Detection of prokaryotic cells with fluorescence in situ hybridization.

Fluorescence in situ hybridization with rRNA targeted oligonucleotide probes is nowadays one of the core techniques in microbial ecology, allowing the identification and quantification of microbial cells in environmental samples in situ. Next to the classic FISH protocol, which uses fluorescently monolabelled probes, the more sensitive CARD-FISH (also known as TSA-FISH), which involves an enzyme catalyzed signal amplification step, is becoming increasingly popular. This chapter describes protocols for both methods. While classic FISH has the advantage of being relatively cheap and easy to do on morphologically diverse samples, CARD-FISH offers a significantly higher sensitivity, allowing the detection of slow growing or metabolically inactive cells, which are below the detection limit of classic FISH. The drawback here is the considerably higher price for the probes and advanced cell fixation and permeabilization requirements that have to be optimized for different target cells.