Mar 25, 2020

Sorting and cultivation of Faecalibacterium prausnitzii from fecal samples using flow cytometry in anaerobic conditions

BioRxiv : the Preprint Server for Biology
S. BellaisVincent Thomas


BackgroundThere is a growing interest in using gut commensal bacteria as next generation probiotics. However, this approach is still hampered by the fact that there are few or no strains available for specific species that are difficult to cultivate. Our objective was therefore to adapt flow cytometry and cell sorting to be able to detect, separate, isolate and cultivate new strains of Extremely Oxygen Sensitive (EOS) species from fecal material, focusing on Faecalibacterium prausnitzii as a proof-of-concept. ResultsA BD Influx(R) cell sorter was equipped with a glovebox that covers the sorting area. This box is flushed with nitrogen to deplete oxygen in the enclosure. Several non-specific staining methods including Wheat Germ Agglutinin (WGA), Vancomycin BODIPY and LIVE/DEAD BacLight were evaluated with three different strains of the EOS species F. prausnitzii. In parallel, we generated polyclonal antibodies directed against this species by immunizing rabbits with heat-inactivated bacteria. Anaerobic conditions were maintained during the full process, resulting in only minor viability loss during sorting and culture of unstained F. prausnitzii reference strains. In addition, staining solutions did not severely impact bacteri...Continue Reading

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