Detection of Mycoplasma contamination in Animal Cell Cultures

Detection of Mycoplasma Contamination

The purpose of detecting mycoplasma contamination in a cell line is validate cell line by proving it to be free of mycoplasmal contamination.

Detection of mycoplasma infections is not possible by routine microscopy. Detection of mycoplasma contamination requires techniques like fluorescent staining, Polymerase Chain Reaction (PCR), Enzyme Linked Immunosorbent Assay (ELISA), immunostaining, autoradiography, or microbiological assay.

Fluorescent staining of DNA by Hoechst stains that involve bis-benzimides is the easiest and is a very reliable method, it displays mycoplasmal infections as a fine particle or filamentous staining over the cytoplasm when observed at 500x magnification. As the DNA staining is done, the nuclei of the cultured cells are also brightly stained by this method and thereby act as a positive control for the staining procedure.

Other microbial contamination are also observed due to fluorescence staining, so low level contamination, very small organisms such as micro-cocci can also be detected. Usually, mycoplasmas do not always reveal their presence by means of macroscopic and visible alterations in the cells or medium. Most mycoplasma contamination, particularly in continuous cell lines, grow slowly without affecting the host cells, but they can alter the metabolism of the culture in different ways. Because mycoplasmas take up thymidine from the medium, infected cultures show abnormal labelling with [³H] thymidine. Immunologic study can be totally frustrated by mycoplasma contamination, as tends to create antibodies against cell surface may raise anti-mycoplasma antibodies. Mycoplasmas alter cell behaviour and metabolic activities in many different ways, so there is a requirement for routine, periodic assays to identify all possible contaminations of cell cultures, particularly the continuous cell lines.

The signs of a chronic mycoplasma infections includes a reduced rate of cell division and proliferation, declined saturation density, and agglutination in suspension during growth. On the other hand, acute infection causes complete deterioration, with a few possibly resistant colonies, although these or any other resulting cell culture or cell lines are not necessarily free from contamination and there are possibilities that they may carry a chronic infection.
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Did you read ? 
Maintenance of Sterility in Animal Tissue Culture.
Prevention of Cross-contamination in Animal cell cultures 
Also read: 

Detection of Mycoplasma Contamination in Animal Cell Cultures.

Viral Contamination in Animal cell cultures.