CO2 incubators provide an optimal environment for cell growth that can also promote the growth of contaminants. Because contamination manifests in different ways, modern CO2 incubators feature a range of specialized solutions for its prevention. This article highlights some of the contamination prevention strategies and technologies to consider when searching for a CO2 incubator.
Contamination can come from direct sources such as contaminated reagents, media, or seed cultures, and from indirect sources like work surfaces, equipment, and personnel. Types of contaminants include bacteria, fungi, yeasts, and mycoplasma, as well as cross-contamination with other eukaryotic cells, all of which vary in terms of how they manifest and how easily they are detected.
Contaminants can come from the incubator if poor aseptic technique leads to unnoticed splashes from cell culture vessels. Once a contaminant has entered the CO2 incubator, the warm, humid environment promotes its spread.
Modern CO2 incubators are equipped with various design solutions and functional measures to prevent contamination. These include the following:
HEPA filters serve to remove particulate matter from the air. However, while they offer protection against contaminants that are larger than 0.2 µm, they are ineffective against mycoplasma due to the small size of these microorganisms. A further limitation of HEPA filters is that they require fan-assisted air circulation in the incubator chamber. This necessitates a more complex interior chamber design, with a duct, seams, and corners in which contaminants can grow, and makes cleaning and disinfection more difficult. Additionally, HEPA filters only trap contaminants. They don’t kill them and can become an important source of contamination if they are not replaced regularly.
Direct exposure to UV light kills air- and water-borne contaminants within the incubator chamber. The UV lamp is usually focused on the directional airflow, which is established using a fan, and on the water in the humidity pan. Drawbacks of UV disinfection are related to fan-assisted air circulation, as previously described, and the fact that the UV light cannot reach every part of the chamber. Also, the impact of UV treatment is reduced when the relative humidity is above 70%, and UV lamps must be replaced periodically to function effectively.
H2O2 nebulization is a form of automated self-disinfection that involves introducing a dilute solution of H2O2 into the incubator chamber, where it creates a vapor. An advantage of H2O2 nebulization is that it is relatively quick (~3 hours, excluding preparation time). However, it requires handling of a toxic reagent and has a high associated risk of recontamination due to manual repositioning of the chamber components and the need to wipe down water vapor once the disinfection process is complete.
High-temperature disinfection is another type of automated self-disinfection. It can use moist-heat, which necessitates a long and tedious procedure and leaves condensed water in the chamber at the end of the cycle, increasing the risk of recontamination. Alternatively, 140–180oC dry-heat disinfection can be run overnight, requires only minimal preparation time, and has the lowest chance of recontamination as the chamber can be used directly afterward.
Active filtering of particles from the air reduces airborne spread of contaminants that are bigger than 0.2 µm
Chamber design is complex with duct, seams, possible welding joints, and concealed corners:
Fan-assisted air circulation:
Direct exposure kills air- and water-borne contaminants
Penetration limited to direct exposure
Influence of humidity on effectiveness
UV lamps must be replaced periodically
Quick (~3 hours, excluding preparation time)
Handling of toxic reagent
Regular cost of reagent
Tedious preparation before and after the procedure
High chance of recontamination due to manual repositioning of interior
Tedious preparation time
High chance of recontamination due to condensed water
Slow (overnight)
Short preparation time
Lowest chance of recontamination as chamber can be used right away after cycle
Long time to complete the disinfection cycle
Besides using proper aseptic technique, implementing a regular cleaning and disinfection schedule will help to prevent contamination. Other best practice recommendations include placing the CO2 incubator on a base with castors to stop dust and dirt entering from the floor, minimizing the frequency and duration of door openings, and using CO2 and N2 gases of “high-grade” quality.
The Eppendorf CellXpert CO2 Incubator is designed to minimize the risk of contamination. It has a seamless chamber with round corners, meaning there are no hidden places to harbor contamination, and is fitted with racking and shelves that are easily removed for cleaning. Other key features include high-temperature (dry-heat) disinfection and fanless air circulation, as well as modern temperature control and CO2 sensing technologies to ensure optimal growth of precious cultures.
To learn more about the Eppendorf CellXpert CO2 Incubator, visit eppendorf.com/CellXpert