基于流式细胞仪高通量分选的深海微生物单细胞培养

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

摘要:

[目的] 建立适用于海洋微生物的流式细胞分选与高通量单细胞培养的方法，通过该方法从印度洋深海样品中分离微生物纯培养菌株。[方法] 利用流式细胞仪单细胞分选功能，以前向角（FSC）和侧向角（SSC）散射光信号代替荧光信号作为分选逻辑，对深海水体和沉积物样品中微生物进行单细胞高通量分选和培养。[结果] 确定了流式细胞分选的区域和条件，发现所建立方法适于分离海洋水体微生物，而不是沉积物微生物。从印度洋深海水体样品中获得61个潜在新菌株，分属于6个新属种，占分离菌株总数的26.29%，其16S rRNA基因序列与已培养的模式菌株相似性为89.79%-95.37%。[结论] 本研究所建立的方法有助于提高发现海洋微生物新物种的效率，获得更多新的海洋微生物资源。

关键词:微生物培养;单细胞;流式细胞仪;深海

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.

Key words:microbial cultivation;single cell;flow cytometry;deep sea