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Viability of yeast form cells of Paracoccidioides brasiliensis after sonication
Maria Fernanda Reis Gavazzoni Dias1, Jacilene Mesquita, Noêmia Rodrigues
1Hospital Universitário Clementino Fraga Filho, Departamento de Dermatologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil. mariafernanda@domain.com.br
Abstract:
To perform in-vitro studies with Paracoccidioides brasiliensis yeast cells it is necessary to avoid the presence of clumps of cells while maintaining their integrity. Because of the multiple budding type of growth, the bud cells are always attached to the mother cell and the yeast cells keep growing, resulting in the formation of large clumps. In order to obtain free cells, the cultures are usually sonicated. The present study shows that sonication induces lesions in a significant number of cells, as evaluated by labelling of the cells with acridine orange and Janus green vital dyes. In some cases labelling was initially observed in only one cell of the clump; however, the other cells also became labelled after a few minutes. These observations were confirmed by scanning and transmission electron microscopy of treated cells. Colony forming units (c.f.u.) on BHI plates also confirmed the decrease in cell viability following sonication.
Insights
Sonication of Paracoccidioides brasiliensis yeast cells for in-vitro studies causes cell damage and reduces viability. This method is not ideal for obtaining intact, viable yeast cells for research.
Area of Science:
- Mycology
- Cell Biology
- Microbiology
Background:
- Paracoccidioides brasiliensis yeast cells form clumps due to their budding growth pattern.
- Obtaining single, intact yeast cells is crucial for accurate in-vitro studies.
- Sonication is a common method used to disaggregate yeast cell clumps.
Purpose of the Study:
- To evaluate the impact of sonication on the integrity and viability of Paracoccidioides brasiliensis yeast cells.
- To determine if sonication is a suitable method for preparing yeast cells for in-vitro experiments.
Main Methods:
- Yeast cell cultures of Paracoccidioides brasiliensis were subjected to sonication.
- Cell integrity was assessed using vital dyes (acridine orange and Janus green).
- Cell viability was confirmed through colony forming unit (c.f.u.) counts on BHI plates and electron microscopy (scanning and transmission).
Main Results:
- Sonication induced significant lesions in a notable number of yeast cells.
- Vital dye labeling indicated compromised cell membranes following sonication.
- Electron microscopy confirmed cellular damage.
- Sonication led to a decrease in viable colony forming units (c.f.u.).
Conclusions:
- Sonication is detrimental to the integrity and viability of Paracoccidioides brasiliensis yeast cells.
- The use of sonication for preparing these yeast cells for in-vitro studies is not recommended.
- Alternative methods should be explored to obtain intact and viable yeast cells.
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