Monitoring of Dynamic Microbiological Processes Using Real-Time Flow Cytometry

<div><p>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 <i>Escherichia coli</i>, (2) fluorescent labeling of heat-induced membrane damage in an autochthonous freshwater bacterial community, (3) the initial growth response of late stationary <i>E. coli</i> 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.</p> </div

Initial growth response of late stationary phase <i>E. coli</i> cells inoculated into fresh growth medium.

<p>(<b>A</b>) Raw RT-FCM data of FSC intensity over time, with marked regions (G1) and (G2) showing data used for panels F. A set FSC value (white dotted line, indicating the line below which 80 % of particles lie at the beginning of the measurement) separates a fraction of small (Fraction A) and large (Fraction B) cells which are used for calculations in panels D and E. (<b>B</b>) Raw data of bacterial GFP fluorescence (ex. 488 nm/em. 530 nm) resulting from the enzymatic cleavage of CFDA. (<b>C</b>) Processed data (1-minute intervals) showing the changes in total cell concentration as well as median cell size (measured with forward scatter). (<b>D</b>) Changes in the cell concentrations of small (Fraction A) and large (Fraction B) cells (from panel A). (<b>E</b>) Changes in the green fluorescence intensity of small (Fraction A) and large (Fraction B) cells (from panel A). (<b>F</b>) FCM overlay density plot of the culture at the start (blue data; (G1)) and end (green data; (G2)) of the experiment. </p

Induction of RecA transcription and GFP expression in <i>E. coli</i> cells exposed to 4 µg/ml ciprofloxacin.

<p>(<b>A</b>) Raw data of forward scatter (FSC) intensity over time, with marked regions (G1) and (G2) showing the data used for panels B and F. (<b>B</b>) Raw data of bacterial GFP fluorescence intensity (ex. 488 nm/em. 530 nm) over time. (<b>C</b>) Processed data (1-minute intervals) of cell concentrations during the experiment, showing four stages with varying growth rates. (<b>D</b>) Quantified changes in median cell size (forward scatter) and GFP fluorescence intensity. (<b>E</b>) Calculated rates of change in cell size and fluorescence intensity. (<b>F</b>) FCM overlay density plot of the culture at the start (blue data; (G1)) and end (red data; (G2)) of the experiment (from panel A). Gating procedures and calculations are detailed in the Materials and Methods section.</p

Treatment of a mixture of <i>Chlamydomonas reinhardtii</i> and <i>Mycrocystis aeruginosa</i> with hypochlorite during 20 minutes.

<p>(<b>A</b>) FCM density plot distinguishing the two populations in the starting mixture based on orange (ex. 488 nm/em. 585 nm) and red auto-fluorescence (ex. 488 nm/em. 670 nm). (<b>B</b>) Raw RT-FCM data of red auto-fluorescence intensity, resulting from the excitation of chlorophyll. Hypochlorite was added after 5 minutes (dotted line) (<b>C</b>) Raw RT-FCM data of orange auto-fluorescence intensity. (<b>D</b>) Processed data (30-second intervals) showing stable cell concentrations for both clusters during the experiment. (<b>E</b>) Changes in the red and orange fluorescence intensities of <i>C. reinhardtii</i>. (<b>F</b>) Changes in the red and orange fluorescence intensities of <i>M. aeruginosa</i>. </p