Purification of bacteria

Although Nycodenz® has been widely used in the purification of a broad range of bacteria from soil samples (and other biological matrices), see Application Sheet C39, there are in addition some other specific examples of the purification of bacteria, which have been primarily grown in either mammalian or non-mammalian cells (or organisms), using both Nycodenz® and OptiPrep™.

Some of these methods are summarized in this Application Sheet. The majority of the published methodology is concerned with obligate intracellular bacteria and two of these, Chlamydophila abortus and Piscirickettsia salmonis are considered in Sections 2 and 3.

To purify the elementary bodies of Chlamydophila abortus Everson et al [1] used a density barrier of 18% (w/v) Nycodenz® containing 0.13 M NaCl, 3 mM KCl, 0.3 mM CaNa2EDTA, 5 mM Tris-HCl, pH 7.2. It was prepared from Nycoprep™ 1.15 (an isoosmotic solution of 27.6% Nycodenz® containing 3mM KCl, 0.3 mM CaNa2-EDTA, 5 mM Tris-HCl, pH 7.4, ρ = 1.15 g/ml). 

This is no longer commercially available so all solutions must be made from Nycodenz® powder as described below. The CaNa2-EDTA in the Nycoprep™ 1.15 was present only to increase solution stability during autoclaving and may not be necessary for the separation. 

It may either be omitted or replaced by Na2- EDTA. The Tris, in the example below, has been replaced by the more cell-friendly buffer Tricine. It is highly probable that Nycodenz® can be replaced by iodixanol, but this has not been verified experimentally.