Prevention of Contaminations in Cell Culture

Contaminations are a serious and frequent issue in cell culture that absolutely has to be avoided. They can lead to false results, and at some stage they can irreversibly and completely destroy the cells in culture. The physiological temperature and humidity in the incubator, as well as the nutrients in the medium, provide excellent conditions for the growth of contaminating microorganisms, such as bacteria, mycoplasma, fungi, and yeast. Also, cross-contamination, which is the unwanted introduction of foreign cells into an existing culture, is a problem that must be taken seriously. If it remains undiscovered, a researcher could work with an entirely different cell line than the initial one without noticing, which makes all results of cell culture assays useless.

Sterility Guidelines

For reproducible experiments and unrestricted cell culture, all possible sources of contamination must be eliminated by maintaining the highest possible level of sterility. This is achieved by following these rules:

• Keep up an aseptic working technique
• Sterilize all media supplements and equipment (e.g., by autoclavation or filtering)
• Disinfect all surfaces (e.g., with 70% isopropanol or ethanol), especially under the hood
• Keep the surfaces free of waste and unnecessary items
• Keep the water bath and the water inside the incubator clean (if necessary, add a decontamination agent)
• Regularly clean the incubator
• Regulate the number of people in the room and use separate clothing and gloves in the cell culture laboratory
• Avoid coughing, sneezing, or talking (humans are the most frequent source of contamination)
• Bind your hair and do not touch your face during cell handling
• Put all cells from new sources into quarantine and perform a quality control
• Examine media and vessels daily for contamination
• Handle only one cell line at a time, and use your own medium to prevent cross-contamination
• Establish a mycoplasma testing routine for all cultures (e.g., by PCR) to prevent mycoplasma contamination

Use of Antibiotics

To protect against bacterial contamination, the antibiotics penicillin and streptomycin can be added to the cell culture medium. However, working without antibiotics increases the validity of the results, because the continuous use of antibiotics can tempt to non-sterile work. Furthermore, contaminations might be hidden and resistances could develop. To circumvent these pitfalls, it is advisable to culture the cells for 2–3 weeks without antibiotics from time to time.

Testing for Contamination

While contamination with microorganisms such as bacteria or fungi is immediately noticeable under the microscope, contamination with other microorganisms such as mycoplasma can remain undiscovered, if not specifically tested for. Mycoplasma-contaminated cell cultures are often infrequent, but may show undesirable functional changes in experiments. For the testing of such microbes, various companies offer kits based on PCR or ELISA.