Fluorescence in situ hybridization

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Fluorescence In Situ Hybridization (FISH) is a fast method for specific identification and quantification of intact cells in their natural environment. FISH is a powerful quantitative methodology and was hailed as a breakthrough for microbial ecology since its introduction. However, it is difficult to apply in some environmental samples. Bacteria in some ecosystems (e.g., aquatic habitats, oligotrophic systems) are small, slow growing, or starving and the signal intensities of hybridized cells are below detection limits or lost in a high fluorescence background. Recently, to increase FISH sensitivity the catalyzed reported deposition (CARD) modification was introduced to solve some of the problems, giving rise to a new methodology called CARD-FISH.



A fluorescent-labelled specific rRNA targeted oligonucleotide probe is used for the direct identification, independent of culture, and quantification of the microorganism (Amann et al., 1995). The use of in situ hybridization for counting and identifying organisms was proposed by Olsen et al. (1986) twenty years ago. The first assays were performed using radioactively labelled oligonucleotides (Giovannoni et al., 1988), and later by using fluorescent probes, which yield superb spatial resolution and can be detected very simply by using epifluorescent microscopy and fluorescent in situ hybridization (FISH) (Amann et al., 1990).

  1. Samples must be fixed immediately after collection.
  2. Microorganisms are made permeable to oligonucleotide probes by fixation with aldehydes (formalin, paraformaldehyde, glutaraldehyde) or alcohols (methanol, ethanol) (Amann et al., 1995).
  3. When a fluorescence probe (labelled with a fluorescence dye, e.g. Cy3 or fluorescein) is placed in contact with permeable cells in fixed physico-chemical conditions, the fluorescent probe hybridizes with the specific intracellular target site in the ribosomes (Amann et al., 1995).
  4. Later, a universal fluorochrome that binds DNA, such as DAPI, is used to evaluate the total number of viable cells in the sample. With epifluorescence microscopy the hybridized cells can be observed and counted, and after changing the filter, the total number of viable cells is counted.


Using DGGE and cloning we can obtain useful qualitative information about the diversity present in the samples, but these methodologies do not provide reliable quantitative data, key information for microbial ecology. To quantify the level of diversity detected by DGGE and cloning, FISH is a very powerful technique. With FISH it is possible to follow microbial population fluctuations.


Difficulties may be encountered when applying FISH to environmental samples from highly eutrophic systems (Pernthaler et al., 2002). Most bacteria in aquatic habitats are small, slow growing, or starving, and the intensity of the signal of hybridized cells is frequently below the detection limits or lost in high background fluorescence. This problem is common with sediments too.

Current use in bioleaching studies

In AMDs and in the Tinto River most of the microorganisms are strict chemolithotrophs, and they are usually associated with minerals and rocks. The study of the microorganisms attached to solid substrates is very interesting, especially for biomining research, but in most of the cases high background fluorescence and a low signal from the hybridized cells is obtained, lowering the efficiency of FISH. To solve this problem amplification of the signal can be introduced in the procedure. Pernthaler et al. (2002) applied catalyzed reporter deposition (CARD) to FISH (CARD-FISH) with excellent results, obtaining detection rates in marine sediments almost one order of magnitude higher than with conventional FISH.

CARD-FISH is tried for detection of microorganisms in direct contact with mineral substrates in bioleaching processes and in the characterization of subsurface microorganisms.

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