Development of a fluorescence in situ hybridization protocol for the identification of micro-organisms associated...

Fluorescence in situ hybridization (FISH) provides a unique tool to study micro-organisms associated with particles and flocs. FISH enables visual examination of micro-organisms while they are structurally intact and associated with particles. However, application of FISH to wastewater and sludge samples presents a specific set of problems. Wastewater samples generate high background fluorescence due to their organic and inorganic content making it difficult to differentiate a probe-conferred signal from naturally fluorescing particles with reasonable certainty. Furthermore, some of the FISH steps involve harsh treatment of samples, and are likely to disrupt the floc structure. This study developed a FISH protocol for studying micro-organisms that are associated with particles and flocs. The results indicate that choice of a proper fluorochrome and labeling technique is a key step in reducing the background fluorescence and non-specific binding, and increasing the intensity of the probe signal. Compared to other fluorochromes tested, CY3 worked very well and enabled the observation of particles and debris in red and probe signal from microbes in yellow. Fixation, hybridization, and washing steps disturbed the floc structure and particle-microbe association. Modifications to these steps were necessary, and were achieved by replacing centrifugation with filtration and employment of nylon filters. Microscope slides generated excellent quality images, but polycarbonate membrane filters performed better in preserving the floc structure.