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A high-throughput ultrasonic spraying inoculation method promotes colony cultivation of rare microbial species
Xizhi Huang1, Pengjie Li1, Mengfan Zhou1
1Britton Chance Centre for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics - Hubei Bioinformatics and Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Abstract:
Current method for obtaining microbial colonies still relies on traditional dilution and spreading plate (DSP) procedures, which is labor-intensive, skill-dependent, low-throughput and inevitably causing dilution-to-extinction of rare microorganisms. Herein, we proposed a novel ultrasonic spraying inoculation (USI) method that disperses microbial suspensions into millions of aerosols containing single cells, which lately be deposited freely on a gel plate to achieve high-throughput culturing of colonies. Compared with DSP, USI significantly increased both distributing uniformity and throughput of the colonies on agar plates, improving the minimal colony-forming abundance of rare Escherichia coli mixed in a lake sample from 1% to 0.01%. Applying this novel USI to a lake sample, 16 cellulose-degrading colonies were screened out among 4766 colonies on an enlarged 150-mm-diameter LB plate. Meanwhile, they could only be occasionally observed when using commonly used DSP procedures. 16S rRNA sequencing further showed that USI increased colony-forming species from 11 (by DSP) to 23, including seven completely undetectable microorganisms in DSP-reared communities. In addition to avoidance of dilution-to-extinction, operation-friendly USI efficiently inoculated microbial samples on the agar plate in a high-throughput and single-cell form, which eliminated masking or out-competition from other species in associated groups, thereby improving rare species cultivability.
Insights
A new ultrasonic spraying inoculation (USI) method enhances microbial culturing by dispersing single cells into aerosols. This high-throughput technique overcomes limitations of traditional methods, significantly improving the detection of rare microorganisms.
Area of Science:
- Microbiology
- Biotechnology
- Environmental Science
Background:
- Traditional dilution and spreading plate (DSP) methods for microbial isolation are labor-intensive and limit the detection of rare species due to dilution-to-extinction.
- Current techniques face challenges in achieving high throughput and uniform distribution of microbial colonies.
Purpose of the Study:
- To introduce and evaluate a novel ultrasonic spraying inoculation (USI) method for microbial culturing.
- To compare the efficiency and throughput of USI against traditional DSP methods.
- To assess the impact of USI on the recovery and cultivability of rare microorganisms.
Main Methods:
- Dispersing microbial suspensions into single-cell aerosols using ultrasonic spraying.
- Depositing aerosols onto agar plates for high-throughput colony culturing.
- Comparing USI with DSP for colony distribution, uniformity, and rare species detection using lake samples.
Main Results:
- USI significantly improved colony distribution uniformity and throughput compared to DSP.
- The minimal colony-forming abundance of rare Escherichia coli was improved from 1% to 0.01% using USI.
- USI increased detected colony-forming species from 11 (DSP) to 23, including seven previously undetectable microorganisms.
Conclusions:
- USI offers an operation-friendly, high-throughput alternative to traditional methods, avoiding dilution-to-extinction.
- The single-cell inoculation format of USI enhances rare species cultivability by reducing competition.
- USI represents a significant advancement in microbial isolation and screening techniques.
