Ca²⁺ in Hybridization Solutions for Fluorescence in situ Hybridization Facilitates the Detection of Enterobacteriaceae

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Fluorescence in situ hybridization (FISH) has been employed to identify microorganisms at the single cell level under a microscope. Extensive efforts have been made to improve and extend the FISH technique; however, the development of a widely applicable protocol is a continuing challenge. The present study evaluated the effects of divalent cations in the hybridization solution on the FISH-based detection of various species of bacteria and archaea with rRNA-targeted probes. A flow cytometric analysis after FISH with a standard hybridization buffer detected positive signals from less than 30% of Escherichia coli IAM 1264 cells. However, the number of cells with positive signals increased to more than 90% after the addition of calcium chloride to the hybridization buffer. Mn²⁺ also had positive effects, whereas Mg²⁺ did not. The positive effects of Ca²⁺ were similarly observed for bacteria belonging to Enterobacteriaceae, including Enterobacter sakazakii IAM 12660^T, E. aerogenes IAM 12348, Klebsiella planticola IAM 14202, and Salmonella enterica subsp. enterica serovar Typhimurium strain LT2. These results indicate that the supplementation of Ca²⁺ to the hybridization buffer for FISH contributes to the efficient detection of Enterobacteriaceae cells.