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Autotrophic ammonia-oxidizing bacteria (AOB) are of vital importance to wastewater treatment plants (WWTP), as well as being an intriguing group of microorganisms in their own right. To date, corroboration of quantitative measurements of AOB by fluorescence in situ hybridization (FISH) has relied on assessment of the ammonia oxidation rate per cell, relative to published values for cultured AOB. Validation of cell counts on the basis of substrate transformation rates is problematic, however, because published cell-specific ammonia oxidation rates vary by over two orders of magnitude. We present a method that uses FISH in conjunction with confocal scanning laser microscopy to quantify AOB in WWTP, where AOB are typically observed as microcolonies. The method is comparatively simple, requiring neither detailed cell counts or image analysis, and yet it can give estimates of either cell numbers or biomass. Microcolony volume and diameter were found to have a log-normal distribution. We were able to show that virtually all (>96%) of the AOB biomass occurred as microcolonies. Counts of microcolony abundance and measurement of their diameter coupled with a calibration of microcolony dimensions against cell numbers or AOB biomass were used to determine AOB cell numbers and biomass in WWTP. Cell-specific ammonia oxidation rates varied between plants by over three orders of magnitude, suggesting that cell-specific ammonia oxidation is an important process variable. Moreover, when measured AOB biomass was compared with process-based estimates of AOB biomass, the two values were in agreement.The quantification of microbial communities and populations is an invaluable aspect of microbial ecology. In principle, the autotrophic ammonia-oxidizing bacteria (AOB) are ideal candidates for the development of quantitative tools. AOB have a coherent phylogeny and defined nutritional requirements and are of profound practical importance in natural and engineered environments.The number of individuals should be the ideal benchmark for quantitative studies. Individual counts can be converted to biomass, biovolume, or proportion of biomass, and results obtained by more indirect methods are typically compared to the number of cells per unit volume (15). Fluorescence in situ hybridization (FISH) represents the "gold standard" for quantification of specific bacterial cells in the environment, against which other methods should be compared. Classical (27) and immunological (20) methods are subject to methodological biases, while nonmicroscopic 16S rRNA-based methods (8, 34) or PCR-based methods (13,14,18,19) deliver a proportion of total cell counts, copy number, or relative signal intensities rather than an absolute number of cells or biomass.A quantitative method may be evaluated with respect to its precision and its accuracy. Wagner et al. (43) originally evaluated the accuracy of FISH counts of AOB by using cell specific oxidation rates, an approach previously used to show that most-probable-number-based methods underestimate AOB numbers...

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