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CO2 Incubators: Preventing Contamination and Using Effective Sterilization Keeps Cultures Growing

87 percent of laboratory staff globally have at least one cell culture contamination event per year

Mike May, PhD

Mike May is a freelance writer and editor living in Texas.

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preventing contamination in CO2 incubatorsIf you’ve ever used a CO2 incubator for long, you’ve probably run into contamination at some point. According to Mary Kay Bates, global cell culture specialist at Thermo Fisher Scientific (Waltham, MA), “Our in-person surveys tell us that 87 percent of [laboratory staff] globally have at least one cell culture contamination event per year, and many have more than one.” She adds that people in the lab usually cause the contamination. “Microorganisms are our constant companions, and we carry trillions on our skin, in our mouth, et cetera,” Bates explains. On top of that, microorganisms jam-pack the air in a lab, with 30 to 1,000 of them in every cubic meter. “So every time the incubator door is opened, there is a risk for contamination,” Bates says. The question is, how do you fight this?

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Other experts also point out the widespread problem of contamination in CO2 incubators. Cheri Gaudet, marketing manager at The Baker Company (Sanford, ME) says, “It’s a widespread and serious concern, a real threat to the integrity of life science research.” Gaudet also points out some other sources of concern, saying, “Many unanticipated or accidental events could introduce contamination within the chamber, include the bumping of a plate or flask, or a dish knocking into a shelf.” She adds, “Of equal concern is cross-contamination, where cultures contaminate one another, or where a contaminated culture is introduced to the chamber and the contamination spreads.”

Controlling contamination

For many CO2 incubators, filtering the air with a HEPA filter provides the key element that fights contamination. Just having a good filter, though, is not enough. “Just saying that a unit offers [HEPA filters] does not mean that the design is effective,” Bates says. “Active airflow circulation is required, but it must be gently distributed so that cultures do not become desiccated.”

The new Thermo Scientific THRIVE active airflow system filters the entire chamber air volume every minute, and it provides ISO 5 cleanroom conditions within five minutes after the door is opened for 30 seconds. As Bates says, “It’s really important to look closely at the technologies and how quickly the incubator gets to clean room conditions, not just that it has HEPA filtration.”

Other aspects of the air in a CO2 incubator also matter. As Gaudet says, “The use of a closed-loop control algorithm to regulate relative humidity can prevent condensation, which is a major contributor to contamination.” She adds, “A closed-loop control algorithm proactively samples the atmosphere inside the chamber and uses the data collected to achieve and maintain the humidity set point.”

To make humidity control as easy as possible, look for a CO2 incubator that uses a nebulizer. “This technology eliminates the need for a water pan and the risk of contamination that typically goes along with it,” Gaudet says. “Also, vertical, unidirectional downward airflow is a newer technology for CO2 incubators; it prevents turbulence that can spread airborne contaminants throughout the chamber.”

Related article: Cell Culture Contamination – Part 3

The inside of a CO2 incubator also matters; for instance, copper surfaces can help. Bates says, “Thermo Scientific CO2 incubators use only 100 percent pure copper, for best quality and easy maintenance.” Details of other incubator parts also matter. As Gaudet points out, “Heated interior glass doors are fogless, which not only helps prevent the growth of contamination on the glass, but eliminates the need to open the door to view cultures, reducing the risk of contamination from airborne particulates.”

Baker’s Cultivo incubator includes fogless interior doors and a collection of other anti-contamination features. “In fact,” says Gaudet, “we call Cultivo ‘The Contamination Preventer,’ because it comes equipped with an arsenal of contamination prevention and control technologies.” That includes InteliCELL, Baker’s proprietary control algorithm that regulates humidity, temperature, and CO2 levels. According to Gaudet, it “virtually eliminates areas of condensation where contaminants can grow and spread.”

Ultimately, the data matter the most. Gaudet says, “To be safe, always ask your supplier for test results to substantiate marketing claims, and examine the test methods and conclusions carefully.”

Details for disinfecting

“The basic task of cleaning an incubator is pretty much unchanged, and it’s important to periodically clean the interior to ensure removal of spilled media, old cultures, dust, and dirt,” Bates says. Thermo Fisher Scientific recommends at least a monthly cleaning and changing the humidifier water every two weeks. When it comes to cleaning, Bates adds, “What is increasingly recognized is the negative effect that volatile organic chemicals, such as bleach or phenols, have on cultured cells, so we recommend only a quaternary ammonium-based disinfectant and/or 70 percent ethanol for manual disinfection following cleaning with a mild dish soap.” She notes that both of these solutions are safe for cells.

Gaudet adds, “Vaporized hydrogen peroxide biodecontamination protocols are now available that—at high enough concentrations, like 15 percent H2 O2 —completely kill a wide variety of contaminating microbes, including spores.” She notes that this speeds up the process from a typical 12 hours or more to less than four hours. Still, she encourages customers to analyze company tests. Also, she says, “Understand what microorganism is of primary concern, and examine the test methods and conclusions to reasonably ensure it will help you meet your goals.”

In addition, Jean Pierre Gomez, an assistant professor at France’s National Centre for Scientific Research (CNRS) in Orleans, sprays the water-bath tub with a disinfectant, AnioSpray, after cleaning.

Some devices provide other options for disinfecting. Bates says, “The new automated Thermo Scientific Steri-Run sterilization cycle provides a complete and fast method to disinfect CO2 incubators, without the need for autoclaving or using harsh chemical disinfectants.” She adds, “Detailed temperature mapping in 47 locations demonstrate that all areas reach the specified temperature, meaning no cold spots where microorganisms could survive, as might be found in other incubators.”

To ensure that a CO2 incubator meets the needs of your lab, carefully consider the features that you need and then make sure that the vendor can substantiate all claims about fighting off protection and disinfecting the device. With the right device and careful methods, you should be able to reduce contamination problems in your lab.

For additional resources on CO2 incubators, including useful articles and a list of manufacturers, visit