Abstract
Gnotobiology has revolutionized the study of microbiota-host interactions. This protocol explains how to generate, maintain, and monitor gnotobiotic mice. Three monitoring methods are presented and compared: bacterial culture, microscopy to visualize the presence (or absence) of bacteria using Gram staining or DNA staining, and 16S rRNA gene amplification and sequencing. The generation and maintenance of gnotobiotic animals should be performed in a germ-free and gnotobiotic facility to guarantee sterility and precision of gnotobiotic conditions. For complete details on the use and execution of this protocol, please refer to McDonald et al., 2020.