Zirconia silica beads

Total RNA was prepared from homogenized vegetation pellets from single animals after digestion with 4 mg/ml proteinase K (Qiagen) for 30 min. S. aureus COL cells were lysed with 0.5 ml zirconia silica beads (Fisher Scientific, Waltham, MA, USA) in a dental amalgamator, and RNA was purified with the RNeasy Mini kit (Qiagen), treated with DNase I (Invitrogen, Grand Island, NY), and stored at -80°C. RNA integrity and absence of eukaryotic RNA were confirmed by denaturing gel electrophoresis. RNA from planktonic cultures was isolated from bacteria harvested from mid-logarithmic (5 h) or stationary phase (18 h) TSB cultures incubated at 37°C.

For analysis of infectious virus from tissues via plaque assay in Vero cells, ½ brain, spleen, both kidneys, heart, liver, and lung were weighed and homogenized in 500 ul of serum-free DMEM or 1x PBS in 2 ml cryovials (Sarstedt) containing 2.3 mm Zirconia/Silica beads (Fisher) on a bead beater at 5300 rpm for 25 seconds. Samples were then clarified at 5000 x g for 10 minutes, and the clarified supernatant transferred to a new tube. Vero cells were plated the previous day in 24-well plates (Corning) at a density of ~1.3 x 10⁵ cells per well. The clarified tissue homogenate supernatants and plasma samples were serially diluted 10-fold in DMEM supplemented with 2% FBS and penicillin-streptomycin. Media was removed from plates, and 200ul of serially diluted sample was plated per well and plates were incubated at 37°C for one hour. After incubation, 0.5 ml of MEM (Gibco) with 1.5% carboxymethyl cellulose (CMC) was overlayed per well, and plates were returned to the 37°C incubator. After 3 days for INKV and SSHV, and 5 days for LACV, TAHV, and JCV, plates were fixed with 10% formaldehyde (Sigma), stained with 0.35% crystal violet, rinsed, air-dried, and plaques counted for each sample and dilution. Sample titers were calculated as PFU/mg tissue or PFU/ml plasma.

A panel of 5 straight-chain SCFAs (acetate, propionate, butyrate, valerate, caproate) and 4 branched-chain SCFAs (isobutyrate, 2-methylbutyrate, isovalerate, 4-methylvalerate) were assessed by LC-MS, as we described previously [39 (link)]. In brief, fecal samples were homogenized in 1:1 acetonitrile/water using a Bead Mill 24 Homogenizer (Fisherbrand, Waltham, MA, USA) in tubes containing 1.0 mm zirconia/silica beads (ThermoFisher Scientific) and centrifuged (15,000× g, 10 min, 10 °C). The supernatant was mixed with internal standard (¹³C₄-butyrate; Cambridge Isotopes Laboratories) and derivatized with 3-nitrophenylhydrazine to convert SCFAs to their corresponding 3-nitrophenylhydrazones prior to LC-MS analysis.