06/01/2022
Congratulations to our application scientist, Yuri Ota, on the acceptance of her abstract for the upcoming ISME18 conference between 14th-19th August in Lausanne, Switzerland. Yuri will present a poster entitled “Combination of droplet-based cultivation and single droplet sorting paves the way for isolating slow growers in soil” in the Soil Microbial Ecology session. The abstract is as follows:
Most environmental microorganisms remain uncultivated. In order to understand their characteristics, it is necessary to culture them. Although the agar plate method and the limit dilution method have been used for culturing environmental microorganisms, low throughput and complicated operation are problems. Droplet-based microfluidic system has been used for microbial cultivation in a high-throughput manner. When cultivating diverse microorganisms within droplets, their growth rates vary in each droplet, including some that do not grow. Therefore, we developed a method for sorting droplets containing growing microorganisms with high sensitivity using a fluorescent nucleic acid probe. We also established the method of dispensing single droplets to analyze the microbial composition within.
Soil microorganisms and a fluorescent nucleic acid probe were encapsulated into water-in-oil droplets. After droplet cultivation, the fluorescent droplets were sorted using a microfluidic-chip cell sorter, On-chip Sort. The microbial compositions of the sorted droplets were analyzed on basis of the 16S rRNA gene sequence. Single droplets were dispensed from a bulk droplet into 96-well plate by using a cell dispenser.
Some soil microorganisms were observed to grow with various rates in the droplets. Droplet sorting on the basis of the fluorescence intensity removed the droplets containing fast-growing microorganisms, and those containing slow-growing microorganisms were collected and re-cultivatied. Single droplets were stochastically dispensed into 96-well plates.
We successfully developed the method for sorting droplets containing growing microorganisms using a microfluidic chip cell sorter and a fluorescent nucleic acid probe, and for dispensing single droplets.