Common pollution conditions and solutions in cell culture - Database & Sql Blog Articles

Common types of biological pollution in cell culture include bacterial contamination, mold contamination, mycoplasma contamination, black worm contamination, fungal contamination, and protozoal contamination. So what are the characteristics of these contaminations in cell culture, and what are the corresponding solutions? Let's go through them together to help you better understand and manage your cell culture process.

Bacteria Under an ordinary inverted microscope, bacteria appear as dark, fine sand-like particles. Depending on the type of bacteria, they may have different shapes, and the culture medium often becomes cloudy and yellow, significantly affecting cell growth.

Solution: Carefully check the sterilization of the vessel. Is there enough steam during autoclaving? Ensure the pressure is sufficient. Items like pipettes that come into contact with the culture medium can cause contamination if they are not properly sterilized. Always check the clarity of the culture solution before use. You can add antibiotics such as tetracycline, gentamicin, or streptomycin to the culture medium for prevention.

2. Mold The culture medium appears clear initially, but after 2–3 days in a 37°C incubator, flocculent impurities appear. Under the microscope, filamentous, clustered floating objects can be seen, along with visible mycelium. While cells may still grow, their viability gradually declines over time.

Solution: Wipe the CO₂ incubator with a copper sulfate solution and add a saturated amount of copper sulfate to the water tray. Alternatively, use a sterile, high-salt disodium hydrogen phosphate solution in the incubator. If mold contamination occurs, transfer all cells temporarily and thoroughly scrub the incubator (including shelves and walls) with peracetic acid. Leave it for an hour, then air out before reusing. Regular cleaning of the incubator is essential, especially during rainy seasons. Other cleaning methods include using 84 disinfectant, washing with water, wiping with 75% alcohol, and UV light exposure. To prevent mold, add 3 μ/ml of amphotericin, nystatin, actinomycin D, or double antibodies to the medium. However, once contaminated, it’s hard to save the cells. It’s recommended to discard them and thoroughly disinfect the environment.

3. Mycoplasma Mycoplasma appears as irregular, polymorphic structures. The culture medium may be slightly turbid. Many domestic serum samples are not tested for mycoplasma, making it one of the most common contaminants in bovine serum. It cannot be removed by filtration. After infection, the cytopathic effect is not obvious, but cells slowly die.

Solution: For important cell lines, it’s crucial to remove mycoplasma. Common methods include antibiotic treatment, antiserum treatment, or a combination of both. Note that mycoplasma lacks a cell wall, so antibiotics targeting cell wall synthesis (e.g., penicillins, vancomycin) are ineffective. They are generally resistant to polymyxin, rifampicin, and sulfonamides. Tetracyclines and macrolides are more effective. Aminoglycosides and chloramphenicol have less inhibitory effects. In severe cases, replace all cultures. Filtration sterilization does not eliminate mycoplasma.

Black Worms These organisms can pass through filter membranes or spread through the air. At low magnification, they appear as small black dots, and under high magnification, they move actively. The culture medium remains clear, and the impact on cell growth is usually minimal. Often, the cell growth state is good, and the number of moving objects doesn’t increase significantly. This phenomenon is commonly found in cells cultured with the same batch of serum.

Solution: Black worms usually don’t significantly affect cell growth. They often disappear naturally when cell proliferation is strong. No special treatment is needed except replacing the serum. If contamination is suspected, increasing the cell density can improve survival rates.

5. Fungi The culture medium is typically clear, but under the microscope, filaments can be observed. Some fungi resemble dead cell debris, but their small pieces are clearly defined, resembling coral-like structures. Unlike cell debris, they grow slowly and develop fine black threads. Once detected, it’s difficult to save the culture.

Solution: Immediately discard the contaminated culture and thoroughly sterilize the culture room, COâ‚‚ incubator, and all equipment.

6. Protozoa The culture medium may appear slightly cloudy. Under the microscope, many tiny, active particles are visible. Although cells can grow, their reproduction slows down, and their appearance becomes unclear and non-transparent. Protozoa compete with cells for nutrients and can form a vicious cycle when their numbers increase.

Solution: There are multiple causes of contamination, including improper disinfection, operational errors, and environmental factors. If the contamination is widespread, it’s best to discard the cells. Otherwise, consider using sterilizing reagents. To check the sterility of the medium, place it in a culture flask without cells and observe at 37°C. If no bacterial growth is observed, the issue likely lies in the operation. Adding a double antibody (streptomycin sulfate and ampicillin) to the culture medium can help prevent contamination. However, this might affect cell state, so it should be removed before transfection or specific experiments to avoid interference.

Possible Causes of Pollution: Contamination can arise from improper disinfection, incorrect procedures, or environmental issues. - The incubator should be regularly disinfected with trioxane or UV light, and wiped with alcohol or Xinjieer. Use distilled water in the incubator. - Factors such as the ultra-clean bench, materials, equipment, culture medium, bottles, and operations can all contribute to contamination. - The fan speed of the ultra-clean bench should not be too high (6–8 grids). Excessive airflow can lead to mold contamination. - After formaldehyde fumigation of the sterile room, neutralize it with equal amounts of ammonia water, and wait a few hours before resuming work.