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Common pollution is as follows:
1. bacteria: Under the ordinary inverted microscope, the bacteria are black and fine sand, which can have different shapes according to the infected bacteria. The culture medium will generally become turbid and yellow, which has obvious influence on cell growth.
Carefully check the sterilization of utensils to see if there is enough time and pressure to deflate when autoclaving! In particular, articles that come into contact with the storage medium, such as pipettes, may pollute the storage medium if they are contaminated twice in a row, so be sure to pay attention! Check whether the culture solution is turbid before the next use!
Corresponding antibiotics can be added to the culture solution for treatment.
2. Mould: The culture solution is clear, and there is no impurity under the inverted microscope. Incubation at 37℃ for 2-3 days is still clear, but flocculent impurities appear. Filamentous floating objects and obvious hyphae can be seen under the microscope. Cells can still grow, but after a long time, their vitality becomes worse.
Wipe the CO2 incubator with copper sulfate solution, then add saturated copper sulfate to the water tray, or add saturated sterilized disodium hydrogen phosphate high-salt solution to the tray of the incubator to prevent mold pollution.
3. Mycoplasma: black, which seems to be polymorphic. General culture medium will be turbid and infected with mycoplasma. Many sera in China are not negative for mycoplasma, and mycoplasma is one of the most common microorganisms in bovine serum. Furthermore, it cannot be removed by filtration. After mycoplasma infects cells, the pathological changes of cells are not obvious, but they just die slowly.
Tylosin, a veterinary drug for the treatment of mycoplasma disease, can be used for cell culture without any adverse reactions. When used by Sigma Company, mycoplasma pollution can be eliminated by culturing in 50ug/ml tylosin culture solution for 6 days or continuously passing for 2 generations. If it is a common antibiotic, it is recommended to use 8ug/ml.
4. Robinia pseudoacacia: It can penetrate the filter membrane or spread through the air. At low magnification, it is a black spot. At high magnification, you can see black bugs swimming around, and the culture medium is not turbid, which generally does not affect much. These cells can still be used. Often the cells grow well, the observed moving animals do not increase significantly, and the color and transparency of the culture medium do not change significantly. Similar phenomena can be found in cells cultured in the same batch of serum. There is no obvious effect on the growth state of cells, and the cells disappear naturally after vigorous proliferation, and no special treatment is needed except replacing serum. It is suggested that if cells are likely to be polluted in this way, the density of seed plates can be increased and the survival rate of cells can be improved.
5. Fungi: Generally, the culture medium is clear and does not change color, and there are filaments under the microscope. Some fungi look like dead cell fragments at first, but many small fragments are very clear, like corals, unlike cell fragments, and gradually grow into tiny black filaments. Fungi grow slowly and are not as easy to be found as bacteria, but once found, cells are polluted and difficult to preserve.
6. Protozoa: The culture medium can be slightly turbid. Under the microscope, there are many tiny spots and a little activity. Although cells can grow, the reproduction speed is obviously slowed down, the cell state is not good, the edge is unclear and the cells are opaque. They can grow with cells, but they will compete with cells for nutrition. This kind of growth is very common, but they are few in number and have advantages in cell stations, so they will not affect the normal growth of cells.
Possible causes of pollution: There are many possible reasons, such as disinfection of liquid preparations, operational problems, environmental problems and so on.
Regarding the aseptic conditions of the culture medium, the culture is based on a culture bottle (without adding cells), and it is observed after a period of trial culture at 37 degrees. If there is no bacterial growth, it is a problem of operation.
Double antibodies (streptomycin sulfate and ampicillin) can also be added to the culture medium in advance. However, the double antibody sometimes affects the state of cells, so it is necessary to remove the double antibody before transfection and detection of some indicators of cells, so as not to affect the experimental results.