How to Prevent Cell-Culture Contamination

Counting the ways to contaminate a cell culture adds up to a big number. Often, scientists won’t even know exactly how a culture got contaminated. The media might get cloudy, cells die, and it’s time to start over. The culprit is usually bacteria, fungi, mold, mycoplasma, adventitious viruses, or yeasts. By knowing more about the most likely sources of contamination, scientists can limit these time-consuming and potentially devastating problems.

“Common viable contamination is often fairly easy to detect within a few days of occurrence due to obvious turbidity and extreme pH changes in the media,” says Ron Jankowski, Vice President of Scientific Affairs at Cook MyoSite.

To get more specific, taking a look at a contaminated culture under a microscope can often determine the cause. “Yeast are unicellular organisms, typically measuring three to four micrometers in diameter,” says Sherwin (Xiaoyu) Zhu, Scientific Support Specialist at Corning Life Sciences. “Under the microscope, the yeast contamination usually looks like small ovals—sometimes with a bud on the oval.” A fungus or mold, on the other hand, can create multicellular filaments that can also be seen under a microscope.

Some contaminants can go unseen. That’s the case for mycoplasma, which is the smallest bacteria—only a few tenths of a micron across. “Mycoplasmas are fastidious in their growth requirements,” Zhu says. “Their small size and lack of a cell wall allow mycoplasmas to grow to very high densities in cell culture often without any visible signs of contamination—no turbidity, pH changes, or even cytopathic effects.”

Detecting mycoplasma requires a test, such as a PCR-based method or specific DNA staining. “Unfortunately, many labs do not perform any type of testing on their cultures to detect any form of contamination unless or until there is an overt reason to do so,” Jankowski explains. “Therefore, various forms of contamination are often present to different degrees and go unnoticed.”

Although scientists might not notice all contamination, the cultures probably will. A contaminant could change the physiology, metabolism, or growth of cells in a culture.

Seeking the source

Preventing contamination starts at the source. Unfortunately, the source can be almost anything: people and even air in a lab, an incubator or biosafety cabinet that’s not clean, unsterile water or reagents, and the list goes on.

Despite typically thinking of biological sources of contamination—from bacteria to yeast and the organisms in between and beyond—chemicals also pose dangers for cultures. According to Nancy Palechor-Ceron, Scientist and Group Leader Manufacturing Science and Technology (MSAT) Cell Biology at ATCC, chemical contaminations can be “caused by the inadvertent use of compromised tissue culture reagents or plasticware containing nonliving substances or particles such as residues of detergents, organic compounds, plastics, and unpurified water.”

Given the wide range of potential problems that can contaminate a culture, sometimes without leaving any obvious signs, the best plan is proactive avoidance of trouble.

Preventing the problems

Step one is keeping a lab clean—keeping everything clean. “Biosafety cabinets, refrigerators, water baths, centrifuges, incubators and other laboratory equipment can become reservoirs of unwanted microorganisms,” Palechor-Ceron says. “They should be regularly disinfected/decontaminated using an appropriate reagent and a validated cleaning method.”

Scientists can also treat cultures to reduce the odds of contamination. “Antibiotics are a common method to prevent bacterial and yeast contamination,” Zhu says. For example, ciprofloxacin is effective against many types of bacteria, including mycoplasma. So, these antibiotics “can be used prophylactically or to treat mycoplasma or bacterial infections,” Zhu says.

As good as an antibiotic approach might sound, it’s only a short-term solution. Long-term use of antibiotics in a culture can cause various problems. For one thing, antibiotics can eventually impact the biology of the cells. “Studies have shown that antibiotics may induce changes in cell gene expression and regulation,” Zhu notes. “For this reason, it is important to understand, test ,and evaluate the effect of antibiotics within the specific cell-culture outcome.”

Protection at purchase

How a lab uses equipment and works with cell cultures play crucial roles in keeping cultures clean, but some of that starts when purchasing products. For instance, Palechor-Ceron says, “Instances of chemical contamination can be decreased by acquiring cell culture reagents from reputable vendors that perform the required quality-control testing and provide supporting documentation to ensure that the reagents meet the essential specifications to be used in cell culture.”

Platforms and processes that reduce interactions with a culture can also decrease the odds of contaminating the cells. “A number of advances focused on more closed-system culturing—rather than open aseptic manipulations—have been introduced that greatly reduce environmental- and operator-sourced contamination,” Jankowski says. “Aside from prevention, advances that have led to quicker, cheaper, and more reliable testing of various contaminants also contribute to the overall reduced risk of having contamination impact the outcome of the cell culturing.”

As indicated here, the long list of potential sources of contamination gets compounded by the many ways to foul up the process. Briefly, cell culture contamination can be less likely when lab personnel understand the likely contaminators, a lab stays as clean as possible, scientists keep an eye out for contaminated cultures and deal with them swiftly, and new products improve rather than amplify the problem.

Having worked in a hood to process many cell cultures—contaminating more than my fair share as I learned the key steps in aseptic technique and how to perform them—technique is near the top of my contamination-prevention list. So, when it comes to successful cell culturing, it’s not just what you do, but how you do it.

Image: Keeping cell cultures free of contamination is crucial in research, especially medical studies that often use HeLa cells like the ones shown at top. Image courtesy of ATCC.