The CO2 incubator provides a simulated human body-like environment for cell cultures and tissues to live and thrive. Unlike the body, however, the incubator is not equipped with a pathogen fighting immune system and must be kept sterile by the user. Pathogens typically enter the incubator in one of a few ways: contamination within cell culture dishes due to poor aseptic technique, in the airflow when the incubator is opened, from the user’s contaminated skin or clothing, or in the water pan. This article highlights tips for avoiding incubator contamination.
Keep chamber doors closed as much as possible—Most modern incubators have an inner chamber door and the finest incubators will have separate doors for each shelf. Since many pathogens are airborne, keeping ambient air from entering the chamber is critical. The easiest way to prevent airborne pathogens from entering is to only open doors when absolutely necessary and for as little time as possible. Positive air pressure within the chamber is also a very effective way to keep outside air from entering the growth chamber, but not all incubators are equipped with this feature.
Watch the sleeves of your lab coat—Your skin and clothing are a likely source of contamination. Exposed skin and the long sleeves of a lab coat or sweatshirt should never come in contact with the incubator chamber or cell culture vessels. It is recommended to pull up your sleeves and use long sterile gloves before handling cell culture materials.
Change and clean the water pan regularly—It is recommended that the water in the pan be changed weekly. Replacement water should be distilled or mineral-free, no purer than 1 mega ohm. Copper disks or disinfectants (without chlorine) added to the water can also help eliminate potential contamination. Never let the water pan run dry, or be overfilled. A maintained water pan is key to preventing contamination.
Decontaminate secondary cell culture containers—When working with many cell culture dishes and flasks at a time, it is common to use a secondary vessel such as a flat short-walled container to carry them. These secondary containers should be thoroughly cleaned with disinfectants that will kill both bacteria and fungi.
Do not overfill the incubator—An incubator filled to the brim with dishes or flasks can disrupt the airflow and cause improper air filtration and disrupt temperature uniformity. A temperature difference within the incubator can cause condensation to occur, which can act as a haven for pathogens. The incubator’s manual should have guidelines for ideal working conditions.
Keep incubator away from contamination sources—Placement of the incubator within the lab space is crucial. If kept under a laminar flow hood, for example, contamination from the underside of the hood or a researcher’s shoes or floor, could easily find its way into the incubator. It is best to install the incubator on a flat surface away from hoods and water baths. Also, the higher the incubator is off the ground, the best for preventing contamination from particles that can be brushed up from walking on the nearby floor.
Know thy incubator—Your incubator may or may not have built-in contamination prevention features. It is important to understand how best to use your particular incubator to prevent contamination. Consult the incubator manual to find if your incubator incorporates the below contamination prevention features.
Positive chamber pressure—It is best if there is a slight positive pressure within the cell culture chamber within the incubator. This eliminates contaminated laboratory air from entering the chamber.
Air filtration—Air within the growth chamber should continuously flow through HEPA filters. It is ideal for the HEPA filter to be located outside of the growth chamber. If located internally, there is a risk of contamination if the motor that blows the air stops. In this case, contaminations within the HEPA filter can fall onto cells. HEPA filters should also be replaced every 6 to 12 months depending on usage.
Removable gasket—The incubator gasket creates the seal around the inner door and is an area where humidity can build resulting in the growth of contamination if left unchecked. Ideally the gasket is removable and easy to clean. If using a v-gasket, beware of the direction of the flap. An outwards pointed flap will collect particles from entering the growth chamber while a flap that points inwards will collect humidity and can act as a breeding ground for contamination.
Rounded corners—Smooth, rounded corners within the incubator can eliminate hiding places for potential contaminants. If your incubator has sharp corners, make sure that the corners are thoroughly cleaned to prevent contamination.
Sterilization protocols—Many incubators have automated high-temperature sterilization cycles for fast and easy complete sterilization within the growth chamber. If there is not an automated sterilization cycle, an external source of dry high-heat or hydrogen peroxide gas can be used. When manually cleaning the chamber, it is recommended that labs create SOPs that include cleaning with a 70% solution of isopropyl alcohol or other disinfectant. UV light can also be used to sterilize but will only be effective in areas where the light shines at sufficient intensity and penetration, missing areas such as the bottoms of the shelves.
Copper interior—Copper has antimicrobial properties that can protect against contaminants introduced by a user or perhaps by the bottoms of culture plates. Chamber shelves can be coated with copper to further reduce the chances of contamination.