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      Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5‐chloromethylfluorescein diacetate

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          Abstract

          Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate ( FDA) and 5‐chloromethylfluorescein diacetate ( CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat‐killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per‐cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live‐dead classification was essentially error free. But overlap between the frequency distributions of living and heat‐killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat‐killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+ CMFDA gave acceptably accurate results for only 8–10 of 24 species (i.e., 33%–42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone.

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          UV-induced DNA damage and repair: a review.

          Increases in ultraviolet radiation at the Earth's surface due to the depletion of the stratospheric ozone layer have recently fuelled interest in the mechanisms of various effects it might have on organisms. DNA is certainly one of the key targets for UV-induced damage in a variety of organisms ranging from bacteria to humans. UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) and their Dewar valence Isomers. However, cells have developed a number of repair or tolerance mechanism to counteract the DNA damage caused by UV or any other stressors. Photoreactivation with the help of the enzyme photolyase is one of the most important and frequently occurring repair mechanisms in a variety of organisms. Excision repair, which can be distinguished into base excision repair (BER) and nucleotide excision repair (NER), also plays an important role in DNA repair in several organisms with the help of a number of glycosylases and polymerases, respectively. In addition, mechanisms such as mutagenic repair or dimer bypass, recombinational repair, cell-cycle checkpoints, apoptosis and certain alternative repair pathways are also operative in various organisms. This review deals with UV-induced DNA damage and the associated repair mechanisms as well as methods of detecting DNA damage and its future perspectives.
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                Author and article information

                Journal
                J Phycol
                J. Phycol
                10.1111/(ISSN)1529-8817
                JPY
                Journal of Phycology
                John Wiley and Sons Inc. (Hoboken )
                0022-3646
                1529-8817
                28 April 2016
                August 2016
                : 52
                : 4 ( doiID: 10.1111/jpy.2016.52.issue-4 )
                : 572-589
                Affiliations
                [ 1 ] Department of OceanographyDalhousie University PO Box 15000 Halifax Nova Scotia B3H 4R2Canada
                Author notes
                [*] [* ]Author for correspondence: e‐mail hugh.macintyre@ 123456dal.ca .
                Article
                JPY12415
                10.1111/jpy.12415
                5074294
                27028462
                08d3fad8-d639-450b-bb43-bdafce7657c4
                © 2016 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 06 August 2015
                : 14 March 2016
                Page count
                Pages: 18
                Funding
                Funded by: Natural Sciences and Engineering Research Council of Canada
                Funded by: Trojan Technologies
                Funded by: Strategic Cooperative Education Incentive Program of Nova Scotia
                Categories
                Regular Article
                Regular Articles
                Custom metadata
                2.0
                jpy12415
                August 2016
                Converter:WILEY_ML3GV2_TO_NLMPMC version:4.9.5 mode:remove_FC converted:21.10.2016

                confidence intervals,false negative,false positive,flow cytometry,mortality,most probable number,viability,vital stain,vitality

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