An Introduction to Fluorescence in situ Hybridization in Microorganisms

Fluorescence in situ hybridization (FISH) is a molecular biology technique that enables the localization, quantification, and identification of microorganisms in a sample. This technique has found applications in several areas, most notably the environmental, for quantification and diversity assessment of microorganisms and, the clinical, for the rapid diagnostics of infectious agents. The FISH method is based on the hybridization of a fluorescently labeled nucleic acid probe with a complementary sequence that is present inside the microbial cell, typically in the form of ribosomal RNA (rRNA). In fact, an hybridized cell is typically only detectable because a large number of multiple fluorescent particles (as many as the number of target sequences available) are present inside the cell. Here, we will review the major steps involved in a standard FISH protocol, namely, fixation/permeabilization, hybridization, washing, and visualization/detection. For each step, the major variables/parameters are identified and, subsequently, their impact on the overall hybridization performance is assessed in detail.