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Common types of biological pollution in cell culture include bacterial contamination, mold contamination, mycoplasma contamination, black rubber insect contamination, fungal contamination, and protozoal contamination. So what are the characteristics of their contamination in cell culture and what are the corresponding solutions? Xiaobian is organized as follows for everyone, I hope to be useful to everyone~
Bacteria
The bacteria are black and fine sand under the ordinary inverted microscope. Depending on the infected bacteria, the bacteria may have different shapes, and the culture solution will generally become cloudy and yellow, which has an obvious influence on cell growth.Solution
: Check the sterilization of the vessel carefully. Is it enough to deflate during autoclaving? The pressure is enough! In particular, items such as pipettes that are in contact with the culture medium may cause contamination of the storage solution if it is contaminated twice. Be sure to pay attention! Check if the culture solution is turbid before the next use! The antibiotic treatment can be added to the culture solution, such as tetracycline, gentamicin, or streptomycin.
2. Mold
: The culture solution is clear, no impurities under the inverted microscope, and cultured in a 37-degree incubator for 2-3 days, still clear, but flocculated impurities appear, and the filament-shaped cluster-shaped floating objects can be seen under the microscope, and obvious Mycelium, cells can still grow, but after a long time, the viability of the cells deteriorates.Solution
: Wipe the CO2 incubator with a copper sulfate solution, and add a saturated amount of copper sulfate to the water tray. Or add a sterile, disodium hydrogen phosphate high-salt liquid to the tray of the incubator to prevent mold contamination.After the CO2 incubator is contaminated with mold, all cells can be temporarily transferred, and the incubator (including the partition, the wall) is scrubbed with peracetic acid. The peracetic acid was placed in the incubator for an hour to flood the steam. After the odor of peracetic acid is dissipated, it is transferred to the cells. The incubator should be cleaned regularly (around February), especially during the rainy season.
Other incubator cleaning methods are: scrub with 84 liquid - scrub with water - 75% alcohol scrub - UV light.
To prevent mold contamination, add 3u/ml of amphotericin or nystatin or actinomycin D or double antibody to the medium; but once the cells are contaminated, it is difficult to save, nystatin or actinomycin D or double It is not helpful to resist, it is recommended to discard the contaminated cells. Thoroughly disinfect the environment. If all cells are contaminated, it may be systemic pollution. Check the medium and equipment. If it is only a single pollution, it may be an operational problem.
3. Mycoplasma
: Black, as if mostly polymorphous, the culture medium is generally turbid, the original infection, many domestic serum are not tested for mycoplasma negative, and mycoplasma is one of the most common microorganisms in bovine serum. And it can't be removed by filtering. After mycoplasma infects cells, the cytopathic effect is not very obvious, but it slowly dies.
Solution
: After mycoplasma contaminates cells, especially important cell lines, it is necessary to remove mycoplasma. Common methods include antibiotic treatment, antiserum treatment, antibiotic plus antiserum and complement treatment. It should be noted that mycoplasma has no cell wall, so antibiotics acting on cell wall biosynthesis, such as lactams and vancomycin, are not sensitive; they are generally resistant to polymyxin, rifampicin and sulfa drugs. The antibiotics most inhibiting mycoplasma are tetracyclines, macrolides, etc., and aminoglycosides and chloramphenicol have less inhibitory effects on mycoplasma. Replace all cultures as necessary. In general, the method of filtration sterilization has no effect on mycoplasma.Black worm
: It can penetrate the filter membrane or spread through the air. It is black dot at low magnification. The black bug can swim and swim at high magnification. The culture solution is also not flawed. Generally, it will not affect too much. use. Often, the cell growth state is good, and the observed moving objects are not significantly increased, and the color and transparency of the culture medium are not significantly changed, and a similar phenomenon can be found in the same batch of serum-cultured cells.Solution
: It will not have a significant effect on the cell growth state, and will naturally disappear after the cell proliferation is strong, and no special treatment is required except for the replacement of serum. It is suggested that if the cells are likely to be contaminated, the plate density of the cells can be increased to increase the survival rate of the cells.5. Fungi
: The general culture solution is clear, does not change color, and has a filament under the microscope. Some fungi begin to resemble dead cell debris, but many of its small pieces are very clear, like corals, unlike cell debris, which is slowly It will grow a very fine black silk. Fungi grow slower and are not as easy to spot as bacteria, but once they are found to be contaminated, it is difficult to save.Solution
: Decisively throw away, then thoroughly sterilize the culture room, CO2 incubator, utensils and culture.6. Protozoa
The culture medium can be slightly turbid. The number of tiny dots under the microscope is very large, and it is slightly active. Although the cells can grow, the reproduction speed is obviously slowed down, and the cell state is not good, the edges are unclear, and the cells are not transparent. They are symbiotic with cells but compete with cells for nutrition. This kind of symbiosis is very common, but their number is small, and the cells are dominant so they do not affect the normal growth of the cells. Only when they reach a certain amount will affect the growth of the cells and eventually form a vicious circle.Solution
There are many possible causes of pollution, such as liquid disinfection problems, operational problems, environmental problems, etc. If there are many cells, throw the resuscitated cells directly. If you are protecting the cells, you can purchase the relevant sterilization reagents.Possible causes of pollution
: There are many possible reasons, such as dosing disinfection problems, operational problems, environmental problems, and so on.Regarding the sterility of the medium, the medium was taken into a culture flask (without cells), and the culture was observed at 37 degrees for a while. If there is no bacterial growth, it is a problem of operation.
It is also possible to add a secondary antibody (streptomycin sulfate and ampicillin) to the culture medium in advance. However, the double antibody sometimes affects the state of the cell, so it is necessary to remove the double antibody before transfection and detection of a certain indicator of the cell to avoid affecting the experimental results.
1. The incubator should be disinfected with trioxane or ultraviolet light regularly, and the incubator should be wiped out with alcohol and Xinjieer. The water in the incubator should be three steamed water.
2, ultra-clean platform \ materials \ equipment \ culture liquid \ culture bottle \ operation and other factors
3, the fan of the ultra-clean platform can not be too large, the fan to 6-8 grid. Otherwise it may also cause mold contamination
4. After the sterile room is fumigated with formaldehyde, it can be neutralized with the same amount of ammonia water, and it can be operated in about a few hours.
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