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Updated: Aug 13, 2026
Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Published on: September 16, 2013
Characterization of Pneumocystis carinii preparations developed for lipid analysis
E S Kaneshiro1, M A Wyder, L H Zhou
1Department of Biological Sciences, University of Cincinnati, Ohio 45221.
Abstract:
Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non-P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10(8)-10(9) organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80-95%.
Insights
Researchers developed a new purification method to isolate Pneumocystis carinii organisms from rat lungs. This technique yields highly pure, viable parasites for further study, crucial for understanding this opportunistic pathogen.
Area of Science:
- Medical Mycology
- Parasitology
- Infectious Diseases
Background:
- Pneumocystis carinii is an opportunistic pathogen causing pneumonia in immunocompromised individuals.
- Previous methods for isolating P. carinii resulted in low purity and viability, hindering research.
- Understanding P. carinii requires pure, viable organism preparations for accurate study.
Purpose of the Study:
- To develop and optimize protocols for purifying Pneumocystis carinii organisms.
- To achieve high purity, recovery efficiency, and organism viability in isolated P. carinii.
- To establish a reliable method for obtaining P. carinii for experimental use.
Main Methods:
- Intratracheal intubation of rats with P. carinii preparations.
- Testing various purification techniques including mucolytic agents, sieving, centrifugation, osmotic shock, and membrane filtration.
- Biochemical characterization using surfactant protein A quantification and protein content analysis.
- Assessing purity via light and electron microscopy, and viability using the calcein acetoxymethyl ester-propidium iodide assay.
Main Results:
- Developed a multi-step purification protocol yielding P. carinii free of intact host cells and with minimal debris (<5%).
- Achieved >99.5% purity as determined by surfactant protein A levels and <6% non-P. carinii protein.
- Obtained high yields (10^8-10^9 organisms/rat) with excellent viability (80-95%).
- Confirmed elimination of host lipids and reduced extracellular markers during purification.
Conclusions:
- A robust and reproducible method for purifying Pneumocystis carinii has been established.
- The developed protocol provides highly pure and viable organisms suitable for various research applications.
- This advancement facilitates further investigation into the biology and pathogenesis of P. carinii infections.
