epimerized to iso-LSD. Under acidic conditions, less than 5% of the LSD was converted to iso-LSD. We also demonstrated that trace amounts of metal ions in buffer or urine could catalyze the decomposition of LSD and that this process can be avoided by the addition of EDTA. This study demonstrates the importance of proper storage conditions of LSD in urine in order to insure proper analytical testing results over time.     

Effects of preservation techniques on in vivo expression of adhesion molecules by aortic valve allografts

Methods of sterilization and preservation of aortic valve allografts influence graft longevity. The effect of storage techniques on valve durability may be mediated by alterations in the immunologic properties of the allograft, which are reflected by expression of leukocyte adhesion molecules. Rat aortic valve grafts were transplanted in the fresh state, after cryopreservation (-196 degrees C), or after storage at 4 degrees C for 1 to 21 days. Syngeneic and strongly allogeneic valves were transplanted for 4 hours to 21 days and were retrieved for immunohistochemical staining for expression of leukocyte adhesion molecules. Unimplanted valves and transplanted syngeneic valves, regardless of storage methods, exhibited little or no expression of leukocyte adhesion molecules. Fresh allogeneic valves expressed all molecules, indicating up-regulation, at all time intervals studied. Cryopreserved allogeneic valves demonstrated no leukocyte adhesion molecules at 4 hours or 2 days and weak reactivity at 10 and 21 days. Allogeneic valves stored at 4 degrees C, regardless of the duration of storage, demonstrated weak expression of all molecules at 10 days and strong expression at 21 days. Expression of leukocyte adhesion molecules requires an allogeneic environment and may precede immune-mediated injury. Reduced expression of leukocyte adhesion molecules resulting from storage may predict a diminished immunologic response. Cryopreservation (-196 degrees C) causes the greatest delay and diminution of expression of leukocyte adhesion molecules.     

Solitary scapula mass: atypical presentation of esophageal adenocarcinoma

The stability of adenosine in various diluents in polypropylene syringes and polyvinyl chloride (PVC) bags at three temperatures was studied. Portions of pooled undiluted adenosine infusion (3 mg/ mL) were stored in 60-mL capped syringes, 20 for each storage condition. Adenosine infusions were prepared by mixing adenosine with 5% dextrose injection, 0.9% sodium chloride injection, lactated Ringer's injection, or 5% dextrose and lactated Ringer's injection to produce a concentration of 0.75 mg/mL. Samples of each infusion were stored in 60-mL capped syringes and 50-mL bags, 20 syringes and 20 bags for each storage condition. Syringes and bags were stored in the dark at 25, 5, and -15 degrees C. At various sampling times, three syringes and three bags of each infusion were removed for visual inspection, pH measurement, and high-performance liquid chromatographic analysis. At 10 and 16 days, fungal growth at 25 degrees C was suspected in the infusions prepared with 5% dextrose injection. For all other samples, there was no evidence of precipitation or change in pH. The concentration of adenosine remained constant in all samples at all storage conditions. Adenosine 3 mg/mL was stable in polypropylene syringes for 7 days at 25 degrees C, 14 days at 5 degrees C, and 28 days at -15 degrees C; adenosine 0.75 mg/ mL in 0.9% sodium chloride injection and in 5% dextrose injection was stable in polypropylene syringes and PVC bags for 16 days at 25, 5, and -15 degrees C; and adenosine 0.75 mg/mL in lactated Ringer's injection and in 5% dextrose and lactated Ringer's injection was stable in syringes and bags for 14 days at 25, 5, and -15 degrees C.     

Physiological role for androgen binding protein-steroid complex in testis?

Rabbit spermatozoa stored at 37 degrees C for 5 hr, 5 degrees C for 5 hr or 5 days, and-196 degrees C for 5 hr, 5 days, or 1 year were used to artificially inseminate does induced to ovulate normal or excess numbers of eggs. Fertility and subsequent embryonic development were examined. Frozen spermatozoa stored for 5 hr fertilized fewer oocytes than those stored for 5 days or longer. Fewer embryos were recovered on Day 6 than on Day 2. Blastocysts were smaller when spermatozoa were stored for 5 days, were frozen or were placed in superovulating does, but no statistically significant differences in the % of fetal survival were found in the groups of normally ovulating does. Embryonic wastage associated with storage of spermatozoa resulted from a reduced fertilization rate and/or increased embryonic mortality before implantation.     

Effects of aluminum sulphate and citric acid ingestion on lipid peroxidation and on activities of superoxide dismutase and catalase in cerebral hemisphere and liver of developing young chicks

IgA antibodies reflecting airways or intestinal mucosal immune responses can be found in saliva and feces, respectively, and IgG antibodies reflecting serum antibodies can be found in saliva. In this study, antibodies were detected in samples of saliva and feces which had been air-dried at room temperature (+20 degrees C) or +37 degrees C, and stored at these temperatures for up to 6 months. In saliva the antibody levels increased, while the antibodies in feces decreased upon storage. The individual IgA antibody concentrations which were adjusted by using the ratios of specific IgA/total IgA were relatively stable in both saliva and feces, and correlated with corresponding antibody levels in samples which had been stored at -20 degrees C. The results indicate that air-dried saliva and feces can be used for semiquantitative measurements of mucosal antibodies, even after prolonged storage at high temperatures and lack of refrigeration.     

A gene essential to brain growth and development maps to the distal arm of rat chromosome 12

Studies were undertaken to determine the stability of nitrobenzodiazepines and their 7-amino metabolites in water and blood. At 22 degrees C nitrazepam and clonazepam were stable in sterile fresh blood containing preservative over 28 days, whereas 25% of flunitrazepam was degraded. At 37 degrees C all three drugs were substantially lost over 9 h (29-51%). There was only a small loss observed for the 7-amino metabolites and no substantial amounts of parent drug and 7-amino metabolite were degraded in water under these conditions. In the absence of preservative substantial amounts (25-50%) of parent drugs were lost in fresh blood over 10 days at 22 degrees C. In bacterially-contaminated postmortem blood all three drugs were completely degraded over 8 h at 22 degrees C with almost all drug completely converted to the respective 7-amino metabolite. These metabolites were also partially degraded (10-20%) over 45 h at 22 degrees C. All 3 nitrobenzodiazepines were stable in blood stored for up to 24 months at -20 degrees C, or 4 degrees C over 10 months. Their respective 7-amino metabolites were, however, relatively unstable at -20 degrees C with a significant loss (29%) after 2 months. At 4 degrees C a 21% loss occurred after 1 month. Freeze/thawing was found not to affect the concentration of nitrobenzodiazepine and 7-amino metabolites. These results show that the nitrobenzodiazepines and their metabolites are unstable chemically and metabolically in blood. We advise that blood collected for the purpose of nitrobenzodiazepine determinations should be preserved with sodium fluoride, stored at -20 degrees C and assayed as soon as practicable, preferably within a week of collection.     

Kinesin and cytoplasmic dynein in spinal spheroids with motor neuron disease

OBJECTIVE: Gene therapy is a promising strategy to modify ischemia-reperfusion injury and rejection after transplantation. We evaluated variables that may affect ex vivo gene transfer to rat lung isografts. METHODS: Left lungs were harvested and perfused via the pulmonary vein with chloramphenicol acetyltransferase complementary deoxyribonucleic acid complexed with cationic liposomes. Several variables were examined: (1) Influence of temperature: In group I (n = 4), grafts were stored for 4 hours at 23 degrees C and transplanted. Chloramphenicol acetyltransferase activity was assessed on postoperative day 2. In groups II and III (n = 4), grafts were stored at 10 degrees and 4 degrees C, respectively. Arterial oxygen tension and inflammatory infiltrate were also determined. (2) Influence of storage time: Grafts were preserved at 10 degrees C for 1, 2, 3, 4 (n = 4), and 10 hours (n = 5). chloramphenicol acetyltransferase activity was assessed on postoperative day 2. (3) Rapidity and duration of transgene expression: Grafts were preserved at 10 degrees C for 1 hour and then transplanted. Chloramphenicol acetyltransferase activity was assessed 2, 4, 6, 12, and 24 hours and 2, 7, 14, 21, and 28 days after implantation. RESULTS: Chloramphenicol acetyltransferase expression was apparently less in lungs transfected at 4 degrees C than in those transfected at 10 degrees and 23 degrees C. Storage for 1 hour at 10 degrees C was sufficient to yield significant expression. Increasing the exposure time to 10 hours did not increase toxicity. There were no differences in arterial oxygen tension between transfected and nontransfected lungs. Chloramphenicol acetyltransferase expression was detected for at least 28 days. CONCLUSION: Ex vivo liposome-mediated transfection of lung isografts can be achieved after a short time of cold storage, with minimal toxicity.     

Effects of vocal loudness on nasalance measures

The effect of storage and high temperatures on the stability of Saccharomyces cerevisiae allergens was studied by immunoblotting. Saccharomyces cerevisiae allergic serum pool and 125I- and galactosidase-labelled anti-IgE were used in the assays. Freeze-dried extracts were reconstituted with saline and with 50% glycerol and then stored at room (+20 degrees C) and refrigerator temperature (+6 degrees C) for different time periods. The stability was better in 50% glycerol at +6 degrees C than at room temperature without glycerol. However, after 1 month, two of the most important allergens of S. cerevisiae, the 48 and 32 kDa protein allergens, lost their IgE-binding capacity even in the extracts stored with 50% glycerol at +6 degrees C. The 45 kDa allergen was, on the other hand, quite stable after storage for 9 months at +6 degrees C. Although the beneficial effect of 50% glycerol was clear, storage at +6 degrees C, even with 50% glycerol should not exceed 1 month for S. cerevisiae extracts. Two commercially available S. cerevisiae extracts in solution with valid expiry dates were also analysed. They had only little allergenic potency, while a freeze-dried extract stored for 8 years showed good allergenic potency. Heating S. cerevisiae extracts resulted in precipitation, the precipitated fraction contained almost all the specific proteins as judged by electrophoresis and IgE detection. The supernatant fraction contained only a few allergens.     

Inhibitory effect of 0 degree C storage on the proliferation of Yersinia enterocolitica in donated blood

BACKGROUND: Yersinia enterocolitica is frequently identified in cases of bacterial sepsis due to red cell transfusion. One of the features that makes Y. enterocolitica particularly dangerous is that, unlike most other bacterial contaminants of blood components, this organism can actively multiply in currently recommended refrigerator temperatures (1-6 degrees C). The effect of a colder than normal storage temperature on Y. enterocolitica growth was investigated to determine whether bacteria growth could be reduced or inhibited at 0 degree C. STUDY DESIGN AND METHODS: Twenty-four units of freshly collected donated blood were obtained. Three sets of 7 units each were inoculated with Y. enterocolitica O:3, Y. enterocolitica O:20, and Y. enterocolitica O:5, 27, respectively. The remaining 3 units served as uninoculated controls. Each of the 24 bags was split into two equal aliquots, with one aliquot stored at 4 degrees C and the other at 0 degree C. Bacteria growth was measured twice weekly for 6 weeks. Endotoxin and hemoglobin levels were also measured at selected intervals. RESULTS: Bacteria growth was detected earlier and in higher concentrations in the aliquots stored at 4 degrees C. Twenty-two of the 42 inoculated aliquots had measureable bacteria growth. Thirteen aliquots had been maintained at 4 degrees C, and nine had been stored at 0 degree C. Sixteen of these 22 aliquots were matched pairs. Exponential growth was detected after 14 to 32 days in the 4 degrees C aliquots and after 28 to 39 days in the 0 degree C aliquots. Final bacteria counts were much higher in the 4 degrees C aliquots (10(5)-10(14) colony-forming units/mL) than in the 0 degree C aliquots (10(1)-10(4) colony-forming units/mL) on Day 42. Endotoxin was present in all 13 of the 4 degrees C aliquots with actively growing Y. enterocolitica. CONCLUSION: Storage of red cells at 0 degree C markedly prolongs the time required for Y. enterocolitica to achieve exponential grwoth and results in lower concentrations of bacteria.     

Analysis of commercial mini-bus accidents

The major challenge in liquid sustained-release oral suspensions is to minimize drug diffusion into the suspending medium and to retain the original properties of the microparticles during storage. Diclofenac wax microspheres prepared by the hydrophobic congealable disperse phase method were formulated as a sustained release suspension and stored at three different temperatures (25, 37 and 45 degrees C) for 3 months, to evaluate the physical and chemical stability of the suspended microspheres. Suspensions of microspheres stored at ambient temperatures were both physically and chemically stable, but at higher temperatures, up to 45 degrees C, there was a decrease in drug release due to scaling and melting on the microsphere surface as observed by scanning electron microscopy. However, on prolonged storage, up to 90 days, especially at 45 degrees C, temperature became a dominant factor causing an increase in drug release. The suspension of diclofenac microspheres was chemically stable for 3 months, while the plain drug suspension exhibited slight degradation.     

Informing the patient

PURPOSE: To investigate the feasibility to use hydroxyethylstarch as an alternative deswelling additive in short-term preservation media. MATERIALS AND METHODS: Corneoscleral discs were prepared from pairs of eye balls of freshly slaughtered pigs. Corneas were stored in MEM-medium containing either 10% or 20% hydroxyethylstarch 450 000 at 4 degrees C in a refrigerator. Subsequently, the tissue was stored for 24 hours in organ culture at 37 degrees C in MEM-medium containing 10% fetal calf serum to detect latent endothelial cell damage. Mate corneas were treated the same except for being stored in Optisol GS during 4 degrees C storage. We determined corneal endothelial cell density, stromal thickness, and glucose concentration in the medium directly after preparation, after short-term storage at 4 degrees C, and after subsequent organ culture at 37 degrees C. Scanning electron microscopy of corneal endothelium was performed at each step during the experimental course. RESULTS: We did not observe any significant differences in endothelial-cell density between experimental groups and control groups. No decrease in endothelial-cell density was observed during the course of experiments. No increase in stromal thickness was determined in any group after short-term storage at 4 degrees C. Corneas stored in medium containing 20% hydroxyethylstarch showed a decrease in stromal thickness after short-term storage. After subsequent organ culture all corneas displayed a uniform stromal swelling. Glucose concentrations in the media decreased in all groups during the experiment. In scanning-electron microscopy we observed a reversible degeneration of cell borders after storage at 4 degrees C. Additionally, corneas stored in Optisol GS showed a reversible cobblestone appearance at this stage of the experiments. CONCLUSION: Hydroxyethylstarch appears to be an alternative to the use of dextran and chondroitin sulfate as a deswelling additive in corneal preservation media.     

Influence of various doses of aspirin (in vivo) on platelet arachidonic acid metabolism (ex vivo) and function

It has been reported that aortic homografts that have been cryopreserved before transplantation remain viable longer as an allograft than tissue stored at 4 degrees C in an antibiotic solution. In the present study, we tested the hypothesis that storage of cardiac valve tissue by cryopreservation or by antibiotic preservation may alter the metabolic status of the tissue. Initially, we collected aortic valves composed of cardiac tissue, aortic root, and valvular tissue from cadaver donors. These specimens were divided into three equal portions, and one portion was analyzed before storage while the other two parts were stored for 3 weeks at either 4 degrees C in an antibiotic solution or at -196 degrees C in liquid nitrogen. All specimens were examined with regard to the following parameters: tissue structure, tissue viability, cell proliferative capacity, metabolic function, and identification of cell-specific antigens. We found no significant alterations in the structure of any of the three tissue components after antibiotic preservation or cryopreservation; however, cell viability and cell number were decreased in all three groups. All tissue samples grew in culture before storage. When we compared activities of the following organellar marker enzymes--lysosomal acid lipase, plasma membrane 5' nucleotidase, mitochondrial cytochrome oxidase, and microsomal neutral alpha-glucosidase--we observed no major differences between tissues stored by either technique. In addition, we observed no loss of enzymic activity as a result of storage. Finally, when cell lines isolated from each tissue specimen were incubated with monoclonal antibodies against cell-specific antigens in an immunoperoxidase assay, all the cell cultures proved to be endothelial cells. These results suggest that although cardiac valve tissue stored by cryopreservation or by antibiotic preservation retained its normal structure and metabolic capabilities, both storage techniques produced significant decreases in cell numbers and viability. However, only endothelial cells from tissue stored by cryopreservation retained the capacity to proliferate in vitro. These findings have important implications for the function of aortic homografts transplanted after storage.     

Analytical methods and stability assessment of liquid yeast derived sucrase

Two independent analytical methods for determining the activity and stability profile of liquid yeast derived sucrase (YS) were established and validated in order to conduct preliminary stability studies as a function of temperature. The methods included a hexokinase-based (HK) enzymatic assay for determining the formation of glucose upon hydrolysis of sucrose by YS, and a direct polarimetric procedure to quantitate YS hydrolysis of sucrose. Both assays were validated with respect to YS dilution, incubation time, sucrose or glucose concentration, linearity of response and within- and between-day variability. A preliminary stability study was conducted over a 24 week period with liquid YS samples stored at -20, 4, 30, 40 and 50 degrees C. Enzymatic activity was monitored as a function of time using both the HK and polarimetric assays. Liquid YS samples stored at -20, 4 and 30 degrees C retained 100% activity after 24 weeks storage, while the samples stored at 40 degrees C lost approximately 70% activity over the same storage period and samples stored at 50 degrees C lost approximately 95% activity after 12 weeks storage. The two methods of analysis gave consistent results over the course of the study.     

Reproducibility of electronic cell counts in milk; a study of 5 further factors

This paper is a sequel to a previous one in which a number of factors likely to influence the accuracy of counting somatic cells in milk was assessed; in the present work the effects of 5 other factors are investigated. In a study of storage time and temperature of milk samples fixed in formalin, a significant increase in cell count occurred after 5-7 d when samples were stored at room temperature (17-23 degrees C), compared with those maintained at 4 degrees C. When manual and mechanical mixing of fixed samples were compared only marginal differences in cell counts were observed. An increase in cell counts followed manual dilution of milk samples in comparison with automatic dilution. The temperature of samples prepared for counting was also studied and no significant variations occurred between mean temperatures of 12-7 and 32-9 degrees C. The final factor evaluated was that of holding time before counting; using 4 cell-count levels it was observed that counts were acceptable up to 1 1/2 h.     

Significance of pre-incubation temperature and inoculum concentration on subsequent growth of Listeria monocytogenes at 14 degrees C

The influence of the bacterial concentration of an inoculum (10(1) or 10(3) cfu ml-1) of two strains of Listeria monocytogenes (Scott A: serotype 4b and V7: serotype 1) and one strain of L. innocua (Lin 11), and the time and temperature at which the inoculum was stored (cold storage: 4 degrees C for 4 weeks, or without cold storage: -20 degrees C before immediate transfer), and the temperature at which cells were pre-incubated (30 degrees C and 14 degrees C) on subsequent growth in Richard's broth at 14 degrees C was investigated. Richard's broth at a pH 5.9 was used to simulate potential growth in soft cheese (camembert type) and an incubation temperature of 14 degrees C was used to simulate storage-temperature ripening of cheese. Enumeration of the number of viable cells was by plate count method, except where viable cell numbers were less that 10(3) cfu ml-1, when the MPN (Most Probable Number) technique was used. With cold storage and an inoculum of 10(3) cfu ml-1 (high bacterial concentration) the pre-incubation temperatures (30 degrees C and 14 degrees C) did not significantly influence the subsequent growth curve: there was no significant lag (less that 21 h) and cell numbers peaked in about 8.5 d. However, with cold storage and an inoculum of 10(1) cfu ml-1 (low bacterial concentration) and a pre-incubation temperature of 30 degrees C a significant shift in the growth curve was observed over that pre-incubated at f14 degrees C, with the appearance of a lag of about 7.7 d. At a pre-incubation temperature of 14 degrees C with the low inoculum concentration, there was a measurable lag of about 1 d. Without cold storage and a pre-incubation temperature of 30 degrees C, there was a lag time of 2.3 d. Storage conditions, pre-incubation temperature and inoculum concentration therefore appear to influence the subsequent growth curve. Importantly, however, the growth curves for cultures from inocula, pre-incubated at either 30 degrees C or 14 degrees C, appeared to involve two distinct values of the exponential growth rate (k): the initial portion of the growth curve described by a low value of k and the subsequent portion by a consistently and significantly greater value. The appearance of two distinct growth phases was reproduced in further data determined for all the studied strains of the microorganism. Further study to explain these unexpected and reproducible findings is being conducted.     

Liquid storage of ram semen: a review

Research from 1930 to 1992 is reviewed with regard to storage of semen at reduced (0-5 degrees C or 10-15 degrees C) and at ambient temperatures. Diluents investigated have included synthetic buffers combined with sugars and egg yolk or its fractions, milk from various sources, glycine, and other substances. Irrespective of the diluent, dilution rate, temperature or conditions of storage, the spermatozoa deteriorate with time of storage. Changes include reduction in motility and morphological integrity of spermatozoa, accompanied by a decline in their survival in the female reproductive tract, reduction in fertility and increased embryonic loss. In critical studies, fertility declined rapidly when semen stored for more than 24 h was used for cervical insemination, but after intrauterine insemination some spermatozoa maintained their fertilizing capacity up to 10 days. Laparoscopic intrauterine or transcervical inseminations could be the means of improvement of fertility. These methods may eliminate the problem of sperm transport through the cervix and ageing of spermatozoa in the reproductive tract, thereby improving fertilization of ova and reducing embryonic loss.     

Laboratory evaluation of the XR-Bond Dentin/Enamel Bonding System

The shear bond strengths of the XR-Bonding System used in conjunction with Herculite composite, to the dentine of forty extracted human permanent first and second molars were determined after the test specimens were stored in physiological saline at 37 degrees C for 48 hours, one week, two weeks and four weeks, respectively. A shear load was applied to the base of the bonded composite cylinders with a knife-edged rod at a crosshead speed of 0.5 mm/minute. The shear bond strengths were expressed in megapascals (MPa). The quantitative microleakage of Class V preparations in dentine (cementum) in forty-eight extracted human maxillary permanent canines restored with the same dentinal bonding system and after storage in physiological saline at 37 degrees C for the same time intervals as for the shear bond strength tests, was determined. On the final day of each time interval the teeth were thermocycled X 500 in a 2 per cent methylene blue solution between 8 degrees C and 50 degrees C with a dwell time of 15 seconds. Microleakage was determined by a spectrophotometric dye-recovery method and expressed in microgram dye/restoration. There was a significant trend for the shear bond strengths to increase with duration of storage (p = 0.01) but the quantitative microleakage was not significantly different (p = 0.75).