TY - JOUR
T1 - An industrial application of multi-parameter flow sytometry: Assessment of cell physiological state and its application to the study of microbial fermentations
AU - Hewitt, Christopher
AU - Nebe-von-Caron, G
PY - 2001/7/1
Y1 - 2001/7/1
N2 - Background: When using traditional microbiological techniques to monitor cell proliferation and viability, stressed, sublethally injured, or otherwise "viable but non-culturable" cells often go undetected. Because of this, such cells often are not considered by mathematical models used to predict bioprocess performance on scale-up and inaccuracies result. Therefore, analytical techniques, decoupled from postsampling growth, are desirable to rapidly monitor individual cell physiologic states during microbial fermentations. Methods: Microbial cells, including Escherichia coli, Rhodococus sp., and Sacharomyces cerevisiae, were taken at various stages from a range of fermentation processes and stained with one of three mixtures of fluorescent stains: rhodamine 123/propidium iodide, bis-oxonol/propidium iodide, or bis-oxonol/ethidium bromide/propidium iodide. An individual cell's physiologic state was assessed with a Coulter Epics Elite analyzer based on the differential uptakes of these fluorescent stains. Results: It was possible to resolve an individual cell's physiologic state beyond culturability based on the functionality of dye extrusion pumps and the presence or absence of an intact polarized cytoplasmic membrane, enabling assessment of population heterogeneity. This approach allows the simultaneous differentiation of at least four functional subpopulations in microbial populations. Conclusions: Fluorescent staining methods used in our laboratories have led to a functional classification of the physiological state of individual microbial cells based on reproductive activity, metabolic activity, and membrane integrity. We have used these techniques extensively for monitoring the stress responses of microorganism in such diverse areas as bioremediation, biotransformation, food processing, and microbial fermentation; microbial fermentation is discussed in this article. (C) 2001 Wiley-Liss, Inc.
AB - Background: When using traditional microbiological techniques to monitor cell proliferation and viability, stressed, sublethally injured, or otherwise "viable but non-culturable" cells often go undetected. Because of this, such cells often are not considered by mathematical models used to predict bioprocess performance on scale-up and inaccuracies result. Therefore, analytical techniques, decoupled from postsampling growth, are desirable to rapidly monitor individual cell physiologic states during microbial fermentations. Methods: Microbial cells, including Escherichia coli, Rhodococus sp., and Sacharomyces cerevisiae, were taken at various stages from a range of fermentation processes and stained with one of three mixtures of fluorescent stains: rhodamine 123/propidium iodide, bis-oxonol/propidium iodide, or bis-oxonol/ethidium bromide/propidium iodide. An individual cell's physiologic state was assessed with a Coulter Epics Elite analyzer based on the differential uptakes of these fluorescent stains. Results: It was possible to resolve an individual cell's physiologic state beyond culturability based on the functionality of dye extrusion pumps and the presence or absence of an intact polarized cytoplasmic membrane, enabling assessment of population heterogeneity. This approach allows the simultaneous differentiation of at least four functional subpopulations in microbial populations. Conclusions: Fluorescent staining methods used in our laboratories have led to a functional classification of the physiological state of individual microbial cells based on reproductive activity, metabolic activity, and membrane integrity. We have used these techniques extensively for monitoring the stress responses of microorganism in such diverse areas as bioremediation, biotransformation, food processing, and microbial fermentation; microbial fermentation is discussed in this article. (C) 2001 Wiley-Liss, Inc.
KW - ethidium bromide
KW - propidium iodide
KW - membrane integrity
KW - bacteria
KW - membrane potential
KW - yeast
KW - multiparameter
KW - bis-oxonol
U2 - 10.1002/1097-0320(20010701)44:3<179::AID-CYTO1110>3.0.CO;2-D
DO - 10.1002/1097-0320(20010701)44:3<179::AID-CYTO1110>3.0.CO;2-D
M3 - Article
C2 - 11429768
SN - 1097-0320
VL - 44
SP - 179
EP - 187
JO - Cytometry
JF - Cytometry
ER -