Sequencing FISH-Separated Microbes from Environmental Samples

Removing the fixation steps to allow recovery of unmodified nucleic acids.

The Science

Most microbes found in environmental samples cannot be cultured in the laboratory making them very hard (or impossible) to study in detail and limiting their exploitation for DOE mission needs in energy, environmental remediation, and carbon cycling. One approach to overcoming this obstacle is to separate individual microbial cells from complex environmental samples using Fluorescence in situ Hybridization (FISH) and to study those with desired characteristics. The standard FISH protocol involved “fixing” cells with paraformaldehyde which complicated subsequent DNA sequencing. Researchers at DOE’s Joint Genome Institute have developed a new protocol that avoids the fixation step. Susan Yilmaz and her colleagues successfully used a variety of fluorescence probes to study freshly-collected, unfixed microbial samples from bioreactor sludge and the termite hind gut. They 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. These promising results constitute a significant technical advance for gaining access to otherwise hard to study microbes. The authors conclude: “This approach should facilitate subsequent genomic sequencing and analysis of targeted populations as DNA is not compromised by crosslinking during fixation”.

BER Program Manager

Ramana Madupu

U.S. Department of Energy, Biological and Environmental Research (SC-33)
Biological Systems Science Division
[email protected]


Yilmaz, S., M. F. Haroon, B. A. Rabkin, G. W. Tyson, and P. Hugenholtz. 2010. “Fixation-Free Fluorescence in situ Hybridization for Targeted Enrichment of Microbial Populations,” The ISME Journal 4. DOI:10.1038/ismej.2010.73.