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Applied and Environmental Microbiology, January 2008, p. 410-415, Vol. 74, No. 2
0099-2240/08/$08.00+0     doi:10.1128/AEM.01812-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Novel Combination of Atomic Force Microscopy and Epifluorescence Microscopy for Visualization of Leaching Bacteria on Pyrite{triangledown}

Stefanie Mangold,1,2 Kerstin Harneit,1 Thore Rohwerder,1 Günter Claus,2 and Wolfgang Sand1*

University of Duisburg-Essen, Biofilm Centre, Aquatic Biotechnology, Geibelstr. 41, 47057 Duisburg, Germany,1 Mannheim University of Applied Sciences, Institute of Technical Microbiology, Windeckstrasse 110, 68163 Mannheim, Germany2

Received 3 August 2007/ Accepted 24 October 2007

Bioleaching of metal sulfides is an interfacial process comprising the interactions of attached bacterial cells and bacterial extracellular polymeric substances with the surface of a mineral sulfide. Such processes and the associated biofilms can be investigated at high spatial resolution using atomic force microscopy (AFM). Therefore, we visualized biofilms of the meso-acidophilic leaching bacterium Acidithiobacillus ferrooxidans strain A2 on the metal sulfide pyrite with a newly developed combination of AFM with epifluorescence microscopy (EFM). This novel system allowed the imaging of the same sample location with both instruments. The pyrite sample, as fixed on a shuttle stage, was transferred between AFM and EFM devices. By staining the bacterial DNA with a specific fluorescence dye, bacterial cells were labeled and could easily be distinguished from other topographic features occurring in the AFM image. AFM scanning in liquid caused deformation and detachment of cells, but scanning in air had no effect on cell integrity. In summary, we successfully demonstrate that the new microscopic system was applicable for visualizing bioleaching samples. Moreover, the combination of AFM and EFM in general seems to be a powerful tool for investigations of biofilms on opaque materials and will help to advance our knowledge of biological interfacial processes. In principle, the shuttle stage can be transferred to additional instruments, and combinations of AFM and EFM with other surface-analyzing devices can be proposed.

* Corresponding author. Mailing address: University of Duisburg-Essen, Biofilm Centre, Aquatic Biotechnology, Geibelstr. 41, 47057 Duisburg, Germany. Phone: 49 203 379 4475. Fax: 49 203 379 4495. E-mail: wolfgang.sand{at}uni-due.de

{triangledown} Published ahead of print on 26 November 2007.

Applied and Environmental Microbiology, January 2008, p. 410-415, Vol. 74, No. 2
0099-2240/08/$08.00+0     doi:10.1128/AEM.01812-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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