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 studies, and high-producing colony selection, all of which require extensive monoclonal screening.

Recently, various single-cell detection and dispensing devices have been designed to significantly enhance the processing speed of microbial samples, while reducing labor and minimizing errors from manual handling⁵. However, these instruments typically address only specific steps within traditional methods, often requiring extensive equipment integration, occupying significant space, and incurring high costs. Therefore, there was a pressing need to develop a low-cost, universally applicable microbial culture and screening platform to compensate for the shortcomings mentioned above.

In our previous work, we successfully developed an automated, high-throughput screening platform, known as the Single-cell Microliter-droplet Culture Omics System (MISS cell, hereafter referred to as "the Omics system")⁶. This platform utilizes droplet microfluidic technology, which holds promise for achieving automation and integration in microbial isolation, cultivation, and picking^7,8,9,10. The Omics system comprises several key modules, including a sampling module, microfluidic chip, droplet detection and collection system, enabling efficient single-cell isolation, cultivation, monoclonal screening, and collection in microbiology research. We have already utilized the Omics system to achieve high-throughput mutagenesis screening of Corynebacterium glutamicum⁶.

Due to the automation and high-throughput screening capabilities of the Omics system, applying it to microbial culturomics is expected to rapidly obtain a large amount of microbial data. In this protocol, we introduced the detailed operational procedure of the MISS cell, with the isolation and cultivation of human gut microbiota as an example to demonstrate the process of microbial single-cell isolation, cultivation, monoclonal detection, and screening. The operation of the Omics system is simple, and researchers only need to follow the software direction for sequential installation of micro-tubing and droplet generation microfluidic chip, parameter settings, and sample preparation.

In the software operation interface, the Omics system is divided into three main functions-isolation, cultivation, and screening. Researchers can select different stages according to the experiment. Furthermore, during the droplet screening stage, researchers can choose from two detection modes: fluorescent signal or optical density. The software provides real-time visualization of the droplet screening process. Finally, researchers have the flexibility to configure parameters such as culture conditions, detected wavelength, and the number of collection wells based on their specific experimental demands, and they can pause the instrument anytime to carry out other operations. The MISS cell is a microbe-friendly, high-throughput monoclonal screening platform with simple operation and minimal reagent consumption.

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 pressure and temperature.
   2. Keep the instrument away from strong electric fields, magnetic fields, and heat radiation sources.
   3. Ensure that the instrument placement area exceeds dimensions of 2,500 mm (D) x 1,500 mm (W) x 2,000 mm (H).
   4. Maintain environmental humidity of the instrument below 60%.

2. Preparations

1. Sequentially turn on the power for the Omics system, the computer, and the Omics system operating software.
2. MISS cell micro-tubing and droplet generation microfluidic chip installation:
   1. Open the door of the droplet generation and cultivation chamber (Figure 1A) and vertically remove the protective cover for the micro-tubing and droplet generation microfluidic chip. Use a disposable syringe to add 10 mL of sterile distilled water to the humidifier inside the droplet cultivation chamber (Figure 1C) and reinstall the protective cover for the micro-tubing and droplet generation microfluidic chip.
   2. Open the sterile packaging of the micro-tubing and droplet generation microfluidic chip and vertically place it directly above the cultivation chamber (Figure 1C).
   3. On the software interface, click on Installation (Figure 2A). At this point, a pop-up window appears with the prompt, Confirm the replacement of micro-tubing and droplet generation microfluidic chip? Click Yes to start the installation.
   4. Take out the air bubble remover and fix it upside down on the air bubble remover placement in the droplet generation and cultivation chamber. Be careful not to press the droplet inlet and outlet tubes of the air bubble remover (Figure 1E).
   5. Attach the droplet outlet tube of the air bubble remover to the clamping valve below it and let it pass through the hole, which is directed to the droplet detection and collection chamber (Figure 1B).
   6. Open the door of droplet detection and collection chamber, vertically insert the detection tube, already connected to the droplet outlet tube, into the detection socket, and ensure that the detection tube is fully inserted (Figure 1D).
      NOTE: When inserting the detection tube, insert it vertically without any bends on the tube.
   7. Tighten the screw that secures the detection tube in a clockwise direction. After confirming that the detection tube is fully inserted and secured, close the door of the droplet detection and collection chamber.
   8. From the micro-tubing and droplet generation microfluidic chip, there are 10 silicone tubes, each labeled with a number (L01-L10). Connect each labeled tube to the corresponding numbered clamp valve (01-10) (Figure 1C).
   9. Connect the quick connector from the micro-tubing and droplet generation microfluidic chip to the corresponding port on the Omics system: C1 to O1, C2 to O2, C4 to O4, and CF to OF.
      NOTE: The installation of the micro-tubing and droplet generation microfluidic chip is complete. C3 does not need to be connected to O3.
   10. After the micro-tubing and droplet generation microfluidic chip installation is completed, a pop-up window appears prompting Droplet tubing clamping valve is opened. Click OK when the micro-tubing and droplet generation microfluidic chip installation is completed. After ensuring all silicone tubes from the micro-tubing and droplet generation microfluidic chip are attached to the corresponding clamp valve, click OK.
3. Instrument initialization
   1. Before performing initialization, click on the Setting interface (Figure 2B) to configure relevant parameters: detection mode (OD-based or fluorescence-based detection; OD here), incubation temperature (37 °C) and time (30 days = 720 h), speed of agitator (20 rpm), the wavelength of OD detection (600 nm), and the excitation and emission wavelength of fluorescence detection.
      NOTE: Parameters used for the isolation and cultivation of human gut microbiota in this protocol are given in parentheses. When setting the parameters, the cultivation temperature should be between 5 °C and 50 °C. When selecting the detection mode, the optical fiber must be replaced if the fiber on the droplet detection module is not the same (see step 2.3.2). When configuring parameters, the oil phase reference value (basic spectral) is automatically identified by the software, eliminating the need for manual adjustments.
   2. The replacement of the detected optical fiber
      1. Remove the detected fiber from the fiber holder (unscrew counterclockwise) and loosen the fiber-fixing screw on the droplet module (Figure 1D).
      2. If the optical fiber will not be used, remove it from the module, insert it into the fiber holder, and tighten it. Insert the detected fiber into the detection port, and tighten the fiber-fixing screw on the module. The replacement of the optical fiber is finished.
   3. Turn to the Home interface and click Initialize to let the Omics system perform a self-check of its components, including the injection pump, temperature settings, waste liquid discharge testing, screening module, and droplet detection module.
      1. During initialization, perform waste liquid discharge testing from the droplet detection module by injecting 1 mL of 75% alcohol into the waste liquid port and observing whether the liquid flows out normally.
      2. For the screening module test, place a 96-well plate on the plate placement and observe whether the plate movement is normal.

3. Droplet generation

1. Collection and processing of human gut microbiota samples
   1. Prepare a chamber pot and stool containers, wash hands, and wear gloves to collect fresh stool samples.
      NOTE: When collecting stool samples, avoid urine contamination as much as possible. It is better to urinate beforehand, and place the stool into a clean, dry container.
   2. Aseptically collect the appropriate amount of mid-segment stool and seal it in sterilized cryovials (approximately 3-5 g per vial). Place the vials on ice immediately for subsequent aliquoting and labeling.
      NOTE: If the stool sample is large or cannot be collected immediately, it should be collected within 2 h at most.
   3. In the anaerobic bench, use a sterile swab or stool sampling tool to collect a mid-segment sample.
      NOTE: The surface layer of stool contains shed intestinal mucosal cells and is prone to external contamination; after exposure to air, some microbial DNA begins to degrade.
   4. Transfer the collected stool samples into 2 mL sterile microcentrifuge tubes or sterile cryovials, with each tube containing 0.5-2.0 g of stool. Prepare two aliquots for freezing per sample.
   5. Resuspend the fresh stool sample in sterile physiological saline solution, with each 100 mg of the stool diluted in 1 mL of solution. Thoroughly mix the stool until no large particles are visible.
   6. After natural sedimentation for 10 min, sequentially filter the supernatant through sterile mesh filters with pore sizes of 200 mesh (0.075 mm), 400 mesh (0.038 mm), and 800 mesh (0.018 mm) to remove undigested food and smaller particulate matter. Finally, collect the filtrate in sterile centrifuge tubes.
   7. Take 10 µL of the filtered fecal suspension and determine the microbial concentration using a hemocytometer and an inverted fluorescence microscope.
      NOTE: After filtering the fecal sample supernatant through different mesh sizes, some smaller fecal particles remain. Therefore, when determining the microbial concentration, active particles observed under the microscope are considered as microorganisms, which only allows for an approximate calculation of the microbial concentration.
   8. Transfer the fecal suspension to a 1.5 mL sterile centrifuge tube and label it with the date, microbial concentration, and sample name. Reserve some for subsequent experiments, and store the rest at 4 °C as future use.
      NOTE: Promptly record sample information (sample name, collection time) to ensure that samples are collected at the same time (considering mammalian intestinal microbial temporal rhythm changes). The entire sample collection and processing should be carried out in an anaerobic environment.
2. Preparation for the initial fecal suspension
   1. Prepare Brain Heart Broth (BHI) medium according to the manufacturer's protocol and sterilize it by autoclaving at 121 °C for 15 min.
   2. Take the fecal suspension from step 3.1.8 and perform serial dilutions with BHI medium to achieve a concentration of ~50 cells/mL.
      NOTE: To fill the sample bottle, prepare at least 40 mL of fecal suspension.
   3. Ensuring a small magnetic stirring bar is at the bottom, pour the diluted fecal suspension into the sample bottle up to the sample adding position. Screw the cap and tighten it. Next, insert the quick connector A into quick connector B to complete the sample loading process (Figure 3A).
   4. Place the sample bottle into the designated position and separate quick connectors A and B from the sample bottle. Connect the quick connector A of the sample bottle to the O3 port on the Omics system, and the C3 connector on the micro-tubing and droplet generation microfluidic chip connect to the quick connector B. Close the door of droplet generation and cultivation chamber (Figure 3B).
3. Droplet generation
   1. Select the desired number of droplet tubings to be generated on the software's home interface (Figure 2A).
      NOTE: Each run can produce up to 10 droplet tubings, with each tube generating approximately 5,000 droplets.
   2. Click Produce in the software's home interface to start single-cell droplet generation.
      NOTE: Confirm whether the discharge of waste liquid pump is working or not. During the droplet generation, each droplet has a volume of 2.0 µL. See the discussion section for a description of droplet generation.
   3. Wait for the beeper alarm to indicate that the droplet generation is finished. Close the clamp on the C3 connector (Figure 1E) and remove the sample bottle.

4. Droplet cultivation

1. Select the same droplet tubing number as during droplet generation on the software's home interface, click Culture, confirm the cultivation time and temperature, and begin the process. Monitor the progress bar on the home interface that shows the progress of cultivation and remaining time.
2. Wait for the beeper alarm to indicate that the droplet cultivation is finished. If the cultivation time must be extended, adjust the time directly on the Setting interface.

5. Droplet screening

1. Press the UV button on the Omics system to turn on ultraviolet (UV) light (Figure 1A), irradiate the droplet detection and collection chamber for 30 min, and then turn off the UV light.
   NOTE: Before turning on the UV light, ensure the door of droplet detection and collection chamber is closed.
2. In a super-clean bench, open all 96-well plates used to collect droplets and stack them on top of each other without lids, numbering them sequentially from bottom to top. Ensure that the top well plate is covered with a lid.
   NOTE: Each run can accommodate up to ten 96-well plates, and the number of plates depends on the total number of droplets.
3. Open the door of the droplet detection and collection chamber, place the well plates in the designated positions (Figure 1D), take out the lid from the top well plate, and close the door of the chamber.
4. Turn on the UV light on the Omics system for 30 min to perform secondary sterilization.
5. Installation of the air bubble remover
   1. Remove the air bubble remover from its placement position, unscrew the cap, and remove the butterfly-shaped screw from the cap (Figure 3C).
   2. Pour 200 mL of the air bubble removal oil into the air bubble remover, screw the bottle tightly with the cap of the air bubble remover on the Omics system, and then fix the remover upside down on the air bubble remover placement. The installation of the air bubble remover is complete.
      NOTE: When fixing the air bubble remover on the placement, ensure that no oil leaks out. If any leakage occurs, tighten the lid.
6. Select the droplet tubing for sorting on the home interface, click Sorting, input the number of well-plates to be collected, and then start the process. After droplet screening begins, observe the process displays area, which shows real-time measurements of droplet optical density (OD) or fluorescence values.
7. Analyze approximately 20-30 droplets to check the OD value. For example, if the majority are found to have an OD value of ~0.2, which corresponds to the OD value of empty droplets, based on the Poisson distribution, set the lower OD threshold to 0.5, and the upper OD threshold to 4.0. The droplets within this range will be automatically collected into 96-well plates (Figure 4A).
   NOTE: According to Beer-Lambert Law, the OD value of empty droplets is determined by the culture medium composition within the droplets. Typically, the lower OD threshold is set 0.2-0.3 units higher than the OD value of empty droplets to ensure a clear distinction between empty droplets and droplets containing microorganisms.
8. Wait for the beeper alarm to indicate that the droplet screening and collection is finished. Open the door of the droplet detection and collection chamber, put the well plate lid on the top well plate, and then take all well plates out of the chamber together to conduct the subsequent sequencing and backup.

6. Data export and display of heatmaps

1. Click Export data to save the collected droplet signal data (Figure 4A,B).
2. Click Heat map, select the droplet collection data file, and observe the OD values of droplets collected in the microplate displayed by the software. Visualize these OD values as a heatmap, where color intensity corresponds to the OD distribution across the wells, providing a clear and intuitive representation of the collected monoclonal OD values (Figure 4A,C).

7. Cleaning of the MISS cell

1. After completing the experiment, select the droplet tubing that requires cleaning, and click Clean to start instrument cleaning.

8. Microbial monoclonal backup and sequencing sample preparation

1. Sequencing sample preparation
   1. In the anaerobic bench, add 100 µL of BHI medium to each well of the collected droplet plate, mix well by pipetting, and then take 10 µL from each well and transfer it all to one 15 mL sterile tube. Vortex to obtain a mixed microbial suspension.
   2. Add 5 mL of phosphate-buffered saline to the mixed microbial suspension, centrifuge at 1,000 × g for 10 min, remove the supernatant, and place the microbial pellet in liquid nitrogen for rapid freezing. Sequencing sample preparation is complete.
   3. Use 16S rDNA amplicon sequencing methods targeting the V3 and V4 domain of the 16S rDNA. The specific sequencing primers used are as follows: 341F: ACTCCTACGGGAGGCAGCA and 806R: GGACTACHVGGGTWTCTAAT.
2. Microbial monoclonal sample cryopreservation:
   1. After collecting 10 µL of the sample from each well in the droplet plates (from step 8.1.1), add 30 µL of glycerol to each well. Place the plates under -80 °C for microbial strain preservation.

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)