Tos propionate agar

Three hundred ninety three of the 453 assessed fecal samples (nearly 87%) were confirmed to contain B. longum subsp. longum DNA employing PCR and a subspecies-specific primer pair^{20 (link)}, of which 177 were selected to retrieve bifidobacteria by cultivation on TOS propionate agar (Eiken Chemical, Tokyo, Japan) supplemented with 50 mg/l mupirocin (Merck KGaA, Darmstadt, Germany). Following incubation at 37 °C for 48 hours under anaerobic conditions, ten colonies per sample were randomly selected and were examined by colony PCR using the above mentioned species-specific primer set. Each identified B. longum subsp. longum isolate was cultivated in Difco Lactobacilli MRS (Becton Dickinson, NJ) supplemented with 0.05% L-cysteine HCl (Kanto Chemical, Tokyo, Japan) at 37 °C for 16 hours under anaerobic conditions before DNA extraction. In this manner a total of 162 B. longum subsp. longum strains were isolated, of which a proportion (based on age representation) was selected for sequencing (see below).

B-3 (MCC1274) lyophilized powder was obtained from the Morinaga Milk Industry (Tokyo, Japan). The live B-3 (B-3L) were suspended in saline just before daily administration. The heat-killed B-3 (B-3HK) were prepared as previously described with slight modifications [18 (link)], by heating B-3 lyophilized powder suspended in saline at 90 °C for 30 min. A lack of viable bacteria was confirmed with anaerobic culture methods using TOS propionate agar (Eiken Chemical, Tokyo, Japan). B-3HK was stored at −20 °C until an administration.