AWWA WQTC60567

The ability to rapidly detect and enumerate ammonia-oxidizing bacteria (AOB) would provide an additional monitoring tool for the nitrification control strategies of chloraminating utilities. Molecular-based assays were developed and applied to samples from several utilities to evaluate the feasibility and diagnostic significance of this approach. Two methods were explored, real-time polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH). The real-time PCR protocol targeted the amoA gene, separately quantifying Nitrosomonas species, Nitrosomonas oligotropha, and Nitrosospira down to detection limits of approximately 80 gene copy numbers. There was no evidence of matrix interference in analyses of samples spiked with a known number of AOB. Weekly monitoring results show the ability to complement routine chemical and physical water quality monitoring with PCR-based AOB enumeration. With the FISH approach, difficulties were encountered in differentiating AOB from non-target cells due to the dim probe-conferred fluorescence of the AOB. The protocol was modified to enhance the fluorescent intensity of AOB using multiple probes and tyramide signal amplification (TSA). Both strategies yielded improved fluorescent intensities, with the TSA FISH modification increasing AOB intensity by approximately four times. Includes 23 references, table, figures.