LabTips: Preventing Microbial Contamination in a Cell Culture Incubator

 <span>LabTips:</span> Preventing Microbial Contamination in a Cell Culture Incubator

CO2 incubators are designed to provide an ideal environment for culturing cells, and most commonly are used to simulate the physiological conditions of mammalian cells. By maintaining a controlled CO2 level at 5%, stable temperature of 37 ºC, and relative humidity of 95%, CO2 incubators are also ideal environments for the growth of microbes, including bacteria, viruses, and fungi.

The risk of microbial contamination of cell cultures occurs throughout the cell culture laboratory, from the biological safety cabinet to the placement of tissue culture flasks, bottles, and plates into the incubator. The biggest source of microbes is laboratory personnel. Microbes are circulated as airborne particles and aerosols and also colonize the layers and structures of the skin.

Figure 1 – Schematic of closed-loop HEPA filtration: A low-velocity air pump (A) continuously draws air from the chamber (B) and circulates the air through a 99.99% capsule HEPA filter (C) before entering the sensor bay (D), where the air is assessed by sensors for CO2, O2, and relative humidity. There must be balance between the satisfactory clearance of introduced particles and the avoidance of desiccation.

Basic good lab practice, including proper aseptic techniques when working with cells and reagents, is the most important factor in reducing the risk of contamination. However, unless the laboratory itself is a cleanroom facility, every time we open an incubator door, microbes that circulate in the air of the laboratory and that colonize us are introduced. Incubator manufacturers have now incorporated a number of new features and design improvements that are directed toward reducing the risk of contaminated cell cultures. Choosing the right equipment that suits your particular laboratory will help control contamination.

Incubator Features That Actively Reduce Contamination

  • Chamber Air Quality—despite the inevitable introduction of microorganisms into the incubator every time the door is opened, continuous closed-loop, high-efficiency particulate air (HEPA) filtration can reduce the number of introduced particles and maintain positive pressure similar to ISO 5 cleanroom conditions (class 100, see Figure 1). In one manufacturer’s study, the concentration of Bacillus subtilis spores introduced via a 1-minute-nebulization challenge into an incubator with continuous closed-loop 99.99% HEPA filtration was markedly reduced to a general cleanliness level of class 100 within 15 minutes, depending on the incubator model.
  • Copper Antimicrobial Touch Surfaces—copper has been used for thousands of years because of its antimicrobial activity. In 2008, the Environmental Protection Agency registered almost 300 different copper surfaces as effective antimicrobials, and there is intense interest in its use for touch surfaces. Bacteria, yeasts, and viruses are killed upon contact with clean copper. If copper surfaces are kept free of wax or other coating agents, they will always be active. Pure copper and copper-alloy surfaces such as those manufactured by CuVerro® are available as options for incubators, and can be used for the entire incubator chamber and as shelving. Laboratory testing has shown that regularly cleaned CuVerro surfaces continue to kill 99.9% of bacteria despite repeated contamination.*
  • Sterilization Cycles—direct-heat incubators can provide high-temperature decontamination to eradicate contaminating agents. Some models offer dual sterilization cycles; the chamber will provide 95 ºC humidified decontamination or 145 ºC dry sterilization. Built-in disinfection cycles do not do away with the need for routine cleaning of the incubator, but can eliminate the need to separately autoclave removable internal components.

Improved Incubator Designs That Reduce the Risk of Contamination

  • Temperature Uniformity—eliminating cold spots decreases the formation of condensates. Water-jacketed incubators provide excellent uniform insulation, and now there are well-insulated direct-heat incubators with heating elements on all sides (including top and bottom) of the chamber, that achieve precise temperature uniformity. Direct-heat incubators that eliminate cold spots have the additional advantageous feature of being able to provide high-temperature decontamination.
  • Easy-to-Clean Growth Chambers—crevice-free construction that minimizes the internal surface area of the chamber makes it easier to clean. In addition, rounded corners without cracks and ridges and the use of polished stainless steel eliminate places that can harbor microbes. Interior components such as fan housings, plenums, shelf supports, and the water reservoir are now designed to be easily removable for autoclaving without the use of tools.
  • Added Protection from External Environment—inner doors are standard features that protect cultures from exposure to the outside environment. Additional protection can be added with the option of divided, gas-tight inner doors that further reduce exposure of the inner chamber to the outside environment by allowing selective access to specific sections of the incubator.

Good Lab Practice and the Lab Environment

In addition to proper aseptic technique, proper clothing that covers street clothes, regularly scheduled housekeeping to reduce dust on floors and other surfaces, and cleaning of equipment routinely used in a tissue culture laboratory are essential for preventing contamination throughout the lab. Ideally, the incubator should be located in an area free of traffic and away from air inlets.

  • Routine Incubator Cleaning—last, but not least, establish and follow a regularly scheduled SOP for thoroughly cleaning the incubator. Remember to use only cleaning solutions and disinfectants that are compatible with the materials used to make the incubator. The water pan should be changed weekly, following the manufacturer’s recommendations. A pinch of copper sulfate can be added to prevent the growth of microbes. Be sure to contact the manufacturer of the copper sulfate for the recommended use in this application.

*Laboratory testing shows that, when cleaned regularly, CuVerro® antimicrobial copper surfaces kill greater than 99.9% of the following bacteria within 2 hours of exposure: MRSA, Staphylococcus aureus, Enterobacter aerogenes, Pseudomonas aeruginosa, and E. coli O157:H7. CuVerro antimicrobial copper surfaces are a supplement to and not a substitute for standard infection control practices and have been shown to reduce microbial contamination, but do not necessarily prevent cross-contamination; users must continue to follow all current infection control practices, including those practices related to cleaning and disinfection of environmental surfaces.

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