Evaluation of tyramide solutions for an improved detection and enumeration of single microbial cells in soil by CARD-FISH.

摘要

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.Digital Object Identifier http://dx.doi.org/10.1016/j.mimet.2012.09.021