Advances in PCR-based detection of mycoplasmas contaminating cell cultures.

M ycoplasmas (the trivial name for microorganisms belonging to the class Mollicutes) are the smallest free-living, self-replicating bacteria, having diameters of 300 to 800 nm. These pleomorph microorganisms have no cell walls. (1~ Because of their small size and flexibility, mollicutes can pass through filters of 450 and 220 nm used commonly in cell culturing. ~,2) Mollicute contamination of primary and continuous eukaryotic cell lines represents a major problem of economic and biological importance in basic research, diagnosis, and biotechnological production. This contamination problem is widespread. Surveys show that 5-87% of cell lines are contaminated. (3-6~ There are currently ~120 mollicute species, (~ but 5 species account for ~>95% of cell contaminations. (3'7-9~ The common contaminants are two bovine mollicutes, Mycoplasma arginini and Acholeplasma laidlawii; two human mollicutes, Mycoplasma orale and Mycoplasma fermentans; and a porcine mollicute, Mycoplasma hyorhinis. (1~ Figure 1 shows a fibroblastic cell line contaminated with M. fermentans detected by scanning electron microscopy. Although contamination originates from laboratory personnel and commercial animal sera used in culture media, the main source of contamination of clean cultures is mollicute-infected cultures.(2,7,11) Mollicutes are capable of altering virtually every property and parameter measured in cell cultures, depending on the contaminating species and on the type of cell infected, leading to unreliable experiments and unsafebiologicals, biopharmaceutical drugs, and virus vaccines. It has been shown that mollicutes affect cell growth and morphology, nuFIGURE 1 Scanning electron microscopy of 3T6 cell line infected with M. fermentans. Arrows indicate the mycoplasmas adsorbed on the cell surface.