1、 Background introduction

The gut microbiota is vast in quantity and complex in composition. The composition and physiological changes of the human gut microbiota are related to various diseases. By studying the gut microbiota, we can better understand its relationship with human health. Currently, the isolation and cultivation of gut microbiota are crucial for this research.

The traditional method of isolating and cultivating gut microbiota is mainly based on the plate culture mode. Due to the large workload, the plate culture mode has the problems of time-consuming, labor-intensive, and inefficient cultivation, and often has poor cultivation results.Droplet microfluidics technologyThe application reports in the isolation and cultivation of gut microbiota are increasing day by day. The method involves monodisperse gut microbiota into individual independent droplets, providing a closed and independent liquid confinement space for the growth of individual strains, greatly promoting microbial growth, avoiding weak strains being covered by dominant strains, and thereby increasing the variety and quantity of cultivable microorganisms.

This experiment uses the Tianmu Bioscience MISS cell instrument to automatically create tens of thousands of independent droplet culture units, dispersing microorganisms into each droplet for independent cultivation. Different types of bacterial individuals can fully grow and proliferate in the droplets, while eliminating the effects of overgrowth and rapid population growth in community culture.The cultivation method based on MISS cell equipment greatly overcomes the shortcomings of traditional plate cultivation methods, providing an important research platform for the identification and discovery of rare groups of intestinal symbiotic bacteria and anaerobic environment microbial communities.

2、 Experimental process of intestinal microbiota isolation and cultivation based on tablet and MISS cell devices

1. Sample pretreatment to determine the concentration on the machine

After collecting fecal samples and diluting them, use a hemocytometer to count and dilute the sample concentration to 10000-30000 CFU/mL with culture medium. The workflow is as follows:

2. Isolation and cultivation of gut microbiota in anaerobic environment using MISS cell equipment

About 20000 droplets were generated in this experiment. After anaerobic cultivation at 37 ℃ for 30 days, detection and sorting were carried out. After 5 hours of sorting, a total of about 4600 bacterial droplets were obtained and subjected to high-throughput sequencing. The workflow is shown in the following figure:

3. Isolation and cultivation of gut microbiota using agar plates

A total of 15 plates were coated in this experiment, collected after anaerobic cultivation at 37 ℃ for 30 days, and subjected to high-throughput sequencing. The workflow is shown in the following figure:

4. Experimental results

(1) Compared with the uncultured fecal solution group, the use of MISS cell equipment for isolation and cultivation resulted in the identification of 10.9% (13/119) more bacterial species (genus level), significantly better than the 0.8% (1/119) on the plate.

(2) Droplet culture increases species richness by 40% (genus level) compared to plate culture, and has good enrichment effects on genera such as Bifidobacterium, Peptoniphilus, Collinsella, Phascolarctobacteria, Enterococcus, Porphyromonas, etc.

3、 Tianmu Bioscience MISS Cell Instrument provides high-throughput and efficient automated solutions for the isolation, cultivation, and screening of strict anaerobic bacteria such as gut microbiota

In this study, the Tianmu Bioscience MISS cell instrument was used to generate 20000 droplets, and approximately 4600 monoclonal droplets were sorted, achieving automated cultivation and sorting of gut microbiota. At the same time, based on the advantages of droplet culture, competitive growth and cross contamination between strains were avoided, significantly improving the richness of enriched strains after cultivation.

MISS cell has achieved high-throughput and automated operations throughout the entire process of gut microbiota experiments, significantly improving microbial cultivation capabilities, significantly reducing the workload of experimental personnel, and improving work efficiency. It provides an efficient automated experimental platform for monoclonal cultivation and high-throughput screening, including strict anaerobic conditions such as gut microbiota, and provides strong support for strain cultivation and screening!