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

This research explores microbial culture omics, using an automatic platform, to enhance microbial isolation, cultivation, and monoclonal screening. The aim is to improve the understanding of human gut, microbiome, and their applications. Technologists advancing microbiome research includes drop of microfluidics, automatic strain isolation and screening platforms, machine learning algorithms, and monoclonal dispensers for high strained culture omics and microbiome analysis.

The mid cell system significantly enhances microview, monoclonal isolation and coloration, generating nearly double number of clone compared with traditional methods, thereby advancing the research capability in gut microview culture mix. This protocol addresses the need of low-cost high-support microbial screening platform, overcoming traditional methods limitations in efficiency and score-ability for culture mix research. Our lab will further focus on the integration of additional droplet manipulation technicals, such as droplet splitting and merging to expand the scope of technological applications.

To begin, open the droplet generation and cultivation chamber door of the MISS cell. Vertically remove the protective cover for the micro-tubing and droplet generation microfluidic chip. Using a disposable syringe, add 10 milliliters of sterile distilled water to the humidifier inside chamber and reinstall the protective cover.

Open the sterile packaging of the micro-tubing and droplet generation microfluidic chip, and vertically place it directly above the cultivation chamber. Then on the software interface, click on installation. When the confirmation popup window appears, click yes to start the installation.

Take out the air bubble remover and fix it upside down on the air bubble remover placement in the chamber. Attach the droplet outlet tube of the air bubble remover to the clamping valve below it. Guide it through the whole leading to the droplet detection and collection chamber.

After opening the door of the chamber, vertically insert the detection tube into the detection socket. Ensure the detection tube is fully inserted. Tighten the screw securing the detection tube in a clockwise direction.

After confirming full insertion and securing, close the door of the chamber. Connect each of the 10 silicone tubes from the microfluidic chip, labeled L1 through L10, to their corresponding numbered clamp valves. Next, connect the quick connector from the microfluidic chip to the corresponding port on the omic system, following proper connections.

Once the chip installation is complete, a pop-up window appears with the message, droplet tubing clamping valve is opened. Click OK when the micro tubing and droplet generation chip installation is completed. After ensuring all tubes are correctly attached to their corresponding clamp valves, click okay.

Before performing initialization, click on the setting interface to configure the relevant parameters, like selective mode, incubation temperature, incubation time, agitator speed, OD detection wavelength, and excitation and emission wavelength for fluorescence detection. Navigate to the home interface and click initialize. The confirmation pop-up window appears click yes to allow the IC system to perform a self-check of its components, including the injection pump, temperature settings, waste liquid discharge testing, screening module, and droplet detection module.

After the initialization is completed, click okay from the pop-up window. To prepare the sample, take the microbial sample and perform serial dilution with the corresponding medium to achieve a concentration of around 50 cells per milliliter to obtain a 40 milliliter sample suspension. Then unscrew the cap of the sample bottle and pour the diluted fecal suspension into the bottle with a magnetic stir bar, up to the sample adding position.

Screw the cap and tighten it securely. Then insert quick connector A into quick connector B to complete the sample loading process. After placing the sample bottle into the designated position, separate quick connectors A and B from the sample bottle.

Connect quick connector A of the sample bottle to the O3 port on the omic system. Connect the C3 connector on the microfluidic chip to quick connector B, and close the door of the chamber. On the software's home interface, select the desired number of droplet tubings to be generated and click produce to start single cell droplet generation.

Wait for the popup window indicating the completion of the droplet creation, and click okay. Close the clamp on the three connector and remove the sample bottle. On the software's home interface, select the same droplet tubing number as used during droplet generation.

Click culture. Confirm the cultivation time and temperature, and begin the process. Wait for the pop-up window, indicating the completion of droplet cultivation.

Press the UV button on the omic system to turn on ultraviolet UV light. Irradiate the droplet detection and collection chamber for 30 minutes before turning it off. In a super clean bench, open all 96 well plates used for droplet collection, 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. Open the door of the droplet detection and collection chamber, and place the well plates in the designated positions. Remove the lid from the top well plate, then close the chamber door.

Turn on the UV light on the omic system again for 30 minutes to perform secondary sterilization. Next, remove the air bubble remover from its placement position. Unscrew the cap and remove the butterfly shaped screw from the cap.

Pour 200 milliliters of air bubble removal oil into the air bubble remover. Secure the cap tightly, then fix the air bubble remover upside-down in its designated placement. On the home interface, select the droplet tubing for sorting.

Click sorting. Input the number of well plates to be collected and start the sorting process. Observe the process display area for real-time measurements of droplet optical density or fluorescence values.

Analyze approximately 20 to 30 droplets to check the OD value. If most droplets have an OD of about 0.2, set the lower OD threshold to 0.5 and the upper OD threshold to four. The system will automatically collect droplets within this range into the 96 well plates.

Wait for the pop-up window indicating the completion of droplet sorting and collection, and then click okay. Open the door of the droplet detection and collection chamber. Place the well plate lid on the top well plate, and remove the plates together.

Click export data to save the collected droplet signal data. Click heat map. Select the droplet collection data file and observe the OD displayed in the microplate format.

Visualize these OD values as a heat map where color intensity corresponds to the OD distribution across the wells, providing a clear and intuitive representation. The species diversity of human gut microbiota obtained from the omic system, the solid plate method, and the initial stool sample were compared at the family level. 34 microbial families were enriched by both the solid plate method and the demonstrated omics method, with the omic system additionally enriching four families.

The omic system also effectively enriched some of the low abundance families. At the genus level, both methods enriched 74 microbial genera, while the MISS cell method additionally enriched 13 genera. Furthermore, two genera initially present at low abundance were effectively enriched, using the OMIC system.

These results showed that the MISS cell culture method provided better growth conditions for those strains that were present in low proportions or had poor growth performance in the original microbial suspension.