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

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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.

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Acknowledgments

This research was supported in part by an internal funding from IOC-FIOCRUZ-PAEF II–IOC-023-18-2-47 and FAPERJ E-26/010.002832/2014. The authors would like to thank to Immunoparasitology Lab., Oswaldo Cruz Institute, FIOCRUZ for the use of CytoFlex Flow Cytometer.

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Author notes

  1. Alvaro L Bertho and Ivano de Filippis have contributed equally to the work.

Authors and Affiliations

  1. National Institute for Quality Control in Health, FIOCRUZ, Laboratory of Reference Microorganisms, Rio de Janeiro, RJ, Brazil

    Rafael Lawson-Ferreira & Ivano de Filippis

  2. Oswaldo Cruz Institute, FIOCRUZ, Laboratory of Immunoparasitology, Rio de Janeiro, RJ, Brazil

    Marta A. Santiago, Vanessa A. Costa & Alvaro L. Bertho

  3. Oswaldo Cruz Institute, FIOCRUZ, Flow Cytometry Core Facility, Rio de Janeiro, RJ, Brazil

    Thaize Q. Chometon, Vanessa A. Costa & Alvaro L. Bertho

  4. National Institute for Quality Control in Health, FIOCRUZ, Quality Department, Rio de Janeiro, RJ, Brazil

    Sergio A. Silva

  5. University of Auckland, School of Biological Sciences, Auckland, New Zealand

    Thaize Q. Chometon

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  1. Rafael Lawson-Ferreira
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Contributions

RLF, MAS and ALB designed and conceived the study; RLF, MAS, TQC, VAC, SAS and ALB, performed acquisition, analysis and interpretation of data; RLF, MAS, TQC, ALB and IF wrote and extensively revised the manuscript. All authors have read and critically revised the manuscript.

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Correspondence to Rafael Lawson-Ferreira.

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Lawson-Ferreira, R., Santiago, M.A., Chometon, T.Q. et al. Flow-Cytometric Method for Viability Analysis of Mycoplasma gallisepticum and Other Cell-Culture-Contaminant Mollicutes. Curr Microbiol 78, 67–77 (2021). https://doi.org/10.1007/s00284-020-02255-1

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