Mycoplasma detection in cell culture Review
Mycoplasmas are the smallest and simplest self-replicating organisms. Due to their seriously degraded genome they cannot perform many metabolic functions, such as cell wall production or synthesis of nucleotides and amino acids.
Mycoplasmas are strictly parasites. They parasitize a wide range of organisms including humans, animals, insects, and plants.
Mycoplasma and Acholeplasma
Mycoplasma and Acholeplasma are Mollicutes, that comprise together more than 100 recognized species. Among them, about 20 species have been described as contaminants of eukaryotic cell cultures. However 5 species (Mycoplasma (M.) arginini, M. fermentans, M. orale, M. hyorhinis and Acholeplasma laidlawii) are isolated in 90-95% of contaminated cell cultures[1].
Mycoplasma contamination of cell cultures occurs through 3 major sources: 1) incoming infected cells, 2) the laboratory personnel, potential carriers of M. orale, M. fermentans, M. salivarium or M. hominis, and 3) the reagents used in cell culture, such as bovine sera (M. arginini, A. laidlawii) or porcine trypsin (M. hyorhinis). Once a contamination is established, bacteria spread by aerosol droplet dispersion. If "Good Laboratory Practices" are not strictly maintained such as regular disinfection, all the equipment (pipettes, laminar flow hoods, incubators) can be contaminated and participate to the spread of mycoplasmal contamination[2].
Mycoplasma contamination
In many cases there are no signs of mycoplasma contamination. In contrast to other microbial contaminants, mycoplasmas do not cause consistent perceptible changes in a cell culture, e.g. rapid pH change or culture turbidity[2,4]. They can grow to very high concentrations, typically 106- 108/ml, but due to their small size (0.1-0.8 µm in diameter) they remain undetectable by microscopic observation. Although invisible, mycoplasmas can cause disastrous effects on eukaryotic cells as they can alter every cellular parameter from proliferation to virus susceptibility and production. The only way to confirm mycoplasma contamination is by routine testing using special techniques.
Mycoplasma detection in cell culture
There are numerous methods for the detection of mycoplasmas among these are direct growth on broth/agar, DNA staining, PCR, ELISA, RNA labeling and enzymatic procedures. However, none of these methods is 100% reliable. Direct growth methods are relatively sensitive to most species but the overall procedure is lengthy (3 weeks), costly and less sensitive to noncultivable species. The PCR method, although rather fast and inexpensive, is limited by its sensitivity and the risk of positive and false negative results.
InvivoGen has developed a mycoplasma detection method that promises to resolve these issues. This method is based on the detection of mycoplasmas by engineered cells that express Toll-like receptor 2, a pathogen recognition receptor that detects mycoplasmas.
PlasmoTest™, InvivoGen's new mycoplasma detection kit, exploits this method. The kit contains all the reagents necessary to perform the assay. PlasmoTest™ is simple and affordable allowing its routine use for presumptive results.
1. McGarrity G. et al., 1992. Mycoplasmas and tissue culture cells. In: Maniloff, J., McElhaney, R.N., Finch, L.R., Baseman, J.B. (Eds.), Mycoplasmas, Molecular Biology and Pathogenesis, American Society for Microbiology, Washington DC, pp.445-54.
2. Lincoln CK,Gabridge MG., 1998. Cell culture contamination: sources, consequences, prevention, and elimination. Methods Cell Biol. 57: 49-65.
3. Razin S. et al., 1998. Molecular biology and pathogenicity of mycoplasma. Microbiol Mol Biol Rev. 62: 1094-1156.
4. Doyle A, Griffiths JB., 1998. The cell: selection and standardization. In: Cell and tissue culture: laboratory procedures in biotechnology. Doyle, A and Griffiths JB, Wiley and Sons, Ltd.35-52.
