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Biological safety cabinets (biosafety cabinets or BSCs) serve as effective engineering controls against biological or infectious agents. Biosafety cabinets are equipped with high efficiency particulate air (HEPA) filters that have 99.97% efficiency for removing particles 0.3 microns in size from the air, and are more efficient for larger or smaller particles. Only externally exhausted BSCs provide for safe use of volatile compounds or volatile radioactive materials. Selection of the correct BSC is based on the classification of the agent, the associated biosafety level for the particular agent, and whether or not other hazardous materials will be used in research.
Place necessary materials in the BSC before beginning work to minimize the number of arm-movement disruptions across the fragile air barrier of the cabinet. The rapid movement of a worker's arms in a sweeping motion into and out of the cabinet disrupts the air curtain and may compromise the partial barrier containment provided by the BSC. Moving arms in and out slowly, perpendicular to the open face of the cabinet reduces this risk. Other staff activities in the room (e.g., rapid movement, opening and closing room doors, etc.) may also disrupt the cabinet air barrier.
Laboratory coats should be worn buttoned over street clothing; latex gloves are worn to provide skin protection. A solid front, back-closing lab gown provides better protection of personal clothing than a traditional lab coat. Gloves should be pulled over the knitted wrists of the gown, rather than worn inside. Elasticized sleeves can also be worn to protect the investigator's wrists.
Before beginning work, the investigator should adjust the stool height so that his or her face is above the front opening. Manipulation of materials should be delayed for approximately one minute after placing the hands and arms inside the cabinet. This allows the cabinet to stabilize and to "air sweep" the hands and arms to remove the surface microbial contaminants. When the user's arms rest flatly across the front grille, room air may flow directly into the work area, rather than being drawn through the front grille, as is desired. Raising the arms slightly will alleviate this problem. The front grille must not be blocked with research notes, discarded plastic wrappers, pipetting devices, etc. All operations should be performed at least four (4) inches from the front grille on the work surface.
Materials or equipment placed inside the cabinet may cause disruption to the airflow, resulting in turbulence, possible cross-contamination, and/or breach of containment. Extra supplies (e.g., additional gloves, culture plates or flasks, culture media) should be stored outside the cabinet. Only the materials and equipment required for the immediate work should be placed in the BSC. BSCs are designed to be operated 24 hours per day.
Cabinet blowers should be operated at least five minutes before beginning work to allow the cabinet to "purge." This purge will remove any particulates in the cabinet. The work surface, the interior walls (not including the supply filter diffuser), and the interior surface of the window should be wiped with 70% alcohol (EtOH) or other disinfectant, as determined by the investigator, to meet the requirements of the particular activity.
All materials should be placed as far back in the cabinet as practical, toward the rear edge of the work surface and away from the front grille of the cabinet. Similarly, aerosol-generating equipment (e.g., vortex mixers, tabletop centrifuges) should be placed toward the rear of the cabinet to take advantage of the air split. Be sure to avoid blocking the air flow at the back of the cabinet. Only materials essential to your work should be inside the BSC.
The surfaces of all materials and containers placed into the cabinet should be wiped with 70% alcohol to reduce the introduction of contaminants to the cabinet environment. This simple step will reduce introduction of mold spores and thereby minimize contamination of cultures. Further reduction of microbial load on materials to be placed or used in BSCs may be achieved by periodic decontamination of incubators and refrigerators.
Active work should flow from the clean to contaminated area across the work surface. Bulky items such as biohazard bags, discard pipette trays and suction collection flasks should be placed to one side of the interior of the cabinet.
Certain common practices interfere with the operation of the BSC. The autoclavable biohazard collection bag should not be taped to the outside of the cabinet. Upright pipette collection containers should not be used in BSCs nor placed on the floor outside the cabinet. The frequent inward/outward movement needed to place objects in these containers is disruptive to the integrity of the cabinet air barrier and can compromise both staff and product protection. Only horizontal pipette discard trays containing an appropriate chemical disinfectant should be used within the cabinet.
Potentially contaminated materials should not be brought out of the cabinet until they have been surface decontaminated. Contaminated materials can be placed into a closable container for transfer to an incubator, autoclave or for other decontamination treatment.
Many common procedures conducted in BSCs may create splatter or aerosols. Standard microbiological practices should always be used when working in a biosafety cabinet. For example, techniques to reduce splatter and aerosol generation will minimize the potential for staff exposure to infectious materials manipulated within the cabinet. Class II cabinets are designed so that horizontally nebulized spores will be captured by the downward flowing cabinet air within fourteen inches of travel. Therefore, as a general rule of thumb, keeping clean materials at least one foot away from aerosol-generating activities will minimize the potential for cross-contamination.
Open flames are not required in the near microbe-free environment of a biosafety cabinet. On an open bench, flaming the neck of a culture vessel will create an upward air current that prevents microorganisms from falling into the tube or flask. An open flame in a BSC, however, creates turbulence that disrupts the pattern of air supplied to the work surface. When deemed absolutely necessary, touch-plate microburners equipped with a pilot light to provide a flame on demand may be used. Internal cabinet air disturbance and heat buildup will be minimized. The burner must be turned off when work is completed. While an open flame is being used in the cabinet, any alcohol or flammable liquid in its vicinity could cause a fire, so additional care must be taken. Small electric furnaces are available for decontaminating bacteriological loops and needles and are preferable to an open flame inside the BSC. Use of pretreated sterile loops is also a good option.
Aspirator bottles or suction flasks should be connected to an overflow collection flask containing appropriate disinfectant, and to an in-line HEPA or equivalent filter. This combination will provide protection to the central building vacuum system or vacuum pump, as well as to the personnel who service this equipment. Vacuum lines are protected with a vacu-guard filter (replaced after 1 year of use or if wetted or noticeably blocked).The aspirator collection flask must contain bleach at all times during work in the BSC so that after the flask is 3/4 full the concentration will be 10:1 bleach solution (hypochlorite). For example, for a two-liter flask, add 150 mL of 12.5% concentrated bleach stock solution. Caution is needed when handling stock bleach. The flask should be labeled with the date, Bleach Added, the percentage and the expiration date (48 hours after the solution is created.). After the expiration period or when the flask is 3/4 full, squirt the inside of the flask with approximately 50 mL of bleach and swirl the flask carefully. Allow the flask to sit for 10 minutes before flushing it down the drain. Rinse and add fresh bleach.
If the flask was not prepared with enough bleach or was created more than 48 hours, inactivation of aspirated materials can be accomplished by placing sufficient concentrated bleach into the flask to kill the microorganisms as they are collected. Once inactivation occurs, liquid materials can be disposed of appropriately as noninfectious waste down the sanitary sewer.
Investigators must determine the appropriate method of decontaminating materials that will be removed from the BSC at the conclusion of work. When chemical means are appropriate, suitable liquid disinfectant should be placed into the discard pan before work begins. Items should be introduced into the pan with minimum splatter, and allowed appropriate contact time as per manufacturer's instructions.
Contaminated items should be placed into a biohazard bag or discard tray inside the BSC. Water should be added to the bag or tray prior to autoclaving.
When a steam autoclave is to be used, contaminated materials should be placed into a clear biohazard bag or discard pan containing enough water to ensure steam generation during the autoclave cycle. The bag should be taped shut or the discard pan should be covered in the BSC prior to removal to the autoclave. The bag should be transported and autoclaved in a leak proof tray or pan.
All containers and equipment should be surface decontaminated and removed from the cabinet when work is completed. At the end of the workday, the final surface decontamination of the cabinet should include a wipe-down of the work surface, the cabinet's sides and back, and the interior of the glass. If necessary, the cabinet should also be monitored for radioactivity and decontaminated when necessary. Investigators should remove their gloves and gowns and wash their hands as the final step in safe microbiological practices. Small spills within the BSC can be handled immediately by removing the contaminated absorbent paper toweling and placing it into the biohazard bag. Any splatter onto items within the cabinet, as well as the cabinet interior, should be immediately wiped with a towel dampened with decontaminating solution. Gloves should be changed after the work surface is decontaminated and before placing clean absorbent toweling in the cabinet. Hands should be washed whenever gloves are changed or removed.
Biosafety cabinets are not to be used for biosafety purposes until they have been certified and meet the appropriate safety specification (e.g., NIH-03-112 or National Sanitation Foundation Standard 49) by a trained technician at the following times:
EH&S schedules annual certification through an outside vendor. Certification records are kept on file in EH&S for each cabinet within Fred Hutch. Contact EH&S for more information.