Flow-Cytometric Method for Viability Analysis of Mycoplasma gallisepticum and Other Cell-Culture-Contaminant Mollicutes
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Flow‑Cytometric Method for Viability Analysis of Mycoplasma gallisepticum and Other Cell‑Culture‑Contaminant Mollicutes Rafael Lawson‑Ferreira1 · Marta A. Santiago2 · Thaize Q. Chometon3,5 · Vanessa A. Costa2,3 · Sergio A. Silva4 · Alvaro L. Bertho2,3 · Ivano de Filippis1 Received: 8 April 2020 / Accepted: 14 October 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract 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.
Alvaro L Bertho and Ivano de Filippis have contributed equally to the work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00284-020-02255-1) contains supplementary material, which is available to authorized users. * Rafael Lawson‑Ferreira [email protected] 1
National Institute for Quality Control in Health, FIOCRUZ, Laboratory of Reference Microorganisms, Rio de Janeiro, RJ, Brazil
2
Oswaldo Cruz Institute, FIOCRUZ, Laboratory of Immunoparasitology, Rio de Janeiro, RJ, Brazil
3
Oswaldo Cruz Institute, FIOCRUZ, Flow Cytometry Core Facility, Rio de Janeiro, RJ, Brazil
4
National Institute for Quality Control in Health, FIOCRUZ, Quality Department, Rio de Janeiro, RJ, Brazil
5
University of Auckland, School of Biological Scie
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