Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques

Specialized microorganisms catalyse central steps of global element cycling, such as nitrogen fixation or the mineralization of organic matter. There is an urgent need for the development of new methods for in situ microbial analysis, which originates from the restricted morphological diversity of prokaryotes and the limited usefulness of cultivation-based methods for quantifying species and genera at a spatial resolution that is relevant for microorganisms. Fluorescence in situ hybridization (FISH) enables reliable quantification of microbial populations in complex environmental samples.FISH probes that target large taxonomic groups, such as the Bacteria, Archaea and Eukarya domains or the Alpha-, Beta- and Gammaproteobacteria classes are popular. Owing to their broad specificity, these probes can be used to analyse samples from many different environments that range from marine and freshwater environments to sediments and soils. They also facilitate an initial, rapid assessment of the dominance of certain taxa in particular environments. Most of these group-specific probes were published more than 10 years ago, when the ribosomal RNA (rRNA) database was less than 10% of its current size.We address the question: which of these old probes are still valid? We checked the probes thoroughly against the comprehensive rRNA datasets of the SILVA project. The good news is that most probes can still be used for initial identification and quantification of microbial populations.Failure to detect cells — that is, a false-negative FISH result — can be due to lack of cell permeabilization, low cellular ribosome content or low efficiency of probe binding based on the higher-order structure of the rRNA.The new, more sensitive FISH assays have the greatest impact in oligotrophic environments, where the indigenous microbiota has low ribosome content, and in samples in which the background fluorescence hampers reliable quantification of less-frequent populations. With good microscopes, even populations of a relative abundance of 1 in 1,000 cells can be accurately quantified.FISH enables studies of microorganisms in their natural contexts. Metagenomics cannot substitute for the information that can be gained by visualizing the identity and activity of single microbial cells in situ. Rather, it will make available huge sequence datasets that will help in improving existing probe sets and facilitate the development of new probes.