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SECTION III
Laboratory Biosafety Level Criteria
The essential elements of the four biosafety levels for activities involving infectious
microorganisms and laboratory animals are summarized in Tables 1 and 2. The levels are
designated in ascending order, by degree of protection provided to personnel, the
environment, and the community.
Biosafety Level 1
BIOSAFETY 1 is suitable for work involving well-characterized agents not known to cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is not required nor generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science.
The following standard and special practices, safety equipment and facilities apply to agents assigned to Biosafety Level 1:
A. Standard Microbiological Practices
1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress.
2. Persons wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the laboratory.
3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas where there is reasonable likelihood of exposure to potentially infectious materials. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.
4. Mouth pipetting is prohibited; mechanical pipetting devices are used.
5. All procedures are performed carefully to minimize the creation of splashes or aerosols.
6. Work surfaces are decontaminated at least once a day and after any spill of viable material.
7. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated at off-site from the laboratory are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility.
8. An insect and rodent control program is in effect.
B. Special Practices: None
C. Safety Equipment (Primary Barriers)
1. Special containment devices or equipment such as a biological safety cabinet are generally not required for manipulations of agents assigned to Biosafety Level 1.
2. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.
3. Gloves should be worn if the skin on the hands is broken or if a rash exists.
4. Protective eyewear should be worn for anticipated splashes of microorganisms or other hazardous materials to the face.
D. Laboratory Facilities (Secondary Barriers)
1. Each laboratory contains a sink for handwashing.
2. The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not appropriate, and should not be used because proper decontamination following a spill extremely difficult to achieve.
3. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
4. Laboratory furniture is sturdy. Spaces between benches, cabinets, and equipment are accessible for cleaning.
5. If the laboratory has windows that open, they are fitted with fly screens.
Biosafety Level 2
BIOSAFETY LEVEL 2 is similar to Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs in that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists, (2) access to the laboratory is limited when work is being conducted, (3) extreme precautions are taken with contaminated sharp items, and (4) certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.
The following standard and special practices, safety equipment, and facilities apply to agents assigned to Biosafety Level 2:
A. Standard Microbiological Practices
1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.
2. Persons wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the laboratory.
3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.
4. Mouth pipetting is prohibited; mechanical pipetting devices are used.
5. All procedures are performed carefully to minimize the creation of splashes or aerosols.
6. Work surfaces are decontaminated at least once a day and after any spill of viable material.
7. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated at off-site from the laboratory are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility.
8. An insect and rodent control program is in effect.
B. Special Practices
1. Access to the laboratory is limited or restricted by the laboratory director when work with infectious agents is in progress. In general, persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. The laboratory director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.
2. The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet specific entry requirements (e.g., immunization) enter the laboratory or animal rooms.
3. When the infectious agent(s) in use in the laboratory require special provisions for entry (e.g., immunization), a hazard warning sign incorporating the universal biohazard symbol is posted on the access door to the laboratory work area. The hazard warning sign identifies the infectious agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates the special requirement(s) for entering the laboratory.
4. Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).
5. When appropriate, considering the agent(s) handled, baseline serum samples for laboratory and other at-risk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the facility.
6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.
7. Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural or policy changes.
8. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.
a. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
b. Syringes which re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.
c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass are decontaminated before disposal, according to any local, state, or federal regulations.
9. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.
10. Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated according to any local, state, or federal regulations before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, state, or federal regulations, before removal from the facility.
11. Spills and accidents which result in overt exposures to infectious materials are immediately reported to the laboratory director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.
12. Animals not involved in the work being performed are not permitted in the lab.
C. Safety Equipment (Primary Barriers)
1. Properly maintained biological safety cabinets, preferably Class II, or other appropriate personal protective equipment or physical containment devices are used whenever:
a. Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals or eggs.
b. High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed rotor heads or centrifuge safety cups are used, and if these rotors or safety cups are opened only in a biological safety cabinet.
2. Face protection (goggles, mask, faceshield or other splatter guards) is used for anticipated splashes or sprays of infectious or other hazardous materials to the face, when the microorganisms must be manipulated outside the BSC.
3. Protective laboratory coats, gowns, smocks, or uniforms designated for lab use are worn while in the laboratory. This protective clothing is removed and left in the laboratory before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices). All protective clothing is either disposed of in the laboratory or laundered by the institution; it should never be taken home by personnel.
4. Gloves are worn when handling infected animals and when hands may contact infectious materials, contaminated surfaces or equipment. Wearing two pairs of gloves may be appropriate; if a spill or splatter occurs, the hand will be protected after the contaminated glove is removed. Gloves are disposed of when contaminated, removed when work with infectious materials is completed, and are not worn outside the laboratory. Disposable gloves are not washed or reused.
D. Laboratory Facilities (Secondary Barriers)
1. Each laboratory contains a sink for handwashing.
2. The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not appropriate, and should not be used because proper decontamination following a spill is extremely difficult to achieve.
3. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
4. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.
5. If the laboratory has windows that open, they are fitted with fly screens.
6. A method for decontamination of infectious or regulated laboratory wastes is available (e.g., autoclave, chemical disinfection, incinerator, or other approved decontamination system).
7. An eyewash facility is readily available.
Biosafety Level 3
BIOSAFETY LEVEL 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done 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 materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. 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 Biosafety Level 3 (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 Biosafety Level 2 facilities. However, the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 must be rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made only by the laboratory director.
The following standard and special safety practices, equipment and facilities apply to agents assigned to Biosafety Level 3:
A. Standard Microbiological Practices
1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.
2. Persons wash their hands after handling infectious materials and animals, after removing gloves, and when they leave the laboratory.
3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the laboratory. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.
4. Mouth pipetting is prohibited; mechanical pipetting devices are used.
5. All procedures are performed carefully to minimize the creation of aerosols.
6. Work surfaces are decontaminated at least once a day and after any spill of viable material.
7. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated at off-site from the laboratory are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility.
8. An insect and rodent control program is in effect.
B. Special Practices
1. Laboratory doors are kept closed when experiments are in progress.
2. The laboratory director controls access to the laboratory and restricts access to persons whose presence is required for program or support purposes. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. Persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. The director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.
3. The laboratory director 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, enter the laboratory or animal rooms.
4. When infectious materials or infected animals are present in the laboratory or containment module, a hazard warning sign, incorporating the universal biohazard 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 laboratory director 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.
5. Laboratory personnel receive the appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).
6. Baseline serum samples are collected and stored for all laboratory and other at-risk personnel. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory.
7. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.
8. Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural changes.
9. The laboratory director is responsible for insuring that, before working with organisms at Biosafety Level 3, all personnel demonstrate proficiency in standard microbiological practices and techniques, and in the practices and operations specific to the laboratory facility. This might include prior experience in handling human pathogens or cell cultures, or a specific training program provided by the laboratory director or other competent scientist proficient in safe microbiological practices and techniques.
10. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.
a. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
b. Syringes which re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.
c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, according to any local, state, or federal regulations.
11. All manipulations involving infectious materials 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.
12. Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials. Contaminated equipment should also be decontaminated before it is sent for repair or maintenance or package for transport in accordance with applicable local, state, or federal regulations, before removal from the facility. Plastic-backed paper toweling used on non-perforated work surfaces within biological safety cabinets facilitates clean-up.
13. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.
14. All potentially contaminated waste materials (e.g., gloves, lab coats, etc.) from laboratories or animal rooms are decontaminated before disposal or reuse.
15. Spills of infectious materials are decontaminated, contained and cleaned up by appropriate professional staff, or others properly trained and equipped to work with concentrated infectious material.
16. Spills and accidents which result in overt or potential exposures to infectious materials are immediately reported to the laboratory director. Appropriate medical evaluation, surveillance, and treatment are provided and written records are maintained.
17. Animals and plants not related to the work being conducted are not permitted in the laboratory.
C. Safety Equipment (Primary Barriers)
1. Properly maintained biological safety cabinets are used (Class II or III - see Appendix A) for all manipulation of infectious materials.
2. Outside of a BSC, appropriate combinations of personal protective equipment are used (e.g., special protective clothing, masks, gloves, face protection, or respirators), in combination with physical containment devices (e.g., centrifuge safety cups, sealed centrifuge rotors, or containment caging for animals).
3. This equipment must be used for manipulations of cultures and of those clinical or environmental materials which may be a source of infectious aerosols; the aerosol challenge of experimental animals; harvesting of tissues or fluids from infected animals and embryonated eggs, and necropsy of infected animals.
4. Face protection (goggles and mask, or faceshield) is worn for manipulations of infectious materials outside of a biological safety cabinet.
5. Respiratory protection is worn when aerosols cannot be safely contained (i.e., outside of a biological safety cabinet), and in rooms containing infected animals.
6. Protective laboratory clothing such as solid-front or wrap-around gowns, scrub suits, or coveralls must be worn in, and not worn outside, the laboratory. Reusable laboratory clothing is to be decontaminated before being laundered.
7. Gloves must be worn when handling infected animals and when hands may contact infectious materials and contaminated surfaces or equipment. Disposable gloves should be discarded when contaminated, and never washed for reuse.
D. Laboratory Facilities (Secondary Barriers)
1. The laboratory is separated from areas which are open to unrestricted traffic flow within the building. Passage through two sets of self-closing doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. A clothes change room (shower optional) may be included in the passage way.
2. Each laboratory contains a sink for handwashing. The sink is foot, elbow, or automatically operated and is located near the laboratory exit door.
3. 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 decontamination.
4. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
5. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.
6. Windows in the laboratory are closed and sealed.
7. A method for decontaminating all laboratory wastes is available, preferably within the laboratory (i.e., autoclave, chemical disinfection, incineration, or other approved decontamination method).
8. A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air from "clean" areas into the laboratory toward "contaminated" areas. The exhaust air is not recirculated to any other area of the building, and is discharged to the outside with filtration and other treatment optional. The outside exhaust must be dispersed away from occupied areas and air intakes. Laboratory personnel must verify that the direction of the airflow (into the laboratory) is proper.
9. The High Efficiency Particulate Air (HEPA)-filtered exhaust air from Class II or Class III biological safety cabinets is discharged directly to the outside or through the building exhaust system. If the HEPA-filtered exhaust air from Class II or III 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)136 that avoids any interference with the air balance of the cabinets or building exhaust system. Exhaust air from Class II biological safety cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every twelve months.
10. Continuous flow centrifuges or other equipment that may produce aerosols are contained in devices that exhaust air through HEPA filters before discharge into the laboratory.
11. Vacuum lines are protected with liquid disinfectant traps and HEPA filters, or their equivalent, which are routinely maintained and replaced as needed.
12. An eyewash facility is readily available.
Biosafety Level 4
BIOSAFETY LEVEL 4 is required for work with dangerous and exotic agents which pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease. Agents with a close or identical antigenic relationship to Biosafety Level 4 agents are handled at this level until sufficient data are obtained either to confirm continued work at this level, or to work with them at a lower level. Members of the laboratory staff have specific and thorough training in handling extremely hazardous infectious agents; and they understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics. They are supervised by competent scientists who are trained and experienced in working with these agents. Access to the laboratory is strictly controlled by the laboratory director. The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. A specific facility operations manual is prepared or adopted.
Within work areas of the facility, all activities are confined to Class III biological safety cabinets, or Class II biological safety cabinets used with one-piece positive pressure personnel suits ventilated by a life support system. The Biosafety Level 4 laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment.
The following standard and special safety practices equipment, and facilities apply to agents assigned to Biosafety Level 4:
A. Standard Microbiological Practices
1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.
2. Persons wash their hands after handling infectious materials and animals; they take a decontaminating shower when they leave the laboratory.
3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the laboratory. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.
4. Mouth pipetting is prohibited; only mechanical pipetting devices are used.
5. All procedures are performed carefully to minimize the creation of aerosols.
6. Work surfaces are decontaminated at least once a day and after any spill of viable material.
7. An insect and rodent control program is in effect.
B. Special Practices
1. Only persons whose presence in the facility or individual laboratory rooms is required for program or support purposes are authorized to enter. Persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. Therefore, persons who may be at increased risk of acquiring infection or for whom infection may be unusually hazardous, such as children or pregnant women, are not allowed in the laboratory or animal rooms.
The supervisor has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. Access to the facility is limited by means of secure, locked doors; accessibility is managed by the laboratory director, biohazards control officer, or other person responsible for the physical security of the facility. Before entering, persons are advised of the potential biohazards and instructed as to appropriate safeguards for insuring their safety. Authorized persons comply with the instructions and all other applicable entry and exit procedures. A logbook, signed by all personnel, indicates the date and time of each entry and exit. Practical and effective protocols for emergency situations are established.
2. When infectious materials or infected animals are present in the laboratory or animal rooms, hazard warning signs, incorporating the universal biohazard symbol, are posted on all access doors. The sign identifies the agent, lists the name of the laboratory director or other responsible person(s), and indicates any special requirements for entering the area (e.g., the need for immunizations or respirators).
3. The laboratory director is responsible for insuring that, before working with organisms at Biosafety Level 4, all personnel demonstrate a high proficiency in standard microbiological practices and techniques, and in the special practices and operations specific to the laboratory facility. This might include prior experience in handling human pathogens or cell cultures, or a specific training program provided by the laboratory director or other competent scientist proficient in these unique safe microbiological practices and techniques.
4. Laboratory personnel receive available immunizations for the agents handled or potentially present in the laboratory.
5. Baseline serum samples for all laboratory and other at risk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory. The decision to establish a serologic surveillance program takes into account the availability of methods for the assessment of antibody to the agent(s) of concern. The program provides for the testing of serum samples at each collection interval and the communication of results to the participants.
6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.
7. Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural changes.
8. Personnel enter and leave the facility only through the clothing change and shower rooms, and shower each time they leave the facility. Personnel use the airlocks to enter or leave the laboratory only in an emergency.
9. Personal clothing is removed in the outer clothing change room and kept there. Complete laboratory clothing, including under garments, pants and shirts or jumpsuits, shoes, and gloves, is provided and used by all personnel entering the facility. When leaving the laboratory and before proceeding into the shower area, personnel remove their laboratory clothing in the inner change room. Soiled clothing is autoclaved before laundering.
10. Supplies and materials needed in the facility are brought in by way of the double-doored autoclave, fumigation chamber, or airlock, which is appropriately decontaminated between each use. After securing the outer doors, personnel within the facility retrieve the materials by opening the interior doors of the autoclave, fumigation chamber, or airlock. These doors are secured after materials are brought into the facility.
11. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments are restricted in the laboratory for use only when there is no alternative, such as for parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.
a. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
b. Syringes which re-sheath the needle, needle-less systems, and other safe devices should be used when appropriate.
c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, according to any local, state, or federal regulations.
12. Biological materials to be removed from the Class III cabinet or from the Biosafety Level 4 laboratory in a viable or intact state are transferred to a nonbreakable, sealed primary container and then enclosed in a nonbreakable, sealed secondary container. This is removed from the facility through a disinfectant dunk tank, fumigation chamber, or an airlock designed for this purpose.
13. No materials, except for biological materials that are to remain in a viable or intact state, are removed from the Biosafety Level 4 laboratory unless they have been autoclaved or decontaminated before they leave the facility. Equipment or material which might be damaged by high temperatures or steam may be decontaminated by gaseous or vapor methods in an airlock or chamber designed for this purpose.
14. Laboratory equipment is decontaminated routinely after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials. Contaminated equipment is also decontaminated before it is sent for repair or maintenance.
15. Spills of infectious materials are contained and cleaned up by appropriate professional staff or others properly trained and equipped to work with concentrated infectious material.
16. A system is set up for reporting laboratory accidents and exposures and employee absenteeism, and for the medical surveillance of potential laboratory-associated illnesses. Written records are prepared and maintained. An essential adjunct to such a reporting-surveillance system is the availability of a facility for the quarantine, isolation, and medical care of personnel with potential or known laboratory-associated illnesses.
17. Materials (e.g., plants, animals, and clothing) not related to the experiment being conducted are not permitted in the facility.
C. Safety Equipment (Primary Barriers)
1. All procedures within the facility with agents assigned to Biosafety Level 4 are conducted in the Class III biological safety cabinet or in Class II biological safety cabinets used in conjunction with one-piece positive pressure personnel suits ventilated by a life support system.
Activities with viral agents that require Biosafety Level 4 secondary containment capabilities can be conducted within Class II biological safety cabinets within the facility, without the one-piece positive pressure personnel suit being used if (a) the facility has been decontaminated, (b) no work is being conducted in the facility with other agents assigned to Biosafety Level 4, (c) all personnel are immunized against the specific agent being manipulated and demonstrate protective antibody levels, and (d) all other standard and special practices are followed.
2. All personnel entering the facility will don complete laboratory clothing, including undergarments, pants, and shirts or jumpsuits, shoes, and gloves. All such personal protective equipment is removed in the change room before showering and leaving the laboratory.
D. Laboratory Facility (Secondary Barriers)
1. The Biosafety Level 4 facility consists of either a separate building or a clearly demarcated and isolated zone within a building. Outer and inner change rooms separated by a shower are provided for personnel entering and leaving the facility. A double-doored autoclave, fumigation chamber, or ventilated airlock is provided for passage of those materials, supplies, or equipment which are not brought into the facility through the change room.
2. Walls, floors, and ceilings of the facility are constructed to form a sealed internal shell which facilitates fumigation and is animal and insect proof. The internal surfaces of this shell are resistant to liquids and chemicals, thus facilitating cleaning and decontamination of the area. All penetrations in these structures and surfaces are sealed. Any drains in the floors contain traps filled with a chemical disinfectant of demonstrated efficacy against the target agent, and they are connected directly to the liquid waste decontamination system. Sewer vents and other ventilation lines contain HEPA filters.
3. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize the horizontal surface area on which dust can settle.
4. Bench tops have seamless surfaces which are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
5. Laboratory furniture is of simple and sturdy construction, and spaces between benches, cabinets, and equipment are accessible for cleaning.
6. A foot, elbow, or automatically operated handwashing sink is provided near the door of each laboratory room in the facility.
7. If there is a central vacuum system, it does not serve areas outside the facility. In-line HEPA filters are placed as near as practicable to each use point or service cock. Filters are installed to permit in-place decontamination and replacement. Other liquid and gas services to the facility are protected by devices that prevent backflow.
8. If water fountains are provided, they are foot operated and are located in the facility corridors outside the laboratory. The water service to the fountain is not connected to the backflow-protected distribution system supplying water to the laboratory areas.
9. Access doors to the laboratory are self-closing and lockable.
10. Any windows are breakage resistant.
11. A double-doored autoclave is provided for decontaminating materials passing out of the facility. The autoclave door which opens to the area external to the facility is sealed to the outer wall, and automatically controlled so that the outside door can only be opened after the autoclave "sterilization" cycle has been completed.
12. A pass-through dunk tank, fumigation chamber, or an equivalent decontamination method is provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the facility.
13. Liquid effluents from laboratory sinks, biological safety cabinets, floor drains (if used), and autoclave chambers are decontaminated by heat treatment before being discharged to the sanitary sewer. Effluents from showers and toilets may be discharged to the sanitary sewer without treatment. The process used for decontamination of liquid wastes must be validated physically and biologically by use of a constant recording temperature sensor in conjunction with an indicator microorganism having a defined heat susceptibility profile.
14. A dedicated non-recirculating ventilation system is provided. The supply and exhaust components of the system are balanced to assure directional airflow from the area of least hazard to the area(s) of greatest potential hazard. The differential pressure/directional airflow between adjacent areas is monitored and alarmed to indicate malfunction of the system. The airflow in the supply and exhaust components is monitored and the components interlocked to assure inward (or zero) airflow is maintained.
15. The general room exhaust air from a facility in which the work is conducted in a Class III cabinet system is treated by a passage through a HEPA filter(s) prior to discharge to the outside. The air is discharged away from occupied spaces and air intakes. The HEPA filter(s) are located as near as practicable to the source in order to minimize the length of potentially contaminated ductwork. The HEPA filter housings are designed to allow for in situ decontamination of the filter prior to removal, or removal of the filter in a sealed gas-tight primary container for subsequent decontamination and/or destruction by incineration. The design of the HEPA filter housing should facilitate validation of the filter installation. The use of pre-certified HEPA filters can be an advantage. The service-life of the exhaust HEPA filters can be extended through adequate filtration of the supply air.
16. A specially designed suit area may be provided in the facility to provide personnel protection equivalent to that provided by Class III cabinets. Personnel who enter this area wear a one-piece positive pressure suit that is ventilated by a life support system. The life support system includes alarms and emergency backup breathing air tanks. Entry to this area is through an airlock fitted with airtight doors. A chemical shower is provided to decontaminate the surface of the suit before the worker leaves the area. The exhaust air from the suit area is filtered by two sets of HEPA filters installed in series. A duplicate filtration unit, exhaust fan, and an automatically starting emergency power source are provided. The air pressure within the suit area is lower than that of any adjacent area. Emergency lighting and communication systems are provided. All penetrations into the internal shell of the suit area are sealed. A double-doored autoclave is provided for decontaminating waste materials to be removed from the suit area.
17. The treated exhaust air from Class II biological safety cabinets, located in a facility in which workers wear a positive pressure suit, may be discharged into the animal room environment or to the outside through the facility air exhaust system. The biological safety cabinets are tested and certified at 12-month intervals. The air exhausted from Class III biological safety cabinets is passaged through two HEPA filter systems (in series) prior to discharge to the outside. If the treated exhaust is discharged to the outside through the facility exhaust system, it is connected to this system in a manner that avoids any interference with the air balance of the cabinets or the facility exhaust system.
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Office of Health and Safety, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Mail Stop F05 Atlanta, Georgia 30333, USA Last Modified: 1/2/97 |
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