Optimizing the setup of a flow cytometric cell sorter for efficient quantitative sorting of long filamentous cyanobacteria

Authors

  • Mark A. van Dijk,

    Corresponding author
    1. Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands
    • Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands
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  • Gerald Gregori,

    1. Université de la Méditerranée, Laboratoire de Microbiologie, Géochimie et Ecologíe Marines, CNRS UMR 6117, 163 Avenue de Luminy, Case 901, Batîment TPR1, 13288 Marseille cedex 9, France
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  • Hans L. Hoogveld,

    1. Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands
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  • Machteld Rijkeboer,

    1. Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands
    Current affiliation:
    1. Centre for Water Management, Laboratory for Hydrobiological Analysis, P.O. Box 17, 8200 AA Lelystad, The Netherlands
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  • Michel Denis,

    1. Université de la Méditerranée, Laboratoire de Microbiologie, Géochimie et Ecologíe Marines, CNRS UMR 6117, 163 Avenue de Luminy, Case 901, Batîment TPR1, 13288 Marseille cedex 9, France
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  • Anthony Malkassian,

    1. Université de la Méditerranée, Laboratoire de Microbiologie, Géochimie et Ecologíe Marines, CNRS UMR 6117, 163 Avenue de Luminy, Case 901, Batîment TPR1, 13288 Marseille cedex 9, France
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  • Herman J. Gons

    1. Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands
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Abstract

Heterogeneity within natural phytoplankton communities makes it very difficult to analyze parameters at the single-cell level. Flow cytometric sorting is therefore a useful tool in aquatic sciences, as it provides material for post-sort analysis and culturing. Sorting subpopulations from natural communities, however, often requires handling morphologically diverse and complex particles with various abundances. Long particles, such as filament-forming cyanobacteria (>100-μm long), prove very difficult to handle. These potentially toxic organisms are widespread in eutrophic systems and have important ecological consequences. Being able to sort filamentous cyanobacteria efficiently and as viable cells is therefore highly desirable when studying factors associated with their toxicity and occurrence. This unconventional sorting requires extensive user experience and special instrument setup. We have investigated the effect of hydrodynamic and electromechanical components of a flow cytometer, and sorting protocol on the quantitative sorting efficiency of these long particles using two filamentous cyanobacterial strains with average lengths of ∼100 and ∼300 μm. Sorting efficiency ranged from 9.4 to 96.0% and was significantly affected by filament length, sorting envelope, drop delay (dd), and for the long species also by tip size, but not by cycle time. Filaments survived sorting and were not damaged. The optimal settings found for the modular MoFlo® cell-sorter to sort the filaments were a 100-μm flow tip at 30 psi (207 kPa) with a three-droplet envelope in Enrich mode while using an extended analysis time of 17.6 μs and an intermediate plate charge and deflection percentage combination of 3,000 V/60%, combined with a dd 0 for the cultures with 100-μm filaments and dd +1 for the culture with 300-μm filaments. To the best of our knowledge, the filaments up to 1063.5 μm sorted in this study are the longest ever sorted. © 2010 International Society for Advancement of Cytometry

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