Microcontact Printing
one may end up selecting cells plus debris or not accounting for cells located just below the layer exposed. Published data [ 10 ] dealing with biofi lm growth indicate that PD2 medium amended with calcium caused a 2-fold growth increase; whereas, we are reporting here only a 1.73-fold growth increase using the same medium and treatments, which could be an indication that we may be missing some information by having a two-dimension SEM image that does not factor in the possibility of layers. T is diff erence might be statistically meaningless considering that all treatments in this proposed method carry the same error.
Conclusion
Figure 4 : The % area of bacterial growth occupied by Xylella fastidiosa measured from SEM images of isolated cells and cells within biofi lm. Area measurements are straightforward and can quickly represent how weak or vigorous was the colonization on each of the modifi ed surfaces, that is, naked gold (blue bar) or thiol-printed areas (red bar). The PD2 medium without additives was the control. Bacterial growth was greater when CaCl 2 was added to the medium. Growth was negatively affected by the addition of EGTA, a calcium chelator.
MCT provided a tunable platform for assessing cell-surface attachment. We have: (a) confirmed that X. fastidiosa cells attach preferentially to surfaces with higher degrees of hydrophobicity; (b) shown that calcium (CaCl 2 ) significantly enhances the tendency of these cells to attach and aggregate; and (c) shown that EGTA reduces attachment and consequently compromises the rest of the colonization process. These results are encouraging and validate previous results. Importantly, the combination of the MCT method and SEM required less time for data acquisition than conventional methods. With appropriate standardization this method may develop as a routine screening step in studying cell surface chemistry and intercellular attachment during the colonization process of many pathogens. In future work we foresee an increase in data generation capabilities allowing: (a) the establishment of a common basis for attachment involving plant and human pathogens and (b) greater understanding of the surface interaction between cells and their environment.
Acknowledgements
We acknowledge the support by NSF through the University of Wisconsin Materials Research Science and Engineering Center (DMR-1121288) and the grant from the USDA National Institute of Food Agriculture and Food Research Initiative (2010-65108-20633) awarded to Auburn University. We also acknowledge suggestions from Vern Robertson and Anthony Laudate, JEOL USA, Inc.
Figure 5 : Idealized diagram (above) to explain the robust growth on the thiol- print side (right side) compared with the pure gold-coated surface (left). Drawings are for illustration purposes only and are not to scale. The hydrophobic thiol layer is likely to hold a greater number of bacterial cells building a solid base because there is less repulsion between bacterial cells. This allows further development of colonies of cells, evolving quickly to a complex biolfi m (city of cells). The image is from the treatment with CaCl 2 (see Figure 4 ) that enhanced colonization. Image width = 60 µm.
subsequent cell-to-cell interactions. Later stages of biofi lm development exhibit deposition of extracellular material around the attached cells [ 9 ].
However, the advantage of performing biofi lm surface analysis also has a limitation. We think that the appropriate time to assess, count, and compare attachment of cells seems to be prior to biofi lm formation. T e reasoning is simple: while trying to count only cells during the thresholding of the image,
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References [1] CN Berger et al ., Environ Microbiol 12 ( 9 ) ( 2010 ) 2385 – 97 . [2] GN Agrios , Plant Pathology , 5th ed. , Elsevier Academic Press , New York , 2005 .
[3] AJ Simpson et al ., Nature 13 ( 406 ) ( 2000 ) 151 – 9 . [4] RM Goulter et al ., Lett Appl Microbiol 49 ( 1 ) ( 2009 ) 1 – 7 . [5] A Kumar et al ., Langmuir 10 ( 5 ) ( 1994 ) 1498 – 1511 . [6] LF Cruz et al ., Appl Environ Microbiol 78 ( 5 ) ( 2012 ) 1321 – 31 .
[7] LF Cruz et al ., Appl Environ Microbiol ( 2014 ), pii: AEM. 02153-14 (in press).
[8] JC Love et al ., Chem Rev 105 ( 4 ) ( 2005 ) 1103 – 69 . [9] B Leite et al ., FEMS 230 ( 2004 ) 283 – 90 . [10] PA Cobine et al ., PLoS ONE 8 ( 1 ) ( 2013 ), e54936 . doi:10.1371/journal.pone.0054936 .
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