Related Experiment Videos
Improved plaque assays for Rickettsia prowazekii in Vero 76 cells
P F Policastro1, M G Peacock, T Hackstadt
1Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840, USA.
Abstract:
Typhus group rickettsiae, including Rickettsia prowazekii and R. typhi, produce visible plaques on primary chick embryo fibroblasts and low-passage mouse embryo fibroblasts but do not form reproducible plaques on continuous cell culture lines. We tested medium overlay modifications for plaque formation of typhus group rickettsiae on the continuous fibroblast cell line Vero76. A procedure involving primary overlay with medium at pH 6.8, which was followed 2 to 3 days later with secondary overlay at neutral pH containing 1 microgram of emetine per ml and 20 micrograms of NaF per ml, resulted in visible plaques at 7 to 10 days postinfection. A single-step procedure involving overlay with medium containing 50 ng of dextran sulfate per ml also resulted in plaque formation within 8 days postinfection. These assays represent reproducible and inexpensive methods for evaluating the infectious titers of typhus group rickettsiae, cloning single plaque isolates, and testing the susceptibilities of rickettsiae to antibiotics.
Insights
Researchers developed new methods for plaque formation of typhus group rickettsiae using continuous cell lines. These assays enable reproducible evaluation of rickettsial infectious titers and antibiotic susceptibility.
Area of Science:
- Microbiology
- Cell Biology
- Virology
Background:
- Typhus group rickettsiae (Rickettsia prowazekii, R. typhi) form plaques on primary fibroblasts but not continuous cell lines.
- Reproducible plaque assays are crucial for quantifying rickettsial infections and drug screening.
Purpose of the Study:
- To establish reproducible plaque formation assays for typhus group rickettsiae on continuous cell lines.
- To enable efficient titration, cloning, and antibiotic susceptibility testing of these pathogens.
Main Methods:
- Modification of medium overlay conditions for Vero76 cells.
- Two-step overlay: initial pH 6.8, followed by neutral pH with emetine and NaF.
- Single-step overlay with dextran sulfate-containing medium.
Main Results:
- A two-step overlay method yielded visible plaques in 7-10 days.
- A single-step overlay method produced plaques within 8 days.
- Both methods proved reproducible and cost-effective.
Conclusions:
- Developed novel, inexpensive, and reproducible plaque assays for typhus group rickettsiae on continuous cell lines.
- These assays facilitate accurate infectious titer determination and antibiotic susceptibility testing.
- Enables cloning of single plaque isolates for further research.