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How to Prevent Biological Contamination in Your CO2 Incubators

How to Prevent Biological Contamination in Your CO2 Incubators

Engineering and user controls help keep contamination at bay

Andy Tay, PhD

Andy Tay, PhD is a freelance science writer based in Singapore. He can be reached at

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Carbon dioxide (CO2) incubators are indispensable for biology work. They provide well-regulated CO2 levels, humidity, and temperature that are ideal for cell and tissue culture. However, these conditions that are optimal for your cells and tissues are also likely to promote the growth of contaminating microorganisms such as fungi, bacteria, mycoplasma, and viruses.

When biological contamination happens, it can adversely affect the progress of research. For instance, some cellular systems like organoids take weeks if not months to grow, and contamination translates to a great loss of productivity. On the other hand, contamination involving mycoplasma is usually not noticeable at the early stage, and is not detected until experimental data show anomalies and have to be discarded. In general, contamination disrupts scientific progress, reduces the reliability of scientific data, and should be minimized, if not prevented.

In a 2010 report, biological contamination was found in close to nine percent of cell cultures in the biopharmaceutical industry. The statistics were even more alarming in academic labs, with reportedly 80 percent of labs in Japan and 65 percent of labs in Argentina having experienced mycoplasma contamination. Due to the prevalence of biological contamination, a variety of control measures have been developed to keep them at bay. Here, we share some of these tips that we have broadly classified into engineering and user controls.

Engineering controls

To minimize biological contamination, manufacturers have introduced a number of engineering solutions.

First, all incubators can be equipped with high-efficiency particulate air (HEPA) filters, on the interior or exterior, that are able to remove particles from outside air with diameters larger than 0.3 μm with more than 99.95 percent efficiency before flowing the filtered air into the incubators. Note that because mycoplasma is between 0.1-0.3 μm, HEPA filters cannot filter them, and thus other engineering controls are needed. Furthermore, HEPA filters should be replaced every six months as they can be clogged with large particles which reduce their filtering efficacy.

Next, newer incubators are equipped with automated heat decontamination and sterilization functions which provide cleaning with the use of heat and steam. Annual inspection is, however, recommended to ensure that this function is well-maintained. Unfortunately, older designs of incubators typically do not come with an automated sterilization system—thus, routine manual cleaning should be performed weekly.

Related Resource: CO2 Incubator Resource Guide

Finally, a number of ingenious designs have been incorporated into incubators to minimize biological contamination and users should try to make use of them. For instance, with the incorporation of rounded corners, users can easily identify and clean spills. Copper is also the material of choice for incubators, including the racks and water pan, due to its antimicrobial properties. Some newer incubators even come with ultraviolet (UV) sterilization. Typically, 15-30 minutes of UV treatment will be sufficient for decontamination, but users should take note that this works only on surfaces that are in contact with UV rays, and cells should be removed from the incubators prior to UV sterilization.

User controls

Researchers are home to a rich ecosystem of microorganisms and are the most likely source of contamination. By following good practices, however, researchers can greatly minimize the risk of introducing biological contaminants into incubators.

First, incubators should be placed as far away from contaminants as possible. It is generally recommended to place incubators above the ground so that microorganisms near the floor and on the shoes of users do not enter the incubator during opening and closing. Incubators should also be placed further away from areas in the lab with high human traffic as the moving air may unintentionally introduce contaminants. Placing incubators away from direct sunlight helps to avoid temperature-induced condensation within the incubators that microorganisms can exploit to grow.

Second, laboratory personnel should practice good protocols when working with incubators. They should never touch the interior of an incubator with unsterilized gloves or loose sleeves or speak into an incubator. Any items being placed inside the incubators should be sterilized with 70 percent ethanol and wiped down thoroughly. It is also important to keep door opening frequency to a minimum—before opening an incubator, users should spray the side of the door and their gloves with 70 percent ethanol to sterilize surfaces that may be contaminated with microorganisms. Importantly, users should also be careful not to overfill an incubator with too many cell culture flasks and well plates as this can disrupt proper airflow and temperature uniformity. When temperature difference happens, condensation may occur within incubators, causing microorganisms to thrive.

Finally, incubators should be cleaned weekly, as this helps to remove potential contaminants early before they become more serious. To clean the interior of the incubator effectively, items in the incubator should first be relocated to another incubator, free from contaminants. Then, with the use of two percent quaternary ammonium disinfectant followed by 70 percent ethanol, the incubator can be properly decontaminated. Contrary to popular practices, manufacturers discourage the use of bleach as disinfectant as it can corrode the metals of incubators. The decontamination process is complete after turning on the heating to dry the interior of the incubator. During cleaning, the water in the pan that helps to maintain incubator humidity should also be changed, and only sterile, distilled water should be used. Tap water should be avoided as it often contains bacteria, and deionized water should be avoided as well as it can corrode the metal pans. Generally, chemicals such as quaternary ammonium disinfectant are also added to the water pan to prevent the growth of microorganisms.

CO2 incubators have greatly transformed the way we perform biological experiments. By providing a well-regulated environment, they facilitate optimal cell and tissue growth, and are a staple in many biology labs. There are multiple sources of biological contamination including the lab environment (air and ground) and users, but with effective engineering and user controls, it is possible to keep contaminants at bay to generate reliable and reproducible scientific data.

For additional resources on co2 incubators including useful articles and a list of manufacturers, visit