This study investigates the differentiation between live and dead Microcystis aeruginosa cells using phycocyanin autofluorescence and a nucleic acid-binding fluorescent probe. The methodology allows for the identification of live cells based on their intact membranes and pigment emission.
Take separate samples of live and dead Microcystis aeruginosa cells.
Mix the samples to create a combined population.
Microcystis contains phycocyanin, an autofluorescent pigment that remains intact in live cells but becomes degraded in dead cells.
Vortex the sample to break the clumps and generate a uniform, single-cell suspension.
Add a cell membrane-impermeable, nucleic acid-binding fluorescent probe, and incubate the sample in the dark to prevent photobleaching.
The probe selectively enters the dead cells through their damaged membranes and binds to the intracellular DNA.
Run the sample through a flow cytometer to measure phycocyanin autofluorescence and nucleic acid probe fluorescence using appropriate excitation wavelength lasers.
The live cells emit strong phycocyanin autofluorescence but show no probe signal.
In contrast, dead cells display reduced pigment emission and elevated probe fluorescence, confirming membrane damage.
This enables the identification of the pigment-rich live cells from the dead cells.
To optimize the molecular probe cell uptake, expose half of the previously prepared monoculture to the appropriate conditions for generating a dead control. Check the variations in the sample micro environment to confirm the death of the culture. And then disaggregate the colony formation as just demonstrated.
Next, select a 488-nanometer laser alongside a detector that can record fluorescence from the green and orange nucleic acid probes. And the 640-nanometer laser to record the phycocyanin signals through their respective detectors.
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