Disruption of Bordetella pertussis in the Ribi cell fractionator. 2. Effect of different suspending media.

Abstract

Using the RCF at various pressures and centrifuging the resulting lysates at 20 000 g, cell envelope and soluble fractions of B. pertussis were obtained. At each pressure the extent of solubilization of several biologically important factors was greatly influenced by the medium in which the cells were suspended during disruption. Disruption at 20 000 – 30 000 psi in tris or tris-EDTA buffers, pH 8.0, gave cell envelopes containing very little of the original DNA, lipopolysaccharide, histamine-sensitizing factor, or protective antigen, whereas disruption in phosphate buffer, pH 7.0, gave cell envelopes rich in these factors. The solubilizing effect of tris on DNA and on cell envelope components could be counteracted by Mg2+ and less efficiently by NaCl or sucrose.