Eliminating mycoplasmas from contaminated cell cultures

Mycoplasmas are bothersome contaminants in cell cultures. Cells derived from various sources are subject to contamination and although many procedures have been suggested to eliminate the contaminating mycoplasmas, there is no simple, rapid and effective method for eliminating them. We have devised a method for the selective killing of mycoplasmas based on the differences between the nucleic acid metabolism of mycoplasmas and that of tissue culture cells. Whereas the nutritional requirements of mycoplasmas for nucleic acid precursors can be met by purine and pyrimidine bases, mammalian cells do not incorporate the free bases. The approach was to selectively incorporate the free base analogue 5-bromouracil (5-BrUra) into the mycoplasmas followed by photosensitization of the DNA containing 5-BrUra by low concentrations of the fluorochrome 33258-Hoechst. Such treatment renders the mycoplasma DNA very susceptible to breaks induced by visible light. The unusually high A + T content in mycoplasma DNA makes these organisms suitable candidates for the induction of breakage by the combined action of 5-BrUra, 33258-Hoechst and light because 33258-Hoechst has a high affinity for A + T base pairs and an even higher affinity for A-BrUra base pairs. We report that Mycoplasma hyorhinis and Acholeplasma laidlawii contaminating Chinese hamster cells were selectively killed by our technique, and cured clones of cells were easily obtained from the treated cell cultures. The technique proved efficient in curing BHK-21 and RAG cells from unknown contaminating mycoplasmas suggesting that this technique could be generally applied to eliminate contaminating mycoplasma strains from mammalian cell culture.