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CO2 Incubators: Contamination and Key Operational Issues

The first question facing lab managers looking for an incubator is whether a dry or humidified incubator will serve their needs. Both designs have their pluses and minuses. Humidified CO2 incubators provide tighter control over cell culture conditions such as temperature, gas mix, and of course humidity.

Angelo DePalma, PhD

Angelo DePalma is a freelance writer living in Newton, New Jersey. You can reach him at

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Product Focus CO2 Incubators

Contamination, operations key issues

Users must seal cultureware in dry incubators to prevent evaporation. Water trays mitigate this requirement somewhat, but may also attract microbial contaminants. For non-humidified designs, experts recommend charging the water pan with sterile distilled water. Tap water contains chlorine, which corrodes stainless steel and copper; deionized (Type 1) water is extremely aggressive in drawing ions from its metal and glass construction components.

The pharmaceutical and biotech industries were the main market entry points for humid incubators. Today, academic, environmental, and food labs also recognize their benefits. Even microplates will not lose fluid to evaporation in humid incubators.

Humidified designs are more costly and complex, and for many applications a dry incubator will serve just fine. Linette Philip, product manager for CO2 incubators at Eppendorf (Enfield, CT), notes that dry incubators make sense for non-biological applications, for example electronics and materials science. “They’re still useful where temperature and atmosphere control are sufficient.”

Linda CO2 Video

Not if, but when

Contamination control is the single most significant operational or maintenance issue for CO2 incubators, says Mary Bates, global cell culture specialist at Thermo Fisher Scientific (Asheville, NC). In her many discussions with cell culture experts from around the world, close to 90 percent say they have experienced at least one contamination in the previous year. Humans are, by far, the predominant source of contaminating microorganisms. Approximately 30 percent of all contamination arises from cell lines, media, and labware, while 70 percent originates with humans or the lab environment.

Except for cases of inadequate sterilization, all contamination originates outside the incubator. “It doesn’t matter whose incubator you have, microorganisms are in our breath and constantly falling off our bodies,” Bates says. “But if I had to pick one factor responsible for repeat contamination, that would be air ducts.”

She advises lab managers to keep incubators away from air ducts. When that is impossible, users should monitor air filters closely, and should consider cleaning ducts as possible contamination sources. In some cases facility managers may be able to turn off or redirect ductwork.

Water in the pan or reservoir should be replaced at least once per week with sterile distilled water. Bates cautions against using ultrapure or deionized water. “It’s great for many applications, but because it contains no salts, it draws ions out of their containers, causing corrosion.” Users should not even consider using bleach or other caustic disinfectants within incubators. “Anything that smells bad to you is bad for cells.” Quaternary ammonium salts are OK because they do not emit volatiles.

Rule by SOP?

Standard operating procedures (SOPs) dominate today’s incubator user base, says Uwe Ross, president at BINDER Inc. (Bohemia, NY). As a consequence, many labs see no need to understand the scientific basis of those protocols.

“An SOP may state that an incubator be emptied and cleaned every two, three, or twelve weeks, and all components autoclaved,” Ross observes. “If that’s what the SOP demands, that’s what the user does. But they should be thinking about why the SOP author selected that particular timeframe, and what events might serve as reasonable triggers for cleaning.”

Every organization has its own SOPs. Universities, pharmaceutical companies, and lab directors use their own or protocols that have been handed down or provided by vendors. But in too many instances SOPs are not applicable to real-world operation. “They’re written by people who know what they’re doing, for individuals who believe they do not need to know why the SOP asks for a certain cleaning timeframe,” Ross says.

Most lab managers would agree, for example, that a good time to clean is when an experiment or process ends, before the next one begins. This effectively eliminates contaminating microbes—known or unknown—that may have arisen during the just-completed experiment. A rigid time-based SOP, Ross says, will not reflect this trigger.

“Clearly our approach to SOPs in the CO2 incubator world are outdated,” Ross concludes. “Labs should instead rely on internal quality practices, and time their cleaning or sterilization to the start of a new process. The consequence will be better results.”

Philip provides a contrary take. “It is a given that lab workers dislike paperwork. But SOPs are extremely valuable in cell culture or any lab for that matter. They simplify operations and provide a level of reproducibility in common tasks like cleaning and validation,” she says. That is not meant to imply that SOPs are written in stone. “SOPs are living documents that should be reviewed periodically.” Annual or semiannual reevaluation of SOPs forces lab managers to examine how the SOP is working based on input from end users, with the ultimate goal of improving the process. “As long as SOPs are allowed to evolve, there is no reason they should infringe on independent thinking or creativity.”

Purchase decisions

Philip advises lab managers to evaluate both short- and long-term needs when acquiring a CO2 incubator. “View this purchase as an investment. Look ahead three to five years. If you anticipate the need to control oxygen or humidity, plan accordingly.”

Cost of ownership is another factor to consider. Many incubators feature some sort of automated or semiautomated cleaning system, but associated energy and material costs may vary significantly. Cleaning that is cost-effective when conducted every three months may not be so on an every-two-weeks cleaning regimen.

Finally, to save time and effort, Philip recommends conducting an initial evaluation online. “So much information is available these days. Take twenty minutes to read it and compare what different vendors have to offer.”

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