Fluorescence in situ hybridization of bacterial cell suspensions.
The use of fluorescence in situ hybridization (FISH) to identify and enumerate specific bacteria within a mixed culture or environmental sample has become a powerful tool in combining microscopy with molecular phylogenetic discrimination. However, processing a large number of samples in parallel can be difficult because the bacterial cells are typically fixed and hybridized on microscope slides rather than processed in solution. In addition, gram-positive cells and certain environmental samples present a unique challenge to achievement of adequate cell fixation and uniform hybridization for optimal FISH analysis. Here, we describe a protocol for FISH in solution that can be performed entirely in suspension, in a microcentrifuge tube format, prior to microscopy. This protocol can be applied to both gram-positive and -negative cells, as well as complex microbial assemblages. The method employs a rapid technique for performing multiple hybridizations simultaneously, which may be used to qualitatively assess the presence of specific phylogenetic groups in bacterial cultures or environmental samples, and/or directly quantify fluorescence by fluorometry or flow cytometry.
Parsley
Larissa C
LC
Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA.
Newman
Molli M
MM
Liles
Mark R
MR
D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D058491:Bacterial Load / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D013535:Suspensions
D013535:Suspensions