Flow-Cytometric Method for Viability Analysis of Mycoplasma gallisepticum and Other Cell-Culture-Contaminant Mollicutes

• Published in: Current microbiology Vol. 78; no. 1; pp. 67 - 77
• Main Authors: Lawson-Ferreira, Rafael, Santiago, Marta A., Chometon, Thaize Q., Costa, Vanessa A., Silva, Sergio A., Bertho, Alvaro L., de Filippis, Ivano
• Format: Journal Article
• Language: English
• Published: New York Springer US 2021; Springer Nature B.V
• Subjects: Biological products; Biomedical and Life Sciences; Biotechnology; Cell culture; Cell Culture Techniques; Cell death; Cell viability; Contaminants; Contamination; Ethanol; Flow Cytometry; Growth rate; Life Sciences; Markers; Microbiology; Mycoplasma; Mycoplasma gallisepticum; Quality control; Reference materials; Statistical analysis; Tenericutes; Titration
• ISSN: 0343-8651; 1432-0991
• DOI: 10.1007/s00284-020-02255-1

Mycoplasma is the smallest self-replicating bacteria, figuring as common contaminant of eukaryotic cell cultures. Production inputs and operator’s manipulation seem to be the main sources of such contamination. Many analytical approaches have been applied for mycoplasma detection in cell cultures and also in biological products. However, unless they were validated, only indicator cell culture and bacteriological culture are considered as compendial methods for quality control of biological products. Nano-flow cytometry has been pointed out as an alternative technique for addressing prokaryotic and eukaryotic cell viability being a substantial tool for reference material production. In this study, a viability-flow-cytometry assay was standardized for
M. gallisepticum
and then applied to other cell-culture-contaminant mycoplasmas. For this,
M. galliseticum
’s growth rate was observed and different treatments were evaluated to establish low viability cultures (cell death-induced control). Distinct viability markers and their ideal concentrations (titration) were appraised. Ethanol treatment showed to be the best death-inducing control. CFDA and TOPRO markers revealed to be the best choice for detecting live and dead mycoplasma frequencies, respectively
.
The standardized methodology was applied to
Mycoplasma arginini
,
M. hyorhinis
,
M. orale
,
Spiroplasma citri
and
Acholeplasma laidlawii
. Significant statistical difference was observed in the percentage of viable cells in comparison to ethanol treatment for
A. laidlawii
in CFDA and in both markers for
M. gallisepticum
,
M. hyorhinis
and
S. citri
. In summary, we standardized a flow cytometry assay for assessing
M. gallisepticum
 − and potentially other species – viability and ultimately applied for reference material production improving the quality control of biological products.