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Intestinal organoids are 3D cell structures that replicate some aspects of organ function and are organized with a polarized epithelium facing a central lumen. To enable more applications, new technologies are needed to access the luminal cavity and apical cell surface of organoids. We developed a perfusion system utilizing a double-barrel glass capillary with a pressure-based pump to access and modify the luminal contents of a human intestinal organoid for extended periods of time while applying cyclic cellular strain. Cyclic injection and withdrawal of fluorescent FITC-Dextran coupled with real-time measurement of fluorescence intensity showed discrete changes of intensity correlating with perfusion cycles. The perfusion system was also used to modify the lumen of organoids injected with GFP-expressing <i>E. coli</i>. Due to the low concentration and fluorescence of the <i>E. coli</i>, a novel imaging analysis method utilizing bacteria enumeration and image flattening was developed to monitor <i>E. coli</i> within the organoid. Collectively, this work shows that a double-barrel perfusion system provides constant luminal access and allows regulation of luminal contents and luminal mixing.