Abstract
We describe a straightforward approach to continuously monitor a variety of highly dynamic microbiological processes in millisecond resolution with flow cytometry, using standard bench-top instrumentation. Four main experimental examples are provided, namely: (1) green fluorescent protein expression by antibiotic-stressed Escherichia coli, (2) fluorescent labeling of heat-induced membrane damage in an autochthonous freshwater bacterial community, (3) the initial growth response of late stationary E. coli cells inoculated into fresh growth media, and (4) oxidative disinfection of a mixed culture of auto-fluorescent microorganisms. These examples demonstrate the broad applicability of the method to diverse biological experiments, showing that it allows the collection of detailed, time-resolved information on complex processes.
Highlights
Flow cytometry (FCM) is a powerful and flexible method to characterize hydrodynamically focussed particles based on light scattering and fluorescence emission, thereby combining large sample sizes with considerable speed of data acquisition and ample information on single-particle level
RecA is a major regulator of the E. coli SOS response, and its expression is induced upon DNA damage [11]
These results suggest the highest rate of cell elongation shortly after addition of the antibiotic, decreasing thereafter as the cells start to respond to the damage
Summary
Flow cytometry (FCM) is a powerful and flexible method to characterize hydrodynamically focussed particles based on light scattering and fluorescence emission, thereby combining large sample sizes with considerable speed of data acquisition and ample information on single-particle level. Adding a temporal dimension to the collected information makes flow cytometry extremely powerful for monitoring dynamic changes in suspended cells without losing the single-particle resolution Studying such processes with conventional techniques is only possible either by compromising on sample size, e.g. in timelapse microscopy, or by losing the single-cell resolution, as it is the case in biochemical analyses, where large numbers of cells are pooled to yield a population average. Such a “real time” or “kinetic” FCM approach (from here on referred to as real-time flow cytometry, RT-FCM) was first applied by Martin and Swartzendruber [4], and has since been used for studying amongst other things the biochemical properties of mammalian cells [5,6,7,8,9] and protists [10], and interactions between abiotic molecules [1]. Recent technical advances in standard flow cytometry instrumentation have made it possible to perform such experiments using small and comparatively cheap devices, in standard research laboratories
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