Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

ABSTRACT Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

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Multiparametric Flow Cytometry for Identification and Fluorescence Activated Cell Sorting of Five Distinct B-Cell Subpopulations in Normal Tonsil Tissue

American Journal of Clinical Pathology ◽

10.1309/ajcpdqnp2u5dzhvv ◽

2011 ◽

Vol 136 (6) ◽

pp. 960-969 ◽

Cited By ~ 30

Author(s):

Malene Krag Kjeldsen ◽

Martin Perez-Andres ◽

Alexander Schmitz ◽

Preben Johansen ◽

Martin Boegsted ◽

...

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Cell Sorting ◽

Fluorescence Activated Cell Sorting ◽

Multiparametric Flow Cytometry ◽

Tonsil Tissue ◽

Cell Subpopulations

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Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to Simulated Food Processing Treatments, Determined Using Fluorescence-Activated Cell Sorting and Plate Counting

Applied and Environmental Microbiology ◽

10.1128/aem.00323-11 ◽

2011 ◽

Vol 77 (13) ◽

pp. 4657-4668 ◽

Cited By ~ 36

Author(s):

Deirdre Kennedy ◽

Ultan P. Cronin ◽

Martin G. Wilkinson

Keyword(s):

Membrane Potential ◽

Cell Membrane ◽

Cell Sorting ◽

Food Processing ◽

Bacterial Species ◽

Membrane Integrity ◽

Control Measures ◽

Cell Membrane Permeability ◽

Fluorescence Activated Cell Sorting ◽

Plate Counts

ABSTRACTThree common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique.

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Rapid identification of bacterial viability, culturable and non-culturable stages by using flow cytometry

10.21203/rs.3.pex-1153/v1 ◽

2020 ◽

Author(s):

Mohiuddin Md. Taimur ◽

Renan Acevedo ◽

Larry A. Sklar ◽

Keith Thomsen ◽

George P. Tegos ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Sorting ◽

Membrane Integrity ◽

Cellular Membrane ◽

Rapid Identification ◽

Plate Count ◽

Gram Negative Bacteria ◽

Cellular Functions ◽

Syto 13 ◽

Syto 9

Abstract The protocol details methods developed to optimize staining procedures and instrument settings for the rapid identification and unbiased enumeration of viable but non-culturable (VBNC) and viable-culturable (VC) cells as well as the membrane integrity of bacteria by flow cytometry (FCM). To detect viability, various Gram-negative bacteria were stained with numerous fluorescent membrane permeable (SYTO 9, SYTO 13, SYTO 17, SYTO 40) and impermeable (propidium iodide, PI) probes, and then quantified using the FCM. FCM data are then integrated with specific plate count results to calculate VC cells in different growth states. The cells were also exposed to heat (72° C) to monitor cellular membrane integrity. At late log phase (at 18 h incubation) of bacterial culture, the culturable, non-culturable and cells with damaged membranes varied from 40-98%, 1 to 64% and 0.7% to 4.5%, respectively. In this study, the cells with damaged cell membranes were considered dead (non-viable). This robust method preserves cell viability and takes a little more than an hour allowing the simultaneous quantification of phenotypes or cellular functions that is compatible with downstream cell-sorting and RNA-based assays.

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