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 approximately 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.