Fluorescence in situ hybridization

This section of the SILVA website presents a comprehensive online resource for information on the identification of individual microbial cells by fluorescence in situ hybridization (FISH) with ribosomal RNA-targeted oligonucleotide probes.

 

The focus is on environmental microbiology and especially marine habitats, but the principles and protocols are also valid for other fields.

You will find ...

Responsibility

The 'FISH & Probes' section of the SILVA Webpage represents the current web-compendium for fluorescence in situ hybridization (FISH) of the Department for Molecular Ecology at the Max Planck Institute (MPI) for Marine Microbiology in Bremen, Germany.

For more information on the group and the current activities, please refer to the official Molecol webpage.

For questions, comments, and suggestions related to the content of this section, please contact Dr. Bernhard Fuchs.

In case of technical remarks related to the website itself, please send an e-mail to contact (at) arb-silva.de.

Practical aspects

Why ribosomal RNA? An introduction to FISH for Microbes ...

  • The ribosomal RNAs are among the most conserved macromolecules in nature:
    • They are found as an integral part of the ribosome with a high copy number in every cell.
    • They have the same function in every linving cell on earth.
  • Resembling a so called "molecular phylogenetic marker" (Zuckerkandl and Pauling, 1965), the comparison of rRNA sequences allows for the reconstruction of microbial phylogeny (Woese, 1987).
  • In Bacteria and Archaea there are three types of rRNAs found in the ribosome, the 5S, 16S, and 23S.
  • Comparative sequence analysis of rRNAs also allows for the identification of short signature sequences which are unique for different groups of microorganisms.
  • These signatures are used as targets for short complementary oligonucleotides, called probes, with a typical length of about 15 to 30 nucleotides.
  • For FISH applications, oligonucleotides are typically labeled with a fluorochrome.
  • In the early days of FISH, the procedure was called “phylogenetic staining” (DeLong et al., 1989), because it allows the enumeration and localization of micro-organisms in their environmental context.
  • FISH is an integral part of the so-called ribosomal RNA approach to microbial ecology and evolution (Olsen et al., 1986).
  • Several reviews are available on the principles of hybridization as well as on methodological aspects and applications of FISH with rRNA-targeted oligonucleotides (Amann et al., 1995; Amann et al., 2001).

Molecular probes for FISH analysis

A prerequisite: A well maintained, comprehensive dataset

Comparative rRNA sequence analysis has become a standard method in microbial taxonomy during the last decade. As a consequence, the SILVA databases now contains more than 800,000 sequences, covering not only pure cultures but mostly rRNA genes of so far uncultured bacteria.

 

The reliability of FISH for the identification of cells in complex environments is strongly dependent on the quality of the rRNA databases. A comprehensive and well maintained database is vital for rational probe design. Powerful software tools are required to manage such amount of information. We recommend the highly integrated software package ARB released by the Technical University of Munich (Ludwig et al., 2004) for database management and probe design.

 

Follow the links below to learn more about molecular probes and their application in FISH and to access our probe-specific tools.

 

And don't forget that probes must also be synthesized! For this a reliable partner is required.

Since nearly a decade we are using the services of biomers.net.

They provide maximum quality, low prices, and always state-of-the-art solutions.

Probe Evaluation

    For probe and primer evaluation you can use:

    • TestProbe, developed by the SILVA Team
    • ProbeCheck, offered by the Department of Microbial Ecology at the University of Vienna
    • delta G calculations can be directly accessed here

    probeBase - a comprehensive and well-documented collection of rRNA-targeted probes

    Offered by the Department of Microbial Ecology at the University of Vienna, Austria, and accessible via Opens external link in new window www.microbial-ecology.net/probebase.

    probeBase is a comprehensive database currently containing >1300 published rRNA-targeted oligonucleotide probe sequences including a detailed characterization of each probe (Loy et al., 2003; Loy et al., 2007).
    probeBase can be searched for target organisms, probe names, probe sequences, probe accession numbers, and references. The "match" tool can be used to rapidly retrieve all published probes targeting your query sequences (e.g. from a rRNA gene clone library) without prior phylogenetic analysis. probeBase is hyperlinked with PubMed, SILVA, RDP-II, and Greengenes to provide additional bibliographic information and up-to-date data on probe specificity.

    &

    are partners of the SILVA database project.

    Probe synthesis

    Due to reasonable prices and various offers, today the synthesis of primers and probes is routinely outsourced to companies dealing with this issue. However, quality of the oligonucleotides applied for in situ hybridization is crucial and therefore reliable partners are required.

     

    Our search for such a partner finally led us to the company

    Opens external link in new window biomers.net, Germany, which serves us with high-quality oligonucleotides including modifications, personal service, and excellent prices for many years now.
     

    For more information on the biomers.net biopolymer synthesis service, please check the

    Opens internal link in current windowProbe synthesis subpage of the SILVA FISH & Probes section

    or directly visit:

    A partner of the SILVA database project.