Viability Analysis of Microcystis aeruginosa Using Flow Cytometry

Abstract

Source: Chapman, I. J., et al., Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry. J. Vis. Exp. (2016)

This video demonstrates the method for distinguishing live and dead Microcystis aeruginosa cells by combining fluorescent probe labeling with flow cytometric analysis of phycocyanin autofluorescence and probe signal.

Protocol

1. Optimization of Molecular Probe Cell Uptake

1. Harvest half of the M. aeruginosa culture from an exponential phase and use it as a 'live' control.
   NOTE: Samples diluted from a high-density culture straight to an exponential phase may affect optimization results through dead cell turnover compared to cultures inoculated from an initial lag/induction phase.
2. Prepare the other half as a 'dead' control by using methods such as 70% ethanol, heating samples at 60 ^oC for 1 hr, paraformaldehyde, or 4% formaldehyde for 30 min. Check variations in the samples' microenvironment (e.g., pH).
   NOTE: The positive, heat-killed, ‘dead’ control in M. aeruginosa is distinguished from a ‘live’ sample through its decrease in phycocyanin signals. Inducing mortality by other methods may not cause the same output and will vary in species.
3. Set up mixed samples using different ratios of 'live' and 'dead' samples (e.g., 0%, 25%, 50%, 100%).
4. Disaggregate colony formation by vortexing or sonication and check pH.
5. Select a 488 nm laser alongside detectors that can record fluorescence from the green (fluorescence channel 1 or FL1, 530 ± 15 nm) and orange (fluorescence channel 2 or FL2, 585 ± 20 nm) nucleic acid probes and the 640 nm laser to record phycocyanin signals through its respective detector.
   NOTE: When bound to DNA, the green nucleic acid probe has an approximate fluorescence excitation wavelength of 504 nm and emission maxima of 523 nm, whilst the orange nucleic acid probe has an excitation wavelength of 547 nm and emission maxima of 570 nm. A 488 nm argon ion solid state laser can be employed to excite both molecular probes, however, a green laser (up to 547 nm) will produce a higher orange fluorescence.
6. As a starting point, introduce the molecular probe at the manufacturer's recommended concentration to the 50% 'live' and 50% 'dead' culture and incubate in the dark.
7. Select a new data cell, place the sample under the sample introduction port (SIP) with thresholds and triggers that will reduce background noise (FSC-H or forward scatter height, 80,000).
8. Create a density plot with FSC-H and SSC-H (side scatter height) parameters, and three histograms. One histogram using the respective molecular probe optical detector channel (FL1 or 2), one to detect phycocyanin emissions (fluorescence channel 4 height or FL4-H), and the other, FSC-H, all on a log scale.
9. Incubate the M. aeruginosa samples with the nucleic acid probes in the dark for up to 60 min, recording in separate data cells at various time points (1, 5, 10, 15, 30, and 60 min).
   NOTE: When adjusting parameters such as pH, check with manufacturers for potential reactions from certain chemicals (for the tested nucleic acid probes, a buffer cannot contain phosphates or high levels of monovalent or divalent cations, as the binding with DNA will be reduced).
10. Apply a software gate to include only the FSC-H histogram (320,000 - 1,500,000) for the target organism's cell size in the respective fluorescence probe channel histogram.
    NOTE: The concentrations used in this protocol were 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 µM, which can be altered by either increasing or decreasing the volume of the M. aeruginosa sample or the initial stock solution of nucleic probes.
11. In the fluorescence probe channel, apply another inclusive software gate to the highest peak in the histogram (green FL1-H, 240,000 - 1,650,000, orange FL2-H, 30,000 - 165,000) and subsequently gate that positive probe fluorescence into the density plot.
12. Run steps 3.1 - 3.11 with 100% "live," 100% "dead," and all mixed culture samples, adjusting the molecular probe concentrations (e.g., x 0.1 to x 10) and/or temperature and pH levels if necessary.
13. Compare the number of positive molecular probe fluorescence signals to the original cell density of 'dead' cells (taken from half the total FSC-H or a 100% 'dead' culture) to find the total percentage of cells stained with the nucleic acid probes.
14. Do this for each time period within each concentration to find the optimal protocol for the highest percentage of cell nucleic probe uptake without producing non-specific staining (use the means in a One-Way analysis of variance, ANOVA, or Kruskal-Wallis one-way analysis of variance if the data is non-parametric).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cyanobacteria Media	Sigma-Aldrich	C3061-500ML	BG-11 Freshwater concentrated solution (x50 dilution)
Decontamination Fluid	BD Biosciences	653155	Run for 2 min when outputs are more than 12 events per second on fast or a flow rate of 66 µl/min. Followed by 2 min of sheath H2O.
Flow Cytometer	BD Biosciences	by request	BD Accuri C6
SYTOX Green	Life Technologies	S7020	Nucleic acid stain – 5 mM solution in DMSO
SYTOX Orange	Life Technologies	S11368	Nucleic acid stain – 5 mM solution in DMSO