Office of Health and Safety

Primary Containment for Biohazards

SECTION V

Preparing a written checklist of materials necessary for a particular activity and placing necessary materials in the BSC before beginning work serves 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 will disrupt the air curtain and may compromise the partial barrier containment provided by the BSC. Moving arms in and out slowly, perpendicular to the face opening of the cabinet, will reduce this risk. Other personnel activities in the room (e.g., rapid movement, open/closing room doors, etc.) may also disrupt the cabinet air barrier.⁵

Laboratory coats should be worn buttoned over street clothing; latex gloves are worn to provide hand 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/her face is above the front opening. Manipulation of materials should be delayed for approximately one minute after placing the hands/arms inside the cabinet. This allows the cabinet to stabilize and to "air sweep" the hands and arms to remove 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. 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 on the work surface at least four (4) inches from the inside edge of the the front grille.

Closure of the drain valve under the work surface should be done prior to beginning work so that all contaminated materials are contained within the cabinet should a large spill occur.

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, and some investigators find that continuous operation helps to control the laboratory's level of dust and other airborne particulates. Although energy conservation may suggest BSC operation only when needed, especially if the cabinet is not used routinely, room air balance is an overriding consideration. In some instances, room exhaust is balanced to include air discharged through ducted BSCs.

Cabinet blowers should be operated at least three to 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% ethanol (EtOH), a 1:100 dilution of household bleach (i.e., 0.05% sodium hypochlorite), or other disinfectant as determined by the investigator to meet the requirements of the particular activity. When bleach is used, a second wiping with sterile water is needed to remove the residual chlorine, which may eventually corrode stainless steel surfaces. Wiping with non-sterile water may recontaminate cabinet surfaces, a critical issue when sterility is essential (e.g., maintenance of cell cultures).

Similarly, the surfaces of all materials and containers placed into the cabinet should be wiped with 70% EtOH 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.

Plastic-backed absorbent toweling can be placed on the work surface (but not on the front or rear grille openings). This toweling facilitates routine cleanup and reduces splatter and aerosol formation³ during an overt spill. It can be folded and placed in an autoclavable biohazard bag when work is completed.

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 (Figure 11). 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 described in Section III. 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 personnel and product protection. Only horizontal pipette discard trays containing an appropriate chemical disinfectant should be used within the cabinet. Furthermore, potentially contaminated materials should not be brought out of the cabinet until they have been surface decontaminated. Alternatively, contaminated materials can be placed into a closable container for transfer to an incubator, autoclave or for other decontamination treatment.

Many procedures conducted in BSCs may create splatter or aerosols. Good microbiological techniques should always be used when working in a biological safety cabinet. For example, techniques to reduce splatter and aerosol generation will minimize the potential for personnel exposure to infectious materials manipulated within the cabinet. Class II cabinets are designed so that horizontally nebulized spores introduced into the cabinet will be captured by the downward flowing cabinet air within fourteen inches²⁴ 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.

The work flow should be from "clean to contaminated (dirty)" (see Figure 11). Materials and supplies should be placed in such a way as to limit the movement of "dirty" items over "clean" ones.

Several measures can be taken to reduce the chance for cross-contamination when working in a BSC. Opened tubes or bottles should not be held in a vertical position. Investigators working with Petri dishes and tissue culture plates should hold the lid above the open sterile surface to minimize direct impaction of downward air. Bottle or tube caps should not be placed on the toweling. Items should be recapped or covered as soon as possible.

Open flames are not required in the near microbe-free environment of a biological safety cabinet. On an open bench, flaming the neck of a culture vessel will create an upward air current which prevents microorganisms from falling into the tube or flask. An open flame in a BSC, however, creates turbulence which disrupts the pattern of HEPA-filtered 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. Small electric "furnaces" are available for decontaminating bacteriological loops and needles and are preferable to an open flame inside the BSC. Disposable sterile loops can also be used.

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 (see Figure 12). This combination will provide protection to the central building vacuum system or vacuum pump, as well as to the personnel who service this equipment. Inactivation of aspirated materials can be accomplished by placing sufficient chemical decontamination solution into the flask to kill the microorganisms as they are collected. Once inactivation occurs, liquid materials can be disposed of as noninfectious waste.

Investigators must determine the appropriate method of decontaminating materials that will be removed from the BSC at the conclusion of the 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. Alternatively, liquids can be autoclaved prior to disposal. 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 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 leakproof tray or pan. It is a prudent practice to decontaminate the exterior surface of bags and pans just prior to removal from the cabinet.

All containers and equipment should be surface decontaminated and removed from the cabinet when work is completed. At the end of the work day, 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 in a manner to prevent contamination of unprotected skin and aerosol generation 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.

Spills large enough to result in liquids flowing through the front or rear grilles require more extensive decontamination. All items within the cabinet should be surface decontaminated and removed. Decontaminating solution can be poured onto the work surface and through the grille(s) into the drain pan.

Twenty to thirty minutes is generally considered an appropriate contact time for decontamination, but this varies with the disinfectant and the microbiological agent. Manufacturer's directions should be followed. The spilled fluid and disinfectant solution on the work surface should be absorbed with paper towels and discarded into a biohazard bag. The drain pan should be emptied into a collection vessel containing disinfectant. A flexible tube should be attached to the drain valve and be of sufficient length to allow the open end to be submerged in the disinfectant within the collection vessel. This procedure serves to minimize aerosol generation. The drain pan should be flushed with water and the drain tube removed.

Should the spilled liquid contain radioactive material, a similar procedure can be followed. Radiation safety personnel should be contacted for specific instructions.

BSCs that have been used for work involving infectious materials must be decontaminated before HEPA filters are changed or internal repair work is done.^4,9-12 Before a BSC is relocated, a risk assessment which considers the agents manipulated within the BSC must be done to determine the need for decontamination. The most common decontamination method uses formaldehyde gas, although more recently hydrogen peroxide vapor¹⁰ has been used successfully. This environmentally benign vapor is useful in decontaminating HEPA filters, isolation chambers and centrifuge enclosures.¹¹

This page last reviewed May 17, 2001

Office of the Director/Administrator
Centers for Disease Control and Prevention
and Agency for Toxic Substances and Disease Registry
Atlanta, GA