Papadimitriou Konstantinos, Pratsinis Harris, Nebe-von-Caron Gerhard, Kletsas Dimitris, Tsakalidou Effie
Laboratory of Dairy Research, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece.
Int J Food Microbiol. 2006 Oct 1;111(3):197-205. doi: 10.1016/j.ijfoodmicro.2006.04.042. Epub 2006 Aug 24.
Flow cytometry in combination with fluorescence probes was applied to rapidly assess the physiological status of Streptococcus macedonicus ACA-DC 198, a newly described member of the lactic acid bacteria group with technologically important features (e.g. lantibiotic production). A sonication procedure was developed for disaggregating typical streptococci chains in order to optimize cell preparations for single cell analysis. Single stained live and dead populations of S. macedonicus cells were clearly resolved based on membrane potential by bis-oxonol [DiBAC(4)(3)], membrane integrity by Propidium Iodide (PI) and enzymatic activity as well as membrane integrity by Carboxyfluorescein Diacetate (cFDA). Further, estimation of both live and dead cells by a cFDA/PI two-colour flow cytometric assay showed excellent correlation with the dead cells in the samples (dead(FCM)=0.9945 dead(S)-0.806, R(2)=0.9986 and live(FCM)=-0.978 dead(S)+98.895, R(2)=0.9992). Finally, the assay was applied to study the physiology of S. macedonicus after acid stress. Interestingly, in situ assessment of the physiological status of stressed S. macedonicus cells by flow cytometry and single cell sorting revealed the coexistence of three distinct subpopulations according to their fluorescence labelling behaviour and culturability, representing intact/culturable, permeabilized/dead and potentially injured cells with the latter exhibiting both metabolic activity and membrane permeabilization as well as decreased culturability.
流式细胞术结合荧光探针被用于快速评估马其顿乳杆菌ACA-DC 198的生理状态，该菌是乳酸菌组新描述的成员，具有重要的技术特性（如产羊毛硫抗生素）。开发了一种超声处理程序来分解典型的链球菌链，以优化用于单细胞分析的细胞制备。基于双羟乙氧羰花青[DiBAC(4)(3)]的膜电位、碘化丙啶（PI）的膜完整性、羧基荧光素二乙酸酯（cFDA）的酶活性以及膜完整性，清晰分辨了马其顿乳杆菌细胞的单染色活细胞和死细胞群体。此外，通过cFDA/PI双色流式细胞术测定法对活细胞和死细胞的估计显示，与样品中的死细胞具有极好的相关性（死细胞(流式细胞术)=0.9945死细胞(平板计数)-0.806，R(2)=0.9986；活细胞(流式细胞术)=-0.978死细胞(平板计数)+98.895，R(2)=0.9992）。最后，该测定法被用于研究酸胁迫后马其顿乳杆菌的生理学。有趣的是，通过流式细胞术和单细胞分选对受胁迫的马其顿乳杆菌细胞的生理状态进行原位评估，发现根据其荧光标记行为和可培养性存在三个不同的亚群，分别代表完整/可培养、通透化/死亡和潜在受损细胞，后者表现出代谢活性、膜通透性以及可培养性降低。
