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Supplementary material for this article is available at www.BioTechniques.com/article/114319.

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BioTechniques 59:94-98 (August 2015) doi 10.2144/000114319 Keywords: segmented filamentous bacteria; SFB; Candidatus Arthromitus; culture; microbiota; commensal

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University of Missouri (MU) Mutant Mouse Resource and Research Center, MU Metagenomics Center, 3 MU Rat Resource and Research Center, 4 Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, and 5Department of Comparative Medicine, School of Medicine, University of Washington, Seattle, WA 1

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Aaron C. Ericsson1,2,3,4, Giedre Turner1,4, Lisa Montoya4, Annie Wolfe4, Stacey Meeker5, Charlie Hsu5, Lillian Maggio-Price5, and Craig L. Franklin1,2,3,4

(8). Thus, to optimize selection of SFB from an endogenous microbial population, mucosal scrapes were collected from thoroughly rinsed ileal tissue from 2- to 3-day post-weaning BALB/cAnNHsd mice (Harlan Laboratories, Indianapolis, IN) that were confirmed to be colonized with SFB via PCR (9). Briefly, immediately following humane euthanasia, approximately 4 cm of distal ileum was excised using aseptic technique and flushed thoroughly with sterile PBS to remove all visible luminal contents. Ilea were placed on a sterile Petri dish and incised longitudinally to expose the mucosal surface. The mucosa was scraped with a sterile scalpel blade to collect epithelial cells and mucosa-associated bacteria. The pooled material recovered from 4 mice was placed in 50 mL of sterile DMEM/ high glucose containing 4 mM L-glutamine, 4500 mg/L glucose, and sodium pyruvate (catalog #SH30243.02; Thermo Scientific, Waltham, MA). Pooled ileal mucosal scrape (IMS) material was plated at 2 mL per well in 24-well polystyrene plates (catalog #08–772–4G, Corning Primaria culture plates; Corning, Durham, NC) and incubated in a non-humidified incubator at 37°C under anaerobic (≥13% CO2,