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How to determine mycoplasma contamination?

Mycoplasma contamination is a common occurrence and labs must be vigilant in monitoring and combating contamination issues.  Mycoplasma are bacteria extremely difficult to detect in cultured cells by visual inspection.  Many labs do not incorporate recurring mycoplasma testing until the lab experiences a contamination incident, at which time cleaning and testing procedures are put in place.

Established labs with experienced cell culture scientists typically have procedures in place to minimize mycoplasma contamination.  Here are common practices employed by those labs:

  • All new cell lines in the lab are cultured and housed in designated quarantine hoods and incubators; these hoods and incubators are also utilized for training aseptic techniques to new employees
  • Backup frozen cell aliquots are tested prior to freezing, along with aliquots being tested as they are brought into culture
  • Routine mycoplasma testing should be incorporated that specifies all cultures to be tested; common testing occurrence is every 2 weeks or quarterly
  • Routine cleaning procedures should be incorporated every 6 months to include extensive decontamination of the hoods, floor, cabinet, water baths and equipment (centrifuges, incubators)


There are multiple commercially available kits to detect mycoplasma contamination utilizing various detection methods: 1) PCR primers specific to mycoplasma genome coupled with agarose gel analysis, 2) fluorescent staining of mycoplasma, or 3) ELISA.

To Treat or Not

If a cell culture is found to be positive, the researcher must decide to throw out the cells or put cells through a lengthy treatment plan.  Typically, it is easiest to toss the contaminated cells in the trash and bring into culture a fresh, frozen aliquot.  If the contaminated cells are precious and there is not a backup, commercially available antibiotics and protocols to eliminate mycoplasma contamination are available.

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