A rapid method to quantify nitrifiers in activated sludge

Quantification of bacteria using Fluorescence In Situ Hybridization (FISH), confocal laser scanning microscopy (CLSM) and image analysis is very time consuming and requires the availability of an expensive microscope. Therefore, a rapid method to quantify nitrifying bacteria in activated sludge using FISH and epifluorescence microscopy was developed. The quantification of the biovolume is based on manual counting of the aggregates formed by nitrifying bacteria and determination of their size. The overall uncertainty of the method was evaluated as a function of the number of analyzed microscopic fields. It was found that 10-15 microscopic fields for ammonia-oxidizing bacteria and 6-8 microscopic fields for nitrite-oxidizing bacteria per sample were optimal regarding effort and accuracy. Accordingly, the time needed for one sample was only 5-15 min, compared to about 1 h for the quantification with CLSM and image analysis. As a consequence, this method also allows for the measurement of extended time series with a reasonable effort. The comparison of the determined biovolume and the measured activity showed an explicit correlation.