Aseptic technique at the beach

/// By Howard Sneider
Last month, while I was on a beach vacation with my family, I disconnected from the daily onslaught of emails and projects for some much-needed rest and relaxation. While I consciously made the decision to not think about work, my subconscious had other plans; I observed a typical scenario on the beach and discovered it was a fantastic metaphor for indiscernible lapses in aseptic technique.

The family arrived at the sandy shore of the Atlantic with our typical beach gear: towels, snacks, some Nerf balls, a bucket, an umbrella and a resealable bag for things that don’t necessarily belong on the beach (like car keys, cellphones, wallet, etc.). The resealable bag was filled with its contents at the car before we got to the beach to ensure that it would not be contaminated with sand. However, as the family fell short of snacks, and a way to pay for more of them, reaching into the resealable bag became a necessity. I dusted off my hands as best I could, withdrew some cash from my wallet and resealed the bag. At the end of the next day, we decided to take a photo with the phone stored in the bag. I rinsed the sand off my hands in the ocean and allowed them to air dry before removing the phone. At the end of each day, I was surprised to see so much sand inside the bag. Where did it come from?

The simple answer is that the sand could have come from anywhere. When I first brought it to the beach, the bag was a closed system and was very much like any number of single-use closed processes that the pharmaceutical and biotech industry has adopted for more than 20 years. (It’s hard to believe the WAVE™ bag is 20 years old.) Opening the resealable bag exposed the inside to the sand on the beach. The sand is a metaphor for the particulates and microbial contaminants typically present in an area not protected by high-efficiency particulate air filtration and clean design.

On the beach, it was important to keep the items in the resealable bag free from contamination while still being able to use them. In biological research and production environments, when manipulating and processing sterile animal tissue and cell culture systems, it is necessary to keep them free from environmental contamination. There are three ways to accomplish this: use a closed system, such as a single-use bag, use an isolator or use a biological safety cabinet (BSC).

A closed single-use bag or isolator provides excellent assurance that the items inside will not be contaminated by the environment. Closed single-use bags, isolators and class III BSCs use impermeable barriers to protect the items inside from external contamination. Single-use bags use a polymer film and specialized connectors to segregate the inside of the bag from the external environment. Isolators use sterilizable pass-throughs to introduce and remove items. The inside chamber of the isolator has filtered air that is circulated turbulently under positive pressure. Isolator interiors are also sterilizable. Class III BSCs are similar to isolators but use unidirectional laminar airflow, usually under negative air pressure. Also, the cabinet is not sterile, and items placed in the pass-throughs must undergo a manual decontamination process.

Class II BSCs can also provide a high degree of sterility assurance, but the installation and operation must adhere to several practices. These types of BSCs create a permeable barrier between the outside and inside of the cabinet by directing high-efficiency particulate air filtered uniform airflows. The airflows are imperceptible, so techniques must be used to ensure the integrity of the barrier. If we could see the contamination like we can see sand on the beach, then it would be easier to be certain that the steps described below are conducted properly and effectively. We would know when a containment breach had occurred, and we could take steps to avoid repeating the same actions in the future. However, because contamination cannot be seen (at the time it occurs), proper design of the space is crucial, and operators must be diligent in their methods to avoid contamination.

Proper placement of the BSC in a room is critical to its function. The air entering the BSC is susceptible to disturbances from air currents generated by people walking close to the BSC, air supply or exhaust registers, heat-generating equipment, other BSCs and opening and closing doors. The BSC should also be placed away from aerosol-generating equipment and should include the clearances around the equipment recommended by the manufacturer. Before a BSC can be used, it must be certified. Because BSC operation may be impacted by the environment in which it is installed, it must be field tested to confirm its performance meets established guidelines, such as NSF/ANSI 49: Biosafety Cabinetry Certification, Occupational Safety and Health Administration, National Institutes of Health/Centers for Disease Control and Prevention and the manufacturer's specifications. This testing must be conducted annually. After installation and certification, the BSC must be maintained on a routine schedule.

Once a BSC is installed, certified and operational, the best practice is to leave it running continuously in order to control the levels of dust and particulate materials in the installed location. BSCs that are hard ducted to the exhaust system must remain on in order to maintain room balance unless other provisions have been made. If it is decided to turn off a BSC, then it should be cleaned prior to use and left to purge for at least 15 minutes before use.

The interior surfaces of BSCs should be decontaminated before and after each use. Always wear a clean lab coat and sterile gloves when working in a BSC; use more rigorous gowning if required in the installed location. Even when cleaning a BSC, the most likely contaminant is a person’s skin and clothing. All interior surfaces should be wiped with a disinfectant. The disinfectant should be effective but also compatible with the materials used inside the BSC. Allowing the disinfectant to wet the surface for up to a half hour before wiping is a time-tested method for effective decontamination. Disinfection can be aided by ultraviolet (UV) lamps before and after each use, but safety and operational procedures must be followed, including cleaning the luminaire and avoiding personal exposure. The UV exposure time can be up to 15 minutes before and after use but never during use.

When large equipment is required within the cabinet, it should be placed in the middle third of the work surface and as far back into the cabinet as ergonomically possible without blocking the rear vents. Aerosol-generating equipment, such as vortex mixers or centrifuges, should be the rear-most items. All other operations should be performed at least 4 inches from the front grill on the work surface and 6 inches from the aerosol-generating equipment. Items inside the BSC that are not touched or have been used and are no longer needed should be placed to one side of the interior of the cabinet. This includes vacuum flasks, biohazard bags, used pipette trays, etc. Arranging the interior in this way ensures that there is a clean zone toward the sash that will not be contaminated by other equipment toward the rear or on the side of the BSC that contains used items.

Before sterile processing, wipe down the exterior of all pipettes, pipette tip boxes, media, materials and equipment with a disinfectant prior to placing them in the biosafety cabinet. The goal is to place everything needed to process the sterile item inside the BSC prior to introducing the sterile item itself; therefore, the sterile item is the last thing to enter the BSC before operations commence. Entry of hands and equipment into the decontaminated BSC should be deliberate and perpendicular to the sash. Movement across the sash will disrupt the airflow, resulting in turbulence. To reduce the likelihood of possible cross-contamination or breach of containment after the materials are placed within BSCs, operations should be delayed for about one minute to allow laminar air flow to wash the items in the hood. Keep all used items in the BSC until operations are complete.

The sash height should be maintained at or below the maximum level to ensure the proper velocity of air into the BSC. Operators should maintain slow, steady movement mostly perpendicular to the sash while working inside the BSC. They should not remove their arms from the BSC until after sterile processing is complete. The movement of air within the BSC is down and to the rear, so open containers should be manipulated so that aerosols and nebulized content can be captured and carried away from the work zone. To achieve this, opened tubes or bottles should not be held in a vertical position but at about a 45-degree angle (or as much as possible without spilling the contents). Petri dishes and tissue culture plates should be shielded by the lid to avoid direct contact with the downward directed airflow. The work surface of the BSC may be lined with disinfectant-soaked absorbent towels to capture spills and splashes that occur while working. Heat sources within the BSC will create turbulence, so using disposable items is preferable to using items that need to be sterilized by a burner.

This brief introduction to the requirements for installing and operating a BSC does not consider the additional requirements for biohazardous operations but demonstrates the requirements and careful preparation required to achieve the best results from a BSC when it is used to ensure the sterility of an item. Hopefully, the metaphor comparing sand on a beach to environmental contaminants in a space provides a clearer understanding of what challenges exist when operating a BSC to create a sterile field.

About the Author

Howard Sneider

Howard Sneider

Howard Sneider is a process engineer at CRB in our Boston, Massachusetts office.
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