AimTo develop a novel validated method for the isolation of Bifidobacterium animalis ssp. lactis BB-12 (BB-12) from faecal specimens and apply it to studies of BB-12 and Lactobacillus rhamnosus GG (LGG) recovered from the healthy human gastrointestinal (GI) tract. Methods and ResultsA novel method for isolating and enumerating BB-12 was developed based on its morphologic features of growth on tetracycline-containing agar. The method identified BB-12 correctly from spiked stool close to 100% of the time as validated by PCR confirmation of identity, and resulted in 97-104% recovery of BB-12. The method was then applied in a study of the recovery of BB-12 and LGG from the GI tract of healthy humans consuming ProNutrients((R)) Probiotic powder sachet containing BB-12 and LGG. Viable BB-12 and LGG were recovered from stool after 21days of probiotic ingestion compared to baseline. In contrast, no organisms were recovered 21days after baseline in the nonsupplemented control group. ConclusionsWe demonstrated recovery of viable BB-12, using a validated novel method specific for the isolation of BB-12, and LGG from the GI tract of healthy humans who consumed the probiotic supplement. Significance and Impact of the StudyThis method will enable more detailed and specific studies of BB-12 in probiotic supplements, including when in combination with LGG.