Automated and High-throughput Microbial Monoclonal Cultivation and Picking Using the Single-cell Microliter-droplet Culture Omics System

Wenhsuan Chou; Xiaojie Guo; Liyan Wang; Xin-Hui Xing; Yi Wang; Chong Zhang

doi:10.3791/67925

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Method Article

Automated and High-throughput Microbial Monoclonal Cultivation and Picking Using the Single-cell Microliter-droplet Culture Omics System

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DOI:

10.3791/67925

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March 14th, 2025

Wenhsuan Chou¹^,² , Xiaojie Guo³ , Liyan Wang³^,⁴ , Xin-Hui Xing¹^,⁵^,⁶^,⁷ , Yi Wang¹^,² , Chong Zhang¹^,²^,⁵

¹Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, ²Key Laboratory of Industrial Biocatalysis, Ministry of Education, Tsinghua University, ³Luoyang TMAXTREE Biotechnology Co., Ltd., ⁴Biobreeding Center, Wuxi Research Institute of Applied Technologies, Tsinghua University, ⁵Center for Synthetic and Systems Biology, Tsinghua University, ⁶Institute of Biomedical Health Technology and Engineering, Shenzhen Bay laboratory, ⁷Institute of Biopharmaceutical and Health Engineering, Shenzhen International Graduate School, Tsinghua University

Summary

This protocol describes how to use the Single-cell Microliter-droplet Culture Omics System (MISS cell) to perform microbial monoclonal isolation, cultivation, and picking. The MISS cell achieves an integrated workflow based on droplet microfluidic technology, which offers excellent droplet monodispersity, high parallel cultivation, and high-throughput biomass detection.

Abstract

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors are hindered by cumbersome procedures and low throughput, impeding the rapid progress of microbial culturomics research. To address these challenges, we had successfully developed the Single-cell Microliter-droplet Culture Omics System (MISS cell), an automated high-throughput platform that utilizes droplet microfluidic technology for microbial monoclonal isolation, cultivation, and screening. This system can generate a large number of single-cell droplets and cultivate, screen, and collect monoclonal colonies in a short time, facilitating an integrated process from microbial isolation to picking. In this protocol, we demonstrated its application using the isolation and cultivation of human gut microbiota as an example and compared the microbial isolation efficiency, monoclonal culture performance, and screening throughput using the solid-plate culture method. The experimental workflow was simple, and reagent consumption was very low. Compared to solid-plate culture methods, the MISS cell could cultivate a greater diversity of gut microbiota species, offering significant potential and value for microbial culturomics research.

Introduction

Microbial culturomics has wide applications in researching beneficial microbes in the food industry, the diversity of environmental microbes, screening for new antimicrobial compounds, and the human microbiome in relation to disease¹^,²^,³^,⁴. Traditional methods, primarily based on solid plates, well plates, or micro-reactors to obtain and pick monoclonal colonies, are easy to operate but suffer from low throughput due to their multiple steps. This limitation hinders applications such as microbial mutagenesis screening, microbial culturomics s....