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 [DiBAC 4 (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 doublestained, 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.

Soy Protein Isolate-Alginate Microspheres for Encapsulation ofEnterococcus faecalis HZNU P2

“…No survival of free E. faecalis HZNU P2 cells was found after 1 h exposure in 1.0 or 2.0 % bile salt solution. The sensitivity of many strains of probiotics to bile salt solutions has been reported by many researchers (Amor et al 2002;Picot and Lacroix 2004;Pan et al 2013;Shi et al 2013a, b;Chen et al 2014;Sathyabama et al 2014). Table 1 shows slight reductions of encapsulated E. faecalis HZNU P2 after 2 h incubation in 1.0 or 2.0 % bile salt solutions.…”

Activity of encapsulated Lactobacillus bulgaricus in alginate-whey protein microspheres

“…The numbers declined more as the bile concentration and incubation time increased. The sensitivity of many probiotics strains to bile concentrations encountered in the human gastro-intestinal tract has been reported by several authors (Chung et al 1999;Amor et al 2002;Picot & Lacroix 2004). Clark and Martin (1994) observed 5.0 log reductions in viable cell counts of B. adolescentis in 2% bile solution after 12 h incubation at 37°C.…”

Limits of propidium iodide as a cell viability indicator for environmental bacteria

“…As PI has been described manifold in literature to penetrate injured membranes and cell walls, the dye is often applied to distinguish living from dead microorganisms. However, although PI uptake was described to occur in compartmented Bifidumbacterium species (20), producing segregate double stained cFDA/PI cells, large quantities of these cells were reproducibly culturable on agar plates, a phenomenon attributed to transiently injured or dead (compartmented, probably as part of a hyphae-like morphology), but recovering cells.…”