Biosafety Level 3 (BL3)

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is conducted with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the manipulation of infectious material are conducted within biological safety cabinets or other physical containment devices or by personnel wearing appropriate personal protective clothing and devices. The laboratory has special engineering and design features. It is recognized, however, that many existing facilities may not have all the facility safeguards recommended for BL3 (e.g., access zone, sealed penetrations, and directional airflow, etc.). In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in laboratories where facility features satisfy BL2 recommendations provided the recommended "Standard Microbiological Practices,'' "Special Practices,'' and "Containment Equipment'' for BL3 are rigorously followed. The decision to implement this modification of BL3 recommendations should be made only by the Principal Investigator.

Standard Microbiological Practices (BL3).

• Work surfaces are decontaminated at least once a day and after any spill of viable material.
• All contaminated liquid or solid wastes are decontaminated before disposal.
• Mechanical pipeting devices are used; mouth pipeting is prohibited.
• Eating, drinking, smoking, storing food, and applying cosmetics are not permitted in the work area.
• Persons wash their hands:
  • (i) after handling infectious materials including organisms containing recombinant DNA molecules, and handling animals, and
  • (ii) when exiting the laboratory.
• All procedures are performed carefully to minimize the creation of aerosols.
• Persons under 16 years of age shall not enter the laboratory.
• If experiments involving other organisms which require lower levels of containment are to be conducted in the same laboratory concurrently with experiments requiring BL3 level physical containment, they shall be conducted in accordance with all BL3 level laboratory practices.

Special Practices (BL3)

• Laboratory doors are kept closed when experiments are in progress.
• Contaminated materials that are to be decontaminated at a site away from the laboratory are placed in a durable leak-proof container which is closed before being removed from the laboratory.
• The Principal Investigator controls access to the laboratory and restricts access to persons whose presence is required for program or support purposes. The Principal Investigator has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.
• The Principal Investigator establishes policies and procedures whereby only persons who have been advised of the potential biohazard, who meet any specific entry requirements (e.g., immunization), and who comply with all entry and exit procedures entering the laboratory or animal rooms.
• When organisms containing recombinant DNA molecules or experimental animals are present in the laboratory or containment module, a hazard warning sign incorporating the universal biosafety symbol is posted on all laboratory and animal room access doors. The hazard warning sign identifies the agent, lists the name and telephone number of the Principal Investigator or other responsible person(s), and indicates any special requirements for entering the laboratory such as the need for immunizations, respirators, or other personal protective measures.
• All activities involving infectious materials including organisms containing recombinant DNA molecules are conducted in biological safety cabinets or other physical containment devices within the containment module. No work in open vessels is conducted on the open bench.
• The work surfaces of biological safety cabinets and other containment equipment are decontaminated when work with infectious materials including organisms containing recombinant DNA molecules is finished. Plastic-backed paper toweling used on non-perforated work surfaces within biological safety cabinets facilitates clean-up.
• An insect and rodent program is in effect.
• Laboratory clothing that protects street clothing (e.g., solid front or wrap-around gowns, scrub suits, coveralls) is worn in the laboratory. Laboratory clothing is not worn outside the laboratory, and it is decontaminated prior to laundering or disposal.
• Special care is taken to avoid skin contamination with contaminated materials; gloves should be worn when handling infected animals and when skin contact with infectious materials is unavoidable.
• Molded surgical masks or respirators are worn in rooms containing experimental animals.
• Animals and plants not related to the work being conducted are not permitted in the laboratory.
• Laboratory animals held in a BL3 area shall be housed in partial-containment caging systems, such as Horsfall units, open cages placed in ventilated enclosures, solid-wall and -bottom cages covered by filter bonnets or solid-wall and -bottom cages placed on holding racks equipped with ultraviolet in radiation lamps and reflectors. Note: Conventional caging systems may be used provided that all personnel wear appropriate personal protective devices. These protective devices shall include at a minimum wrap-around gowns, head covers, gloves, shoe covers, and respirators. All personnel shall shower on exit from areas where these devices are required.
• All wastes from laboratories and animal rooms are appropriately decontaminated before disposal.
• Vacuum lines are protected with high efficiency particulate air/HEPA filters and liquid disinfectant traps. (See schematic in Appendix 3.)
• Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for the injection or aspiration of fluids containing infectious materials including organisms that contain recombinant DNA molecules. Extreme caution should be used when handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. Needles should not be bent, sheared, replaced in the needle sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse.
• Spills and accidents which result in overt or potential exposures to infectious materials including organisms containing recombinant DNA molecules are immediately reported to the Biological Safety Officer, Institutional Biosafety Committee, and NIH/ORDA. Appropriate medical evaluation, surveillance, and treatment are provided and written records are maintained.
• Baseline serum samples for all laboratory and other at-risk personnel should be collected and stored. Additional serum specimens may be collected periodically depending on the agents handled or the function of the laboratory.
• A project-specific biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and follow the instructions on practices and procedures.
• Alternative Selection of Containment Equipment (BL3)
  Experimental procedures involving a host-vector system that provides a one-step higher level of biological containment than that specified may be conducted in the BL3 laboratory using containment equipment specified for the BL2 level of physical containment. Experimental procedures involving a host-vector system that provides a one-step lower level of biological containment than that specified may be conducted in the BL3 laboratory using containment equipment specified for the BL4 level of physical containment. Alternative combination of containment safeguards may be considered. Refer to the CDC/NIH Guidelines for these safeguards.

Containment Equipment (BL3).
Biological safety cabinets (Class I, II, or III) or other appropriate combinations of personal protective or physical containment devices (e.g., special protective clothing, masks, gloves, respirators, centrifuge safety cups, sealed centrifuge rotors, and containment caging for animals) are used for all activities with infectious materials including organisms containing recombinant DNA molecules which pose a threat of aerosol exposure. These include: manipulation of cultures and of those clinical or environmental materials which may be a source of aerosols; the aerosol challenge of experimental animals; the harvesting of infected tissues or fluids from experimental animals and embryonate eggs; and the necropsy of experimental animals.

Laboratory Facilities (BL3)

• The laboratory is separated from areas which are open to unrestricted traffic flow within the building. Passage through two sets of doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. Physical separation of the high containment laboratory from access corridors or other laboratories or activities may be provided by a double-doored clothes change room (showers may be included), airlock, or other access facility which requires passage through two sets of doors before entering the laboratory.
• The interior surfaces of walls, floors, and ceilings are water resistant so that they can be easily cleaned. Penetrations in these surfaces are sealed or capable of being sealed to facilitate decontaminating the area.
• Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
• Laboratory furniture is sturdy and spaces between benches, cabinets, and equipment are accessible for cleaning.
• Each laboratory contains a sink for hand washing. The sink is foot, elbow, or automatically operated and is located near the laboratory exit door.
• Windows in the laboratory are closed and sealed.
• Access doors to the laboratory or containment module are self-closing.
• An autoclave for decontaminating laboratory wastes is available preferably within the laboratory.
• A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air into the laboratory through the entry area. The exhaust air is not recirculated to any other area of the building, is discharged to the outside, and is dispersed away from the occupied areas and air intakes. Personnel shall verify that the direction of the airflow (into the laboratory) is proper.
• The high efficiency particulate air/HEPA filtered exhaust air from Class I or Class II biological safety cabinets is discharged directly to the outside or through the building exhaust system. Exhaust air from Class I or II biological safety cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every twelve months. If the HEPA-filtered exhaust air from Class I or II biological safety cabinets is to be discharged to the outside through the building exhaust air system, it is connected to this system in a manner (e.g., thimble unit connection that avoids any interference with the air balance of the cabinets or building exhaust system.

Biosafety Level 4 (BL4)

The description for BL4 will not be included here as such facility is currently unavailable at UCD. Please refer to the NIH Guidelines for a full description of a maximum containment facility.

Laboratory Facilities

Secondary Barriers - Laboratory Design

The laboratory must include specific design features which can only be put into place during the initial construction or remodel of a work area. Therefore it is necessary to consider what the current as well as future needs might be of a given area. It is not possible to plan or anticipate all future needs, so it is necessary to provide minimally for a standard laboratory suite.

The standard or basic teaching laboratory is denoted as a biosafety level 1 (BSL 1) laboratory. The biosafety level 2 laboratory is often referred to as a modified basic laboratory, as it has the same design features as the BSL 1 laboratory with the added requirement of a biosafety cabinet as a standard feature.

The containment laboratory is the type of facility specified by the CDC/NIH Biosafety guidelines for Biosafety Level 3 infectious agents. It is a restricted lab with specific design features which must be accomplished during a construction phase. These features are identified earlier in this section.

The maximum containment facility is required for infectious agents and pathogens which are in the biosafety level 4. It is not anticipated that these pathogens would be handled at the UCD campus. Currently there are very few such facilities in the nation. One of these is located at the Center for Disease Control in Atlanta, Georgia and another is located at Fort Detrick in Maryland.

Primary Barrier - Equipment
The primary barriers are those which are enclosed by the secondary barrier, These primary barriers include equipment and instrumentation which would be used to contain biohazardous materials at the source. Examples of these are: biosafety cabinets, closed ventilated animal cages, safety centrifuge cups and safety blenders. Of these, the biological safety cabinet is the single most important containment device.

Biosafety Cabinets - Tissue Culture Hoods
Biological safety cabinets are classified as Class I, Class II, or Class III cabinets. The most common type biosafety cabinets used at UCD are Class II, Type A and Type B2. Biosafety cabinets should not be confused with clean air benches or glove boxes. Clean air benches offer no protection to the environment or personnel. Room air is pulled into the cabinet by a fan motor. The air then passes through a HEPA or standard air filter before the air passes over the product. Glove boxes are a totally enclosed, ventilated cabinet of gas tight construction, which is maintained under negative pressure and provides for worker protection. Operations are performed through attached rubber gloves. Other terms often used are laminar flow benches and tissue culture hoods. Please note that these offer little protection to the operator. For consistency, this manual will refer to biosafety cabinets and types as described below.

The common Biosafety Cabinet features are as follows:

• A Class I is a ventilated cabinet for personnel protection having an inward flow of air away from the operator. This class of cabinet does not provide protection for the product. The exhaust air from this cabinet is filtered through a high efficiency particulate air/HEPA filter. This cabinet is used in three operational modes: (i) with a full-width open front, (ii) with an installed front closure panel (having four 6-inch diameter openings) without gloves, and (iii) with an installed front closure panel equipped with arm-length rubber gloves. The face velocity of the inward flow of air through the full-width open front is 75 feet per minute or greater.
• A Class II cabinet is a ventilated cabinet for personnel and product protection having an open front with inward air flow for personnel protection, and HEPA filtered mass recirculated air flow for product protection. The cabinet exhaust air is filtered through a HEPA filter. The face velocity of the inward flow of air through the full-width open front is 75 feet per minute or greater. Design and performance specifications for Class II cabinets have been adopted by the National Sanitation Foundation, Ann Arbor, Michigan. Class II biosafety cabinets are subdivided into three types.
  • Class II, Type A - This cabinet type should maintain a minimum average inflow velocity of 75ft/min, have HEPA filtered downflow air where a portion of the air is exhausted from the cabinet and the remainder is supplied to the work surface. All the air is HEPA filtered regardless whether it is exhausted to the room or is the downflow portion of air to the work surface. The plenum and ducts are maintained at positive pressure. This type of cabinet is most commonly used for tissue and cell culture and recombinant work.
  • Class II, Type B1 - This cabinet maintains an average inflow velocity of 100 ft/min. This type exhausts 70% of the exhausted air to a dedicated building exhaust system and recirculates 30%. All air is HEPA filtered. A Type B1 cabinet may be used with BL1-3 agents with minute quantities of volatile toxic chemicals or radionuclides.
  • Class II, Type B2 - The type cabinet is 100% exhaust with no recirculation. It requires a supply air fan and forces filtered air to the work surface. An external building exhaust is required. The cabinet must maintain a minimum average air inflow velocity of 100 ft/min. A Type B2 cabinet can be used for BL 1-3 agents treated with volatile toxic chemicals and radionuclides.
  • Class II, Type B3 - The Type B3 cabinet should maintain a minimum average inflow velocity of 100 ft/min through the work access opening. This cabinet is the same cabinet as the Type A, except that it can be exhausted to a building exhaust system and the plenum and ducts are at a negative pressure.
• A Class III cabinet is a closed-front ventilated cabinet of gas tight construction which provides the highest level of personnel protection of all biosafety safety cabinets. The interior of the cabinet is protected from contaminants exterior to the cabinet. The cabinet is fitted with arm-length rubber gloves and is operated under a negative pressure of at least 0.5 inches water gauge. All supply air is filtered through HEPA filters. Exhaust air is filtered through two HEPA filters or one HEPA filter and incinerator before being discharged to the outside environment. National Sanitation Foundation Standard 49. 1976. Class II (Laminar Flow) Biohazard Cabinetry, Ann Arbor, Michigan.

Containment Centrifuges A containment centrifuge is one which is equipped with a containment feature which protects the laboratory atmosphere from the release of potentially infectious aerosolized materials. Aerosolization of the product in a centrifuge can occur when a bottle or tube leaks or ruptures. A containment device can be a secondary gasket to seal the rotor or centrifuge lid or safety cups and canisters which would contain a ruptured tube and/or specimen. For more detailed information contact your centrifuge manufacturer or the Biosafety Officer at X6754.