Investigating Bacterial Adhesion and Detachment on Plant Surfaces Under Flow Conditions

<p>Observations of bacteria and the surfaces will be performed in a flow channel mounted to a microscopy setup. To be able to track bacteria adhesion and motion in top view and side views, two different flow channels will be created. Flow generated by a syringe pump at typical speeds of 0.1 to 50 ml/min will result in flow speeds ranging from 1 and 500 mm/s, leading to Reynolds numbers ranging between 0.5 and 250. Though E. coli bacteria are capable of swimming at speeds of about 30 ?m/s, the flow speeds in the channel will be a few orders of magnitude higher, making independent bacteria motion negligible. At 1 µm width and up to 6 µm length E. coli would have Reynolds numbers between 1 x 10-3 and 3 in the free flow at the speeds given above. For easier visualization and tracking of different cultures of E. coli, differentialy colored fluorescent proteins will be used, such as Fluorescein, FM 1-43, SYPRO Ruby, Fura Red and FM 4-64. As all of these proteins have an excitation wavelength in the same range (450 - 500 nm) but emit at a different wavelengths (517, 578, 618, 670, 751 nm, respectively), it will be possible to capture them all at the same time with the appropriate fluorescence filter set. The goal will be to distinguish different bacteria according to their different colors, thus enabling easier separation of bacteria in clusters and tracking of their motion and of attachment/detachment from distinct surface regions. We will be able to then distinguish between attachment/detachment taking place in grooves vs. that taking place in plateaus or clusters of stacked bacteria identified at the onset of the experiment. The first phase of the project will be focused on visualizing bacteria on real plant surfaces. For this purpose, leaves will be contaminated with defined amounts of fluorescent E. coli. After an incubation period to allow bacteria to settle and adhere in a natural distribution, sections of the leaves will be placed in the flow channel and observed at different flow velocities. Additionally, the leaf surfaces will then be replicated by a two-step molding process with polyvinyl siloxane as negative mold material and epoxy resin as positive mold material. This will allow for a precision higher than 4 nm and will allow for the use of well defined, identical surfaces in different experiments. At the end of the study, a complete technical report, describing methods, all results with data tables, images, statistical analysis, conclusions, and recommendations for further work will be provided to USDA in electronic format.<p>