title: Evaluation of tyramide solutions for an improved detection and enumeration of single microbial cells in soil by CARD-FISH.
abstract: Several tyramide solutions were evaluated for the application of fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) in soil. Fluorescently labeled tyramide solutions were synthesized and compared to commercially available tyramides for the detection and quantification of single microbial cells. Among the tyramide solutions tested, a succinimidyl ester of fluorescein diluted with dimethylformamide containing iodophenolboronic acid (SFX-DMF-IPBA) gave the best results, yielding highly reproducible cell numbers and detection rates of archaea and bacteria along with negligible non-specific signals. The addition of organic and inorganic compounds to the amplification reagents had a positive impact on the detection of prokaryotic cells. The applicability of SFX-DMF-IPBA for CARD-FISH in soil was further evaluated in soils of different texture. Cell numbers and detection rates of bacteria and archaea remained on a high level independent of the clay or organic matter content. Based on the results obtained in this study, the choice of the tyramide solution used for CARD-FISH has a significant influence on the detection and quantification of single microbial cells in soil. Therefore, we suggest the application of the presented tyramide signal amplification procedure including the tyramide solution SFX-DMF-IPBA for comprehensive CARD-FISH studies investigating the abundance and spatial distribution of soil microorganisms.
authors.affiliation: University of Bremen, Bremen, Germany.
authors.forename: Hannes
authors.initials: H
authors.lastname: Schmidt
authors.forename: Thilo
authors.initials: T
authors.lastname: Eickhorst
authors.forename: Rolf
authors.initials: R
authors.lastname: Tippkötter
mesh_terms: D001105:Archaea / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D001419:Bacteria / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation / Q000379:methods*; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*; D012988:Soil Microbiology