(751d) High Throughput in Situ Cultivation and Isolation of Unculturable Bacteria Using Microfluidic Devices

Microorganisms can be found everywhere, from the air we breathe, to the food we eat. It is widely known that the majority of microorganisms cannot grow in artificial media that is prepared in laboratories. This problem was first realized over a century ago with the phenomenon known as “great plate count anomaly” when the direct bacterial counts from environmental samples did not correlate with the number of resulting colony forming units (CFUs). Approaches, such as rRNA sequencing, have revealed that there is an immense number of uncultivated species in nature.

Over the past several years, microfluidic devices have been used for self-sorting and isolation of bacterial species. Microfluidic devices have had success, but they use complex instrumentation and need to remove the sample from the original environment for processing, which potentially introduces sample bias and loss of diversity. Other approaches use a polymer microfluidic device with sub-micrometer constrictions to isolate single bacterial species from heterogeneous mixtures, however there is a limitation on the number of isolation chambers that fit on a given device, which leads to the use of a significant number of devices to isolate a sufficient number of bacteria to be able to compare results with conventional culture techniques.

In this project, we use new materials and devices geometries to improve on the existing state-of-the- art. Using a SOI (silicon on insulator) wafer, it is possible to increase the density of holes on a device and also further remove sample collection bias.

The new design will have 24 sub-micrometer constrictions in the SOI wafer, and it will be connected to a polycarbonate plastic part (isolation chamber) where the bacteria will be cultured after isolation.

Once the bacteria are trapped in the constriction, it will reach the isolation chamber by cell division. This way, only one species can enter one isolation chamber. This method is very important and practical because it is an autonomous process and can be easily deployed for a variety of applications. It can be used in isolating and obtaining pure cultures from environmental samples and in water purification processes.