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.
Jung
Hsuan
H
Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taipei, ROC.
Wang
Shih-Yee
SY
Yang
I-Wen
IW
Hsueh
Ding-Wei
DW
Yang
Wei-Ju
WJ
Wang
Tzu-Hao
TH
Wang
Hsin-Shih
HS
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D006801:Humans; D009174:Mycoplasma / Q000187:drug effects / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction
D000900:Anti-Bacterial Agents