Density gradient centrifugation

Percoll density gradients were conducted as per Patrick and Reid²⁹. Briefly, a stock solution of Percoll (Sigma Aldrich, St. Louis, MO, USA) was prepared by dilution with 1.5 mol NaCl at a ratio of 9:1(v/v). Solutions containing 80%, 70%, 60%, 50%, 40%, 30%, 20% and 10% Percoll in 0.15 mol NaCl were further prepared from the stock solution. A 1.2-ml volume of each of these solutions was carefully layered into a 13.2 ml polycarbonate tube to produce a step gradient with 80% Percoll at the bottom and the 10% Percoll at the top. One milliliter of each bacterial culture (OD₆₀₀ = 2) suspended in 0.15 mol NaCl was applied to the top of the 10% layer and the gradient was then centrifuged for 1 h at 10,000 × g at 4 °C in a SW41Ti rotor using a Beckman centrifuge (Optima L-100XP, Beckman Coulter, Inc. CA, USA). The gradient was visualized using fuchsine stained layers as the marker (M).