Multiple physiological states of a Pseudomonas fluorescens DR54 biocontrol inoculant monitored by a new flow cytometry protocol

Authors

  • Tommy Harder Nielsen,

    1. Section of Genetics and Microbiology, Department of Ecology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark
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  • Ole Rüdiger Sjøholm,

    1. Section of Genetics and Microbiology, Department of Ecology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark
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  • Jan Sørensen

    1. Section of Genetics and Microbiology, Department of Ecology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark
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  • Editor: Christoph Tebbe

  • Present address: Tommy Harder Nielsen, Division of Vaccine, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.

Correspondence: Jan Sørensen, Section of Genetics and Microbiology, Department of Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. Tel.: +45 3533 2626; fax: +45 3533 2606; e-mail: jan@life.ku.dk

Abstract

A new fluorescence staining and flow cytometry protocol was developed to monitor several physiological states in biocontrol strain Pseudomonas fluorescens DR54 during storage survival in a stationary-phase culture, preparation of clay carrier for seed formulation, and establishment in a sugar beet spermosphere. The high load of impurities in the environmental samples was dealt with by adding a density-gradient purification step to the staining protocol. Staining by SYBR Green, combined with either propidium iodide or ethidium bromide (EB)+DiBAC(4)3, was used to quantify the total cell population and further divide this population into: (1) intact cells with an unaffected membrane and energy metabolism. (2) De-energized cells unable to maintain membrane export (EB exclusion). (3) Depolarized cells unable to maintain membrane potential. (4) Permeabilized cells with a damaged membrane. During both stationary-phase storage and steps for preparation of formulation carrier, loss of intact P. fluorescens DR54 cells was quantitatively accounted for by depolarized and permeabilized states. Surviving inoculum cells subsequently proliferated on the germinating seeds, but with a surprisingly high abundance of de-energized cells. The new protocol is the first for flow cytometry to include a recording of both intact and several subpopulations of physiologically affected bacteria in complex, environmental samples with high impurity loads.

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