, Brooklyn, NY), to record pH of the processed RF and media. VFA concentrations in rumen fluid and its preparations were determined by capillary gas chromatography of their butyl esters, as described previously [15, 16], on an Agilent 6890 N gas chromatograph
(Agilent Technologies, Inc., Santa Clara, CA). Culture conditions, and processing for proteomics RF preparations from Samples A and B were analyzed separately per experiment set, and each analysis in turn was conducted in duplicate. In Experiment I, 5 ml LB, dRF, or fRF media were aliquoted separately into 85, 16 × 150 mm tubes. Of these, five tubes VS-4718 per media were used as uninoculated controls. The remaining 80 tubes were inoculated with O157. To create anaerobic culture conditions, half of these tubes were transferred into the anaerobic Coy Chamber for 72 hrs, sealed and inoculated within the chamber and then removed. The log-phase O157 culture, re-Selleckchem Autophagy inhibitor suspended in 0.9% OICR-9429 saline was inoculated to a starting OD600 0.05-0.06, into all the 80 tubes, which were then incubated at 39°C with shaking, along with the uninoculated control tubes. O157 was grown to an OD600 of 0.8-1.0, before harvesting cells from each
tube by centrifugation at 7,000 rpm, 15 min at 4°C. Bacterial cells from like media, whether derived from RF-samples A or B, were pooled together and washed three times with an equal volume of ice-cold sterile phosphate buffered saline (PBS; pH 7.4), and processed to obtain cell lysate and pellet fractions for bottom-up proteomic analysis . In Experiment II, uRF was included to the media (LB, dRF, fRF) being evaluated and aliquoted as described above. However, the O157 inoculum diluted in saline to the starting OD600 0.05-0.06 was placed in sterile dialysis tubing (Spectra/Por Oxymatrine Type F, PVDF: 80,000 kDa cut off; Serva Electrophoresis,
Heidelberg, Germany) and suspended within the uRF containing tubes . This was to ease the recovery of O157 from the complex uRF milieu and the colony counts recovered from the tubings matched those obtained by magnetic recovery of O157 from directly inoculated uRF (data not shown). O157-innoculated LB, dRF, fRF, and uRF were incubated for 48 h, anaerobically, before harvesting cells and processing for proteomic analysis  using iTRAQ. For this experiment, bacterial cells from like media were pooled together but kept separate between preparations derived from RF-samples A and B. The culture conditions used in Experiment II correlated with ruminal conditions and feed turnover rates [19–21]. In both experiments, OD600 of each tube was recorded relative to uninoculated control tubes, centrifuged at 10,000 rpm for 10 min to remove any sediments or particulate matter which could interfere with the spectrophotometer reading. In addition, pH, and colony counts (on LB agar) were determined from the five uninoculated and ten inoculated tubes at different time points, for comparison.