High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates

Abstract

c This paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation of Escherichia coli and antimicrobial susceptibility testing of Pseudomonas aeruginosa PAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including four Mycobacterium isolates and a previously unknown fluoranthene-degrading Blastococcus species.

Harnessing microfluidic streak plate technique to investigate the gut microbiome of Reticulitermes chinensis

“…In this study, we continued our previous efforts to cultivate microbes from the termite gut (Chen et al., ; Fang et al., , ) by application of the newly developed MSP method (Jiang et al., ). As a microfluidic technology (Ma et al., ; Tandogan et al., ), MSP method enables high‐throughput single‐cell cultivation of diverse bacterial groups and even rare species from environmental samples (Jiang et al., ).…”

“…In this study, we continued our previous efforts to cultivate microbes from the termite gut (Chen et al., ; Fang et al., , ) by application of the newly developed MSP method (Jiang et al., ). As a microfluidic technology (Ma et al., ; Tandogan et al., ), MSP method enables high‐throughput single‐cell cultivation of diverse bacterial groups and even rare species from environmental samples (Jiang et al., ). Comparing with other cultivation tools such as extinction‐culturing‐based method (Colin et al., , ; Connon & Giovannoni, ), MSP technology has higher throughput as one culture‐plate can harbor thousands of droplets, whereas the extinction cultivation method carried only a few hundreds of wells.…”

Dynamic Sessile‐Droplet Habitats for Controllable Cultivation of Bacterial Biofilm

“…To culture biofilms in precisely confined conditions with constant hydrodynamic conditions during a long time period, we followed several criteria to design the microfluidic system: i the size and shape of biofilms should be precisely controlled during growth; ii the device should permit continuous supplement of culture media; iii the device should allow appliance of controllable and predictable hydrodynamic condition to the biofilms; iv the biofilms should harbor distinct boundary for studying the expansion or dispersal behaviors. To fulfill these criteria, we introduced the DSH method based on droplet microfluidic technologies . The device consisted of a top polydimethylsiloxane (PDMS) layer with microchannel and a bottom glass plate with arrays of hydrophilic micropatterns of different sizes ( Figure A).…”

“…Here we demonstrated a novel method for cultivation of bacterial biofilms in sessile droplets, which were named as the dynamic sessile‐droplet habitat (DSH). The method is constructed based on droplet microfluidic technique . Arrays of sessile droplets attached on the bottom surface of the microchannel were designed for seeding bacterial cells and providing space and nutrients for growth of biofilms spatially constrained to them.…”

Advances in Microfluidics Applied to Single Cell Operation

“…Meanwhile, interactions between cells also can be simulated. In recent years, microfluidic cell culture equipment has been used in tissue engineering, diagnostics, drug screening, immunology, cancer research, and stem cells proliferation and differentiation . Taylor introduced the first microfluidic system for complete mammalian cell culture.…”