An industrial application of multiparameter flow cytometry: Assessment of cell physiological state and its application to the study of microbial fermentations

Background: When using traditional microbiological techniques to monitor ce ll proliferation and viability, stressed, sublethally injured, or otherwise "viable but non-culturable" cells often go undetected. Because of this, su ch cells often are not considered by mathematical models used to predict bi oprocess performance on scale-up and inaccuracies result. Therefore, analyt ical techniques, decoupled from postsampling growth, are desirable to rapid ly monitor individual cell physiologic states during microbial fermentation s. Methods: Microbial cells, including Escherichia coli, Rhodococus sp., and S acharomyces cerevisiae, were taken at various stages from a range of fermen tation processes and stained with one of three mixtures of fluorescent stai ns: rhodamine 123/propidium iodide, bis-oxonol/propidium iodide, or bis-oxo nol/ethidium bromide/propidium iodide. An individual cell's physiologic sta te was assessed with a Coulter Epics Elite analyzer based on the differenti al 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, enabli ng assessment of population heterogeneity. This approach allows the simulta neous differentiation of at least four functional subpopulations in microbi al populations. Conclusions: Fluorescent staining methods used in our laboratories have led to a functional classification of the physiological state of individual mi crobial cells based on reproductive activity, metabolic activity, and membr ane 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.