Resolution of viable and membrane-compromised bacteria in freshwater and marine waters based on analytical flow cytometry and nucleic acid double staining
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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DOI：
10.1128/aem.67.10.4662-4670.2001
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发表时间：
2001-10-01
影响因子：
4.4
通讯作者：
Denis, M
中科院分区：
文献类型：
--
作者：
Grégori, G;Citterio, S;Denis, M
细胞膜的完整性是表征活细胞（活性或非活性）并将其与受损和膜受损细胞区分开来的公认标准。该信息对于研究自然环境中微生物群落的功能非常重要，以便将通过各种方法测量的大量活动分配给有效负责观察的非常活跃的细胞。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染色测定方法，它使我们能够区分活细菌与受损和膜受损的细菌，并分出噪音和碎屑。该方法源自 S. Barbesti 等人的工作。 (Cytometry 40:214-218, 2000)这是对培养的细菌进行的。该方法的原理是同时使用渗透性（SYBR Green；分子探针）和非渗透性（碘化丙啶）探针，并利用两种探针对核酸进行染色时在它们之间发生的能量转移。不透性探针对透性探针荧光的完全猝灭将指示具有受损膜的细胞，部分猝灭将指示具有轻微损坏膜的细胞，并且缺乏猝灭将表征被鉴定为存活的完整膜细胞。在本研究中，这种方法已适用于地中海地区淡水和海水中的细菌。它快速且易于使用，并表明大部分低 DNA 含量的细菌可以由活细胞组成。诚然，局限性源于自然环境中存在的不明物种的未知行为，这可能偏离荧光染料的既定渗透特性。
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.