The use of multi-parameter flow cytometry to compare the physiological response of Escherichia coli W3110 to glucose limitation during batch, fed-batch and continuous culture cultivations

Hewitt, Christopher J.; Caron, G. Nebe-von; Nienow, Alvin W.; McFarlane, Caroline M.

doi:10.1016/s0168-1656(99)00168-6

Cited by 89 publications

(80 citation statements)

References 27 publications

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“…Regrowth of the injured cells following sorting confirmed that a fraction of the stressed cells adopted a latent state in which they could not reproduce but could be induced to a physiologically active state after recovery (1,6,15,16). The percent recovery of injured cells (40%) and the percent viable cells (47%) did not reach (11,23). The electroporated PIand cF-stained bifidobacterium cells were able to grow on agar plates (data not shown), showing that the concentration of PI (5 g/ml) used in our experiment was not toxic for bifidobacteria.…”

Section: Discussionmentioning

confidence: 75%

“…Multicolor FCM has been successfully used to assess physiological heterogeneity within bacterial populations during bacterial fermentations (10,11) and to gain insight into the mechanism of action of antibiotics on Staphylococcus aureus and M. luteus (25). Our multiparameter FCM results clearly illustrated the succession of cell changes that occurred in a bile salt-stressed bifidobacterium population and revealed physiological heterogeneity within the cell population (23).…”

Section: Discussionmentioning

confidence: 86%

“…FCM offers a powerful tool for analyzing a cell population at the single-cell level, since it can be used both to identify and enumerate bacterial populations from environmental samples and to characterize functional properties of the individual cell (14,31,34). Moreover, it allows simultaneous measurement of different physical and biochemical parameters and hence offers substantial information on the dynamics and physiological heterogeneity of a bacterial population (10,11,23). In addition, FCM offers the ability to physically separate selected cells by cell sorting for further molecular and physiological analysis (15,37).…”

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confidence: 99%

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

Amor

Breeuwer

Verbaarschot

et al. 2002

Appl Environ Microbiol

252

108

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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.

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Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 86%

mentioning

confidence: 99%

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

Amor

Breeuwer

Verbaarschot

et al. 2002

Appl Environ Microbiol

252

108

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“…When cells were exposed to substances accumulated in the culture broth, severe physiological stress was generally detected in high-density fedbatch cultures but not in continuous cultures, despite the cultivation conditions being comparable (specific growth rate, pH, temperature, and substrate). Hewitt et al (24,25) observed a decrease in the viability of nonrecombinant E. coli of approximately 15% in fed-batch cultures and an absence of dead cells in batch and continuous cultures. They concluded that this phenomenon is due either to increased glucose limitation or to the accumulation of toxic by-products in fed-batch cultures.…”

Section: Discussionmentioning

confidence: 99%

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Hyka

Züllig

Ruth

et al. 2010

Appl Environ Microbiol

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Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut ؉ (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.Changes to the product quantity and quality as well as the robustness of bioprocesses can be triggered by a number of factors which affect the physiological state of the microorganisms. The expression of a foreign gene, the processing of the recombinant protein, and the exposure of the cell to metabolites, inductors, substrates, unfavorable environmental conditions, high cell density (HCD), and/or "aging" can all result in markedly different physiological responses (17).The interpretation of bioprocess data often fails to reflect the actual state of individual cells within a population. It is based typically on performance characterization using concentration measurements and the simplifying assumption of uniform ("averaged") performance of each cell. This practice (39) provides an example of the failure to distinguish between a homogenous population of equally compromised microorganisms and a heterogeneous population of cells each performing differently. An understanding of the state of the individual cells is critical in achieving high efficiency in recombinant protein production. Only by knowing the number of (vital) cells that express the target molecule at the highest possible rate can the number of such cells within the population be maximized and the propo...

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“…Thus if the CMP does not overcome the affinity of the probe to the intracellular target or a temporary "leakage" across the cytoplasmic membrane exists, the dye accumulates inside the cell. The control applied to test for CMP independent accumulation of a dye is to collapse the CMP using decouplers (Hewitt et al, 1999). However, this method fails if the cells do not stain because of their outer membrane or active dye export or because of uptake due to temporary "leakiness".…”

Section: Rhodamine 123mentioning

confidence: 99%

Multi-parameter flow cytometry and cell sorting reveal extensive physiological heterogeneity in Bacillus cereus batch cultures

et al. 2011

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The use of multi-parameter flow cytometry to compare the physiological response of Escherichia coli W3110 to glucose limitation during batch, fed-batch and continuous culture cultivations

Cited by 89 publications

References 27 publications

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

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

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Multi-parameter flow cytometry and cell sorting reveal extensive physiological heterogeneity in Bacillus cereus batch cultures

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