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Detection of fibrils associated with Rickettsia rickettsii
Abstract:
The ultrastructural appearance of the "halozone" formed at the interface between the spotted fever agent Rickettsia rickettsii and the cytoplasm of persistently infected cultured vole cells (Microtus pennsylvanicus) was studied by transmission electron microscopy. In sections of epoxy-embedded specimens stained with uranyl acetate and lead citrate, the halozone appeared clear and devoid of ultrastructural features. However, when unembedded preparations of whole infected cells were examined at 1,000 kV, fine structural features were observed within the halozone. These features, associated with the rickettsial outer membrane, were more clearly detectable when the infected cells were extracted with the detergent Triton X-100 before fixation. Under such conditions, long extensions of the rickettsial outer membrane, microfilament-like structures attached to that membrane, and extensive attachments between adjacent rickettsiae were seen. The fine structural features within the rickettsial halozone were also seen at 75 kV when unembedded sections were prepared from polyethylene glycol-embedded specimens. Thus, epoxy-embedding medium obscures the fine structural features within the halozone surrounding the rickettsiae in infected cells.
Insights
Ultrastructural analysis revealed hidden features within the halozone surrounding Rickettsia rickettsii in infected cells. Epoxy embedding obscured these details, which were visible using high-voltage electron microscopy on unembedded samples.
Area of Science:
- Microbiology
- Cell Biology
- Electron Microscopy
Background:
- The spotted fever agent Rickettsia rickettsii infects host cells, forming a "halozone" at the host-pathogen interface.
- Understanding the ultrastructure of this halozone is crucial for comprehending Rickettsia-host cell interactions.
Purpose of the Study:
- To investigate the ultrastructural characteristics of the halozone surrounding Rickettsia rickettsii in persistently infected vole cells.
- To determine the effect of sample preparation methods on the visibility of halozone features.
Main Methods:
- Transmission electron microscopy (TEM) of infected Microtus pennsylvanicus cells.
- Examination of both epoxy-embedded and unembedded cell preparations.
- High-voltage (1,000 kV and 75 kV) electron microscopy.
- Cell extraction with Triton X-100 prior to fixation.
Main Results:
- Epoxy-embedded specimens showed a clear halozone devoid of ultrastructural detail.
- Unembedded preparations, especially after Triton X-100 extraction, revealed fine structures within the halozone.
- Observed features included extensions of the rickettsial outer membrane, associated microfilament-like structures, and inter-rickettsial attachments.
- Polyethylene glycol embedding also allowed visualization of these features in unembedded sections.
Conclusions:
- Standard epoxy embedding obscures critical fine structural details of the Rickettsia halozone.
- High-voltage electron microscopy and specific sample preparation techniques (unembedding, detergent extraction) are essential for visualizing the true ultrastructure of the halozone.
- These findings provide new insights into the physical interactions between Rickettsia rickettsii and its host cell cytoplasm.