Abstract
Fluorescence in situ hybridization with rRNA targeted oligonucleotide probes is nowadays one of the core techniques in microbial ecology, allowing the identification and quantification of microbial cells in environmental samples in situ. Next to the classic FISH protocol, which uses fluorescently monolabelled probes, the more sensitive CARD-FISH (also known as TSA-FISH), which involves an enzyme catalyzed signal amplification step, is becoming increasingly popular. This chapter describes protocols for both methods. While classic FISH has the advantage of being relatively cheap and easy to do on morphologically diverse samples, CARD-FISH offers a significantly higher sensitivity, allowing the detection of slow growing or metabolically inactive cells, which are below the detection limit of classic FISH. The drawback here is the considerably higher price for the probes and advanced cell fixation and permeabilization requirements that have to be optimized for different target cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
DeLong, E.F., Wickham, G.S., and Pace, N.R. (1989) Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 243, 1360–1363.
Pernthaler, A., Pernthaler, J., and Amann, R. (2002) Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol. 68, 3094–3101.
Schonhuber, W., Fuchs, B., Juretschko, S., and Amann, R. (1997) Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification. Appl. Environ. Microbiol. 63, 3268–3273.
Zwirglmaier, K., Ludwig, W., and Schleifer, K.H. (2004) Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization – RING-FISH. Mol. Microbiol. 51, 89–96.
Lee, N., Nielsen, P.H., Andreasen, K.H., Juretschko, S., Nielsen, J.L., Schleifer, K.-H., et al. (1999) Combination of fluorescent in situ hybridization and microautoradiography – a new tool for structure-function analyses in microbial ecology. Appl. Environ. Microbiol. 65, 1289–1297.
Ouverney, C.C. and Fuhrman, J.A. (1999) Combined Microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ. Appl. Environ. Microbiol. 65, 1746–1752.
Loy, A., Horn, M., and Wagner, M. (2003) probeBase: an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res. 31, 514–516.
Pruesse, E., Quast, C., Knittel, K., Fuchs, B.M., Ludwig, W., Peplies, J., Glöckner, F.O. (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 35, 7188–7196.
DeSantis, T.Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E.L., Keller, K., et al. (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 72, 5069–5072.
Cole, J.R., Chai, B., Farris, R.J., Wang, Q., Kulam-Syed-Mohideen, A.S., McGarrell, D.M., et al. (2007) The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data. Nucleic Acids Res. 35(suppl 1), D169–D172.
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai T., Steppi, S., Jobb, G., Förster, W., Brettske, I., Gerber, S., Ginhart, A.W., Gross, O., Grumann, S., Hermann, S., Jost, R., König, A., Liss, T., Lüssmann, R., May, M., Nonhoff, B., Reichel, B., Strehlow, R., Stamatakis, A., Stuckmann, N., Vilbig, A., Lenke, M., Ludwig, T., Bode, A., Schleifer, K.H. (2004) ARB: a software environment for sequence data. Nucleic Acids Res. 32, 1363–1371.
Loy, A., Tischler, R.A.P., Rattei, T., Wagner, M., and Horn, M. (2008) probeCheck: a central resource for evaluating oligonucleotide probe coverage and specificity. Environ. Microbiol. 10, 2894–2898.
Fuchs, B.M., Wallner, G., Beisker, W., Schwippl, I., Ludwig, W., and Amann, R. (1998) Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 64, 4973–4982.
Behrens, S., Rühland, C., Inácio, J., Huber, H., Fonseca, A., Spencer-Martins, I., Fuchs, B.M., Amann, R. (2003) In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes. Appl. Environ. Microbiol. 69, 1748–1758.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zwirglmaier, K. (2010). Detection of Prokaryotic Cells with Fluorescence In Situ Hybridization. In: Bridger, J., Volpi, E. (eds) Fluorescence in situ Hybridization (FISH). Methods in Molecular Biology, vol 659. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-789-1_27
Download citation
DOI: https://doi.org/10.1007/978-1-60761-789-1_27
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-788-4
Online ISBN: 978-1-60761-789-1
eBook Packages: Springer Protocols