微生物单细胞分离方法研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2022-1506

摘要/Abstract

摘要：

自然界中大多数微生物处于未培养状态，被称为 “微生物暗物质”。随着微生物单细胞分离方法的不断更新，利用新技术、新方法应对微生物纯培养的挑战获得了重要进展，这些新的分离及培养策略对推动微生物资源学的发展具有重要意义。尽管宏基因组学和基因组学数据相关成果日益增多，但微生物单细胞的分离与培养对于系统研究微生物的生态功能、遗传进化等仍至关重要。本文主要概述了目前使用的或正在研发的膜扩散培养法、微流控分选、荧光激活细胞分选、单细胞拉曼分选、光镊技术、显微操作技术等单细胞分离技术的原理与应用，及其在微生物单细胞分离和培养方面的优点与不足，同时展望了这些单细胞分离技术未来的发展和应用前景。

关键词: 微生物暗物质, 单细胞分离, 未培养微生物, 微生物选择性分离

Abstract:

Most microorganisms in nature are in an uncultured state，which are called “microbial dark matter”. With the development of technology innovations in microbial single-cell separation, significant progress has been made in applying new technologies and methods to the challenges of microbial pure-culture. These new separation and culture strategies will greatly promote the development of microbial resources research. Despite the increasing achievements related to metagenomics and genomics, the isolation and cultivation of single microbial cells are still essential to systematically study their ecological functions, genetic evolution, and so on. This paper mainly summarizes the principle and application of membrane diffusion culture, microfluidic sorting, fluorescence activated cell sorting, single cell Raman sorting, optical tweezers technology, micromanipulation technology and other single cell separation technologies currently used or under development, as well as their advantages and disadvantages in microbial single cell isolation and culture. At the same time, the paper also discusses development and application prospects of single-cell separation technologies in the future.

Key words: microbial dark matter, single cell separation, uncultured microbes, selective isolation of microorganisms

张坤, 闫畅, 田新朋. 微生物单细胞分离方法研究进展[J]. 生物技术通报, 2023, 39(9): 1-11.

ZHANG Kun, YAN Chang, TIAN Xin-peng. Research Progress in Microbial Single Cell Separation Methods[J]. Biotechnology Bulletin, 2023, 39(9): 1-11.

图/表 4

图1 单细胞分离方法 a：膜扩散培养法[18]；b：微流控技术[24]；c：单细胞拉曼分选[25]；d：荧光激活细胞分选[26]；e：光镊技术[27]；f：显微操作技术[28]

Fig. 1 Single cell separation methods a: Membrane diffusion culture method[18]. b: Microfluidic technology[24]. c: Single cell Raman sorting[25]. d: Fluorescence activated cell sorting[26]. e: Optical tweezers technology[27]. f : Micromanipulation technology[28]

图2 基于Live-FISH技术的单细胞分离方法 a: Live-FISH标记及活细胞染色；b: 复染后的流式细胞分选

Fig. 2 Single cell separation method based on Live-FISH technique a: Live-FISH labeling and live cells staining. b: Flow cytometry after counterstain

图3 基于反向基因组学的微生物靶向分离

Fig. 3 Targeted isolation of microorganisms based on reverse genomics

表1 微生物单细胞分离方法的优点及不足

Table 1 Advantages and disadvantages of single cell separation methods for microorganisms

分离方法 Separation method		优点 Advantages	不足 Disadvantages
高通量分离方法	膜扩散培养法	原位培养微生物，增加可培养率	耗时久，工作量大
High throughput separation methods	微流控技术	高通量，结构简单，成本低	通用性及特异性低
	单细胞拉曼分选	分选效率高，无创，无标记	仪器要求较高，拉曼光谱比较弱
	荧光激活细胞分选	高通量，效率高，灵敏度高	成本高，仪器要求较高，损伤细胞
靶向分离方法	Live-FISH	靶向分离细胞效果较强	耗时久，细胞破坏性大，成活率低
Targeted separation methods	反向基因组学	靶向分离细胞，适用性广泛	需要依赖抗体标记和流式分选
其他方法	光镊技术	精度高、效率高	会损伤细胞，成本高
Other methods	显微操作技术	适用范围广，可分离选定细胞	效率低，容易损伤细胞

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