(398bg) In situ Isolation of Bacteria Using Microfluidic Devices

The microbial world is mostly unexplored, much less understood. Identifying and studying new bacterial species can yield new information about diseases, result in the discovery of new drugs, and reveal the missing pieces of the microbial world. New species are difficult to study however because most organisms do not grow in standard laboratory growth media. To address the current limitations to isolating and cultivating bacteria, a nanofluidic device was developed to isolate bacterial species based on their size in complex samples.

A polydimethylsiloxane (PDMS) polymer microfluidic device containing seven sub-micrometer constrictions with new non-parallel wall geometries was developed. The constrictions were smaller than the diameter of a bacterial cell in one or two dimensions. Two devices were placed in river water samples collected from the Charles River in the summer. When bacterial cells entered the main entrance of the device, fresh food in the isolation chambers created a gradient, which chemotactically attracted microorganisms toward the constrictions. The microorganism was trapped in the non-linear constrictions as it tried to reach the chamber. The cells divided, and each progeny advanced further through the constriction. After several divisions cells from a single species should enter the isolation chamber through the constriction.

The two devices isolated 11 different bacterial species using a total of only 14 constrictions and chambers: Brevundimonas nasdae, Brevundimonas staleyi, Devosia psychrophila, Microbacterium maritypicum, Pseudomonas extremaustralis, Pseudomonas lurida, Pseudomonas marginalis, Pseudomonas rhodesiae, Pseudomonas trivialis, Pseudomonas veronii and Sphingobium xenophagum. In contrast, 22 different bacterial species were obtained using conventional plate culture techniques, but required multiple dilutions to isolate.

Of the 11 species isolated using the microfluidic device, only one species overlapped with the bacteria collected with the conventional technique. The microorganisms collected with the devices are generally considered not to be abundant species in river water. With increasing numbers of isolation chambers tested, the species richness is expected to increase as well.

This study validates the use of a microfluidic device to capture and isolate bacterial cells not obtained through plate culture techniques, giving us further bacterial diversity to study. Future work includes looking at different sample sources, such as bacteria isolated from soil and swamps.