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

Common types of biological contamination in cell culture include bacterial contamination, mold contamination, mycoplasma contamination, black worm contamination, fungal contamination, and protozoan contamination. Understanding the characteristics of each type and knowing how to prevent or treat them is crucial for maintaining a healthy cell culture environment.

Bacteria

Under an ordinary inverted microscope, bacteria appear as small black particles resembling sand. The shape may vary depending on the type of bacteria. The culture medium often becomes cloudy and yellow, which significantly affects cell growth.

Solution:

Ensure proper sterilization of all equipment, especially during autoclaving. Check if the pressure is sufficient. Items like pipettes that come into contact with the culture medium should be carefully handled to avoid secondary contamination. Always inspect the culture medium for turbidity before use. Adding antibiotics such as tetracycline, gentamicin, or streptomycin can help prevent bacterial infections.

2. Mold

The culture medium appears clear under the microscope, but after 2–3 days in a 37°C incubator, flocculent impurities and filamentous structures may appear. Mycelium can be observed, and although cells may still grow, their viability may decline over time.

Solution:

Clean the CO2 incubator using a copper sulfate solution and place a saturated amount in the water tray. Alternatively, use a high-salt sterile solution like disodium hydrogen phosphate. If mold contamination occurs, move the cells temporarily, clean the incubator thoroughly with peracetic acid, and allow it to air out before reusing. Regular cleaning is essential, especially during the rainy season. Other cleaning methods include using 84 disinfectant, alcohol, and UV light. To prevent mold, add antifungal agents like amphotericin B, nystatin, or actinomycin D to the medium. However, once contaminated, it’s often difficult to save the cells—discarding them is usually the best option.

3. Mycoplasma

Mycoplasma appears as polymorphic, dark particles. The culture medium may be slightly turbid, and it's common in bovine serum. It cannot be removed by filtration. The cytopathic effect is subtle, but it gradually harms the cells.

Solution:

Mycoplasma removal is critical, especially for important cell lines. Treatments include antibiotics (tetracyclines, macrolides), antiserum, or combinations. Avoid antibiotics targeting cell walls (e.g., penicillins). Replace all cultures if necessary. Filtration has no effect on mycoplasma.

4. Black Worms

These small, black dots can move under high magnification and are not easily visible. They don’t typically affect cell growth, but they may persist in the same batch of serum-cultured cells.

Solution:

No special treatment is needed unless the contamination is severe. Increasing the cell density can help improve survival rates. Replacing the serum may also be beneficial.

5. Fungi

The culture medium is generally clear, but filaments may appear under the microscope. Fungal growth is slow and often hard to detect, but once found, it's difficult to salvage.

Solution:

Immediately discard the contaminated cultures and thoroughly sterilize the lab, incubator, and equipment.

6. Protozoa

These tiny organisms may cause slight cloudiness in the medium and reduce cell growth rate. Although they compete for nutrients, they rarely interfere with normal cell function unless present in large numbers.

Solution:

If contamination is suspected, discard the affected cultures. Use sterilizing reagents to protect remaining cells. Regular monitoring and proper handling are key to preventing contamination.

Possible Causes of Contamination:

Contamination can arise from improper disinfection, operational errors, or environmental factors. If many cultures are affected, consider replacing the medium and checking the sterility of all materials. Adding double antibiotics (e.g., streptomycin and ampicillin) can help, but remove them before transfection to avoid interference with experimental results.

Preventive Measures:

Regularly disinfect the incubator with trioxane or UV light. Wipe it with alcohol or Xinjieer. Use distilled or triple-distilled water in the incubator. Ensure the laminar flow hood fan runs at 6–8 levels to avoid excess airflow. After formaldehyde fumigation, neutralize it with ammonia water before resuming work.