Elucidating the Effectiveness of Antibiotic Treatments in Eliminating the Black Dots Contamination from Vero Cell Line Culture

The present study was conducted to identify the effectiveness of various antibiotics and antibiotic combinations in eliminating the black dots contamination in cell culture and to identify its effect on cell growth kinetics. Isolation of contaminant organism (Black dots) observed in our laboratory, was attempted by inoculation of harvested cell supernatant and pellet into Nutrient broth, Luria broth, and Pleuropneumonia-like organisms' broth but no visible growth was observed. To analyze the effect of antibiotics in elimination of contamination, the cell monolayer was incubated with different antibiotics and combination of antibiotics till 120 hrs. Withdrawal effect of antibiotics was observed by further incubation cells in maintenance media for another 48 hrs. To investigate the effect of different antibiotics on cell growth kinetics, cell seeding was done in media containing antibiotics. A subset of cells was harvested every 24 hrs and counted till 120 hrs. Among all antibiotics, Ampicillin reduced the contamination, the Piperacillin and Tazobactam combination diminished the contamination to a great extent. At the studied concentrations of the antibiotic trio of piperacillin, tazobactam, and ampicillin, black dot contamination was eliminated and found 100% effective in inhibiting contamination within 120 hrs of the study period; but, after 48 hrs of antibiotic withdrawal, contamination reappears in cell culture. The growth kinetics showed no significant impact of antibiotics treatment on the cell growth curve. The growth kinetics showed that cell growth was not interfering significantly by individual or combination of antibiotics. Therefore, in the presence of black dot, a combination of three antibiotics (piperacillin, tazobactam, and ampicillin) should be effective in cell culture.