Abstract
This chapter presents a protocol for the phylogenetic identification of microorganisms in environmental samples (water and sediments) by means of fluorescence in situ hybridization (FISH) with ribosomal RNA-targeted oligonucleotide probes and signal amplification (catalyzed reporter deposition [CARD]). The FISH probes are labeled with the enzyme, horseradish peroxidase (HRP). A subsequent deposition of fluorescently labeled tyramides results in substantially higher signal intensities of target cells than after FISH with probes directly labeled with fluorochromes. Sample preparation and cell permeabilization strategies for various microbial cell wall types are discussed. The custom labeling of tyramides with different fluorochromes is described. A sequential multicolor CARD-FISH protocol is outlined for the simultaneous detection of different phylogenetic groups.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Amann, R. I., Ludwig, W., and Schleifer, K. H. (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143–169.
Amann, R. I. and Schleifer, K. H. (2001) Nucleic acid probes and their application in environmental microbiology, in Bergey’s Manual of Systematic Bacteriology. Vol. 1, 2 ed. (Garrity, D., Boone, D. R., and Castenholz, R. W., eds.), Springer Verlag, New York.
Amann, R., Fuchs, B. M., and Behrens, S. (2001) The identification of microorganisms by fluorescence in situ hybridisation. Curr. Op. Biotech. 12, 231–236.
Gieseke, A., Purkhold, U., Wagner, M., Amann, R., and Schramm, A. (2001) Community structure and activity dynamics of nitrifying bacteria in a phosphate-removing biofilm. Appl. Environ. Microbiol. 67, 1351–1362.
Ishii, K., Mußmann, M., MacGregor, B., and Amann, R. (2004) An improved fluorescence in situ hybridization protocol for the identification of bacteria and Archaea in marine sediments. FEMS Microbiol. Ecol., 50, 203–212.
Mußmann, M., Ishii, K., Rabus, R., and Amann, R. (2005) Diversity and distribution of cultured and uncultured Delta Proteobacteria in an intertidal mud flat of the Wadden Sea. Appl. Environ. Microbiol., 7, 405–418.
Stahl, D. A. and Amann, R. (1991) Development and application of nucleic acid probes in bacterial systematics, in Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt, E. and Goodfellow, M., eds.), Wiley & Sons Ltd., Chichester, England, pp. 205–248.
Teira, E., Reinthaler, T., Pernthaler, A., Pernthaler, J., and Herndl, G. J. (2004) Combining microautoradiography and catalyzed reporter deposition fluorescence in situ hybridization (MICRO-CARD-FISH): a method applicable even to meso-and bathypelagic prokaryotes. Appl. Environ. Microbiol. 70, 4411–4414.
Morita, R. Y. (1997) Bacteria in oligotrophic environments, starvation-survival lifestyle. Vol. 1. Chapman Hall, New York.
Pernthaler, A., Pernthaler, J., and Amann, R. (2002) Fluorescence in situ hybridization and catalyzed reporter deposition (CARD) for the identification of marine bacteria. Appl. Environ. Microbiol. 68, 3094–3101.
Sekar, R., Pernthaler, A., Pernthaler, J., Warnecke, F., Posch, T., and Amann, R. (2003) An improved protocol for the quantification of freshwater Actinobacteria by fluorescence in situ hybridization. Appl. Environ. Microbiol. 69, 2928–2935.
Bobrow, M. N., Harris, T. D., Shaughnessy, K. J., and Litt, G. J. (1989) Catalyzed reporter deposition, a novel method of signal amplification: application to immunoassays. J. Immunol. Meth. 125, 279–285.
Kersten, H. M. J., Poddighe, P. J., and Hanselaar, G. J. M. (1995) A novel in situ hybridization signal amplification method based on deposition of biotynilated tyramide. J. Histochem. Cytochem. 43, 347–352.
Raap, A. K., van de Corput, M. P. C., Vervenne, R. A. W., van Gijlsvijk, R. P. M., Tanke, H. J., and Wiegant, J. (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin-or fluorochrome tyramides. Hum. Mol. Genet. 4, 529–534.
Raap, A. K. (1998) Advances in fluorescence in situ hybridization. Mutation Res. 400, 287–298.
Speel, E. J. M., Ramaekers, F. C. S., and Hopman, A. H. N. (1995) Cytochemical detection systems for in situ hybridization, and the combination with immunocytochemistry: who is still afraid of red, green and blue. Histochem. J. 27, 833–858.
Jansen, M., Hopman, A. H. N., Bot, F. J., et al. (1999) Morphologically normal, CD30-negative B-lymphocytes with chromosome aberrations in classical Hodgkin’s disease: the progenitor cell of the malignant clone? J. Pathol. 189, 527–532.
Braissant, O. and Wahli, W. (1998) A simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mRNAs on tissue sections. Biochemica 1, 10–16.
Speel, E. J. M., Hopman, A. H. N., and Komminoth, P. (1999) Amplification methods to increase the sensitivity of in situ hybridization, Play CARD(S). J. Histochem. Cytochem. 47, 281–288.
van Gijlsvijk, R. P. M., van de Corput, M. P. C., Bezrookove, V., Wiegant, J., Tanke, H. J., and Raap, A. P. (2000) Synthesis and purification of horseradish peroxidase labeled oligonucleotides for tyramide-based fluorescence in situ hybridization. Histochem. Cell Biol. 113, 175–180.
Bobrow, M. N., Adler, K. E., and Roth, A. (2002) Enhanced catalyzed reporter deposition. U.S.A. patent US 6,372,931 B1.
Boetius, A., Ravenschlag, K., Schubert, C. J., et al. (2000) A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623–626.
Dubilier, N., Mülders, C., Ferdelman, T., et al. (2001) Endosymbiotic sulphate-reducing and sulphide-oxidizing bacteria in an oligochaete worm. Nature 411, 298–302.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Pernthaler, A., Pernthaler, J. (2007). Fluorescence In Situ Hybridization for the Identification of Environmental Microbes. In: Hilario, E., Mackay, J. (eds) Protocols for Nucleic Acid Analysis by Nonradioactive Probes. Methods in Molecular Biology, vol 353. Humana Press. https://doi.org/10.1385/1-59745-229-7:153
Download citation
DOI: https://doi.org/10.1385/1-59745-229-7:153
Publisher Name: Humana Press
Print ISBN: 978-1-58829-430-2
Online ISBN: 978-1-59745-229-8
eBook Packages: Springer Protocols