Anaerocult a pouches

Fecal samples were serially 10-fold diluted in Oxoid Maximum Recovery Diluent (ThermoFisher) and plated onto Oxoid Bile Aesculin Agar (BEA, enumeration of enterococci), De Man-Rogosa-Sharpe agar (MRS, enumeration of lacobacilli), Columbia agar with 5% defibrinated sheep blood, 0.01 g/L hemin, 0.01 g/L vitamin K (enumeration of a range of anaerobic bacteria), and Reinforced Clostridial (RCM) agar supplemented with 5 g/L propionic acid, 2.5 g/L lactulose and 0.01 g/L riboflavin (enumeration of bifidobacteria). Plates were incubated for 48–72 h anaerobically with Anaerocult A pouches in anaerobic jars (Merck Millipore) at 37°C, and colonies were counted. Representative colony types were subjected to Gram staining and streaked out in aerobic and anaerobic conditions to confirm approximate identity of the isolates. As expected, gram-positive facultative anaerobic cocci were recovered from bile aesculin agar, gram-positive facultative anaerobic rods were seen on MRS agar. Columbia agar produced a variety of mostly gram-negative strictly anaerobic rods consistent with Bacteroidales order morphology. Isolates from supplemented RCM agar were mostly irregular and bifid-shaped gram-positive rods.

Fecal samples were collected between 2016 and 2017 from 13 healthy men and women (UCC employees and students, residents of Cork, Ireland), in accordance with study protocol APC055³³ (link) approved by the Cork Research Ethics Committee. Samples were anonymized by APC Microbiome Ireland research nurses and issued with unique subject-specific identifiers (Table S1). One of the subjects (916) was sampled twice. More extensive participant metadata is available from the original cohort study publication.³³ (link) Samples were collected in participants homes and delivered to the lab in tightly closed containers within 2–3 h after voiding. Immediately upon delivery samples were aseptically aliquoted into 1 g portions (in triplicates) and placed into individual Sterilin containers. Samples were then incubated anaerobically with Anaerocult A pouches in anaerobic jars (Merck Millipore) at 37°C and frozen at −80C at the indicated time points.