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.

FIG. 1.

Dual-parameter dot plot of the side scatter intensity versus DiBAC₄(3) fluorescence of B. lactis. Untreated cells (A), cells incubated with 15 μM CCCP (B), and cells that had been heat killed at 70°C for 30 min (C) were stained with 1 μM DiBAC₄(3) and analyzed by FCM. Data show the effects of the ionophore CCCP on the membrane potential of B. lactis and a high correlation between DiBAC₄(3) fluorescence and the side scatter signal. Control cells are gated on region R1 and display little green fluorescence, while cells gated on region R2 show a marked increase in green fluorescence as a result of the addition of 15 μM CCCP or heat treatment (70°C for 30 min).

FIG. 2.

Fluorescence histogram overlays of B. lactis DSM 10140 (A) and B. adolescentis DSM 20083 (B) stained with 1 μM DiBAC₄(3) for 4 min at 37°C. The x axis represents the corrected DiBAC₄(3) fluorescence for the cell size, obtained by calculating the log ratio of the green fluorescence of DiBAC₄(3) to the side scatter intensity. Results are shown for control untreated cells (a), for cells exposed to 0.05% (b), 0.1% (c), 0.2% (d), or 0.25% (e) dBS, and for cells that were heat treated at 70°C for 30 min (f).

FIG. 3.

Fluorescence histograms of B. lactis and B. adolescentis stained with 5 μg of PI/ml or 10 μM cFDA following exposure to different concentrations of dBS.

FIG. 4.

Viability assessment of bile salt-stressed cells of B. lactis DSM 10140 (A) and B. adolescentis DSM 20083 (B) by FCM and plate counts. Cells were harvested at the mid-exponential phase and then exposed to 0, 0.05, 0.1, or 0.2% dBS for 10 min at 37°C in anaerobic potassium phosphate buffer (50 mM; pH 7; containing 1 mM DTT). Results are expressed as percentages of cells stained with either cF (•), PI (▴), or DiBAC₄(3) (▪) and as the percentage of survival as determined by plate counts (⧫).

FIG. 5.

Multiparameter dot plots of B. lactis DSM 10140 representing PI fluorescence versus cF fluorescence. Cultures were exposed for 10 min to 0, 0.05, 0.1, 0.2, or 0.25% dBS in anaerobic potassium phosphate buffer (50 mM; pH 7; containing 1 mM DTT) for 10 min at 37°C. Subsequently, all samples were stained simultaneously with 10 μM cFDA and 5 μg of PI/ml and were analyzed by FCM. Three main subpopulations, corresponding to viable cF-stained cells (upper left quadrant), injured cells double stained with PI and cF (upper right quadrant), and dead PI-stained cells (lower right quadrant), can be readily differentiated.

FIG. 6.

Viability assessment of B. lactis DSM 10140 (A) and B. adolescentis DSM 20083 (B) using multiparameter FCM and the plate count method. Results illustrate the different physiological states of bile salt-stressed cells, consisting of viable, active cells stained only with cF (▪), injured cells stained with both cF and PI (⧫), dead cells stained only with PI (▴), and culturable cells determined by the plate count method •.