Staining with an Acridine Orange Derivative for the Detection of Mycoplasmas in Cell Cultures

• Fischer 0., Dagmar Zendulkova: Staining With an Acridine Orange Derivative for the Detection of Mycoplasmas in Cell Cultures. Acta vet. Brno, 62,1993: 49-53. 24 cell cultures were examined for the presence of mycoplasmas by fluorescence methods using 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA) or bisbenzimide 33258 (Hoechst), and by culture in liquid media containing glucose or arginine and, under anaerobic conditions, on solid media. Mycoplasmas were detected in 17.9,25.5 and 28.6 per cent of the cultures by staining with AMHA, staining bisbenzimide and by culture, respectively. The differences in sensitivities of the methods were not significant. Disadvantages of staining with AMHA were discussed. Fluorescence, staining, mycoplasma contamination Detection of mycoplasmas in cell cultures by fluorescence methods is based on the visualization of nucleic acids of mycoplasmas. The stains used include bisbenzimide (Chen 1977), olivomycin (Mikhailova et aI. 1982) and 4'-6-diamidine-2-fenylindol (OAPI) (Russell et al. 1975). J ayat-Vignoles et aI. (1990) described the use of a new acridine orange derivative 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA) for this purpose. In our laboratory, checks of the presence of mycoplasmas in cell cultures have been performed using the culture and the bisbenzimide 33258 (Hoechst) methods. Staining with AMHA was examined to extend the set of the detection methods, and the results were compared with those of the bisbenzimide and the culture methods • . Materials and Methods The examined cell cultures (Table 1) came from the cell culture bank and other laboratories of the Veterinary Research Institute, Bmo, as well as from laboratories outside the institute. Altogether 117 samples of 24 cell cultures, of which 23 were monolayer cultures and 1 (myeloma line FO) was· a semisuspension culture, were examined. The monolayers were grown in a closed system in Mueller, Legroux or Roux flasks in Eagle's Minimal Essential Medium supplemented with 10 per cent of fetal calf serum, penicillin (100 I.U. per 1 ml) and streptomycin (100}tg per 1 ml). The .cells were released enzymatically before re-seeding them by a solution containing 0.1 to 0.2 per cent of chymotrypsin or trypsin, and 0.02 per cent of versene. The propagation of hepatoma cell lines was described by Hankinson (1979). Before the examination by fluorescence, the cells were inoculated into test tubes containing pieces of slides (5 x 20 mm) and 2 ml of growth medium, and incubated at 37°C. A cell suspension density was chosen that would not produce a complete monolayer during 3 to 5 days of growth. The propagation of the semi-suspension myeloma cell line FO and its co-culture with Vero cells, used as an indicator, were described earlier (Fischer et aI. 1991). The acridine orange derivative AMHA was kindly supplied by Prof. H. W. Zimmermann from the Institut fUr physikalische Chemie der Universitiit in Freiburg, FRG. Stock solution of AMHA in distilled water (10 mM) was stored iIi the dark at +4 °C and working solutions were prepared before staining. The concentration 5}tM and an exposure period