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Plaque assay for Rickettsia rickettsii
Abstract:
A plaque technique for the assay of Rickettsia rickettsii is described. The method employs primary chick or green monkey kidney monolayer cell cultures with either an agarose or special Noble agar overlay. Plaques were counted in 6 days and resultant titers correlated well with ld(50) end points obtained by a standard assay in embryonated eggs. Identification of the plaque-forming organisms was accomplished by direct observation of rickettsiae-like bodies in the monolayer lesions, inhibition of plaques by antibiotics, sensitivity of plaques to specific immune serum, and failure to cultivate other microorganisms from the infected cells. Versatility of the test was demonstrated by assaying samples of rickettsiae from several different sources commonly used in our laboratory. These included infected yolk sacs, various cell cultures, and infected guinea pig tissue. Sufficient numbers of viable rickettsiae were present in the cells of a single lesion to permit direct recovery.
Insights
A new plaque assay for Rickettsia rickettsii using cell cultures offers a reliable method for quantifying these bacteria. This technique provides accurate titers comparable to traditional assays and allows for direct recovery of viable Rickettsia rickettsii.
Area of Science:
- Microbiology
- Cell Biology
- Infectious Diseases
Background:
- Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever, a serious tick-borne illness.
- Accurate quantification of Rickettsia rickettsii is crucial for research and diagnostics.
- Existing methods for Rickettsia rickettsii assay can be labor-intensive or require specialized equipment.
Purpose of the Study:
- To describe a novel plaque assay for the enumeration of Rickettsia rickettsii.
- To validate the accuracy and reliability of this new plaque assay.
- To demonstrate the versatility of the plaque assay for various sample types.
Main Methods:
- Utilized primary chick or green monkey kidney monolayer cell cultures.
- Employed agarose or Noble agar overlay for plaque formation.
- Incubated cultures for 6 days to count plaques and determine titers.
- Identified plaque-forming organisms via microscopy, antibiotic sensitivity, immune serum neutralization, and microbial culture.
Main Results:
- The plaque assay demonstrated good correlation with traditional LD(50) endpoint assays in embryonated eggs.
- Identification methods confirmed the plaque-forming agents as Rickettsia rickettsii.
- The assay successfully quantified Rickettsia rickettsii from diverse sources including yolk sacs, cell cultures, and guinea pig tissues.
- Viable Rickettsia rickettsii could be directly recovered from single lesions.
Conclusions:
- The described plaque assay is a valid and reproducible method for quantifying Rickettsia rickettsii.
- This technique offers a versatile alternative for Rickettsia rickettsii titrations across different sample matrices.
- The assay facilitates direct recovery of viable bacteria, aiding further research and diagnostics.