Development of a Fluorescence In Situ Hybridization protocol for simultaneous analysis of Gram-positive and Gram-negative bacteria

Abstract

In the present study several FISH procedures, varying on the fixatives used (50,80 and 96% ethanol aqueous solutions), were investigated for analyzing both Gram-positive and Gram-negative bacteria cells harvested at different growth stages. The universal bacterial EUB338 Cy3-labeled probe were used and FISH signals were analyzed by: i) flow cytometry, in terms of percentage of fluorescent cells, average and total fluorescence intensities, and ii) by epifluorescence microscopy in terms of visualization. The FISH signals obtained for cells in different growth phases with the three fixatives were correlated with RNA content and cell viability.