Cell Culture Contamination

To identify cell culture contamination, you must have knowledge of the morphology of cells you are studying and the potential contaminants the culture may have been exposed to. With this information, you can decide which methods will be most effective for testing and monitoring your cell cultures for contamination. This is essential to ensure that you catch any potential contamination before it becomes unmanageable. 

However, some biological contaminants, such as mycoplasma, can be extremely difficult to detect even with regular monitoring and require alternative identification and treatment.

Learn more about mycoplasma contamination

Cell culture contamination can be identified by performing tests such as cell analysis through microscopy, microbial testing, cell line authentication, and immunostaining. Various methods may be necessary to determine which contaminant is present or if there are multiple contaminants present in the cell culture. 

A culture should be tested for contamination if you suspect that it was exposed to a potential contaminant. Testing for contaminants should also be routine procedure in your regular culturing protocols. It is recommended to test cell lines or primary cultures prior to beginning an experiment and to routinely analyze all cell cultures for potential contamination.

Bacteria are a large and ubiquitous group of unicellular microorganisms. They are typically a few micrometers in diameter and can have a variety of shapes ranging from spheres to rods and spirals. Bacteria are one of the most common contaminants in cell culture because of their ubiquity, size, and fast growth rates.

Bacterial contamination in cell cultures is easily detected by visual inspection within a few days of it becoming infected.

• Infected cultures usually appear cloudy (i.e., turbid), sometimes with a thin film on the surface.
• Sudden drops in the pH of the culture medium are also frequently encountered.
• Under a low-power microscope, the bacteria appear as tiny, moving granules between the cells, and observation under a high-power microscope can resolve the shapes of individual bacteria.

 The simulated images below show an adherent 293 cell culture contaminated with E. coli.

While not as common as microbial contamination, extensive cross-contamination of many cell lines with HeLa and other fast growing cell lines is a clearly-established problem with serious consequences.  Obtaining cell lines from reputable cell banks, periodically checking the characteristics of the cell lines, and practicing good aseptic technique are practices that will help you avoid cross-contamination.  DNA fingerprinting, karyotype analysis, and isotype analysis can confirm the presence or absence of cross-contamination in your cell cultures.

Video 2:  Sterile Technique:  This video is focused on the steps you should take to prevent contamination of your cell culture.

Viruses are microscopic infectious agents dependent on a host cell for replication. Their extremely small size makes it difficult to detect and remove them from cultures and reagents. Since most viruses have very stringent requirements for their host, they usually do not adversely affect cell cultures from species other than their host. However, using virally infected cell cultures can present a serious health hazard to the laboratory personnel, especially if human or primate cells are cultured in the laboratory.

Viral infection of cell cultures can be detected by electron microscopy, immunostaining with a panel of antibodies, ELISAs, or PCR with appropriate viral primers.

Yeasts are unicellular eukaryotic microorganisms in the kingdom of Fungi, ranging in size from a few micrometers (typically) up to 40 µm (rarely).

Like bacteria, yeast contamination in cell culture turns the medium turbid, especially if the contamination is in an advanced stage. There is very little change in the pH of a culture contaminated by yeast until the contamination becomes heavy—at which stage the pH usually increases. Under microscopy, yeast appears as individual ovoid or spherical particles, that may bud off smaller particles.

Molds are eukaryotic microorganisms in the kingdom of Fungi that grow as multicellular filaments called hyphae. A connected network of these filaments contains genetically identical nuclei and are called a colony or mycelium. 

Similar to yeast, mold contamination in cell culture presents with a stable pH level in the initial stages of contamination, then rapidly increases as the culture becomes more heavily infected and turbid. Under microscopy, the mycelia usually appear as thin, wisp-like filaments, and sometimes as denser clumps of spores. Spores of many mold species can survive extremely harsh and inhospitable environments in their dormant stage, only to become activated when they encounter suitable growth conditions.

Antibiotics and antimycotics should not be used routinely in cell culture, because their continuous use encourages the development of antibiotic-resistant strains and allows low-level contamination to persist, which can develop into full-scale contamination once the antibiotic is removed from media and may hide mycoplasma infections and other cryptic contaminants. Further, some antibiotics might cross react with the cells and interfere with the cellular processes under investigation.

Antibiotics should only be used as a last resort and only for short term applications, and they should be removed from the culture as soon as possible. If they are used in the long term, antibiotic-free cultures should be maintained in parallel as a control to help investigate cryptic infections.

The decision to use antibiotics to prevent cell culture contamination should be based on the individual researcher's needs and experience. The following table is a general guide for use of Gibco antibiotics in cell culture media. Solutions that use one or more antibiotics in conjunction with an antimycotic are also available. For all media types, optimal concentrations of antibiotics and antimycotics should be determined empirically. Please always reference antibiotic and antimycotic product information sheets to help determine working concentrations.

When an irreplaceable culture becomes contaminated, researchers may attempt to eliminate or control the contamination.

• First, determine if the cell culture contamination is bacteria, fungus, mycoplasma, or yeast. 
• Isolate the contaminated culture from other cell lines.
• Clean incubators and laminar flow hoods with a laboratory disinfectant, and check HEPA filters.
• Antibiotics and antimycotics at high concentrations can be toxic to some cell lines; therefore, perform a dose response test to determine the level at which an antibiotic or antimycotic becomes toxic.

The following is a suggested procedure for determining toxicity levels and decontaminating cultures:

1. Dissociate, count, and dilute the cells in antibiotic-free medium. Dilute the cells to the concentration used for regular cell passage.
2. Dispense the cell suspension into a multi-well culture plate or several small flasks. Add the antibiotic of choice to each well in a range of concentrations. 
3. Observe the cells daily for signs of toxicity such as sloughing, appearance of vacuoles, decrease in confluency, and rounding.
4. When the toxic antibiotic level has been determined, culture the cells for two to three passages using the antibiotic at a concentration one- to two-fold lower than the toxic concentration.
5. Culture the cells for one passage in antibiotic-free media.
6. Repeat step 4.
7. Culture the cells in antibiotic-free medium for 4 to 6 passages to determine if the contamination has been eliminated.

While not as common as microbial contamination, extensive cross-contamination of many cell lines with other fast growing cell lines is a clearly established problem with serious consequences. Obtaining cell lines from reputable cell banks, periodically checking the characteristics of the cell lines, and practicing good aseptic technique are practices that will help you avoid cross-contamination. DNA fingerprinting, karyotype analysis, and isotype analysis can confirm the presence or absence of cross-contamination in your cell cultures.

Hassan, S., Ahmad, F. (2020). The relevance of antibiotic supplements in mammalian cell cultures: Towards a paradigm shift. Gulhane Medical Journal. 62. 224-230. 10.4274/gulhane.galenos.2020.871.