TY - JOUR

T1 - A high-throughput ultrasonic spraying inoculation method promotes colony cultivation of rare microbial species

AU - Huang, Xizhi

AU - Li, Pengjie

AU - Zhou, Mengfan

AU - Li, Yiwei

AU - Ou, Xiaowen

AU - Chen, Peng

AU - Guggenberger, Georg

AU - Liu, Bi Feng

N1 - Funding Information: We gratefully thank Professor Foster Kevin of Oxford University for his constructive comments. We gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant number 41807051, 21775049, 31700746), China Postdoctoral Fund (Grant number 2019M662635) and Hubei Provincial Natural Science Foundation of China (Grant number 2020CFB578).

PY - 2021/2/24

Y1 - 2021/2/24

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85099234375&partnerID=8YFLogxK

U2 - 10.1111/1462-2920.15386

DO - 10.1111/1462-2920.15386

M3 - Article

C2 - 33400374

AN - SCOPUS:85099234375

VL - 23

SP - 1275

EP - 1285

JO - Environmental microbiology

JF - Environmental microbiology

SN - 1462-2912

IS - 2

ER -