Fecal collection tube

Fecal samples of a human donor were collected as part of a study approved by the ethics committee of the University of Potsdam, Germany (no. 11/2016). The first fecal sample of the day was collected using a feces catcher (Servoprax, Germany). Three aliquots of different fecal areas were immediately transferred to a fecal collection tube (Sarstedt, Germany) with a perforated lid. The tube was kept under anoxic conditions in a plastic box containing an AnaeroGen sachet (Thermo Fisher Scientific, Germany) at 4 °C. Further processing was performed in an anaerobic chamber (MACS Anaerobic Workstation, Don Withley Scientific, UK). One gram of the fecal sample was homogenized in anoxic phosphate-buffered saline (PBS, pH 7.0, Table 4) by vortexing with glass beads (c. 3 mm; Roth, Germany) to yield a 10% fecal suspension. The fecal suspension was further diluted to 1% in a Hungate tube containing anoxic PBS supplemented with 3.18 mM sterile filtered Ti(III) nitrilotriacetate (Sigma-Aldrich, Germany) as a reductant. Subsequently, the fecal slurry aliquots were centrifuged (14,000× g, 4 °C, 5 min) and the supernatant frozen until further processing.
For spiking experiments, the fecal supernatants were serially diluted using 50% acetonitrile and 0.1% formic acid in water to obtain final dilutions of 1:10,000.

Following the obtainment of informed consent, the subjects were provided with a fecal sample collection kit along with the instructions for sample collection. Subjects collected freshly voided fecal samples (≈0.5 g) into the fecal collection tube (Sarstedt AG & Co., Numbrecht, Germany) containing 2 ml RNAlater (an RNA stabilization solution; Ambion, Austin, TX) (for microbiota analysis) and an empty tube (for organic acid analysis and pH measurement). Samples were placed immediately in a cooling box containing refrigerants and were transported (within 24 h) to the Yakult Central Institute where these were stored immediately after collection at 4°C (tubes with RNAlater) and -20°C (tubes without RNAlater) in a Bio-safety Level II laboratory until further processing.

Fresh fecal specimens were collected from five healthy adult male subjects for spiking tests, and from 124 infants and 221 young adults for analytical validation of the qPCR assays (details of the subjects are provided in subsequent sections). Immediately after defecation, a spoonful of feces (0.5 to 1.0 g) was collected into a fecal collection tube (Sarstedt AG & Co., Numbrecht, Germany), and the tubes were stored at −80 °C until use for the pretreatment for qPCR. Primary treatment of feces for total DNA extraction was done as follows. Each fecal sample was weighed, suspended in 9 volumes of sterile PBS (−) to make 10-fold (v/w) dilution, and was washed twice with PBS (−) by centrifugation at 12,000 × g at 4 °C for 5 min. About equal volume of glass beads (2.5 mm, diameter), as that of fecal sample, were added to the diluted samples, and the mixture was subjected to vigorous vortex to make a uniform fecal homogenate which was then divided into 200 μl aliquots and stored at −80 °C until DNA extraction.