Control of Pleuropneumonia-like Organisms in Cell Culture
1The Upjohn Company, Kalamazoo, Michigan 49001.
Abstract:
Mammalian cell culture systems were maintained free of mycoplasmas by using a 3-day agar plate test as a weekly routine to monitor the conditions of the cells. If contaminated cell cultures were found, they were discarded and replaced from a pleuropneumonia-like organism (PPLO)-free cell bank. PPLO-free lines were established by treatment with various antibiotics. The KB cell line was freed of mycoplasmas by treatment for 1 week with a mixture of chlortetracycline, kanamycin, and chloramphenicol. L-929 cells were cleared of contamination with either spectinomycin or tylosin, and a synovial cell line was cleared with lincomycin or tylosin. Each cell line, after eradication of the contaminant, was stored in liquid nitrogen. A number of agents were tested to determine minimal inhibitory concentration against three known and three unidentified mycoplasmas. Chlortetracycline and tetracycline were found to be highly active against all strains, whereas tylosin, spectinomycin, and lincomycin, though less active, were equally useful because of their low toxicity against cells. Kanamycin was highly active against three strains, but inactive at high levels against the KB cell contaminants. A disc plate test was used to check isolated cell contaminants for sensitivity to various agents.
Insights
Maintaining mycoplasma-free mammalian cell cultures is crucial. Weekly testing and antibiotic treatment, like chlortetracycline and tylosin, effectively eradicate mycoplasma contamination, ensuring reliable cell line integrity.
Area of Science:
- Cell Biology
- Microbiology
- Biotechnology
Background:
- Mycoplasma contamination poses a significant threat to mammalian cell culture systems.
- Routine monitoring and effective eradication strategies are essential for maintaining cell line integrity and experimental reproducibility.
Purpose of the Study:
- To establish and maintain mycoplasma-free mammalian cell lines.
- To evaluate the efficacy of various antibiotics for mycoplasma eradication.
- To determine the minimal inhibitory concentrations (MICs) of different antimicrobial agents against various mycoplasma strains.
Main Methods:
- Weekly monitoring of cell cultures using a 3-day agar plate test.
- Discarding contaminated cultures and replacing them from a mycoplasma-free cell bank.
- Treating contaminated cell lines with specific antibiotic combinations (e.g., chlortetracycline, kanamycin, chloramphenicol; spectinomycin; tylosin; lincomycin).
- Testing antibiotic efficacy by determining MICs against known and unidentified mycoplasma strains.
- Utilizing a disc plate test to assess contaminant sensitivity to antimicrobial agents.
Main Results:
- Successful eradication of mycoplasma from KB, L-929, and synovial cell lines using specific antibiotic treatments.
- Chlortetracycline and tetracycline demonstrated high activity against all tested mycoplasma strains.
- Tylosin, spectinomycin, and lincomycin showed lower activity but were valuable due to low cellular toxicity.
- Kanamycin was effective against some strains but not against KB cell contaminants at high levels.
Conclusions:
- A routine weekly agar plate test is effective for monitoring and preventing mycoplasma contamination in cell cultures.
- Specific antibiotic treatments can successfully eradicate mycoplasma from contaminated cell lines.
- The choice of antibiotic for mycoplasma eradication should consider both efficacy and cellular toxicity.
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