Visualization of lipopolysaccharide aggregates by freeze-fracture and negative staining

The morphology of Escherichia coli 0111:B4 lipopolysaccharide (LPS) in aqueous medium was studied by freeze-fracture and negative staining. Samples processed by freeze-fracture showed LPS aggregates that were mainly spherical or elliptical and of rather homogeneous size. Negative staining, however, showed a more heterogeneous population, although globular structures revealed by both procedures had a similar size. Considering the mechanisms involved in the processing of the samples, we suggest that the genuine shape of LPS aggregates is more likely to be globular, thus being artefactual those forms visualized by negative staining, traditionally associated with the organization of LPS in aqueous suspension. Key words: lipopolysaccharide, freeze-fracture, negative staining Gram-negative bacteria express at their outer membrane an amphiphilic macromolecule, termed lipopolysaccha-ride (LPS), that is responsible for a wide spectrum of pathophysiological effects in susceptible hosts, including humans.1 ' The organization of the LPS aggregates formed in aqueous suspensions is related to their biological activities. Thus changes in LPS size by sonication or association with plasma proteins, lead to LPS structures with modifications in their endotoxic activities.23 ' Isolated LPS has been usually analyzed by negative staining4 ' and the structures visualized by this technique are still considered to be the real organization of LPS aggregates in suspension. It has been suggested that, as a consequence of the staining treatment, there are possible modifications in the LPS structures5'; thus, non-disturbing techniques, such as synchrotron diffrac-tion, are being used in the study of the LPS supramolecular states.6' In this work we have compared the structural characteristics of E. coli 0111:B4 LPS in aqueous media, prepared either by freeze-fracture or negative staining