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Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata

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Abstract

Membrane integrity has been used as a criterion for the definition of cell viability. In the present work, staining conditions (time and dye concentration) for the evaluation of membrane integrity in a fluorescence microplate reader, using the membrane-impermeant nucleic-acid dye SYTOX Green, were optimized. Incubating Pseudokirchneriella subcapitata algal cells with 0.5 μmol/l SYTOX Green for 40 min allowed a clear discrimination between live (intact plasma membrane) and dead cells (with compromised plasma membrane). Algal cell suspensions, labelled with SYTOX Green, exhibited a green fluorescence proportional to the fraction of the cells with a permeabilized plasma membrane. The optimized staining conditions were used to assess the toxicity of 1-pentanol on P. subcapitata in a short-term exposure (6 h) assay. The loss of membrane integrity in the cell population increased with the concentration of 1-pentanol. The 6-h EC10 and EC50 values were 7,617 mg/l 1-pentanol (95 % confidence limits 4,670–9,327) and 12,818 mg/l 1-pentanol (95 % confidence limits 10,929–15,183), respectively. The developed microplate-based short-term assay can be useful in the high-throughput screening of toxics or environmental samples using the alga P. subcapitata.

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Acknowledgments

The authors thank the Fundação para a Ciência e a Tecnologia (FCT) through the Portuguese Government for their financial support of this work through the grant PEST-OE/EQB/LA0023/2011 to IBB. Manuela D. Machado gratefully acknowledges the post-doctoral grant from FCT (SFRH/BPD/72816/2010).

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Authors and Affiliations

  1. Bioengineering Laboratory, Chemical Engineering Department, Superior Institute of Engineering of Porto Polytechnic Institute, Rua Dr António Bernardino de Almeida, 431, 4200-072, Porto, Portugal

    Manuela D. Machado & Eduardo V. Soares

  2. IBB-Institute for Biotechnology and Bioengineering, Centre for Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal

    Manuela D. Machado & Eduardo V. Soares

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  1. Manuela D. Machado
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  2. Eduardo V. Soares
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Correspondence to Eduardo V. Soares.

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Machado, M.D., Soares, E.V. Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata . Appl Microbiol Biotechnol 95, 1035–1042 (2012). https://doi.org/10.1007/s00253-012-4185-y

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  • DOI: https://doi.org/10.1007/s00253-012-4185-y

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