Abstract
Mycoplasma often surreptitiously contaminates cell cultures without detection, mainly parasitizing the cell surface and thus interfering to some extent with the cells. Direct DNA staining of cell cultures often yields equivocal results and will only reliably detect heavily contaminated cultures. Interpretation is difficult in cases where degraded DNA from host cells may produce small spots of fluorescence under microscopy that mimic mycoplasma. To quickly and directly screen for mycoplasma contamination, we stained cells that were either contaminated with mycoplasma or treated with antibiotics using a combination of DNA and cell membrane fluorescent dyes. The contamination of mycoplasma could be accurately assessed by determining its colocalization with the surface of the plasma membrane. This approach minimized interference from cytoplasmic DNA components and greatly improved the accuracy of using DNA staining alone for mycoplasma detection. This study aimed to develop a colocalization method for the rapid detection of mycoplasma in culture samples, facilitating early diagnosis and treatment. It could serve as a valuable tool for the swift identification of contamination in cell cultures.