Conical shaped polypropylene tube

A given pulverized frozen human fecal sample (353±184 mg; mean±SD) was transferred to an anaerobic Coy chamber (atmosphere 75% N₂, 20% CO₂, 5% H₂) in a 2mL Axygen screw topped tube. The tube was then opened and its contents were transferred to a 50 mL conical shaped polypropylene tube (Falcon). The fecal material was suspended in 10 mL of sterile PBS supplemented with 0.1% L-cysteine (Sigma) by vortexing with sterile 2 mm-diameter glass beads. The suspension was passed through a nylon 100 μm mesh filter (BD) and the filtrate was mixed with an equal volume of 30% glycerol in PBS/0.1% cysteine. Aliquots (1.2 mL) of this suspension were placed amber glass vials, each of which was sealed with a crimp top, and frozen at −80°C. Tubes were thawed, and transferred into gnotobiotic isolators (with surface sterilization achieved by treatment with Clidox). Aliquots (200 μL) were then introduced into each germ-free mouse in a given experimental group by oral gavage. A total of 38 animals were used for this study (n=4–5 mice/donor microbiota). This size of each treatment group was not based on a formal power calculation but was informed by our previous work described in ref. 8 . There was no randomization of mice for this study; male C57BL/6J animals in each group were age- and weight-matched prior to gavage. Investigators were not blinded with respect to the donor microbiota.