Cultivation-Independent, Semiautomatic Determination of Absolute Bacterial Cell Numbers in Environmental Samples by Fluorescence In Situ Hybridization
AUTOR(ES)
Daims, Holger
FONTE
American Society for Microbiology
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=93375Documentos Relacionados
- Detection of Atopobium vaginae in Postmenopausal Women by Cultivation-Independent Methods Warrants Further Investigation
- Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria
- Sperm Identification in Maize by Fluorescence in Situ Hybridization.
- Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization
- Estimation of the State of the Bacterial Cell Wall by Fluorescent In Situ Hybridization