Introduction to cell line contamination identification, prevention and treatment issues
Ordinary microbial contamination: The culture medium is turbid, and microbial contamination is observed under high-power microscope; discard according to regulations and be fully and strictly sterilized.
Mycoplasma contamination: It cannot be observed with a microscope. According to experience, it shows slow cell growth, floating cells, etc. It should be identified through PCR and other methods. If mycoplasma contamination is found, it should be discarded according to regulations. The laboratory must be fully disinfected and sterilized in a timely manner to prevent the spread. . (Some routine cytology experiments are not affected by mycoplasma contamination and can continue to use cell passaging. In order to prevent the spread of contamination, other high-potency antibiotics can be selectively added to the culture medium and operated in an isolation laboratory and used independently. All utensils and culture media, etc.).
Mycoplasma contamination detection: Mycoplasma contamination is the most common in cell culture and is difficult to detect. However, mycoplasma contamination can significantly affect the results of cell function interference experiments. Mycoplasma is the smallest microorganism currently known between bacteria and viruses that can live independently. It has no cell wall, is highly polymorphic in shape, has a minimum diameter of 0.2um, and can pass through a filter. At present, more than 100 domestic cell lines/lines have been tested, and the situation is not optimistic. The contamination rate reaches 30% ~ 60%. Mycoplasma contamination often occurs when the research team introduces cell lines/lines from abroad. After mycoplasma contaminates cells, it inhibits cell growth by affecting the synthesis of DNA and RNA in cells and rapidly consuming amino acids in the culture medium, and can reduce the fusion rate of cells. Therefore, when using various cell lines for experimental research, it is first necessary to prove whether the cells used are contaminated by mycoplasma. After mycoplasma contamination, the culture medium will not become turbid, and the cell lesions will be slight or inconspicuous, making it difficult to detect. At present, the methods for detecting mycoplasma include the following: (a) phase contrast microscopy; (b) hypotonic lichen red staining method; (c) fluorescent staining method; (d) enzyme labeling method; (e) PCR method (advantages : High sensitivity, short detection time, small sample volume, only 50ul of cell culture supernatant is needed; Disadvantages: higher cost of primers and preparations).