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How to prevent contamination in cell culture?

Views: 0     Author: Site Editor     Publish Time: 2023-07-04      Origin: Site

This article mainly introduces common types of cell contamination.


Work area:

  1. Is the ventilation hood for cell culture set up correctly?

  2. Are there any airflows or direct entry channels in the area where the cell culture ventilation hood is located?

  3. Is the workbench clean?

  4. Have only the items required for the experiment been placed on the workbench?


  5. Have you wiped the workbench with 70% ethanol before starting work?

  6. Do you regularly clean and disinfect the incubator, refrigerator, freezer, and other laboratory equipment?


Personal hygiene:

  1. Have you washed your hands?

  2. Have you worn personal protective equipment?

  3. If you have long hair, is it tied back?

  4. Are you using a pipette to handle liquids?

  5. Reagents and culture media

  6. Have you sterilized all reagents, media, and solutions prepared in the laboratory using appropriate methods?

  7. Before placing containers, culture bottles, culture plates, and culture dishes on the work surface, have you wiped their exteriors with 70% ethanol?

  8. Have you tightened the lids of reagent bottles, culture bottles, and other containers when not in use?

  9. Have all culture plates been placed in sterile, airtight bags?

  10. Are there any signs of contamination in the reagents, such as cloudiness, floating particles, unpleasant odors, or abnormal colors? If so, have you cleaned or discarded them?


Operation:

  1. Are you operating slowly, carefully, and paying attention to aseptic techniques?

  2. Have you wiped the surfaces of pipettes, reagent bottles, and culture bottles with 70% ethanol before placing them in the cell culture ventilation hood?

  3. Have you placed the lids facing downwards in the work area?

  4. Are you using sterile glass pipettes or disposable sterile plastic pipettes to handle liquids?

  5. Are you using sterile pipettes only once to avoid cross-contamination?

  6. Have you avoided touching the tip of the pipette to any non-sterile items, including the outer edge of bottle neck threads?

  7. If liquid spills occur, have you immediately absorbed it and wiped the area with 70% ethanol?



It should be noted that the placement and arrangement of items on the super clean bench, as well as personal hygiene such as long hair, are easily overlooked. Too many items on the super clean bench can cause interference to the air flow and air pressure, so everyone needs to pay attention.


"Know yourself and your enemy, and you will never be defeated in a hundred battles." Knowing how to prevent contamination, we also need to know how to identify it. After all, contaminated cells need to be discarded promptly, otherwise, if the contaminants collect all the gems, it will be...


Cell culture contamination is mainly divided into two categories: chemical contaminants, such as impurities in culture media, serum, and water, endotoxins, plasticizers, and detergents; and biological contaminants, such as bacteria, fungi, yeast, viruses, mycoplasma, and cross-contamination of other cell lines.




Bacterial contamination:

Bacteria come in various shapes, such as spherical, rod-shaped, and spiral-shaped. Bacteria and fungi together make up the most commonly encountered biological contaminants in cell culture. When cells are contaminated with bacteria, it can often be detected within one or two days by simple observation with the naked eye, and the pH value of the culture medium may suddenly decrease. The image below shows 293 cells contaminated with E. coli and growing as adherent cells.





Fungal contamination:

In the initial stage of fungal contamination, the pH of the culture medium remains stable, but as the contamination worsens, the pH rapidly increases, causing the medium to become turbid. Under the microscope, the hyphae often appear as thin bundles, and sometimes as dense clusters of spores. Spores can withstand extremely harsh and unfavorable environments during dormancy. Therefore, when fungal contamination occurs, it is necessary to promptly discard the cells to prevent large-scale contamination in the incubator.




Yeast contamination:

Similar to bacterial contamination, the culture medium becomes turbid when contaminated with yeast, especially during late-stage contamination. The pH of the culture medium changes very little after contamination with yeast, and only increases when the contamination is severe. Under the microscope, yeast appears as individual oval or spherical particles, some of which may bud into daughter cells. The image below shows 293 cells contaminated with yeast.




Viral contamination:

Viruses have extremely small sizes, making it difficult to detect and remove them from the reagents used in cell culture. As most viruses have very strict requirements for their hosts, they generally do not have adverse effects on cell cultures from host species other than their own. However, the use of cell cultures infected with viruses can pose serious health threats to experimental operators.



Mycoplasma contamination:

Mycoplasma is considered the smallest self-replicating organism. Due to its extremely small size, detection of mycoplasma is very difficult, and there are often no obvious signs of infection unless the density is very high. Some mycoplasma can even persist in cell cultures without causing cell death, but they can alter the behavior and metabolism of cells in the culture system. The possible manifestations of chronic mycoplasma infection include a decrease in cell proliferation rate, a decrease in nuclear density, and cell aggregation in suspension culture. The most effective methods for detecting mycoplasma contamination are fluorescence staining, ELISA, PCR, immunostaining, radiographic self-development, or microbiological determination techniques to periodically test the culture.



Cross-contamination:

Although not as common as microbial contamination, widespread cross-contamination between many cell lines and rapidly growing cell lines such as HeLa cells is a clear problem. Obtaining cell lines from reputable cell banks, regularly checking the properties of cell lines, and using good aseptic techniques are conventional methods to help avoid cross-contamination.




Use of antibiotics:

Antibiotics should not be routinely used in cell culture because their overuse can promote the development of antibiotic-resistant strains, leading to persistent low-level contamination. Once antibiotics are removed, this low-level contamination can eventually develop into large-scale contamination, and continued use of antibiotics can also mask mycoplasma infections and other types of contamination. Therefore, antibiotics can only be used as a last resort against contamination and should only be used for a short period of time, and should be removed as soon as possible.


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