CAtalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) for Complex Environmental Samples

CARD-FISH technique allows us to increase microbial cell detection compared to traditional FISH assays. Specific nonfluorescent oligonucleotide probes targeting 16S rRNA genes are employed and are chemically activated by the binding of tyramide molecules, with the latter able to generate a cascade of fluorescence signals, improving sensitivity and reducing background noise. The technique has been successfully applied for the detection of microorganisms in different environmental matrices and under different growth conditions (including those where cells are characterized by low physiological activity and low ribosome content). This chapter presents a straightforward procedure to execute CARD-FISH analysis, from sample preparation and fixation, to microscopic visualization, along with relevant technical notes.