Gordon Lab Bacterial Growth Protocol

Abstract

Animal Husbandry. All experiments using mice were performed using protocols approved by the animal studies committee of Washington University. NMRI-KI mice (10) were maintained in flexible plastic film isolators under a strict 12 h light cycle, and fed an irradiated standard low-fat, high plant polysaccharide chow (LF/PP, diet 2018 from Harlan Tecklad, www.tekladcustomdiets.com) or a high fat, high-sugar (HF/HS) Western-style diet (Harlan Teklad 96132) or a corresponding control low fat, high-sugar (LF/HS; Harlan Teklad 03317). Animals were colonized via gavage with 108 CFU from an overnight culture of B. thetaiotaomicron or a log-phase culture of E. rectale. Gavage with E. rectale was repeated on 3 successive days using cells from separate log-phase cultures begun from separate colonies. Cecal contents were flash frozen in liquid nitrogen immediately after animals were killed.
Quantitative (q) PCR Measurements of Colonization. A total of 100–300 mg of frozen cecal contents from each gnotobiotic mouse was added to 2 mL tubes containing 250 L of 0.1 mm-diameter zirconia/silica beads (Biospec Products), 0.5 mL of Buffer A (200 mM NaCl, 20 mM EDTA), 210 L of 20% SDS, and 0.5 mL of a mixture of phenol:chloroform:isoamyl alcohol (25:24:1; pH 7.9; Ambion). Samples were lysed with a bead beater (BioSpec; ‘‘high’’ setting for 4 min at room temperature). The aqueous phase was extracted after centrifugation (8,000 g at 4 °C for 3 min), and the extraction repeated with another 0.5 mL of phenol:chloroform:isoamyl alcohol and 1 min of vortexing. DNA was precipitated with 0.1 volume of 3M sodium acetate (pH 5) and 1 volume of isopropanol (on ice for 20 min), pelleted (14,000 g, 20 min at 4 °C) and washed with ethanol. The resulting pellet was resuspended in water and 1/2 (for E. rectale mono-associations) or 1/10 of the DNA (for B. thetaiotaomicron- colonized samples) cleaned up further using a DNAEasy column (Qiagen).

qPCR was performed using (i) primers specific to the 16S rRNA gene of B. thetaiotaomicron (11) or to the Clostridium coccoides/E. rectale group (forward: 5-CGGTACCTGACTAAGAAGC-3; reverse: 5-AGTTT(C/T)ATTCTTGCGAACG3) (12), and (ii) conditions described previously for B. thetaiotaomicron (11). The amount of DNA from each genome in each PCR was computed by comparison to a standard curve of genomic DNA prepared in the same manner from pure cultures of each bacterial species. Data were converted to genome equivalents by calculating the mass of each finished genome (2.8 105 genome equivalents (GEq) per ng E. rectale DNA, and 1.5 105 GEq per ng B. thetaiotaomicron DNA).