Application of flow cytometry for ecological monitoring of the rumen microbial ecosystem

Abstract

Flow cytometry in combination with fluorescently labeled ribosomal RNA oligonucleotide probes was used for enumeration and monitoring of ruminal bacteria. The polyanionic azo dye Trypan Blue was used for discrimination between live bacterial cells and inorganic particles and the separation was further improved by lysozyme treatment and sonication. Cy3-labeled universally conserved probe EUB338 and FITC-labeledPrevotella bryantii specific probe PBB14 were used forin situ hybridization in mixed culture experiments and in samples of crude rumen fluid. The results were analyzed by flow cytometry. The separation ofP. bryantii andButyrivibrio fibrisolvens, another ruminal bacterium, in mixed culture experiments was satisfactory and enabled monitoring of these bacteria in a test system.P. bryantii cells were detected in crude rumen fluid samples only after supplementation with pure culture cells; this implicates a low concentration ofP. bryantii cellsin vivo (less than 100/nL,i.e. 10⁵ per mL).