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首页> 外文期刊>Applied and Environmental Microbiology >Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining
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Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining

机译：基于分析流式细胞仪和核酸双重染色的淡水和海水中活菌和膜受损细菌的分离

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The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.
机译：细胞膜的完整性是表征活（活性或非活性）细胞并将其与受损的和膜受损的细胞区分开的公认标准。该信息对于研究自然环境中微生物的功能至关重要，以便将通过各种方法测得的大量活动分配给有效负责观察的活跃细胞。为了实现淡水和海水中细菌的这一任务，我们提出了一种基于分析流式细胞术的核酸双重染色测定法，它使我们能够将存活的细菌与受损的和膜受损的细菌区分开来，并分清噪音和碎屑。此方法源自S. Barbesti等人的工作。 （Cytometry 40：214–218，2000）是对培养的细菌进行的。这种方法的原理是同时使用渗透性（SYBR Green;分子探针）和渗透性（碘化丙啶）探针，并利用两个探针对核酸染色时它们之间发生的能量转移。不渗透探针对渗透探针荧光的完全淬灭将指向具有受损膜的细胞，部分淬灭将指示具有轻微受损膜的细胞，而缺乏淬灭则将被鉴定为存活的完整膜细胞表征为特征。在本研究中，这种方法已经适应了地中海地区淡水和海水中的细菌。它快速且易于使用，显示出大部分具有低DNA含量的细菌可以由活细胞组成。当然，局限性是由于存在于自然环境中的身份不明物种的未知行为而引起的，这可能与针对荧光染料的既定渗透性不同。

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