Protocol for preparing fiber-defined simulated media for growth profiling of diverse human gut bacterial isolates

Dietary fiber availability shapes gut microbial growth, yet standardized approaches for comparative growth profiling under defined fiber conditions remain limited. Here, we present a protocol for preparing fiber-defined simulated media based on yeast extract-casitone-fatty acid (YCFA) basal medium. We describe steps for anaerobic culturing of human gut bacterial isolates and inoculum normalization. We detail procedures for quantifying growth using optical measurements across contrasting fiber conditions. This scalable framework supports controlled investigation of diet-microbe interactions and microbial physiology.