title: Fixation-free fluorescence in situ hybridization for targeted enrichment of microbial populations.
abstract: We modified the standard ribosomal RNA-targeted fluorescence in situ hybridization (FISH) protocol by removing the fixation steps to allow recovery of unmodified nucleic acids. Using this method, hybridized cells could be visualized in two bioreactor sludges and termite hindgut samples by epifluorescence microscopy. We then targeted one bacterial and one archaeal population in the sludge samples with group-specific oligonucleotide probes using in-solution fixation-free FISH and sorted hybridized populations using fluorescence-activated cell sorting (FACS). We could show that sorted populations were highly enriched for the target organisms based on 16S rRNA gene sequencing, thus confirming probe specificity using the modified FISH protocol. This approach should facilitate subsequent genomic sequencing and analysis of targeted populations as DNA is not compromised by crosslinking during fixation.
authors.affiliation: Microbial Ecology Program, DOE Joint Genome Institute, Walnut Creek, CA, USA.
authors.forename: Suzan
authors.initials: S
authors.lastname: Yilmaz
authors.forename: Mohamed F
authors.initials: MF
authors.lastname: Haroon
authors.forename: Brian A
authors.initials: BA
authors.lastname: Rabkin
authors.forename: Gene W
authors.initials: GW
authors.lastname: Tyson
authors.forename: Philip
authors.initials: P
authors.lastname: Hugenholtz
mesh_terms: D000818:Animals; D001105:Archaea / Q000235:genetics / Q000302:isolation & purification*; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D019149:Bioreactors / Q000382:microbiology; D005434:Flow Cytometry / Q000379:methods; D041981:Gastrointestinal Tract / Q000382:microbiology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D020049:Isoptera / Q000382:microbiology; D008828:Microbiological Techniques / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D019642:RNA, Archaeal / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012722:Sewage / Q000382:microbiology
chemical_list: D019642:RNA, Archaeal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012722:Sewage