Office of Health and Safety

Primary Containment for Biohazards

SECTION III

The similarities and differences in protection offered by the various classes of biosafety cabinets are reflected in Table 1. Please also refer to Table 2 and Section IV for further considerations pertinent to BSC selection and risk assessment.

The Class I BSC provides personnel and environmental protection, but no product protection. It is similar in air movement to a chemical fume hood, but has a HEPA filter in the exhaust system to protect the environment (Figure 2). In the Class I BSC, unfiltered room air is drawn across the work surface. Personnel protection is provided by this inward airflow as long as a minimum velocity of 75 linear feet per minute (lfpm) is maintained⁵ through the front opening. Because of the product protection provided by the Class II BSCs, general usage of the Class I BSC has declined. However, in many cases Class I BSCs are used specifically to enclose equipment (e.g., centrifuges, harvesting equipment or small fermenters), or procedures (e.g. cage dumping, aerating cultures or homogenizing tissues) with a potential to generate aerosols that may flow back into the room.

The Class I BSC is hard-ducted to the building exhaust system, thimble-connected, or recirculated back into the room depending on use. If it is hard-ducted, the building exhaust fan provides the static pressure necessary to draw room air into the cabinet. Cabinet air is drawn through a HEPA filter as it enters the exhaust plenum. Sometimes a second HEPA filter is installed in the building exhaust system.

A steel panel with 8" arm holes to allow access to the work surface can be added to the Class I cabinet. The restricted opening results in increased inward air velocity, thereby increasing worker protection. For added safety, arm-length gloves can be attached to the panel. Makeup air is then drawn through an auxiliary air supply opening (which may contain a filter) and/or around a loose-fitting front panel. To permit access to the cabinet interior with the panel installed, a double-door air lock is attached on either side of the cabinet. Consideration must be given to the chemicals used in a BSC with HEPA filters as some chemicals can destroy the filter medium, housings and/or gaskets causing the loss of containment.

As biomedical researchers began to use sterile animal tissue and cell culture systems, particularly for the propagation of viruses, cabinets were needed that also provided product protection. In the early 1960's, a principle evolved stating that unidirectional air moving at a steady velocity along parallel lines (i.e., "laminar flow") would aid in the capture and removal of airborne contaminants.³¹ Biocontainment technology also incorporated this laminar flow principle with the use of the HEPA filter to provide a particulate-free work environment. This combination serves to protect the laboratorian from the potentially infectious microorganisms being manipulated¹⁸ and provid necessary product protection.

The Class II (Types A, B1, B2, and B3)²⁴ biological safety cabinets provide personnel, environmental and product protection. Air flow is drawn around the operator into the front grille of the cabinet, which provides personnel protection. In addition, the downward laminar flow of HEPA-filtered air provides product protection by minimizing the chance of cross-contamination along the work surface of the cabinet. Because cabinet air exhaust is passed through a certified exhaust HEPA filter, it is contaminant-free (environmental protection), and may be recirculated back into the laboratory (Type A BSC) or exhausted out of the building (Type B BSC).

HEPA filters are effective at trapping particulates and infectious agents, but not at capturing volatile chemicals or gases. Only BSCs that are exhausted to the outside should be used when working with volatile toxic chemicals (see Table 2). In certain cases a charcoal filter may be added to prevent release of toxic chemicals into the atmosphere.

All Class II cabinets are designed for work involving microorganisms assigned to biosafety levels 1, 2 and 3.⁶ Class II cabinets provide the microbe-free work environment necessary for cell culture propagation, and also may be used for the formulation of nonvolatile antineoplastic or chemotherapeutic drugs.³⁰

1. The Class II, Type A BSC - An internal blower (Figure 3) draws sufficient room air through the front grille to maintain a minimum calculated or measured average inflow velocity of at least 75 lfpm at the face opening of the cabinet. The supply air flows through a HEPA filter and provides particulate-free air to the work surface. Laminar airflow reduces turbulence in the work zone and minimizes the potential for cross-contamination.

The downward moving air "splits" as it approaches the work surface; the blower draws part of the air to the front grille and the remainder to the rear grille. Although there are variations among different cabinets, this split generally occurs about half-way between the front and rear grilles, and two to six inches above the work surface.

The air is then discharged through the rear plenum into the space between the supply and exhaust filters located at the top of the cabinet. Due to the relative size of these two filters, approximately 30% of the air passes through the exhaust HEPA filter and 70% recirculates through the supply HEPA filter back into the work zone. Most Class II, Type A cabinets have dampers to modulate this 30/70 division of airflow.

An unducted Class II Type A BSC is not to be used for work involving volatile or toxic chemicals. The buildup of chemical vapors in the cabinet (by recirculated air) and in the laboratory (from exhaust air) could create health and safety hazards (see Section IV).

It is possible to duct the exhaust from a Type A cabinet out of the building. However, it must be done in a manner that does not alter the balance of the cabinet exhaust system, and thereby disturbing the internal cabinet air flow. The typical method of ducting a Type A cabinet is to use a "thimble",¹³ or canopy hood, which maintains a small opening (usually 1 inch) around the cabinet exhaust filter housing (Figure 4). The volume of the exhaust must be sufficient to maintain the flow of room air into the space between the thimble unit and the filter housing.⁰ The thimble must be removable or be designed to allow for operational testing of the cabinet (see Section VI). The performance of a cabinet with this exhaust configuration can be affected by fluctuations in the building exhaust system.

"Hard-ducting" (i.e., direct connection) of Class II Type A cabinets to the building exhaust system is not recommended unless a dedicated exhaust fan system with a dynamic flow balancing mechanism is provided. The building exhaust system must be precisely matched to the airflow from the cabinet in both volume and static pressure. However, fluctuations in air volume and pressure that are common to all building exhaust systems make it difficult to match the airflow requirements of the cabinet. A competent in-house maintenance and engineering staff is required to achieve this.

2. The Class II, Type B1 BSC - Some biomedical research requires the use of small quantities of certain hazardous chemicals, such as carcinogens. The powdered form of these carcinogens should be weighed or manipulated in a chemical fume hood or a static-air glove box equipped with a double-door airlock.. Carcinogens used in cell culture or microbial systems require both biological and chemical containment.¹⁹

The Class II, Type B cabinet originated with the National Cancer Institute (NCI)-designed Type 2 (later called Type B) biological safety cabinet (Figure 5A), which was designed for manipulations of minute quantities of these hazardous chemicals with in vitro biological systems. The National Sanitation Foundation (NSF) Standard 49 definition of Type B1 cabinets²⁴ includes this classic NCI design Type B, as well as cabinets without supply HEPA filters located immediately below the work surface (Figure 5B), and/or those with exhaust/recirculation downflow splits other than 70/30%.

The cabinet supply blowers draw room air (plus a portion of the cabinet's recirculated air) through the front grille and then through the supply HEPA filters located immediately below the work surface. This particulate-free air flows upward through a plenum at each side of the cabinet and then downward to the work area through a back-pressure plate. In some cabinets there is an additional supply HEPA filter to remove particulates that may be generated by the blower/motor system.

Room air is drawn through the face opening of the cabinet at a minimum inflow velocity of 100 lfpm. As with the Type A cabinet, there is a split in the down-flowing air stream just above the work surface. In the Type B cabinet, approximately 70 percent of the downflow air exits through the rear grille, passes through the exhaust HEPA filter, and is discharged from the building. The remaining 30 percent of the downflow air is drawn through the front grille. Since the air which flows to the rear grille is discharged into the exhaust system, activities that may generate hazardous chemical vapors or particulates should be conducted towards the rear of the cabinet.²⁷

Type B1 cabinets must be hard-ducted, preferably to a dedicated exhaust system, or to a properly-designed laboratory building exhaust. As indicated earlier, blowers on laboratory exhaust systems should be located at the terminal end of the duct work. A failure in the building exhaust system may not be apparent to the user, as the supply blowers in the cabinet will continue to operate. A pressure-independent monitor should be installed to sound an alarm and shut off the BSC supply fan, should failure in exhaust air flow occur. Since this feature is not supplied by all cabinet manufacturers, it is prudent to install a sensor in the exhaust system as necessary. To maintain critical operations, laboratories using Type B BSCs should connect the exhaust blower to the emergency power supply.

3. The Class II, Type B2 BSC - This BSC is a total-exhaust cabinet; no air is recirculated within it (Figure 6). This cabinet provides simultaneous primary biological and chemical containment. Consideration must be given to the chemicals used in BSCs as some chemicals can destroy the filter medium, housings and/or gaskets causing loss of containment. The supply blower draws in room air or outside air at the top of the cabinet, passes it through a HEPA filter and down into the work area of the cabinet. The building or cabinet exhaust system draws air through both the rear and front grills, capturing the supply air plus the additional amount of room air needed to produce a minimum calculated or measured inflow face velocity of 100 lfpm. All air entering this cabinet is exhausted, and passes through a HEPA filter (and perhaps some other air-cleaning device such as a carbon filter) prior to discharge to the outside. Exhausting as much as 1200 cubic feet per minute of conditioned room air makes this cabinet expensive to operate.

Should the building or cabinet exhaust fail, the cabinet will be pressurized, resulting in a flow of air from the work area back into the laboratory. Cabinets built since the early 1980's usually have an interlock system (installed by the manufacturer) to prevent the supply blower from operating whenever the exhaust flow is insufficient; systems can be retrofitted if necessary. Exhaust air movement should be monitored by a pressure-independent device.

4. The Class II, Type B3 BSC - This biological safety cabinet (Figure 7) is an exhausted Type A cabinet having a minimum inward airflow of 100 lfpm. All positive pressure contaminated plenums within the cabinet are surrounded by a negative air pressure plenum. Thus, leakage from a contaminated plenum will be into the cabinet and not into the environment.

5. Special applications - Class II BSCs can be modified to accommodate special tasks. For example, the front sash can be modified by the manufacturer to accommodate the eye pieces of a microscope, or the work surface can be designed to accept a carboy, a centrifuge, or other equipment that requires containment. A rigid plate with arm holes can be added if needed. Good cabinet design, microbiological aerosol tracer testing of the modification, and appropriate certification (see Section VII) are required to ensure that the basic systems operate properly after modification. Maximum containment potential is achieved only through strict adherence to proper practices and procedures (see Section V).

The Class III biological safety cabinet (Figure 8) was designed for work with microbiological agents assigned to biosafety level 4, and provides maximum protection to the environment and the worker. It is a gas-tight (1x10^-5 cc/sec leak rate) enclosure with a non-opening view window. Access for passage of materials into the cabinet is through a dunk tank (that is accessible through the cabinet floor) or double-door pass-through box (such as an autoclave) that can be decontaminated between uses. Reversing that process allows for safe removal of materials from the Class III biosafety cabinet. Both supply and exhaust air are HEPA filtered. Exhaust air must pass through two HEPA filters, or a HEPA filter and an air incinerator, before discharge to the outdoors. Airflow is maintained by a dedicated independent exhaust system exterior to the cabinet, which keeps the cabinet under negative pressure (usually about 0.5 inches of water gauge).

Arm-length, heavy-duty rubber gloves are attached in a gas-tight manner to ports in the cabinet and allow for manipulation of the materials isolated inside. Although these gloves restrict movement, they prevent the user's direct contact with the hazardous materials. The trade-off is clearly on the side of maximizing personal safety. Depending on the design of the cabinet, the supply HEPA filter provides particulate-free, albeit somewhat turbulent, airflow within the work environment.

Several Class III cabinets can be joined together in a "line" to provide a larger work area. Such cabinet lines are custom-built; the equipment installed within the cabinet line (e.g., refrigerators, small elevators, shelves to hold small animal cage racks, microscopes, centrifuges, incubators, etc.) is generally custom-built as well. Furthermore, Class III cabinets are usually only installed in maximum containment laboratories that have controlled access and require special ventilation or other support systems (such as steam for autoclaves). The reader should consult more definitive literature on these systems.^16,21,23

Horizontal laminar flow "clean benches" (Figure 9A) are not BSCs. They discharge HEPA-filtered air across the work surface and toward the user. These devices only provide product protection. They can be used for certain clean activities, such as the dust-free assembly of sterile equipment or electronic devices. These benches should never be used when handling cell culture materials or drug formulations, or when manipulating potentially infectious materials. The worker can be exposed to materials (including proteinaceous antigens) being manipulated on the clean bench, which may cause hypersensitivity. Horizontal air flow "clean benches" should never be used as a substitute for a biological safety cabinet.

Vertical laminar flow clean benches (Figure 9B) also are not BSCs. They may be useful, for example, in hospital pharmacies when a clean area is needed for preparation of intravenous drugs. While these units generally have a sash, the air is usually discharged into the room under the sash, resulting in the same potential problems as the horizontal laminar flow clean benches.

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