A rapid and reproducible method for generating germ-free Drosophila melanogaster

Microbial communities exert a profound influence on various facets of animal behavior and physiology, making the comprehension of their interactions with hosts or the environment essential. Drosophila melanogaster, a widely recognized model organism, has been pivotal in elucidating host-microbe interactions. Despite the existence of several protocols for generating germ-free (GF) Drosophila, their reproducibility has been constrained by the technical difficulty of maintaining airtight conditions in centrifuge tubes. In this study, we introduce a refined method for the production of GF Drosophila, complemented by a straightforward verification process to ascertain its efficacy. We propose an innovative strategy employing bio-reaction tubes equipped with a 0.22 μm filter membrane cap, which facilitates the rearing and maintenance of GF flies, thereby streamlining the procedure and enhancing the efficiency of model construction.