Bacterial populations display high heterogeneity in viability and physiological activity at

Bacterial populations display high heterogeneity in viability and physiological activity at the single-cell level, especially under stressful conditions. stresses [1], [2], [3] that result in a significant cell-to-cell discrepancies in Rac-1 viability and physiological state, becoming more pronounced under stressful conditions. 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]. Population-based methods, such as respiration measured by the overall oxygen uptake 71939-50-9 manufacture or estimation of photosynthesis performance, provide averaged information on the population’s physiological state without considering the 71939-50-9 manufacture properties of single cells, and may result in faulty interpretation of population development and its stress response. Therefore, a versatile approach that estimates multiple physiological parameters at the single-cell level is required for reliable information on the state of the cells in inhomogeneous populations. The use of fluorochromes for physiological assessment of bacteria provides accurate information about the state of individual cells in populations [6], [7]. A number of fluorescence-based assays that reflect various physiological functions are available for detecting cell viability and activity, such as assessment of membrane integrity and potential, intracellular pH, respiration intensity, intracellular enzymatic activity, etc. [7], [8], [9]. In studies of physiological heterogeneity in populations of microorganisms the fluorochrome staining techniques are often based on detection of only one particular cell function, although multiparameter techniques for bacteria 71939-50-9 manufacture and yeasts have also 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]. The cell is a complex system that responds to a fluctuating environment by modifying its structural organization and by changing its multiple physiological parameters. We consider that a living, healthy and active cyanobacterial cell is primarily characterized by plasma membrane and genome integrities, detectable metabolic activity, and significant content of pigments for effective photosynthetic performance. Under stressful conditions, and due to apoptosis, cells may sustain one or several kinds of damage to their subcellular structures, and changes in their physiological activities. For the detection and estimation of metabolic activity an assay based on energy dependent processes is required. Respiration is closely bound to the cellular activity [16] and accurately reflects overall energy metabolism of cells. Therefore, detecting respiration is preferable to indirect techniques based on active transport of fluorochromes into the cells, fluorogenic assays for intracellular enzymatic activity, or analysis of photosynthetic performance. Such an estimate may be achieved by employing tetrazolium salts that act as artificial electron acceptors in reaction with the respiratory chain, therefore directly competing with molecular oxygen, and this reaction detects metabolically active cells [17]. The loss of plasma membrane integrity provides a 71939-50-9 manufacture good estimate for bacterial cell viability as it plays a key role in the operation of the whole cell. The maintenance of its integrity is one of the main features discriminating dead or severely injured cells from living cells. Fluorescence assays intended for estimating membrane integrity are based on the passive exclusion of particular dyes (e.g. propidium iodide, SYTOX Green) by cells with structurally integral membranes. The presence of genetic material is another inherent prerequisite of viability. In cyanobacterial cell, DNA is organized as a compact structure (nucleoid), which is usually located at the center of the cell. The absence of nucleoid or its visibly severe degradation is an obvious.