Rapid assessment of the physiological status of Streptococcus macedonicus by flow cytometry and fluorescence probes

• Published in: International journal of food microbiology Vol. 111; no. 3; pp. 197 - 205
• Main Authors: Papadimitriou, Konstantinos, Pratsinis, Harris, Nebe-von-Caron, Gerhard, Kletsas, Dimitris, Tsakalidou, Effie
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.10.2006; Elsevier
• Subjects: acid treatment; antibiotics; bacteriology; Biological and medical sciences; Bis-oxonol; Carboxyfluorescein diacetate; cell culture; cell cycle; Cell Membrane Permeability - drug effects; Cell Membrane Permeability - physiology; Cell Survival; Coloring Agents - metabolism; Flow cytometry; Flow Cytometry - methods; Fluoresceins - metabolism; fluorescence; Fluorescent Dyes - metabolism; Food industries; Food microbiology; Fundamental and applied biological sciences. Psychology; Heterogeneity; iodides; lactic acid bacteria; membrane permeability; Membrane Potentials - drug effects; Membrane Potentials - physiology; microbial physiology; Propidium - metabolism; Propidium iodide; rapid methods; Single cell; single cell protein; Streptococcus; Streptococcus - growth & development; Streptococcus - physiology; Streptococcus macedonicus; Stress; Thiobarbiturates - metabolism; Time Factors; Flow cytometry; Heterogeneity; Streptococcus macedonicus; Single cell; Propidium iodide; Carboxyfluorescein diacetate; Stress; Bis-oxonol; Fluorescence; Streptococcaceae; Streptococcus; Bacteria; Micrococcales
• ISSN: 0168-1605; 1879-3460
• DOI: 10.1016/j.ijfoodmicro.2006.04.042

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.