Mycoplasmas are the smallest self breeding prokaryotes from the class of Mollicutes. They have no cell wall. Due to the presence of cholesterol in their cell membranes, they are extremely deformable and can thus pass through the usual sterile filters (0.2 μm). Mycoplasmas are parasitic, intra- and extracellular living bacteria. Contaminations of cell cultures with mycoplasmas are common and often difficult to detect as they do not always produce dramatic effects. Mycoplasmas can spread rapidly to other cultures (including liquid nitrogen). Of all infections, 95% are caused by M.orale, M.arginii, M.fermentans, M.salivarum, M.hyorhinis and A.laidlawii. Mycoplasmas are parasites! They deprive nutrients from the cells which they can not produce by themselves because of their small genome.
The effects of mycoplasma contamination on the cells are diverse: Mycoplasmas produce toxins that damage the cells Induction of chromosomal aberrations Changes in membrane antigenicity Changes in transfection efficiency Increased cell death rate Inhibition of cell proliferation through nutrient deprivation Inhibition of cell metabolism Changes in DNA, RNA and protein biosynthesis Influence on virus production Activation of immune cells (lymphocytes)
Contaminations can significantly affect the reproducibility and validity of research results, as well as the profitability of production. Cell cultures should therefore be routinely tested for mycoplasmas. The detection of mycoplasma can be done by different methods.
Detection methods of mycoplasma contaminations: Detection methods:
qPCR (according to Ph. Eur.):
The polymerase chain reaction (PCR) makes it possible to enrich a specific fragment a millionfold from small amounts of heterogeneous DNA. The primers used include mycoplasma-specific, highly conserved sequences of 16S ribosomal RNA (rRNA). The real-time quantitative PCR (qPCR for short) is a method for amplifying DNA. It is based on the principle of "conventional" PCR. The quantification is carried out by means of fluorescence measurements, which are recorded in real time after each PCR cycle. Fluorescence increases proportionally with the amount of PCR products. The PCR can be performed directly from the sample (e.g., serum, medium) or from the cell culture supernatant. If the qPCR is performed according to Ph. Eur., the DNA is isolated from a sample (200 μl) or from the cell culture supernatant (200 μl) and then the qPCR is carried out.
Fluorescence detection with DAPI:
DAPI (4,6'-diamidino-2-phenylindole) is a DNA-binding fluorescent dye. It preferentially binds to AT-rich sequences in the minor groove of double-stranded DNA. Mycoplasma contamination can be detected by staining the mycoplasma DNA with the fluorochrome DAPI. The mycoplasmas appear as uniformly shaped, small, brightly glowing spots or accumulations of those outside the indicator cells (usually at the cell membrane). Since DAPI does not stain the RNA and mitochondrial DNA is barely visible, the background fluorescence is very low.
Detection of mycoplasma-specific enzymes:
A biochemical, highly sensitive and specific test kit is used to measure the activity of mycoplasma-specific enzymes. Living mycoplasmas are lysed and their enzymes are released. The released carbamate kinase and acetate kinase are incubated with the substrate mix. This leads to a conversion from ADP to ATP. The formed ATP in the sample is measured via a luciferase reaction. If mycoplasmas are present in the sample, a significant [increase] of the ATP level occurs, which leads to an intensified light reaction.
Product recommendation:
From a scientific and economic point of view, a sustained removal of mycoplasma from cell culture is absolutely essential. Not always can infected cell cultures be discarded and replaced with new ones. Infected cells also can not be treated with the standard antibiotics since the efficiency of this antibiotic treatment is usually insufficient. Mycorase 50x Solution (PAN-Biotech, Product number P06-02100) is a reagent for the permanent elimination of mycoplasma in cell culture. | 
