Fluorescence In Situ Hybridization of Bacterial Cell Suspensions
| Autor: | Molli M. Newman, Larissa C. Parsley, Mark R. Liles |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Microscope
Bacteria medicine.diagnostic_test In situ hybridization Biology Molecular biology Fluorescence Bacterial Load General Biochemistry Genetics and Molecular Biology Fluorescence spectroscopy Bacterial cell structure Flow cytometry law.invention Suspensions law Microscopy medicine Biophysics In Situ Hybridization Fluorescence Fluorescence in situ hybridization |
| Zdroj: | Cold Spring Harbor Protocols. 2010:pdb.prot5493 |
| ISSN: | 1559-6095 |
| DOI: | 10.1101/pdb.prot5493 |
| Popis: | INTRODUCTIONThe use of fluorescence in situ hybridization (FISH) to identify and enumerate specific bacteria within a mixed culture or environmental sample has become a powerful tool in combining microscopy with molecular phylogenetic discrimination. However, processing a large number of samples in parallel can be difficult because the bacterial cells are typically fixed and hybridized on microscope slides rather than processed in solution. In addition, Gram-positive cells and certain environmental samples present a unique challenge to achievement of adequate cell fixation and uniform hybridization for optimal FISH analysis. Here, we describe a protocol for FISH in solution that can be performed entirely in suspension, in a microcentrifuge tube format, prior to microscopy. This protocol can be applied to both Gram-positive and -negative cells, as well as complex microbial assemblages. The method employs a rapid technique for performing multiple hybridizations simultaneously, which may be used to qualitatively assess the presence of specific phylogenetic groups in bacterial cultures or environmental samples, and/or directly quantify fluorescence by fluorometry or flow cytometry. |
| Databáze: | OpenAIRE |
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