Detection and treatment of mycoplasma contamination in cultured cells

Background: Mycoplasmas, the smallest and simplest prokaryotes that reside in endosomes of mammalian cells, are widespread contaminants found in cell cultures. About 30% of all cell cultures, varying from 15 to 80%, are reportedly contaminated with mycoplasmas. Here, we present our experience in successfully detecting and treating mycoplasmal infection in various cell lines. Methods: The nested polymerase chain reaction (PCR) detection and microscopic examination, including phase-contrast, fluorescent, as well as differential interference contrast, were used for detecting potential mycoplasma contamination of cell lines used in our laboratory. As soon as mycoplasma was identified, antibiotic treatment was initiated. Results: Mycoplasmal contamination was detected in six of 15 cell lines using the nested PCR amplification of mycoplasma DNA, which was further demonstrated using 4, 6-Diamidino-2-phenylindole (DAPI) staining and fluorescent microscopy. Alternate treatment with two antibiotics, macrolide (tiamulin) and tetracycline (minocycline), effectively eliminated mycoplasma, which was validated by both PCR and microscopic studies. Conclusions: The nested PCR using genomic DNA extracted from cultured cells as templates is a rapid and sensitive method for detecting mycoplasma contamination. Treatment with combined antibiotics can completely eradicate mycoplasmal infection from cultured cells. For the ease of use, PCR and/or DAPI staining appear suitable for detecting potential mycoplasmal contamination in laboratories that rely heavily on the cell culture system.