Methodological Aspects of Fluorescence In Situ Hybridization

Abstract

The current status of fluorescence in situ hybridization (FISH) is critically examined ten years after the initial application of fluorescently labeled, rRNA-targeted oligonucleotide probes as “phylogenetic stains” by Edward DeLong and coworkers in 1989 for the in situ identification of whole fixed bacterial cells in natural samples. The method has in the meanwhile found numerous applications including the identification and enumeration of bacteria in human feces. Still, however, the principal problems that need to be solved before a FISH assay is successfully applied have remained the same. These include e.g.: (i) Permeabilization of the cell envelope for free probe diffusion to the intracellular target molecules, mostly 16S rRNA, by a fixation protocol that preferentially also maintains the cell morphology.(ii) A selection of the probe target site that takes into consideration that not all rRNA sites are equally accessible. In this respect, the predictive power of a complete in situ accessibility map of 16S rRNA of Escherichia coli will be discussed.(iii) Low cellular ribosome content as found in many environmental samples automatically results in weak probe-conferred staining. Methods to increase the signal strength will be discussed together with issues of instrumentation and automation. Depending on the samples of interest and the questions to be addressed a high quality epifluorescence microscope with optional image analysis, a confocal laser scanning microscope or a flow cytometer may be the instruments of choice.