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Cell Culture Contamination: Identifying Mycoplasma, Fungus, and Bacteria in a Growth Medium

February 2021

Colleagues working in a science lab

Contamination in Cell Cultures

Medical research is an ever-evolving field that continues to contribute to the advancement of human health. A key component of this type of research is the process of cell culturing, which involves the isolation and expansion of a cell population. The cell culture process begins with the extraction of a target sample. Once the desired cells have been acquired, they must be purified to minimize contamination. When the cells have been properly isolated, they are introduced to a medium in which they can rapidly reproduce and multiply. This process creates a larger population of the target cell for experimentation.

Culturing cells is a difficult task due to the level of attention necessary to care for a cell population. Any contaminants that find their way into the sample will also take advantage of the nutrient-heavy medium and reproduce quickly, stealing resources from the target cell population. A contaminated sample leads to wasted time, money, and effort. Not to mention it puts other effort’s cultures at risk if they were being stored in the same area.

Minor Contaminants

There are a variety of different organisms that can ruin a sample, and some are more harmful than others. Typically, the easier a contaminant is to detect, the less likely it is to ruin a cell culture. Bacteria and fungi are the easiest to detect and treat.

Detection

Determining the cause depends on what type of bacteria or fungi contaminates the sample. Certain bacteria infiltrate the sample as a result of improper aseptic procedures, such as the researchers not cleaning their hands or properly sanitizing the equipment. When the cell culture is contaminated by yeast or mold, it could have to do more with the storage area. If the fume hood or incubation environment is in some way tainted, the particles can find their way into the sample.

Contaminants can be detected in a multitude of ways:

  • Distinct bacteria can often be spotted moving around the population
  • Fungi will cause clumps, colonies, or budding (yeasts) on the media surface
  • The media will become cloudy
  • The researcher can perform a side-by-side comparison to a small sample of fresh cells under a microscope

Depending on whether or not the cell culture media contains phenol red, a common pH indicator, the color of the sample can also reveal a contamination. Phenol red causes the solution to appear red at a pH of 8.2 or above, and yellow at 6.4 or below. If the media begins to turn orange/yellow, there could be a cell culture contamination.

Solution

While these can be easily detectable with the correct protocol, to the naked eye a contaminant may appear to be normal cell debris. If the bacteria or fungi is missed in the early stages, researchers could get all the way to the end before they realize their results are not usable

The best way to avoid minor contamination is to be thorough with aseptic techniques. If the sample is already compromised, it’s recommended the experiment be restarted from scratch. If the cells are very rare, it is possible to treat this level of infection with antibiotics, but it can be harmful to the population.

Major Contaminants

Viruses and chemicals are more difficult to detect and eliminate. Although these are less common than bacteria and fungi, they cause a much larger problem for the overall procedure and cannot be dealt with as easily. 

Detection

When it comes to the detection of unwanted chemicals in a cell culture, it’s more about noting possible accidents that could have occurred. If a chemical spill hasn’t been properly cleaned up, for example, there’s a decent chance that the solution will become contaminated.

Viral contaminants can be detected by using a few different methods, such as ELISA, PCR, and immunostaining, but these require more effort and resources.

Other organisms like parasites and those found on insects or arachnids are very difficult to detect, so it’s essential to protect a solution through proper aseptic techniques.

Solution

Unfortunately, even if a major contaminant is discovered early in the cell culture process, there is currently no foolproof method for removing them without damaging the original sample. If any of these organisms or chemicals are discovered, the researcher should start from scratch.

Mycoplasma

The most common form of contamination in cell culture is mycoplasma. Mycoplasma is a unique form of bacteria that has no cell walls.

Detection

Mycoplasma is very difficult to detect. However, since it is such a large problem, researchers have created some techniques for identification:

  • PCR and ELISA
  • Fluorescent DNA stains
  • Laboratory testing by an external, certified facility

Mycoplasma is most commonly spread through cross-contamination of cultures, so it’s necessary to find out whether or not it has entered a laboratory space. Contamination of one sample could spread to contaminate all other samples within the same area.

Solution

To be rid of a mycoplasma contamination, everything must be cleaned and heated in an autoclave, a device that uses steam to sterilize equipment. Given the severity of the issue, professional cleaning services can also be hired solely for the purpose of eradicating mycoplasma. Commercial kits are also available but often only provide a temporary solution and are not as effective. 

Common Questions

Why shouldn’t I always use antibiotics to clear minor contaminations?

Long term use of antibiotics can lead to resistant strains that allow some contamination to go undetected. Antibiotics also have an effect on the behavior of cells, so it’s important to only use them as a last resort when using cells that are difficult to re-obtain.

Does phenol red reveal all types of contaminants?

No, the presence of phenol red will only help in certain situations when the pH levels of the overall solution are altered. Contamination will not cause any color change unless it changes the acidity levels of the sample. However, it may make the solution cloudy.

What are some ways to avoid contamination before it becomes a problem?

Using sterilized equipment that has been properly cleaned and sanitized will drastically reduce the chances of contamination. Make sure the storage area is clean and protected from external organisms. When interacting with the cell culture, do things as quickly and efficiently as possible to decrease the potential of a mishap or infection. 

Akadeum’s Microbubbles

The cell culture process relies heavily on the isolation of target cells from a host organism. Regardless of the experiment, the original population needs to be free of contaminants and substantial enough to reproduce in the cell culture medium. There are multiple methods of cell separation that can be used to extract a clean sample and eliminate unwanted substances, and Akadeum’s microbubble technology is a fast and efficient method of sample preparation that maintains the health and physiology of cells of interest.

Traditional methods, like magnetics or cell sorting, require extensive training and expensive equipment to carry out the cell separation procedure. Akadeum, however, uses microbubbles to float marked cells to the top of a solution for collection or removal. This process is gentle on fragile cells and can remove more than 99% of residual blood cells in less than 10 minutes. It’s called buoyancy-activated cell separation (BACS™) because it harnesses the buoyant properties of the bubbles to get the job done using only the power of gravity; eliminating the need to expose delicate cell types to harsh chemicals like lysis buffers or external forces like rare earth magnets.

Whether you’re isolating cells with the intention of culturing them or for other downstream processing, Akadeum’s BACS™ microbubbles offer a fast and easy workflow that delivers a highly-enriched sample for downstream processing. Our RBC Depletion product eliminates residual RBC contamination from biological samples in 10 minutes, while our cell isolation products like our T Cell and B Cell Isolation Kits use a 30-minute workflow that is exceptionally gentle on target cells and that can exponentially decrease sort times. If you have any questions about the microbubble technology and how it is solving long-standing headaches in sample preparation, contact our team today.

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