Nutrient broth no 2 nb

Reagents: Sodium dihydrogen phosphate (99.99%) and di-sodium hydrogen phosphate anhydrous (99.99%) were acquired from Merck (Steinheim, Germany). o-dianisidine dihydrochloride (D9154), horseradish peroxidase (P8375-5KU), hydrogen peroxide (>30%), and dialysis membrane (D6191) were acquired from Sigma-Aldrich (St. Louis, MO, USA), which is a part of Merck. Potassium permanganate, sodium oxalate, hydrochloric acid, Baird Parker agar base (BP), and egg yolk emulsion with potassium tellurite were acquired from VWR International Eurolab, which is part of Avantor (Llinars del Vallés, Cataluña, Spain). D-(+)-Glucose (99.99%) was acquired from Panreac (Barcelona, Spain). Nutrient broth no. 2 (NB) and Ringer’s solution were acquired from Oxoid, which is part of Thermo Fisher (Basingstoke, Hampshire, UK). Water was deionized using a Milli-Q water purification system (Wasserlab, Navarra, Spain).
Apparatus: We used the Varian Cary Bio 400 spectrophotometer (Varian, part of Agilent Technologies, Santa Clara, CA, USA) and fluorometer Varioskan LUX microplate reader (Thermo Fisher, Kanderl, Germany). We also used sterile 96-well round-bottomed polystyrene microtiter plates (Brand, Wertheim, Germany).

Growth assays were performed in TSB (Oxoid), nutrient broth no.2 (NB; Oxoid),basic medium (BM: 1% soy peptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose and 0.1% K₂HPO₄, pH 7.2), or chemically defined medium^{72 (link)} as described previously^{20 (link)}. Overnight bacterial cultures (OD₆₀₀ of ~13) or bacteria grown to the exponential phase OD₆₀₀ of ~2.5) were diluted to an OD₆₀₀ of ~0.01 in plain medium or medium supplemented with AFAs (50 to 200 µM), cholesterol (50 to 100 µM), MVs (1 µg/ml), and/or recombinant Lip2 (1 µg/ml). Bacteria were then grown in a 96-well plate (U-bottom) at 37 °C with linear shaking at 567 cpm (3-mm excursion) for 24 h. The OD₆₀₀ was measured every 15 min with an Epoch 2 plate reader (BioTek). Areas under growth curves were computed with GraphPad Prism 9.5.1.