Microbial floc stabilization and preparation for structural analysis by correlative microscopy

In the analysis of microbial flocs from activated sludge it is important to stabilize these structures and their components for structural studies sufficiently to assess, minimize and conceptually balance artifacts, particularly during manipulation. By employing multi-technique stabilization and immediate preservation it is possible to analyze a single sample by correlative microscopy (conventional optical microscopy (COM), scanning confocal laser microscopy (SCLM), and transmission electron microscopy (TEM)). This approach minimizes variability associated with multiple sampling. Floc samples were collected using plankton chambers consisting of reservoirs with a removable circular microscope slide. Flocs which come to rest on the slide are stabilized within low melting point agarose. The solidified gel is a clear, highly porous and resilient medium amenable to further staining, washing, sub-sampling or direct microscopic analysis. Stabilization in agarose was found not to significantly influence floc size distribution. The use of agarose was found to be compatible with SCLM and TEM techniques and minimized perturbations. Agar-embedded samples were easily infused with Nanoplast, a hydrophilic melamine resin, which stabilizes material in its natural state. This facilitates the ultrastructural analysis of the three-dimensional fibrillar architecture of the floc matrix. The matrix is found to consist of complex pores bounded by fibrils of 4–6 nm diameter.