This Article Full Text Full Text (PDF) Other Versions of this Article: AEM.02884-06v1 73/8/2653    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Müller, R. Articles by Ruhl, S. Search for Related Content PubMed PubMed Citation Articles by Müller, R. Articles by Ruhl, S. Agricola Articles by Müller, R. Articles by Ruhl, S.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, April 2007, p. 2653-2660, Vol. 73, No. 8
0099-2240/07/$08.00+0     doi:10.1128/AEM.02884-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Fluorescence-Based Bacterial Overlay Method for Simultaneous In Situ Quantification of Surface-Attached Bacteria

Institute of Physical and Theoretical Chemistry, University of Regensburg, Regensburg, Germany,¹ Department of Prosthodontics,² Department of Operative Dentistry and Periodontology, Dental School, University of Regensburg, Regensburg, Germany³

Received 13 December 2006/ Accepted 12 February 2007

For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, Streptococcus mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications.

Published ahead of print on 16 February 2007.