High definition method for imaging bacteria in microconfined environments on solid media

In this paper, the methods and devices used to confine, observe and characterize the growth of bacteria on an agar based nutrient media are described. Selected strain E. coli MG1655 [1, 2] was successfully inoculated in a confinement structure composed of a microchannel (7 µm × 60 µm × 6.345 mm) with two culture chambers of 3 mm in diameter on both ends. The microchannel was fabricated on a standard microscope glass slide using a mask-less photolithographic technique and chemical etching. Isolation and manipulation of the confined bacteria was achieved by means of a custom designed 3D printed test cell. Observation was performed on an optical transmission microscope enhanced with a customized automation system. Growth characterization was performed by calculating the surface area colonized by the bacteria through image processing and analysis. The discussion focuses on the comparison of the growth rate within the confinement structure compared to traditional cell counting methods and the description of an observed, but inconsistent, scouting behavior. Finally, we discuss on the possible uses of the reported work and extend a call for introducing this system in current bacteriology research.