Co-Culture of Gut Bacteria and Metabolite Extraction Using Fast Vacuum Filtration and Centrifugation

This protocol describes a robust method for the extraction of intra and extracellular metabolites of gut bacterial mono and co-cultures. In recent years, the co-culture techniques employed in the field of microbiology have demonstrated significant importance in regard to understanding cell-cell interactions, cross-feeding, and the metabolic interactions between different bacteria, fungi, and microbial consortia which enable the mimicking of complex co-habitant conditions. This protocol highlights a robust reproducible physiologically relevant culture and extraction protocol for the co-culture of gut bacterium. The novel extraction steps are conducted without using quenching and cell disruption through bead-cell methods, freeze-thaw cycles, and sonication, which tend to affect the physical and biochemical properties of intracellular metabolites and secretome. The extraction procedure of inoculated bacterial co-cultures and monocultures use fast vacuum filtration and centrifugation. The extraction methodology is fast, effective, and robust, requiring 4 h to complete.