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An Aufwuchs Chamber Slide for High-Resolution Confocal Laser Scanning Microscopy and Stereo Imaging of Microbial Communities in Natural Biofilms

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Abstract

Aufwuchs chamber slides were constructed by attaching a silicone rubber gasket to a glass slide with epoxy cement. For biofilm growth, the slides were suspended in Cayuga Lake near Ithaca, NY, for 27 days. Biofilms in the chamber were stained with 0.05% acridine orange. After rinsing, the chamber was filled with molten 1% agarose to stabilize filaments and delicate polymer structures at the biofilm surface. Areas of biofilm ~0.5 mm thick on the inner face of the wall of the chamber were selected for side-on optical sectioning in a confocal laser scanning microscope (CLSM). Stacks of high-resolution optical images captured by the CLSM z-sectioning software, were used to create left-right stereo image pairs. At low magnification the stereo pairs showed 3-D details of the microbial landscape in the mature biofilms. Channels, pores, and other structural features of the biofilm matrix were observed in peripheral regions. Higher magnification images revealed the 3-D distribution of specific biofilm components such as filaments of sheathed bacteria projecting outward into the liquid milieu, and organic coatings, including bacterial cells on the surfaces of mineral particles.

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Acknowledgements

NSF Grant BES-97067715 supported this work. We thank Yarrow Nelson for help with sampling and general assistance with other aspects of this project. This paper is dedicated to Peter Hirsch in celebration of his 75th birthday. It is published in loving memory of Margaret Anne Ghiorse, who died during its preparation, and who shared our admiration of Peter’s unusual ability to, in the words of poet William Blake, “see a world in a grain of sand and a heaven in a wild flower.”

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  1. B. S. Eaglesham

    Present address: Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA

Authors and Affiliations

  1. Department of Microbiology, Cornell University, Ithaca, NY 14853, USA

    B. S. Eaglesham & W. C. Ghiorse

  2. School of Civil and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA

    L. W. Lion

Authors
  1. B. S. Eaglesham
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  2. L. W. Lion
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  3. W. C. Ghiorse
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Correspondence to W. C. Ghiorse.

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Eaglesham, B., Lion, L. & Ghiorse, W. An Aufwuchs Chamber Slide for High-Resolution Confocal Laser Scanning Microscopy and Stereo Imaging of Microbial Communities in Natural Biofilms . Microb Ecol 47, 266–270 (2004). https://doi.org/10.1007/s00248-003-1042-7

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  • DOI: https://doi.org/10.1007/s00248-003-1042-7

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