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

Abstract

Summary 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.

Microfluidics for multiscale studies of biomolecular condensates

“…Lastly, more microfluidic setups developed for studying cells could be used to study condensates as well. Different environments can be created with high-throughput and precision using surface-wettability-guided assembly [68,69] and microfluidic spraying [70].…”

“…Different environments can be created with high-throughput and precision using surface-wettability-guided assembly 68,69 and microfluidic spraying. 70 3 The kinetics of biomolecular condensates Cells and their condensates operate out-of-equilibrium. 71 Phase diagrams, which display the thermodynamic behaviour, therefore only provide part of the picture.…”

“…Different environments can be created with high-throughput and precision using surface-wettability-guided assembly 68,69 and microfluidic spraying. 70…”

Reaching unreachables: Obstacles and successes of microbial cultivation and their reasons

“…One such factor is acoustic vibration, which is useful as a cultivation enhancement in several biotechnological studies ( Bochu et al, 2003 ; Avhad and Rathod, 2015 ; Huang et al, 2017 ). By causing (i) cavitation and repairable damage in microbial cells, (ii) loosening of microbial aggregates in liquid cultures, and (iii) an increase in cell membrane permeability, ultrasonic low-intensity waves (∼20 kHz) can increase the substrate intake in microbial cells and subsequently enhance microbial proliferation ( Huang et al, 2017 , 2021 ), and thus can help in the cultivation of the unreachables.…”

“…Cultures can be sprayed onto medium plates instead of being spread with a hockey stick. This procedure effectively compartmentalizes microorganisms in droplets, hence the aggregation of cells and interspecies competition, once they land on the medium, is significantly reduced ( Huang et al, 2021 ). Gao and co-workers developed a microbe observation and cultivation array (MOCA) that allows the recovery of microbes on a small scale and does not require any complex equipment ( Gao et al, 2013 ).…”