Isolation of deep-sea microorganisms by flow cytometry-based high-throughput cell sorting and single cell cultivation

Abstract:

[Objective] To test a single cell cultivation method involving flow cytometry-based high-throughput cell sorting and single cell cultivation in the isolation of microorganisms from deep-sea samples. [Methods] The flow cytometer sorted to microbial cells of interest based on the size and complexity of the particles using forward versus side scatter gating (SSC vs. FSC) without the need for fluorescence labeling. Then, the single cells were cultivated in 96-well plates and further transferred to agar plate for scale-up cultivation and taxonomic identification. The performance of this method to cultivate microorganisms from deep-sea water and sediment samples was evaluated. [Results] An optimal sort region was chosen to sort microbial cells from the deep-sea samples, following by high-throughput single-cell broth cultivation. A total of 61 potential novel microbial strains, which belong to 6 novel genus or species, were obtained from deep-sea samples from the Indian Ocean. The novel strains accounted for 26.29% of the total isolates and shared 89.79%-95.37% similarity at the 16S rRNA gene sequence level. [Conclusion] The FCM-based high-throughput cell sorting and single-cell cultivation method is more suitable for sorting and cultivating of sea-water microorganisms. This methed may help increase the efficiency of identifying novel species from deep seas.