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Effects of volume, culture media and type of culture dish on in vitro development of hamster 1-cell embryos.We examined effects of medium volume and two different culture media (HECM-3 and HECM-4) on in vitro development of hamster embryos. Groups of 5 to 8 1-cell embryos were cultured for 72 h in either < or =100 or > or =100 microl volumes. In the first experiment, embryos were cultured in Petri dishes with 2, 5, 20, 50 or 100 microl of medium using the two media (2 x 5 factorial experiment). Optimal volumes for morula and blastocyst development were 100 microl of HECM-3 and > or =50 microl of HECM-4; in HECM-4, > or =20 microl volumes were suitable whereas in HECM-3 < or = 50 microl volumes were unsuitable. In the second experiment, embryos were cultured in 100, 200, 500 and 1000 microl of HECM-3 and HECM-4 using organ culture dishes. Controls were 100 microl drops in Petri dishes. In organ culture dishes, blastocyst development was < or =6% in HECM-3 and 33-41% in HECM-4, and suitable volumes for development to at least morulae were > or =200 microl of HECM-3, and > or =100 microl of HECM-4. In both experiments development to morula and blastocyst stages with 100 microl volume in Petri dishes was significantly higher with HECM-4 (96 and 85% in Experiment 1 and 2, respectively) than that with HECM-3 (52 and 40% in Experiment 1 and 2, respectively; P < 0.05). These results indicate that attention should be paid to both type and volume of medium and interaction with type of culture dish for optimizing development of embryos in vitro.
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Alterations in both the hematopoietic microenvironment and the progenitor cell population follow the recovery from myeloablative therapy and bone marrow transplantation.Experimental data are conflicting, but suggest that after recovery from bone marrow transplantation (BMT), alterations in clonogenic growth and myeloid microenvironment remain. To further characterize this abnormality, light-density marrow cells from 15 patients who were in remission from hematologic malignancies and had undergone BMT (eight allogeneic and seven autologous) were studied in two culture systems after their marrows had reconstituted and were compared to normal. The preconditioning regimen for transplantation was fractionated total-body irradiation (TBI) 12 Gy, cyclophosphamide 120 mg/kg, and total nodal irradiation 6 Gy. Bone marrow mononuclear cells (MNCs) from the patients and controls were divided in two fractions; stromal layers (SL) were prepared from the first fraction. To examine the attributes of the stroma, the petri dish surface covered by the SL was measured after 5 weeks in culture, and representative layers were trypsinized and stained with Sudan black, butirate esterase, and acid and alkaline phosphatases. Stromal layers were also studied for their support in the development of blastic colonies (CFU-BI). From the second fraction, the CD34+ population was selected with immunomagnetic beads, and 1 x 10(4) progenitors from patients or controls were seeded onto the opposite group of preformed stroma. Stroma-adhesive precursors were scored for the formation of CFU-BI (> 20 cells) on day 5 of culture. Nonadherent selected CD34+ cells were recovered by standardized washing and quantitated in clonogenic assays (CFU-GM). The median patient age was 26 (SD 6.65) years, and eight of 15 patients were female. The median infused bone marrow (BM) MNC number was 0.9 x 10(8)/kg (SD 0.31). Grafts were studied at a median of 37 (SD 48.43) months from transplant. SL from the patients failed to reach confluence by 5 weeks (median dish area covered 55.5% [SD 32.38] vs. control: 100% [SD 2.35]; p = 0.0001). BMT CD34+ progenitors gave 19.5 (SD 42.2) CFU-Bl, significantly lower than those from normal individuals (127 [SD 62.2]; p = 0.01) when panned on control stroma, while control CD34+ cells were poorly supported on BMT layers (corrected for surface, median 2.5 [SD 42.2] CFU-Bl; p = 0.039). Although numbers of stroma nonadherent CFU-GM were not different between the groups (median BMT 56 [SD 54.5] vs. control 62.5 [SD 60.76]; p > 0.05), the ratio of CFU-Bl to CFU-GM showed a significant reduction in adherent CD34+ progenitors in BMT patients (median 0.28 [SD 0.44] vs. normal 2.09 [SD 1.3]; p = 0.04). None of the values were significantly different between patients receiving allogeneic or autologous grafts. We conclude that post-BMT stroma is defective and supports CFU-Bl growth poorly. Moreover, we documented a significant reduction within the CD34+ cells in the adherent primitive clonogenic precursors that was compensated by a proportional increase in the more mature CFU-GM.
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In vitro fertilization rate of horse oocytes with partially removed zonae.Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 micro M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under x 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (2/57), 12 (7/58), 52 (31/60), and 86% (44/51) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (2/2), 57 (4/7), 58 (18/31), and 34% (15/44), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 11/49), and increased to 38% (21/55) at 5 h, to 46% (23/50) at 10 h, and to 56% (27/48) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.
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Test models for hygienic handrub and hygienic handwash: the effects of two different contamination and sampling techniques.Two methods for artificial contamination of hands and two sampling techniques to recover the test organisms were compared for their effects on the results of two post-contamination hand treatments: a handrub with two portions of 3 ml of 2-propanol 60% v/v for 1 min, and a handwash with liquid soap 20% w/v for 1 min followed by a 15 s rinse. The two contamination methods involved a short immersion of the hands (up to the middle of the mid-hand) in a suspension of the test organism followed by either air-drying (3 min) or drying by rubbing the hands' vigorously against each other (3 min) in a standardized way. The two sampling techniques consisted of rubbing the fingertips in either 10 ml trypticase soy broth (TSB) against the bottom of a Petri dish; or 100 ml TSB against glass beads contained in a bowl. Sixteen volunteers were randomly allotted to four blocks of four. They carried out the four possible combinations of two treatments and two contamination methods in a series of four tests arranged in a Latin-square design. In addition, the two sampling techniques were compared with each other concurrently by sampling of the right and left hand each with a different one of the two techniques. The alcoholic handrub reduced the release of test organisms significantly (2P less than 0.005) more effectively, by 1.1-1.3 x log10, than did the handwash with liquid soap, regardless of the contamination or sampling method. Whereas the two recovery techniques yielded virtually identical results in corresponding situations, the method of artificial contamination affected the mean reduction factors, strongly.(ABSTRACT TRUNCATED AT 250 WORDS)
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Validation of conditions for efficient detection of HPRT and APRT mutations in suspension-cultured Chinese hamster ovary cells.Conditions for reliable and efficient assay of mutations affecting the activity of HPRT (hypoxanthine phosphoribosyltransferase EC 188.8.131.52) and APRT (adenine phosphoribosyltransferase EC 184.108.40.206) have been determined for a strain of CHO (Chinese hamster ovary) cells that has been adapted for rapid growth both in suspension culture and in monolayer. To facilitate measurement of mutation at the aprt locus, clones were derived that are presumptively heterozygous at that locus. At a limiting concentration of 8 microgram/ml of azaadenine, 14/16 of the resistant clones picked and tested had approximately 1/2 of the APRT activity of the wild-type cells. One such clone, strain AA8, was chosen for further studies and found to be readily mutable to resistance to 80 microgram/ml azaadenine. Most of the highly resistant colonies isolated (21/24) had very low in vitro APRT activity. The optimal conditions for detection of TGr and AAr mutations were determined for two critical parameters, expression time and cell density. Cultures treated with mutagen either in monolayer or in suspension were allowed to express mutations in suspension. The expression of mutations induced by UV light, EMS, and ICR-191 was complete by 3 days for AAr and by 4-5 days for TGr. The time required to reach a maximal frequency of mutants was essentially independent of the type of mutagen and the level of survival after treatment. Induced mutation frequencies for both loci were notably stable during the time intervals examined. With respect to cell-density conditions, both markers were detected at frequencies that were independent of the cell inocula over the range of 1 x 10(5) to 1 x 10(6) cells per 100-mm petri dish (i.e. 1.6 x 10(3) to 1.6 x 10(4) cells/cm2) containing 20 ml of medium. These results were obtained with both mutagenized populations and with reconstructed mixtures obtained by adding drug-resistant cells to varying numbers of wild-type cells. The rapid expression of mutations for both markers, particularly AAr, combined with the advantage that large inocula can be plated for selection of mutants, make this CHO strain an attractive system for the simultaneous measurement of mutations at the autosomal aprt and X-linked hprt loci.
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[The Influence Of Ultraviolet Irradiation Upon The Development And Infectivity Of Hookworm Larvae]The eggs and rhabditoid larvae of canine hookworm were irradiated with ultraviolet rays for one hour at a distance of 10, 20, 30, and 40 cm. The infective stage larvae of the same parasites were irradiated for l, 3, 5 and 14 hours from the same distances. The infective larvae were also exposed under direct sunlight for l, 2, 3 and 4 hours. PARASITES: Ancylostoma caninum was used. Eggs were collected in vitro from female adult worms. The worms were kept at 37 degrees C in petri-dish filled with Kreb's Ringer solution. There was an average of two cell stages, and they were used as early as possible before the morula stage. Rhabditoid larvae were obtained by culture of the above eggs for twenty-four hours in 25 degrees C incubator. The larvae reached the infective stage in seven days culture at the same condition. IRRADIATION OF ULTRAVIOLET RAY: Kingston ultraviolet light (100 volt, 10 watt, 50 cycles, 0.230 ampere) was used. The potential U.V.R. power was 1.8 watts. The distances between the material and the light were 10, 20, 30 and 40 cm at a temperature of 25 degrees C in each case. The samples were smeared on the tile in order to keep them in saturated moisture. Fully wetted ten ply gauze was laid underneath the tile. The tile was surrounded by 2 x 5 cm rectangular piece of glass in order to prevent the spread of the larvae to the outside. All of the samples received irradiation for one hour and were cultured for a period of seven days. The hatching of the egg and the development of the larvae were observed. For the purpose of the study, the infectivity and pathogenicity of the irradiated samples, were inoculated into mice orally. The lungs, livers and carcass were examined three days after the infection. A routine pathological examination of the organs was also carried out. In order to study the eggs productivity, the larvae were given to the proper host, dog. The eggs in the feces were examined from three to 6 weeks after infection, both quantitatively and qualitatively. As a supplementary experiment, the infective larvae of canine hookworm were exposed four hours under direct sunlight (September 25), and the infectivity and pathogenicity of the host were examined. HATCHING, DEVELOPMENT AND INFECTIVITY OF IRRADIATED EGGS: Hatchability of the irradiated group for one hour according to the distance from the light to the sample were 48.0 % at 10 cm, 60.3 % at 20 cm, 85.2 % at 30 cm and 88. 2 % at 40 cm respectively. None of them developed to the infective stage. They remained rhabitoid for several days and were destroyed. None was found alive in the host. 93.0 % of the control group hatched and developed to the infective stage. DEVELOPMENT AND INFECTIVITY OF IRRADIATED RHABDITOID LARVAE: None of the irradiated group reached the infective stage. Under irradiation they coiled and died soon after straightening out again. Only the group irradiated at the distance of 40cm survived for six days. They finally granulated. There was no manifestion of irradiated larvae alive in the host tissue. LIFE SPAN, INFECTIVITY, PATHOGENICITY AND EGG-PRODUCTIVITY OF THE IRRADIATED INFECTVE STAGE LARVAE: All were destroyed in the group of fourteen hours irradiation at 40 cm distance. Thirteen precent survived in the five hours irradiation group at the same distance. The survivability of larvae was reduced by the period of irradiation and at the shortest distance. The infectivity to mice was only 0.8 % at 30 cm, and 8.2 % at 40 cm in the three hour irradiation group. The recovery of the infected larvae from the host tissues was reduced as the irradiation period was increased and the distance shortened. The pathogenicity was paralleled with the vitality of the irradiated larvae. From the groups of one hour irradiation and ten cm distance, three hour irradiation and ten to thirty cm distance, the egg-productivity was all negative. But as the irradiation period decreased and the distance lengthened the egg-productivity tended closer to normal. The infective stage larvae which were exposed to direct sunlight were destroyed within three hours, but survived 81 % in the one hour exposure group and 20 % in the two hour exposure group. The summary of the results is as follows: 1. The hatching of eggs was reduced to half for one hour irradiation at the ten cm distance. Even hatched larvae did not develop to infective stage. 2. Infectivity was inhibited by the irradiation to at the ten cm distance for one hour. About ten percent of the irradiated infective stage larvae were recovered from the infected animal among the group of 40 cm distance for one hour. The egg-productivity became lower in the group of one hour irradiation at 40 cm distance. 3. The pathogenicity of the irradiated group was mild compared to the control group. 4. The direct sunlight destroyed the infective stage larvae within three hours. In general, the ultraviolet ray showed the inhibitory action in the hatching, development, pathogenicity and egg-productivity of the hookworm. The grade was paralleled with the period of irradiation and reversed to the distance between the light and samples.
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