Microcontact Printing
of Health ImageJ soſt ware (
http://imagej.nih.gov/ij/ ). Images were converted into 8-bit grayscale format and submitted to a thresholding routine in order to separate defi nite cell forms from the image background ( Figure 3 ).
Results Biofi lm studies . Processed data revealed that X. fastidiosa
biofi lm had more bacterial cells in thiol-printed areas compared to non-printed areas, an indication that hydrophobicity is an important factor for X. fastidiosa biofi lm formation ( Figure 4 ). T e colonized area % corresponded to the fraction of the total area with bacterial cell forms. T e experiment was repeated with similar results. Eff ect of added calcium . We also observed that supple- mentation of the divalent calcium (Ca 2+ ) resulted in even more biofi lm and more cells within the biofi lm architecture. T us, in media supplemented with calcium, the biofi lm exhibited more cells per area, and, comparatively, less debris accumulation ( Figures 4 and 5 ).
Figure 2 : Microcontact thiol printing (MCT). (a) Hydrophobicity results from forming a monolayer of octadecanethiol molecules, with thiol moieties from the octadecanethiol bound to gold or silver and the tail of the molecules extending from the surface. (b) and (c) together indicate differential biofi lm growth for the letters of US penny print when compared to the background. The fi rst three letters of “United” are shown in the SEM image (c) obtained using an off-center backscattered electron detector.
Effect of calcium chelator . Media with calcium chelator EGTA exhibited a reduced number of cells within the biofilm; however, the presence of EGTA was not enough to prevent some biofilm formation ( Figure 4 ). The influence of calcium on X. fastidiosa biofilm formation was confirmed and is a result of calcium bridging; the phenomenon was demonstrated in nutritional media aggregation and biofilm studies [ 9 ].
Discussion T e present work evaluates for the fi rst time the importance
Figure 3 : MCT was used to print a custom-designed microfl uidic channel pattern on a gold substrate as experimentation evolved because gold coating is easier to obtain. (a) Secondary electron image showing bacterial growth of X. fastidiosa cells at the interface between thiol-coated (bottom) and gold only coated (top). SEM image width = 60 µm. (b) Light optical microscope image of the custom- designed pattern used. (c) Threshold image generated by the software Image J (NIH). In the process, bacterial cells were colorized (red) to distinguish them from the background. The % area of bacterial growth occupied was calculated and plotted in Figure 4 . The area in this case corresponds to a non-thiol printed (gold only) surface (similar to top area on (a)).
SEM analysis . T e treatments were later analyzed by scanning electron microscopy (SEM) at the JEOL USA, Inc. facilities in Peabody, MA. Samples were imaged in the JSM-6610 SEM. A low accelerating voltage (between 1 and 3 keV) was used to avoid charging because the treatments to be examined did not receive a conductive surface coating. Most of the images were obtained in a way to highlight diff er- ences between regions outside and inside the thiol-printed areas. Biofi lm formation and/or cell colonization were assessed by processing the data with the National Institutes
2015 September •
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of calcium in a X. fastidiosa biofi lm using an artifi cially modifi ed surface. X. fastidiosa biofi lm seems to be aff ected by: (a) the xylem vessel’s internal surface chemistry, (b) the chemical composition of the xylem fl uid ( Figure 4), (c) the presence of morphological structures, and (d) other unknown factors (not evaluated in this work). In a recent study at Auburn University, in vitro studies [ 6 ] demonstrated that X. fastidiosa mutants responded positively to the presence of calcium in the medium and induced more biofi lm formation; the authors concluded that strong cell-to-cell attachment and biofi lm formation are directly related to calcium concentrations. In another recent investigation, it was verifi ed that a number of ions accumulate in developed biofi lms of X. fastidiosa , including a two- fold increase in calcium [ 10 ]. Our results strongly support a multi-layered stepwise progression toward biofi lm formation: (1) free cells, (2) cell aggregation, and (3) adhesion to surfaces mediated by free calcium. Moving forward, calcium accumu- lation seems to play a key role in vessel plugging because calcium ions can form insoluble minerals (for example, calcium phosphates and calcium oxalates) resulting in biofi lm hardening. T is intersection of surface science and biological techniques provides a unique platform for examining biofi lm formation from a surface chemistry perspective. Functionalization of the substrate surface chemistry allows us to probe the cell-surface interactions that aff ect biofi lm formation. Biofi lm development appears to begin with the random attachment of cells to the surface, followed by
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