High-throughput cultivation of fecal derived gut microbiota

Anaerobic cultivation of fecal microbiota is a promising approach to investigate how gut microbial communities respond to specific intestinal conditions and perturbations. Here, we describe a practical protocol using 96-deepwell plates to cultivate stool-derived gut microbiota. Our protocol addresses key challenges in high-throughput culturing, including a thorough assessment of the impact of gas phase on medium chemistry, a modular medium preparation process to enable testing of several conditions in parallel, a medium formulation designed to maximize the compositional similarity of fecal cultures with the donor microbiota, and the creation of a step-by-step protocol detailing all practical procedures from material preparation to sample handling for analyses. Finally, we validated the protocol by demonstrating that cultivated fecal microbiota responded similarly to dietary fibers (resistant dextrin, soluble starch) and drugs (ciprofloxacin, 5-fluorouracil) as reported in vivo. This high-throughput cultivation protocol can facilitate culture-dependent studies, accelerate the discovery of gut microbiota-diet-drug-host interactions, and pave the way to personalized and microbiota-centered interventions.