Selective killing of mycoplasmas from contaminated mammalian cells in cell cultures

Abstract

Mycoplasmas are common contaminants of animal cells in cell cultures^1–3. About 25 Mycoplasma and Acholeplasma species have been identified as cell culture contaminants but the most frequent are Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini and Acholeplasma laidlawii³. These species are responsible for almost 85% of the contaminations in cell cultures³. We have devised a method for the selective killing of mycoplasmas in contaminated cell cultures, based on the differential nucleic acid metabolism of mycoplasma and mammalian cells. Mycoplasmas are unusual in that they have a nutritional requirement for nucleic acid precursors which can be met by purines and pyrimidine bases or by nucleosides^4–6. Mammalian cells, on the other hand, incorporate very little free pyrimidines^7,8 and for that reason incorporation of free bases such as uracil has been used to detect mycoplasmas in contaminated cell cultures⁹. One of the free base analogues which can be incorporated selectively into nucleic acids of mycoplasmas is 5-bromouracil (5-BrUra). Visible light induces breaks in 5-BrUra-containing DNA¹⁰ and this photosensitivity can be greatly increased by binding of the fluorochrome 33258-Hoechst to DNA¹¹. Its unusually high content of A + T makes the mycoplasma DNA³ an excellent candidate for the induction of breakage by the combined action of 5-BrUra, 33258-H and light because 33258-H has a high affinity for A−T base pairs and an even higher affinity for A–BrUra base pairs¹². We report here that treating them in this way provides a practical and simple method for the selective killing of contaminating mycoplasmas; they are probably killed by the breakage of their DNA.