Testing and Validation of a Microfluidic Device and Laser Tweezers for Use in Single-Cell Isolation for Cultivation

SMSU Event Center BC

Of the many species of bacteria and archaea that are known, few have been successfully grown in pure culture. This is due to a variety of reasons, including the inability of some microbes to grow on solid media or their slow growth rates and low abundance in natural samples. Optofluidic cell sorting, a method for viewing and separating live microbial cells via microscopy, is one technique that can be used to isolate cells for cultivation, but is not often implemented due to the level of difficulty and associated costs. Based on a previous design used for single-cell genomics, a microscope was adapted for single-cell isolation using optical trapping (laser tweezers) and a microfluidic device made from polydimethylsiloxane (PDMS). The goal of this study was to address several concerns associated with this technique: maintaining sterility, preventing cells from sticking to the inner surfaces of the microfluidic device, validating single-cell isolation, and determining cell survival rates. To validate that single, viable cells could reliably be obtained, control sorting experiments were performed using E. coli. Confirming that the technique can be reliably applied for isolation of viable microbes allows for use in future studies for isolation of uncultivated thermophiles, including members of the candidate phyla Aigarchaeota, Fervidibacteria, and Calescamantes that are currently maintained in mixed enrichment cultures in the laboratory. Obtaining pure cultures or defined co-cultures of members of these candidate phyla will allow for a better understanding of their metabolic capabilities.