Rapid quantification of bacteria in activated sludge using fluorescence in situ hybridization and epifluorescence microscopy

A rapid quantification method for bacteria in activated sludge has been developed, based on fluorescence in situ hybridization (FISH) and epifluorescence microscopy. Samples are hybridized on slides and analyzed by direct microscopic observation. Abundance categories were designed based on digital images of the target organisms. These rating systems were developed for the filamentous bacteria Microthrix parvicella and for different morphotypes of nocardioform actinomycetes, but can easily be adapted to other types of microorganisms. Due to the quantification by direct microscopic observation, this method is suitable for samples that are difficult to be processed by semi-automated image analysis techniques, such as samples containing fluorescent debris, cells of different fluorescence intensities and target organisms that need partial enzymatic digestion prior to FISH. In contrast to commonly used rating systems consisting of photographs, the newly developed categories allow to quantitatively compare results of different categories and different organisms. The uncertainties of the results were calculated by a non-parametric bootstrap procedure; a thorough uncertainty analysis was performed including sample variability and operator subjectivity.