Influence of swabbing solution and swab type on DNA recovery from rigid environmental surfaces.

Determination of the metagenome has become an important component of forensic identification, which requires efficient environmental sampling techniques. Therefore, in this study, we compared the efficiency of sample collection using swabbing with cotton swabs and three types of medical swabs (S7, S22, S24) along with three different solutions: phosphate-buffered saline (PBS), 1% Tween 20 + 1% glycerol in PBS (TG), and GS commercial solution (Noble Bio, Hwaseong, Republic of Korea). Combinations of the three solutions with the three types of swabs were tested at different volumes (cotton swab, S7: 0, 30, 50, 70 µL; S22, S24: 0, 70, 100, 130 µL). Escherichia coli and Staphylococcus aureus were selected as representative environmental microbial samples, and the number of colony-forming units (CFUs), DNA concentration, and DNA copy numbers were compared across groups. The sampling process had a clear effect on the efficiency of extraction, which allowed for determination of a more efficient sample sampling method. In particular, cotton swabs showed 2-10-fold greater CFUs of both species than the medical swabs, and resulted in significantly greater amounts of extracted DNA. TG was found to be the most efficient solution for bacterial DNA extraction, with higher CFUs and DNA obtained than with the other three solutions at all volumes tested. This study highlights the need for a standardized sampling method that can be applied to all environmental samples, especially for microbial quantification, and provides valuable reference data for the efficient collection of environmental samples for metagenomic analyses in microbial-based forensic assessments.

Sensitivity of spore germination and germ tube elongation of Saccharina japonica to metal exposure.

The sensitivity of early life stages of the brown seaweed Saccharina japonica to six metals (Cd, Cu, Hg, Ni, Pb, Zn) and two waste-water samples were investigated and a new toxicity bioassay developed. The two endpoints used were spore germination and germ tube elongation with an exposure time of 24 h. Optimal test conditions determined for photon irradiance, pH, salinity and temperature were darkness, pH 8, 35‰ and 15°C, respectively. The toxicity ranking of five metals was: Hg (EC(50) of 41 and 42 µg l(-1)) > Cu (120 and 81 µg l(-1)) > Ni (2,009 and 1,360 µg l(-1)) > Zn (3,024 and 3,897 µg l(-1)) > Pb (4,760 and 4,429 µg l(-1)) > Cd (15,052 and 7,541 µg l(-1)) for germination and germ tube elongation, respectively. The sensitivities to Cd, Cu and Ni were greater in germ tube elongation than in germination process. When tested against two different waste-water samples (processed animal and printed circuit board waste-water) values of EC(50) were between 21.29 and 32.02% for germination and between 5.33 and 8.98% for germ tube elongation. Despite differences in their chemical composition, the toxic effects of waste-water samples, as indicated by EC(50) values, did not differ significantly for the same endpoints. The CV range for both germination and germ tube elongation was between 4.61 and 37.69%, indicating high levels of precision of the tests. The results compare favourably with those from more established test procedures employing micro- and macroalgae. The advantages and potential limitations of the bioassay for the assessment of anthropogenic impacts on coastal ecosystems and commercial cultivation areas in near-shore environments are discussed.

Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantation.

BACKGROUND: Previous studies showed that immature oocytes stored in ovarian tissue could develop to the mature stage after transplantation. However, the quality and competency of the oocytes developed in xenografted ovarian tissue have never been investigated. As a pilot study to investigate this uncharted issue, we evaluated microtubule organization and chromatin configuration of human oocytes harvested from xenografted frozen-thawed ovarian tissue. METHODS: Frozen-thawed human ovarian tissue was transplanted into severe combined immunodeficient mice. All animals were stimulated with gonadotrophin from 20 weeks after transplantation. Grafts were recovered 36 h after hCG administration. The oocytes were retrieved from the antral follicles (>2 mm diameter), cultured in vitro, stained for microtubule and chromatin localization. RESULTS: Five oocytes from 21 female mice and seven oocytes from nine male mice were retrieved. Immunocytochemical examinations of these oocytes after in vitro maturation revealed only two developed to the metaphase II stage. Most oocytes were between prophase and metaphase with abnormal microtubule organization and chromatin configuration. CONCLUSIONS: Immature oocytes in stored human ovarian tissue can grow to maturity in host animals after xenotransplantation. Retrieval of oocytes from the xenograft can be carried out and is reproducible. However, many oocytes, grown in host animals and further matured in vitro, showed aberrant microtubule organization and chromatin patterns.