Purification of Treponema pallidum from Infected Rabbit Tissue: Resolution into Two Treponemal Populations

Virulent Treponema pallidum organisms, extracted from infected rabbit testes, were subjected to velocity sedimentation in discontinuous gradients of Hypaque, a high density, low viscosity material. After centrifugation of extracts at 20 C for 45 min at 100,000 x g, treponemes separated into two distinct bands based upon their relative velocities, although some variation was observed in the densities of the two bands and the number of treponemes per band. Rabbit tissue components sedimented more rapidly. Dark field and electron microscopy of preparations after velocity sedimentation indicated that treponemes retained general structural characteristics and no tissue contamination occurred in the treponemal fractions. Purification of treponemes in Hypaque resulted in their loss of motility and infectivity based upon animal inoculation. Antigenicity with respect to reactivity with antibody was preserved as shown by the high fluorescence intensity of treponemes in the fluorescent treponemal antibody adsorption test.