A Novel Staining Protocol for Multiparameter Assessment of Cell Heterogeneity in Phormidium Populations (Cyanobacteria) Employing Fluorescent Dyes

Daria Tashyreva,Josef Elster,Daniela Billi,Lucas J. Stal

doi:10.1371/journal.pone.0055283

Abstract

Bacterial populations display high heterogeneity in viability and physiological activity at the single-cell level, especially under stressful conditions. We demonstrate a novel staining protocol for multiparameter assessment of individual cells in physiologically heterogeneous populations of cyanobacteria. The protocol employs fluorescent probes, i.e., redox dye 5-cyano-2,3-ditolyl tetrazolium chloride, ‘dead cell’ nucleic acid stain SYTOX Green, and DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole, combined with microscopy image analysis. Our method allows simultaneous estimates of cellular respiration activity, membrane and nucleoid integrity, and allows the detection of photosynthetic pigments fluorescence along with morphological observations. The staining protocol has been adjusted for, both, laboratory and natural populations of the genus Phormidium (Oscillatoriales), and tested on 4 field-collected samples and 12 laboratory strains of cyanobacteria. Based on the mentioned cellular functions we suggest classification of cells in cyanobacterial populations into four categories: (i) active and intact; (ii) injured but active; (iii) metabolically inactive but intact; (iv) inactive and injured, or dead.

Highlights

Bacterial populations, including pure cultures in laboratory studies, display high heterogeneity in morphological and physiological activity
Further staining with higher concentrations of the dye did not increase fluorescence intensity. This treatment, as long as it passes through the membranes of the dead cells, confirmed the applicability of SYTOX Green dye to Phormidium cultures, since it is the inactive control
Since we discovered that deposition of into their corresponding colored (INT) and CTC formazans possibly inflicted damage to plasma membranes of studied cyanobacteria, a careful investigation of their effect on staining with membrane-impermeant SYTOX Green dye was required

Summary

Introduction

Bacterial populations, including pure cultures in laboratory studies, display high heterogeneity in morphological and physiological activity. In natural microbial communities this variability is high due to the non-homogeneous physical character of natural environments, irregularity in nutrient distribution and competition between species [4,5]. Populationbased methods, such as respiration measured by the overall oxygen uptake or estimation of photosynthesis performance, provide averaged information on the population’s physiological state without considering the properties of single cells, and may result in faulty interpretation of population development and its stress response. In studies of physiological heterogeneity in populations of microorganisms the fluorochrome staining techniques are often based on detection of only one particular cell function, multiparameter techniques for bacteria and yeasts have been established [10,11,12,13]. In cyanobacterial research similar studies, including those where the application of fluorescence dyes are used, are rare and mostly concern unicellular species [14,15]

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Conclusion