Fluorescence in situ hybridisation

April 13, 2011

Fluorescence in situ hybridisation (FISH) is a powerful tool to make hidden cellular and smaller molecular structures visible. This is achieved by using different probes connected to different light emitting substances (for example fluorophores). Common are probes which bind to DNA. However, in the picture below (Fig. 1) Markus Petzold and me performed a FISH analysis to identify methane producing microbials (methanogens) from within a microbial enrichment sample by making use of probes which attached to certain sequences of 16S ribosomal RNA (rRNA). 16S rRNA is highly conserved and specific among species and enabled us via tripple crossprobing to identify a methanogenic species within the sample. This species seems to be the archeon Methanosarcinales.

 16S rRNA FISH analysis of 29 day granular WWTP methanogenic sludge enrichment

Fig. 1: FISH colored aggregates of microorganisms from a granular sludge day 29 methane enrichment. (A) Methanogenic Methanosarcinales are depicted in purple together with eubacteria coloured in green. In (B) aggregates of archea with in light purple Methanosarcinales are shown.

In crossprobing three different colours are used that stand for one condition each. Examples of conditions that help to identify microbial species from a sample are for example domain, phylum and species. Unique colour combinations (we used Cy3, Cy5 and Fluos in different combinations) make it thus possible to denote a species, a phylum or a domain which is present within the sample.

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