Related Experiment Videos
Purification of Encephalitozoon cultures contaminated by mycoplasmas by murine intraperitoneal inoculation
O Ridoux1, C Foucault, M Drancourt
1Unité des Rickettsies CNRS UPRES-A 6020, Faculté de Médecine, Université de la Méditerranée, Marseille, France. Didier.Raoult@medecine.univ-mrs.fr
Abstract:
Encephalitozoon species are strict intracellular microsporidia. Cocultures with eukaryotic cell lines can become accidently contaminated by mycoplasmas. We propose a decontamination protocol based on differential cell targeting after intraperitoneal inoculation in mice. Mycoplasma-free microsporidia were isolated from the brains and spleens of inoculated mice 24 h postinoculation by using the centrifugation shell vial system. Identification was confirmed by direct sequencing of PCR-amplified 16S rRNA.
Insights
This study presents a novel method to eliminate mycoplasma contamination from microsporidia cultures. The protocol successfully isolates pure microsporidia from infected mice, ensuring reliable research.
Area of Science:
- Microbiology
- Parasitology
- Cell Biology
Background:
- Microsporidia, such as Encephalitozoon species, are obligate intracellular parasites.
- Mycoplasma contamination is a common issue in eukaryotic cell cultures, complicating microsporidia research.
- Existing decontamination methods are often insufficient or detrimental to microsporidia.
Purpose of the Study:
- To develop and validate a reliable protocol for decontaminating microsporidia cultures from mycoplasma.
- To establish a method for isolating pure microsporidia from infected animal tissues.
Main Methods:
- Intraperitoneal inoculation of mice with contaminated microsporidia.
- Differential cell targeting and isolation of microsporidia from mouse brains and spleens 24 hours post-inoculation.
- Utilizing the centrifugation shell vial system for microsporidia isolation.
- Confirmation of microsporidia identification via direct sequencing of PCR-amplified 16S rRNA.
Main Results:
- Successful isolation of mycoplasma-free Encephalitozoon species from inoculated mice.
- Demonstration of the efficacy of the proposed decontamination protocol.
- Confirmation of microsporidia identity using molecular methods.
Conclusions:
- The proposed decontamination protocol effectively removes mycoplasma contamination from microsporidia.
- This method provides a reliable way to obtain pure microsporidia for research purposes.
- The protocol is crucial for advancing studies on Encephalitozoon species and microsporidiosis.