Background: Phytoplankton conversion of light on the upper limits of the ocean consists of half of the photosynthesis on the Earth. The population densities indicate the health of not only the phytoplankton, but the entire aquatic ecosystem. The isolation and sorting of aquatic samples using flow cytometry allows for quick and effective population analysis. The forward scatter on the MoFlo Astrios allows for differentiation of small and large particles from 0.2 to 30 μm on FSC. This design provides researchers greater flexibility to isolate and sort specific phytoplankton of different sizes while utilizing the 7 laser, 42 parameter MoFlo Astrios.
Methods: Plankton species; Chlorella, Phormidium inundatum, Phormidium persicinum, Cryptomonas, Rhodoos, Synechococcus, Skeletonema, Fremyella, were acquired from the UTEX: The Culture of Algae and grown in photobioreactors and cultured in specialized salt and fresh water media. Plankton species Prochlorococcus marinus and Emiliana huxlei were grown in sterile 2 L containers supplied with 0.2 um filtered air in salt water with fertilizer. Instant Ocean, ½cup per gallon of deionized water, was added to the culture with Microalgae Grow Mass Pack with Silicate. Cells were harvested by gentle centrifugation, roughly 300 x g for 5-10 minutes. The supernatant was decanted/aspirated and the pellet resuspended in a sterile saline solution to achieve 1x106 cells/mL. Plankton were then stained with SYTOX Green (Invitrogen, S7020), at a maximum concentration of 5 μM for 20 minutes after vortexing. Samples were filtered with a 70 μM Partec filter and kept on ice before flow cytometric analysis. Flow Cytometry: The MoFlo AstriosEQ was configured with 7 lasers and setup with a 100 μm tip to accommodate the larger phytoplankton (Cryptomonas). Cells were selected on their “live” status by being highly fluorescent in the red channels (chlorophyll) and low in the green channels (Sytox -). For small particle analysis, Prochlorococcus and Synechococcus were simultaneously analysed on FSC-Log parameters. Populations were sorted based on fluorescence and size as a 6-way sort into 5 mL tubes. The plankton were sorted at 25K eps to collect at least 100,000 events per each population using sort mode Purify 1-2.
Results: Plankton populations were distinguishable using fluorescence and scatter patterns on both log and linear scales simultaneously. With the Astrios optical flexibility, the plankton fluorescence spectra were optimized for signal to noise. Isolation of the Prochlorococcus, Synechococcus and other plankton species using cell sorting achieved 99% purity for all populations.
Conclusions: The forward scatter and optical collection design of the MoFlo AstriosEQ provide flexibility to detect large and small populations and sort them with high purity.