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

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 disease^1,2,3,4. 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....

Protocol

All study procedures are compliant with all relevant ethical regulations. Procedures were approved by Science and Technology Ethics Committee of Tsinghua University. For studying human gut microbiota, stool samples were collected from a healthy adult with no significant medical conditions, who gave written informed consent.

1. Instrument installation

1. Place the Omics system instrument in a clean or sterile environment (such as a sterile room or anaerobic bench). The instrument is a precision device, and when placing it in the facility, consider the following:
   1. Maintain the instrument under normal press....

Results

The human gut microbiota, constituting the predominant microbial community, is estimated to harbor approximately 4 × 10¹³ microorganisms in the gut, showcasing its vast numbers and complex composition¹¹. In this study, we aimed to isolate and culture gut microbiota and used the solid plate method as a control to demonstrate the high-throughput performance of the MISS cell.

First, we used the same fecal suspension to compare the single-cell isolation.......

Discussion

This protocol outlines the operation of the MISS cell for automated and high-throughput microbial monoclonal isolation, cultivation, detection, and collection. Compared to traditional methods by which only ~20%-30% of gut microbiota could be isolated and cultured^2,12, the number of monoclonal clones obtained using the Omics system was 1.97 fold higher than those obtained from solid plates. This comparison reveals that the MISS cell has advantages in single-cell i.......

Materials

Name	Company	Catalog Number	Comments
100 mm Petri dish	Merck KGaA, Darmstadt, Germany	P5731-500EA	For solid plate preparation
30 mL Stool Containers	Boen Healthcare Co., Ltd	611101	For collecting the stool samples
37 °C constant temperature incubator	Shanghai Yiheng Technology Co., Ltd.	LRH-150	Cultivate the solid plate in the incubator
96-well Clear Flat Bottom Polystyrene TC-treated Microplates	Corning	3599	For well plate movement detection and droplet collection
Agar	Becton, Dickinson and Company	214010	For solid plate preparation
Air bubble removal oil	Luoyang TMAXTREE Biotechnology Co., Ltd.	MISS cell-S-oil	The oil in the air bubble remover during droplet screening
Air bubble remover	Luoyang TMAXTREE Biotechnology Co., Ltd.	MISS cell-S	Exclude the gas phase between droplets before performing droplet detection and collection
Anaerobic bench	Argon and Nitrogen Space Equipment Business Department, Haiyu Town, Changshu City	VGB-4CM	For aseptic operation and UV sterilization under anaerobic condition
Autoclave	Puhexi Health and Medical Equipment Co., Ltd.	MLS-830L	For autoclaving BHI medium, EP tube, and so on.
Brain Heart Infusion (BHI) Broth	Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd	HB8297-1	Components of the BHI medium The ingredient list: 38.5 g/L BHI Broth in distilled water
Cell Spreader	Merck KGaA, Darmstadt, Germany	HS8151	Inoculate the microbial solution onto the solid plate
Centrifuge tube, 15 mL	Beijing Xinhengyan Technology Co., Ltd	HB53397	For microbial solution preparation
Computer	Lenovo	E450	Software installation and MISS cell control
Cryovial	Thermo Fisher	2.0 mL	For stool preservation
Distilled water	Beijing Mreda Technology Co., Ltd.	M306444-100ml	Add into humidifier to keep the humidity in droplet cultivation chamber
EP tube	Thermo Fisher	2.0 mL	For collecting the stool samples
Fluorescent inverted microscope	Olympus Life Science (LS)	CKX53	Check and calculate the microbial concentration
Glycerol	GENERAL-REAGENT	G66258A	For strain preservation
Hemocytometer	Acmec	AYA0810-1ea	Calculate the microbial concentration
KCl	Ambeed	A442876	Components of phosphate buffered saline (PBS solution)The ingredient list: 8 g/L NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in distilled water
KH2PO4	MACKLIN	P815661	Components of phosphate buffered saline (PBS solution)The ingredient list: 8 g/L NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in distilled water
Mesh filter	Anping Jiufeng Wire Mesh Manufacturing Co., Ltd	200 mesh (0.075 mm), 400 mesh (0.038 mm), 800 mesh (0.018 mm)	Remove undigested food and smaller particulate matter from the stool samples
Micro-tubing and droplet generation microfluidic chip	Luoyang TMAXTREE Biotechnology Co., Ltd.	MISC-B2	For droplet generation and droplet incubation
MISS cell oil	Luoyang TMAXTREE Biotechnology Co., Ltd.	MISS cell-BOS-B	The oil phase for droplet microfluidics
MISS cell software	Luoyang TMAXTREE Biotechnology Co., Ltd.	MISS cell V3.2.4	Perform experimental operations on the MISS cell instrument
Na2HPO4	Solarbio	D7292	Components of phosphate buffered saline (PBS solution)The ingredient list: 8 g/L NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in distilled water
NaCl	GENERAL-REAGENT	G81793J	Components of the physiological saline solution The ingredient list: 9 g/L NaCl in distilled water
Pipette	eppendorf	2.5 μL, 10 μL, 100μL, 1000μL	For liquid handling
Polytetrafluoroethylene tube	Shenzhen WOER Heat-shrinkable Material Co., Ltd.	3401000141	For droplet incubation. This material was already included in micro-tubing and droplet generation microfluidic chip
Sample bottle	Luoyang TMAXTREE Biotechnology Co., Ltd.	MISS cell-bottle	Sampling of microbial solution
Single Cell Microliter-droplet Culture Omics System (MISS cell)	Luoyang TMAXTREE Biotechnology Co., Ltd.	MISS cell-G3f	Performing the microbial monoclonal isolation, cultivation, detection and collection
Superspeed Centrifuge	Thermo Fisher	Sorvall Lynx 4000	Prepare the microbial solution for sequencing
Syringe	Jiangsu Zhiyu Medical Instructment Co., Ltd	10 mL	Draw the distilled water and inject it into the humidifier in droplet cultivation chamber
Ultra low temperature refrigerator	SANYO Ultra-low	MDF-U4086S	For strain preservation (-80 °C)