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

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 of 15 minutes, as recomm~up~p qA J ayat-Vignoles et a1.(1990), were used in pilot experi-• ments, but the fluorescence was very weak only.Therefore, the concentration was increased to 10 pM and the exposure period prolonged to 30 minutes.
The slides with cell islets were rinsed with isotonic phosphate buffer PBS (pH 7.2), stained with 10 pM AMHA for 30 minutes, washed with PBS for another 15 minutes, mounted into glycerol buffer (pH 5.5) (Machatkova et a1. 1986), and viewed in a Zeiss fluorescence microscope at a magnification of 252 x .
Staining with bisbenzimide 33258 (Hoechst) was performed using a modification (Machatkova et a1.1986) of Chen's (1977) method.The incomplete cell monolayers were rinsed with PBS (pH 6.5 to 6.8) and the cells were fixed with methanol for 15 minutes.Mter air-drying, the cells were stained with 0.001 per cent solution of bisbenzimide 33258 in methanol for 10 minutes, rinsed with distilled water, mounted into glycerol buffer (PH 5.5) and viewed as described for the AMHA-stained cultures.
The examination by culture was preceded by at least 3 passages in a streptomycin-free medium.Both liquid media, containing glucose or arginine, and solid media were used.The culture on solid media passed under anaerobic conditions (Jurmanova and Machatkova 1986;Jurmanova et al. 1990).
. The results were processed by the Student's t-test.

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Mycoplasmas were demonstrated in 13 of the 24 cell cultures (Table 1).17.9, 25.5 and 28.6 per cent of the samples were positive by the AMHA, bisbenzimide and culture methods, respectively.The differences in seIlsitivities of the methods were not significant (P > 0.05).
Discolouration of liquid media, resulting from the fermentation of glucose or arginine, became evident within 10 to 14 days of incubation of mycoplasma-infected cell cultures.Staining with bisbenzimide or AMHA visualized mycoplasmas as minute, intensively fluorescent, yellow-green particles in the vicinity of large, intensively fluorescent, yellow-green nuclei (Fig. 2 and 4); only fluorescent cell nuclei were observed in mycoplasma-free cell cultures (Fig. 1 and 3).
The two staining methods differed in their intensities and persistences of fluorescence.
While nucleoli were clearly distinguishable in the fluorescent nuclei, the background was dark and cytoplasma was stained in sporadic cells and only very weakly in cell cultures stained with bisbenzimide, no nucleoli were recognisable and the background and cytoplasma were bright yellow-green in those stained with AMHA (Fig. 1 to 4).
The fluorescence persisted longer in the bisbenzimide-stained than in the AMHA-stained cell cultures.

Discussion
It follows from the description of the two fluorescence methods that staining with Al\IHA is more time-consuming than that with bisbenzimide (45 vs. 25 minutes).Regarding the shorter persistence of fluorescence of AMHA-stained cultures, • Jayat- Vignoles et al. (1990) recommended to use an anti-fading solution.
In our experiments this step was replaced by mounting of the preparations into glycerol buffer as described by Machatkova et al. (1986).A more intensive and persistent staining was achieved by increasing the concentration of AMHA from 5 to 10 pM and prolonging the exposure period from 15 to 30 minutes.
While only nuclei and nucleoli are stained by bisbenzimide, and the fluorescence of cytoplasma, if present; is regarded as an unwanted artifact (Fischer et al. 1991), cytoplasm of all cells is stained by AMHA.J ayat- Vignoles et al. (1990) explained this phenomenon by the ability of AMHA to stain both nuclear DNA and cytoplasmatic RNA.Minute fluorescent particles can be overlooked easily on a bright background and therefore the fluorescence• of cytoplasm is unwanted.Staining with bisbenzimide is apparently more suitable, because the background is dark and the minute mycoplasmas can be recognized easily.This is important especially in weakly contaminated cell cultures.
Regarding the difficulties associated with the detection of mycoplasmas in• cell • cultures, parallel use of at least two methods should be preferred to relying upon the results of any single method.
No significant differences in sensivities were demonstrated between the two staining methods.However, AMHA staining had more disadvantages than staining with bisbenzimide.PouZiti derivatu akridinove oran!e k detekci mykoplazmat v bune~Dych kulturach .

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Number of positive and total number of samples are given in the numerator and denominator, respectively.••Visible colonies of mycoplasmas developed on solid media after 14 days of

Table 1
Comparison of staining and culture methods for detection of mycoplasmas in cell cultures