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Quantification and assessment of viability of Pneumocystis carinii organisms by flow cytometry
S E Lapinsky1, D Glencross, N G Car
1Division of Pulmonology, University of the Witwatersrand Medical School, Parktown, Johannesburg, South Africa.
Abstract:
Analysis of drug efficacy in animal models of Pneumocystis carinii pneumonia requires an accurate method of quantification of organisms, as well as a means of assessing viability. Lung homogenates were prepared from a colony of athymic nude F344 rats experiencing a spontaneous outbreak of P. carinii pneumonia. With the fluorescent nucleic acid stain propidium iodide, flow cytometric analysis was able to quantify P. carinii cysts and trophozoites reproducibly. As this stain is excluded by living cells, this method was also used to assess the viability of organisms. Application of this technique to analysis of bronchoalveolar lavage specimens was demonstrated.
Insights
Flow cytometry with propidium iodide staining accurately quantifies Pneumocystis carinii organisms and assesses viability in rat pneumonia models. This method is crucial for evaluating drug efficacy in Pneumocystis pneumonia research.
Area of Science:
- Immunology
- Infectious Diseases
- Microbiology
Background:
- Accurate quantification and viability assessment of Pneumocystis carinii are essential for studying drug efficacy in animal models.
- Spontaneous Pneumocystis carinii pneumonia outbreaks in athymic nude F344 rats provide a relevant model system.
Purpose of the Study:
- To develop and validate a flow cytometry-based method for quantifying Pneumocystis carinii organisms.
- To assess the viability of Pneumocystis carinii using a specific fluorescent stain.
Main Methods:
- Preparation of lung homogenates from rats with Pneumocystis carinii pneumonia.
- Utilizing propidium iodide, a fluorescent nucleic acid stain, for flow cytometric analysis.
- Applying the technique to bronchoalveolar lavage specimens.
Main Results:
- Flow cytometry reproducibly quantified Pneumocystis carinii cysts and trophozoites.
- Propidium iodide exclusion by live cells allowed for accurate viability assessment.
- The method was successfully applied to bronchoalveolar lavage samples.
Conclusions:
- Flow cytometry with propidium iodide is a reliable method for quantifying Pneumocystis carinii and assessing its viability.
- This technique enhances the analysis of drug efficacy in Pneumocystis pneumonia animal models.
- The method is applicable to both lung homogenates and bronchoalveolar lavage specimens.