Peptide Nucleic Acid-Fluorescence In Situ Hybridization (PNA-FISH) for the Detection and Spatial Location of Microorganisms.

Fluorescence in situ hybridization (FISH) applied to microorganisms is a 30-year-old technology that has evolved to become one of the most established techniques in molecular biology. Traditionally, DNA probes have been used for in situ hybridization. However, peptide nucleic acid (PNA) probes have emerged as a promising alternative, offering better hybridization performance and making FISH procedures simpler and more robust. In this chapter, we describe a universal PNA-FISH protocol for the detection of microorganisms in different types of samples, including enriched suspensions, histological samples, and microbial communities, using microscopy or flow cytometry. This protocol can be easily applied to different microorganisms as a way of identifying in situ relevant microorganisms (including pathogens) and assess their spatial distribution in different types of samples.