This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hunter, R. C.
Right arrow Articles by Beveridge, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hunter, R. C.
Right arrow Articles by Beveridge, T. J.
Agricola
Right arrow Articles by Hunter, R. C.
Right arrow Articles by Beveridge, T. J.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, May 2005, p. 2501-2510, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2501-2510.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Application of a pH-Sensitive Fluoroprobe (C-SNARF-4) for pH Microenvironment Analysis in Pseudomonas aeruginosa Biofilms

Ryan C. Hunter* and Terry J. Beveridge

Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Received 24 September 2004/ Accepted 26 November 2004

An important feature of microbial biofilms is the development of four-dimensional physical and chemical gradients in space and time. There is need for novel approaches to probe these so-called microenvironments to determine their effect on biofilm-specific processes. In this study, we describe the use of seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms. C-SNARF-4 is a fluorescent ratiometric probe that allows pH quantification independent of probe concentration and/or laser intensity. By confocal scanning laser microscopy, C-SNARF-4 revealed pH heterogeneity throughout the biofilm in both the x,y and x,z planes, with values ranging from pH 5.6 (within the biofilm) to pH 7.0 (bulk fluid). pH values were typically remarkably different than those just a few micrometers away. Although this probe has been successfully used in a number of eukaryotic systems, problems have been reported which describe spectral emission changes as a result of macromolecular interactions with the fluorophore. To assess how the biofilm environment may influence fluorescent properties of the dye, fluorescence of C-SNARF-4 was quantified via spectrofluorometry while the probe was suspended in various concentrations of representative biofilm matrix components (i.e., proteins, polysaccharides, and bacterial cells) and growth medium. Surprisingly, our data demonstrate that few changes in emission spectra occur as a result of matrix interactions below pH 7. These studies suggest that C-SNARF-4 can be used as a reliable indicator of pH microenvironments, which may help elucidate their influence on the medical and geobiological roles of natural biofilms.

* Corresponding author. Mailing address: Department of Microbiology, CBS, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Phone: (519) 824-4120, ext. 58904. Fax: (519) 837-8702. E-mail: rhunte01{at}uoguelph.ca.

Applied and Environmental Microbiology, May 2005, p. 2501-2510, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2501-2510.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Venn, A. A., Tambutte, E., Lotto, S., Zoccola, D., Allemand, D., Tambutte, S. (2009). From the Cover: Imaging intracellular pH in a reef coral and symbiotic anemone. Proc. Natl. Acad. Sci. USA 106: 16574-16579 [Abstract] [Full Text]  
  • Wu, J., Xu, H., Tang, W., Kopelman, R., Philbert, M. A., Xi, C. (2009). Eradication of Bacteria in Suspension and Biofilms Using Methylene Blue-Loaded Dynamic Nanoplatforms. Antimicrob. Agents Chemother. 53: 3042-3048 [Abstract] [Full Text]  
  • Schooling, S. R., Hubley, A., Beveridge, T. J. (2009). Interactions of DNA with Biofilm-Derived Membrane Vesicles. J. Bacteriol. 191: 4097-4102 [Abstract] [Full Text]  
  • Palmer, R. J. Jr., Stoodley, P. (2007). Biofilms 2007: Broadened Horizons and New Emphases. J. Bacteriol. 189: 7948-7960 [Full Text]  
  • Sandoz, K. M., Mitzimberg, S. M., Schuster, M. (2007). From the Cover: Social cheating in Pseudomonas aeruginosa quorum sensing. Proc. Natl. Acad. Sci. USA 104: 15876-15881 [Abstract] [Full Text]  
  • Villain-Guillot, P., Gualtieri, M., Bastide, L., Leonetti, J.-P. (2007). In Vitro Activities of Different Inhibitors of Bacterial Transcription against Staphylococcus epidermidis Biofilm. Antimicrob. Agents Chemother. 51: 3117-3121 [Abstract] [Full Text]  
  • Webster, P., Wu, S., Gomez, G., Apicella, M., Plaut, A. G., St. Geme, J. W. III (2006). Distribution of Bacterial Proteins in Biofilms Formed by Non-typeable Haemophilus influenzae. J. Histochem. Cytochem. 54: 829-842 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»