Broadly speaking, all foreign ingredient or component changes in the cell culture system, should be regarded as pollution, cell pollution is not completely eliminated, but can reduce the frequency and the seriousness of the consequences.
Cell pollution can be divided into physical, chemical and biological pollution according to pollution sources.
(1) physical pollution:
Physical pollution by influencing the biochemical composition in cell culture system, which affects the cell metabolism, such as temperature, radiation, vibration, radiation (uv) or fluorescence affect cells.
Common examples are: the incubator temperature is too high, the equipment that can cause mechanical vibration is placed around, the reagent is put in the refrigerator that has glass door medium long term receives illumination etc.
Physical pollution is often overlooked or generally classified as chemical pollution.
(2) chemical pollution:
Many chemicals in the culture can cause cell pollution.
Chemicals don't always inhibit cell growth, and certain chemicals, such as hormones, promote cell growth.
Unpurified substances, reagents, water, serum, growth auxiliary factors, and containers of stored reagents can all become sources of chemical contamination.
In order to avoid the contamination of water by metal ions, organic molecules, and intracellular toxins, it is necessary to use ultra-pure water that contains no impurities when cleaning containers.
All substances used in cell culture should be of high purity and have been identified by the authorities.
At the same time, proper configuration and storage of medium and reagents are also very important. Standard operation procedures should be taken to avoid the errors of liquid volume calculation and the mixtures of similar compounds.
(3) microbial contamination:
The causes of microbial contamination can be divided into two stages: experimental preparation and experimental operation.
The reasons for the preparation stage include: the sterilization of experimental consumables is not thorough;
The cleanliness of cell room is not sufficient and the risk of microbial contamination is increased.
Ultra-clean table cleanliness is not enough, the disinfection is not thorough enough, the ultraviolet ray does not have the preexposure enough time, the surface does not use alcohol wipe;
No sterile latex gloves;
The reagent itself has been contaminated and has not been detected.
The pollution in the experimental operation is often not rigorous enough, and careless and careless, such as: hands passed from the top of the sterile reagent;
The liquid is not operated properly and the liquid is exposed to the front of the pipe-shifting device;
The suction fluid of the pipette was excessive;
No replacement, less pollution, etc.
Different microbial pollutants have different effects on cells, and the effects of mycoplasma and viruses on cell morphology and skills are long-term, slow and potential.
And mold and bacteria multiply rapidly and can inhibit cell growth or produce toxic substances in a very short period of time.
1. Mould pollution: many kinds of fungus, many of which are aspergillus, white candida, yeast, black mould, spore fungus, etc.
After mould pollution, most of them form pale yellow or white floating objects in the medium, which are generally visible to the naked eye and can be easily found. In the short term, the medium is not cloudy.
Under the inverted microscope, the cells can be seen crisscrossing through the silk, twigs or tubular mycelium and floating in the medium.
Many strains of mycelium can be seen under high magnification in a chain of strains.
Candida and yeast strains are ovoid, scattered on cells and cells.
Treatment: identify mold contamination. If the cell type is not particularly valuable, give up and disinfect the experimental link thoroughly.
As a last resort, you can try anti-fungal and anti-fungal B to clear the pollution, and the effect is hard to guarantee.
2. Bacterial contamination: bacterial contamination is more common in e. coli, white staphylococcus, pseudomonas, and so on.
The use of antimicrobial medium can prevent and eliminate the contamination of individual and small amounts of bacteria.
Once bacterial contamination is found, it is easy to find that in most cases the culture medium turns yellow, indicating that there is a large amount of acidic material, and there is obvious turbidity.
Sometimes the static medium is not mixed at first, but with a little concussion, a lot of turbidity floats.
Under the inverted microscope, there is a large amount of spherical particles floating in the medium. Sometimes, there are a lot of bacteria on the cell surface and surrounding. The cell stops growing and is poisoned.
If necessary, a small amount of medium smear stain should be taken to determine the type of bacteria;
Some of the culture changes are not obvious and there are suspected pollution, but also can be used in the broth culture medium in a small amount of culture medium, 37 degrees of culture to detect.
Treatment: 5~10 times of the usual dosage of antibiotics is used for shock therapy. It is used for 24~48 hours to change the normal culture medium, sometimes it is effective in early pollution.
3. Mycoplasma pollution: mycoplasma is a kind of between bacteria and virus (minimum diameter is 0.
2um) and living independently of microorganisms.
About 1 percent can be passed through the filter.
Mycoplasma - cell wall morphology is highly pleomorphic.
Can be round, silk or pear shaped.
Mycoplasma forms are changeful and the internal structure is not easy to be seen in the light environment.
Most mycoplasma are suitable for the existence of alkaloids (PH7).
0) the tolerance of acid tolerance.
Sensitive to heat, not sensitive to general antibiotics.
The mycoplasma is contaminated with cells.
The medium is not cloudy.
In most cases, the pathological changes of the cell are slight or not obvious, and the subtle changes can be alleviated by the transfer and exchange of the fluid, so it is easy to be ignored.
But individual severe, can cause cell to proliferate slowly.
Even from the culture vessels.
Fluorescence staining can be done to determine the presence of mycoplasma contamination: fluorescent dyes that can be combined with DNA specificity can be used to stain the DNA in the mycoplasma, which is then observed with fluorescence microscopy.
Treatment: the market has many kinds of original body cleaning kit, the effect is better.
(4) cross contamination of cells
Cell pollution is not the same as bacterial contamination. Cell pollution is caused by the combination of various cells in the culture process and the use of equipment or liquid.
This pollution does not exist in microorganisms, but it causes chaos among different kinds of cultured cells, cell growth characteristics and morphological changes.
The contaminated cells were not pure and could not be studied experimentally.
Control and treatment: in the process of multiple kinds of cell culture operation, all instruments should be strictly distinguished, and each cell should be operated in order to avoid the chaos caused by the operation.
When the liquid or the transfer operation is carried out, the dropper or the head should be replaced in time to avoid taking the cells to the culture bottle;
All from elsewhere or cell lines should be built by early reserves, have a plenty of cryopreserved once suspected cross-contamination, can be used as cell genetics appraisal, if discover the original cell genetic change, can be used in early recovery of cryopreserved cells.
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