Detection of nitrifying bacteria in activated sludge by fluorescent in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for eubacteria (EUB338), ammonium- oxidizing bacteria (Nsm156) and nitrite-oxidizing bacteria (Nb1000) were us ed for the rapid detection of nitrifying bacteria in the activated sludge o f a pilot nitrifying reactor by whole-cell, fluorescent in situ hybridizati on (FISH). Emission scanning and synchronous scanning fluorescence spectrom etry were used to measure the hybridization. The binding of the probes at a temperature significantly lower than the melting temperature of the hybrid s was conventionally considered as non-specific. Total binding of the probe s at a temperature significantly higher than the melting temperature of the hybrids was conventionally considered as the sum of non-specific and speci fic binding (hybridization). Non-specific binding of the oligonucleotide pr obes with a biomass of activated sludge was 37% of the total binding of the EUB338 probe, 54% of the total binding of the Nsm156 probe, and 69% of the total binding of the Nb1000 probe. The ratio of the specific binding of th e Nsm156 and Nb1000 probes was 2.3:1. The ratio of the numbers of ammonium- oxidizing bacteria to nitrite-oxidizing bacteria, determined by microbiolog ical methods, was 2.4:1. Measuring fluorescent in situ hybridization by flu orescence spectrometry appears to be a practical tool for monitoring the mi crobial communities that contain nitrifying bacteria. However, a method tha t accounts for the non-specific binding of the probes more easily and relia bly should be developed for practical application.