Fluorescence in situ hybridization for the identification of environmental microbes.
This chapter presents a protocol for the phylogenetic identification of microorganisms in environmental samples (water and sediments) by means of fluorescence in situ hybridization (FISH) with ribosomal RNA-targeted oligonucleotide probes and signal amplification (catalyzed reporter deposition [CARD]). The FISH probes are labeled with the enzyme, horseradish peroxidase (HRP). A subsequent deposition of fluorescently labeled tyramides results in substantially higher signal intensities of target cells than after FISH with probes directly labeled with fluorochromes. Sample preparation and cell permeabilization strategies for various microbial cell wall types are discussed. The custom labeling of tyramides with different fluorochromes is described. A sequential multicolor CARD-FISH protocol is outlined for the simultaneous detection of different phylogenetic groups.
Pernthaler
Annelie
A
Molecular Ecology Department, Max-Planck-Institute for Marine Microbiology, Bremen, Germany.
Pernthaler
Jakob
J
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D002021:Buffers; D004783:Environmental Microbiology; D006735:Horseradish Peroxidase; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008828:Microbiological Techniques / Q000379:methods*; D015336:Molecular Probe Techniques; D015345:Oligonucleotide Probes / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification
D002021:Buffers; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D006735:Horseradish Peroxidase