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

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.