Cultivation-independent, semiautomatic determination of absolute bacterialcell numbers in environmental samples by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotid e probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria c an quickly be detected by FISH, a reliable method to determine absolute num bers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environme ntal samples by a combination of FISH, confocal laser scanning microscopy,, and digital image analysis. The quantification is based on an internal sta ndard, which is introduced by spiking the samples with known amounts of Esc herichia coli cells. This method was initially tested with artificial mixtu res of bacterial cultures and subsequently used to determine the concentrat ion of ammonia-oxidizing bacteria in a municipal nitrifying activated sludg e. The total number of ammonia oxidizers was found to be 9.8 x 10(7) +/- 1. 9 x 10(7) cells ml-(1). Based on this value, the average in situ activity w as calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia ox idizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating t he utility of this quantification method for enumerating bacteria in sample s in which cells are not homogeneously distributed.