Applied and Environmental Microbiology, December 2001, p. 5810-5818, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5810-5818.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Lehrstuhl für Mikrobiologie, Technische Universität München, 85350 Freising, Germany,1 and Department of Microbial Ecology, Institute of Biological Sciences, University of Aarhus, 8000 Aarhus, Denmark2
Received 2 July 2001/Accepted 24 September 2001
Fluorescence in situ hybridization (FISH) with rRNA-targeted
oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable
method to determine absolute numbers 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 environmental
samples by a combination of FISH, confocal laser scanning microscopy,
and digital image analysis. The quantification is based on an internal
standard, which is introduced by spiking the samples with known amounts
of Escherichia coli cells. This method was initially
tested with artificial mixtures of bacterial cultures and subsequently
used to determine the concentration of ammonia-oxidizing bacteria in a
municipal nitrifying activated sludge. The total number of ammonia
oxidizers was found to be 9.8 × 107 ± 1.9 × 107 cells ml
1. Based on this value, the
average in situ activity was calculated to be 2.3 fmol of ammonia
converted to nitrite per ammonia oxidizer cell per h. This activity is
within the previously determined range of activities measured with
ammonia oxidizer pure cultures, demonstrating the utility of this
quantification method for enumerating bacteria in samples in which
cells are not homogeneously distributed.
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