Bifidobacterial (B. longum and B. adolescentis) Colonization of the C. elegans Digestive Tract

The method of Garsin et al. (2001), with minor modifications, was used to estimate the approximate numbers of bacterial cells in the worms. Ten worms were selected, and the surface bacteria were removed by washing the worms 4–5 times with 5 μl of M9 buffer on agar plates. Each worm was transferred to a 0.5 ml centrifuge tube containing 20 μl of M9 buffer and mechanically disrupted with a Microtube pestle (Scientific Specialties, Inc., Lodi, CA, USA). The volume was then adjusted to 100 μl with M9 buffer (serially dilute and spread) and the numbers of bacteria [colony-forming units (cfu)] were estimated by spreading the culture on MRS agar plates. The plates were incubated at the appropriate temperature until the colonies were observed. The same colonies were washed well with M9 buffer and immediately fixed and observed with SEM. Genomic DNA was isolated from some bacteria and subjected to 16S rRNA amplification and alignment.