**2.1. Isolation and cultivation of bifidobacteria resistant to acidic pH and bile salts**

### *2.1.1. Isolation via stress-shock procedure*

Selection of acid and bile resistant bifidobacteria has been based on the stressing isolation method developed by Chung *et al*., (1999). Faecal samples are collected from infants and/or adults. The tube containing the faecal sample is promptly screened for the isolation of resistant strains, as follows: Faecal samples (0.8 g each) are inoculated into 8 ml of Transga-lactooligosaccharide-propionate (TP) medium as an enrichment medium for the bifidobacteria. After an anaerobic incubation for 12 h at 37 °C, 0.8 ml of the incubated cultures is transferred into fresh TP medium with pH adjusted to 2.0 and incubated anaerobically for another 12 h at 37 °C. After the acid exposure, an aliquot (0.8 ml) of the incubation medium is transferred into fresh TP medium supplemented with 1.5% ox-gall, and the incubation continued for another 2 h at 37 °C. The resulting incubation medium is serially diluted and plated on TP agar, to select colonies of the resistant bifidobacteria strains. To isolate reference strains, serially diluted *Bifidobacterium* cells grown in the regular TP medium are plated on TP agar medium. In most of the isolation studies, *B*. *adolescentis, B. longum, B. infantis, B. bifidum* and owner identified *Bifidobacterium* strains (commonly called "own isolates" in microbiology) are used as reference strains. The reference strains are utilized for the convenience of comparison to the resistant strains. Microscopic analysis (1000 with immersion oil) is routinely performed to confirm *Bifidobacterium* morphology.

In addition, *Bifidobacterium* cells are examined for their biochemical and morphological characteristics according to the Bergey's Manual of Determinative Bacteriology. The cultures are grown in Man, Rogosa, and Sharpe (MRS) medium under anaerobic conditions, in a microprocessor-controlled anaerobic chamber. Cultures are incubated for 18 h at 37 °C and stored at 3 – 5 °C between transfers. For the fermentation test, 0.5 ml of 10% substrate solutions (which were membrane filtered through 0.45 µM filter), are added to 9.5 ml of Peptone Yeast-extract Fildes (PYF) basal medium (Mitsuoka, 1990). After 2.5 days of strictly anaerobic incubation, the pH of the growth medium is measured. Tubes showing pH values below 5.5 are considered to be positive for fermentation. The presence of acetate and lactate in the fermented PYF containing glucose medium is assayed by using gas chromatography (GC) or high performance liquid chromatography (HPLC).
