Cell culture contamination is a common problem that can have serious consequences for biology labs. In addition to time and financial losses from discarded cultures and disrupted experiments, contamination also has the potential to lead to erroneous identifications and inaccurate results. The CO2 incubator is one area where cultures may be exposed to contaminants, especially considering that the growth conditions for cell cultures can also promote the propagation of unwanted microbes. While some contamination can be prevented through best practices such as regular cleaning, maintaining filters and keeping incubator doors closed as much as possible, additional decontamination technologies can provide more robust protection against a range of unwanted microorganisms.

Many CO2 incubators have built-in, automated processes for decontaminating the chamber, and it is beneficial to understand the different technologies that can be used to eliminate contaminants such as bacteria, yeast and mold. Weighing the benefits of features such as high heat, hydrogen peroxide (H2O2) vapor or ultraviolet (UV) decontamination cycles can help decide whether these technologies may be worth an added cost and which approach is best suited for your laboratory’s needs.

Moist vs. Dry Heat Decontamination

Heat-based decontamination programs in incubators can be divided into moist heat and dry heat methods. Moist heat sterilization can be performed at lower temperatures (~90°C) and thus does not always require the removal of some heat-sensitive components like sensors, but typically involves longer cycles ranging from 15 hours to 24 hours or longer.1,2 Moist heat treatment causes non-vegetative bacterial spores to germinate, which can reduce their heat resistance and allow them to be killed at lower temperatures.3 One of the risks of moist heat sterilization is the risk of recontamination if condensed water is not properly cleared out of the chamber after the decontamination cycle.Additionally, moist heat programs in CO2 incubators use lower temperatures than the 121°C used for autoclave steam sterilization and may not be as effective against the heat-resistant spores of species like Bacillus subtilis and Bacillus stearothermophilus in comparison to methods that use higher temperatures.

Dry heat sterilization involves much higher temperatures (~180°C) and can be completed in a shorter cycle (less than 15 hours). At these temperatures, dry heat sterilization is typically seen as a “tried and true” method; for example, dry heat methods between 160-180°C are often used to sterilize medical equipment in accordance with multiple pharmacopeias and other national standards.2,3 This heat is sufficient to kill the spores of common heat resistant species like B. subtilis and B. stearothermophilus, but any heat sensitive components will need to be removed from the chamber before the decontamination cycle. The success of dry heat sterilization relies on high temperatures reaching every area of the chamber, and both the United States and European Pharmacopeias require the use of a fan or blower to constantly circulate air during the cycle. A lack of uniform heat distribution and the presence of “cold spots'' in the incubator pose the risk of some bacteria surviving the decontamination cycle and recolonizing the chamber.3

Not all incubators are designed to generate or withstand the high heats needed for dry heat sterilization, so some models may only offer moist heat programs, while others may have both options available. It should also be noted that water jacket incubators cannot be decontaminated using high-heat methods. Whichever high heat sterilization method is used, prospective buyers should always look for proof of efficacy of an incubator’s decontamination process. Under US and EU Pharmacopeia standards, manufacturers should be able to demonstrate a 6 log reduction in spores of a specific heat-resistant bacterial strain, such as Bacillus subtilis var. niger, using their method.

H2O2 Decontamination

While high heat sterilization is the most common automated decontamination process found in CO2 incubators, H2O2 vapor decontamination – using a nebulizer within the chamber – is a popular alternative as it requires a much shorter run time than heat-based methods.4 Many labs will only run their heat-based decontamination processes overnight in order to limit instrument downtime during the day; with a shortened runtime of around 3 hours, H2O2 decontamination can allow for more flexibility to decontaminate during the workday if needed. H2O2 is effective against a wide range of microbes, including those that may be more resistant to heat treatments, and does not require the removal of components that are vulnerable to heat damage.

One of the main disadvantages of H2O2 decontamination is the safety risk of H2O2 exposure.5 Hydrogen peroxide should always be handled with care and proper procedures should be followed to avoid spills or inhalation of the H2O2vapor. Additionally, residual vapor inside the chamber could compromise cell cultures. Some incubators that include H2O2 decontamination processes also include a UV system to provide additional protection and decompose the vapor into water and oxygen once decontamination is complete. The costs and labor associated with replenishing the H2O2 disinfectant should also be taken into consideration.

UV Decontamination

Short-wavelength UV radiation is a commonly used sterilization method in laboratories and medical settings, acting as a germicide by damaging the nucleic acids of bacteria, viruses and other microorganisms. CO2 incubators may include a built-in UV lamp as a decontamination measure, but it should be noted that UV radiation is not suitable as a standalone means of sterilizing the incubator chamber. The construction of a typical incubator chamber does not allow for the UV light to reach every surface; additionally, the germicidal abilities of UV radiation are reduced when the relative humidity is above 50%.6 For these reasons, incubators offering built-in UV decontamination should still undergo additional, thorough decontamination processes using other sterilization methods.

UV light may be used inside an empty incubator chamber or the lamp may be placed away from the chamber behind a plenum to protect cell cultures, where it can be used to continuously disinfect circulating air or the surface of the humidity reservoir.7 The daily usage and estimated lifetime of the lamp should be taken into consideration to account for any costs associated with replacing the lamp over time.

References

  1. "Total Thermal Disinfection of CO2 Incubators," Application Note by D. Jäger and R. Leimbeck, Thermo Scientific (2009). https://tools.thermofisher.com/content/sfs/brochures/D17298~.pdf
  2. "Cell cultivation without contamination," White Paper by Jens Thielmann, BINDER. https://pages.binder-world.com/en/cell-cultivation-without-contamination
  3. "Beat Cell Culture Contamination with High Temperature Sterilization," Blog Post by Cindy Neeley, Thermo Fisher Scientific (2016). https://www.thermofisher.com/blog/cellculture/beat-cell-culture-contamination-dry-heat-sterilization/
  4. "Buyer's Guide: CO2 Incubators," Article by Michelle Taylor, Labcompare (2021). https://www.labcompare.com/10-Featured-Articles/576162-Buyer-s-Guide-CO2-Incubators/
  5. "Why is automated heat decontamination a better solution for sterilizing my CO2 incubator than an automated chemical disinfection, such as in situ hydrogen peroxide vapor?," Thermo Scientific Smart Notes (2014). https://tools.thermofisher.com/content/sfs/brochures/SmartNote-PFCO2AUTDCONSN-1013.pdf
  6. "CO2 Incubators – Best Practices for Selection, Set-up and Care," White Paper by Ines Kristina Hartmann and Jessica Wagener, Eppendorf (2020). https://www.eppendorf.com/product-media/doc/en/151861/Eppendorf_CO2-Incubators_White-Paper_029_CO2-Incubators_Best-Practices-Selection_Set-up-Care.pdf
  7. "HEPA Filter vs. Ultraviolet Light," White Paper, PHC Corporation. https://www.phchd.com/global/-/media/biomedical/global/WhitePaper/MCO/09_HEPA-Filter-vs_web.pdf?rev=1749922c9a164f58bfbf339436a37296&sc_lang=en