Modified bifidobacterium agar

Aliquots of stool were stored at −80 °C prior to DNA extraction to culture bacteria of interest, such as those associated with health and low genetic risk. Samples showing the highest relative abundance of significant Bifidobacterium ASVs were serially diluted in 1X phosphate buffered saline and plated on pre-reduced Modified Bifidobacterium Agar (Becton, Dickinson and Company) without the addition of lactulose. Individual colonies were isolated from the two lowest dilution plates and culture stocks were stored in a 15% glycerol solution at −80 °C. DNA from each isolate was extracted using the E.Z.N.A Bacterial DNA kit (Omega Bio-tek, Doraville, CA) and the full length 16 S rRNA gene sequence was amplified by PCR using universal primers 27F and 1492R⁵⁴ . The resulting PCR products were Sanger sequenced to determine the identity of the isolates (Eton Bioscience, Inc., San Diego, CA). Resulting sequences were compared to the GenBank nucleotide database for identification using BLAST⁵⁵ .

An aliquot (5 g) of each fermented fecal batch was mixed with 45 mL of the sterilized NaCl solution (0.9% w/v) and homogenized. Decimal dilutions were carried out. Viable bacterial cells were counted in the following culture media: plate count agar (total aerobes); Wilkins–Chalgren anaerobe agar (total anaerobes); MRS agar (LAB); M17 agar (streptococci); violet red bile glucose agar (coliforms); reinforced Clostridium agar (clostridial microbes); and modified Bifidobacterium agar (Becton Dickinson; Le Pont de Claix, SA, France) (fecal bifidobacteria). Except for modified Bifidobacterium agar, other media were purchased from Oxoid Ltd. (Basingstoke, Hampshire, England). Except for Wilkins–Chalgren anaerobe agar and modified Bifidobacterium agar, other media were aerobically incubated. For culturing conditions, the adopted times and temperatures varied according to manufacturers’ instructions.