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

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.

TOL plasmid carriage enhances biofilm formation and increases extracellular DNA content in Pseudomonas putida KT2440

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Combined use of flow cytometry and microscopy to study the interactions between the gram-negative betaproteobacterium Acidovorax facilis and uranium(VI)

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Understanding the simultaneous biodegradation of thiocyanate and salicylic acid by Paracoccus thiocyanatus and Pseudomonas putida

“…Staining procedures were performed as previously reported Alonso et al (Alonso et al 2012). Total counts of cells were determined by staining with SYBRgreen (SYBRgreen; Invitrogen), a green fluorescent cell-permeable DNA probe (Foladori et al 2010;Nielsen et al 2009), to differentiate the microorganisms from other particles in samples (background). Fluorescent microspheres (Perfect Count; Cytognos, Spain) were used as internal standards in each sample, following the supplier's recommendations for ratiometric counting (Quirós et al 2007).…”