Flow cytometry‐based method to detect and separate Mycoplasma hyorhinis in cell cultures

Abstract Mycoplasma hyorhinis is a frequently observed contaminant in cell cultures, and its detection and purification pose considerable challenges. Fragments or other cell components are similar in size to those of Mycoplasma ; therefore, distinguishing them is difficult. In this study, we used Hoechst staining in combination with carboxyfluorescein succinimidyl ester (CFSE) to label Mycoplasma . The trigger threshold was set in the Hoechst Blue channel rather than in the default forward scatter channel, utilizing the differences in DNA content between Mycoplasma and fragments. Subsequently, we identified and isolated it at single‐cell resolution via flow cytometry and successfully sorted infectious Mycoplasma in cell culture. Simultaneously, we validated the accuracy and feasibility of this approach using polymerase chain reaction, fluorescence confocal microscopy, and cryo‐electron microscopy. This methodology enabled the diagnosis of Mycoplasma at extremely low concentrations, significantly enhancing the detection efficiency and facilitating the isolation and purification of parasitic Mycoplasma in cell culture instead of pure Mycoplasma culture in artificial media for subsequent studies.