Laboratory maintenance of Rickettsia rickettsii

Abstract

This unit includes protocols for the laboratory maintenance of the obligate intracellular bacterium Rickettsia rickettsii, including propagation in mammalian cell cultures, as well as isolation, counting, and storage procedures. Regulations for working with R. rickettsii in biosafety level 3 containment are also discussed.

Figure 1

Giménez staining of rickettsiae-infected Vero 76 cells. The black arrow points to an individual bacterium.

Figure 2

Diff-Quik staining of rickettsiae-infected Vero 76 cells. The black arrow points to an individual bacterium.

Figure 3

Rickettsiae, partially purified from Vero 76 cells. The black arrow points to an individual bacterium, and the red arrowhead points to cellular debris. (A) Giménez staining. (B) Diff-Quik staining. Scale bar = 10 μm

Figure 4

Ultracentrifuge tubes with rickettsiae purified from Vero 76 cells by Renografin density gradient ultracentrifugation. The rickettsiae were purified from six 150cm² flasks of heavily infected cells. (A) Tubes after the first run through the Renografin gradient. The black arrow points to the band of viable rickettsiae at the 32%–36% Renografin interface. The red arrowhead points to the debris at the SPG-32% Renografin interface. (b) Tubes after the second run through the Renografin gradient. The black arrow points to the band of viable rickettsiae at the 32%–36% Renografin interface.

Figure 5

Giménez staining of rickettsiae purified from Vero 76 cell by two rounds of Renografin density gradient ultracentrifugation.