Mycoplasma detection in a mouse cell line and possible contamination source

The detection of cintaminating mycoplasma in a NS-1 myeloma cell line was studied comparing three methods The probable source of mycoplasma contamination were some batches of commercial sera. Fetal Bovine Sera (FBS), mouse myeloma NS-1 cells and Vero cells (African green monkey) were cultured on PPLO medium. The other methods involved the use of an indicator cell culture system (Vero cells) and the DNA-fluoroehrome staining technique. The bacteriological procedure for the isolation of mycoplasma was successful with NS-1 and Vero cells, but not with FBS. However, myeroplasma colonies with typical "fried egg" appearance were only observed with Vero cells. Moreover, the number of colonies isolated could be appreciated only after 18 days of growth. Vero tested, showed cytopathic effect (CPE). Initially, dark grasules appeared in the cytoplasm of the cells. At the 5th or 7th day, cell membranes showed small finger-like projections and vacuolization. By the 3rd or 4th week, the CPE was more pronunced, Monolayers of Vero cells were also grown on coverslips for 2 to 5 days and were stained with Hoeehts DNA fluorescent stain. The cells showed discrete zones of fluorescence in the cytoplasm and nuclei. The fluorescent spots were time-dependent Hoechst technique appears to be the method of choice, since it is more efficient, less time consuming and simpler.