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Summary: The cost of supply cylinders can be expensive for smaller gnotobiotic facilities on strict budgets. Here, we present an alternative approach to enter supplies into germ-free isolators that does not require large cylinder drums (supply cylinders). We describe procedures for autoclaving double-bagged packs of supplies, sealing the packs aseptically into zip lock bags in biosafety cabinet, and entering supply packages into isolators. Our gnotobiotic facility has been using this protocol since 2018 and has been effective in maintaining germ-free status. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.