Detection of Mycoplasmas in Cultures of Myeloma Cell Lines and Hybridomas by Fluorescence Method Using Vero Cells as Indicators

Abstract

Four myeloma cell lines and four hybridomas were examined for the presence of mycoplasmas by the fluorescence method using bisbenzimide 33258 (Hoechst). The cell suspensions under examination were cultivated for 3 days together with Vero cells as indicators and then examined by fluorescence microscopy. Mycoplasma contamination was detected in 2 myeloma lines. The results were confirmed by culture methods and by scanning electron microscopy. Mixed cultures, bisbenzimide, scanning electron microscopyFluorescence (Chen, 1977) is often used for the detection of mycoplasmas and other contaminant microorganisms in cell cultures. The method is based on chemical bound of the fluorochrome bisbenzimide 33258 (Hoechst) to DNA. The fluorescence microscopy visualizes not only nuclear' DNA of the examined cells, but also DNA present in the contaminant microorganisms.In our laboratory, routine examinations of monolayer cell cultures using fluorescence method' by Chen (1977), modified by Machatkova et al. (1986) are performed. In correctly stained preparations, only the nuclei of examined cells and organellae of contaminant microorganisms. containig DNA show bright, greenish-yellow fluorescence. Weak fluorescence, sometimes observed in the cytoplasm of examined cells is considered to be an artifact. Mycoplasmas and bacteria are the' most frequent contaminant microorganisms, but we have demonstrated that mould and yeast: contaminations are also detectable by the fluorescence method (Fischer et al. 1989).While the fluorescence method is reliable and, above all, much faster than culture methods for the examination of monolayer cell cultures, it cannot be used for direct examinations of semi-sus-. pension cultures of myeloma cells and hybridomas. Staining with bisbenzimide results in a very' intensive fluorescence of myeloma and hybridoma cells and fluorescence of the much smallercontaminant microorganissms can be overlooked easily (Smirnova and Fridlyanskaya 1985) .. Regarding the threat of mycoplasma contamination of myeloma cell lines and hybridomas maintained in our laboratory (Granatova et al. 1988), we tried to detect mycoplasms in these semi-sus--pension cultures by the fluorescence method using Vero cells as indicators. Materials and MethodsMyeloma cells and hybridomas (Table 1) were cultured in an open system in H~aeus thermo-· stat at 37°C in an atmosphere containing 5 % COg. The medium RPM! 1640 (USOL, Prague)· supplemented with nonessential amino acids (USOL, Prague) was used. The content of an ampule was added to 1 OOOml medium containing· 2 mM/ml L-glutamine, 2 mM/ml· sodium pyruvate, 40 I'g/ml gentamycine and 10 % bovine fetal serum (Cooperative farm, Sokolnice). Before reseeding~.the cells were released from the walls of Mueller flasks by agitating. The reseeding ration was 1 :3., Vero indicator cells (ATCC.CCL 81), isolated from kidney of Cercopithecus aethiops, were cul-

The Use of Ciprofloxacin for Decontamination of LSCC-H 32 Cell Line of Chicken Embryonal Fibroblasts Contaminated by Mycoplasma arginini

“…Mycoplasmas in cell cultures in antibiotic-free media propagate well and can be detected' by a combination of the fluorescence method and culture methods . Scanning electron microscopy serves only as a supplementary method, because it is very difficult to differentiate mycoplasmas adhered to cell surfaces and cell microvilli (Fischer et al 1991). The sensitivity of decontaminated cell line LSCC-H 32 to avian viruses will be the subj ect of another research project.…”

“…Mycoplasmas were detected in the LSCC-H 32 culture prior to, during and after decontamination by culture in liquid media supplemented with glucose, urea or arginine, culture in a solid medium Ourmanova and Machatkova 1986), the fluorescence method by Chen (1977), modified by Machatkova et al (1986) and scanning electron microscope Tesla BS 300 (Fischer et al 1991). Indirect immunofluorescence method (Rosendal and Black 1972) was used for the determination of mycoplasma species.…”