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Biosafety Levels

A. Introduction

This section describes the various biosafety levels which consist of combinations of laboratory practices and techniques, safety equipment and laboratory facilities that are commensurate with the potential hazard posed by the infectious agents used in the laboratory. Section B describes three general biosafety levels that are appropriate for most experimentation.

Section C describes three working levels, to be followed when using experimental animals. They are designated Animal Biosafety Levels. The levels to which the various agents are assigned can be found in Section D. 

Please note that work with agents above biosafety level 3 is handled on a case-by-case basis. Please contact EH&S for regulations regarding this type of experimentation.

The lab supervisors and instructors are directly responsible for the safe operation of the laboratory. Their knowledge and judgment are critical in assessing risks and appropriately applying the recommendations of these guidelines. The recommended biosafety level to which a particular agent is assigned represents minimal conditions under which the agent can ordinarily be safely handled. Special characteristics of the agents used, the training and experience of personnel and the nature or function of the laboratory may further influence the supervisor in applying these guidelines. 

Work with known agents should be conducted at the biosafety level recommended unless specific information is available to suggest that virulence, pathogenicity, antibiotic resistance patterns, etc., are significantly altered to require more or allow less stringent practices to be used. As a general policy, clinical, field and environmental specimens should be handled at the level recommended for most pathogenic agents the clinical diagnosis or other evidence suggests is likely to be present. For example, sputa submitted for tuberculosis examination should be handled from the outset as potentially infectious (biosafety level 3). Personnel working with specimens or tissues submitted for rabies examination should be immunized and take appropriate precautions to prevent parenteral or aerosol exposures. 

Personnel working with specimens or tissues of domestic and wild animals should be aware of known or potential zoonotic infections and should be immunized if vaccines are available (see Agent Listing and Appendix 5 for vaccination recommendations). Personnel wishing to work at a biosafety level lower than that specified in this manual are required to have prior approval to do so. The request, with supporting materials, should be submitted to EH&S. 

B. General Specifications

Biosafety Level I (BL1) is suitable for experiments involving agents of no known or minimal potential hazard to laboratory personnel and the environment. The laboratory is not separated from the general traffic patterns of the building. Work is generally conducted on open benchtops and special containment equipment is not required or generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by personnel with general training in microbiology or related science. BL 1 practices, safety equipment and facilities are those appropriate for undergraduate and secondary educational training and teaching laboratories and for other facilities working with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans or not known to colonize in humans. (Bacillus cereus, Naegleria fowleri, and canine distemper virus are representative of those microorganisms assigned to BL 1.) Note however, that many agents not ordinarily associated with disease processes or colonization in humans are opportunistic pathogens and may cause infection for the young, the aged and for immunosuppressed or immuno-incompetent individuals. Vaccine strains that have undergone multiple in vivo passages should not a priori be considered avirulent.  

 

Biosafety Level I

  1. Standard Microbiological Practices
    1. Laboratory doors are kept closed when experiments are in progress.
    2. Work surfaces are decontaminated daily and after each spill of viable material. (See Section IV for a list of common disinfectants.) 
    3. All contaminated liquids or solid wastes are decontaminated before being disposed of or otherwise handled (See Section VI). 
    4. Mechanical pipetting devices are used; mouth pipetting is prohibited. Eating, drinking, smoking, storing food and applying cosmetics are not permitted in the work area. 
    5. Persons will wash their hands after handling viable materials or animals and before they leave the laboratory. 
    6. All procedures must be carefully performed to minimize the creation of aerosols. 
    7. The wearing of laboratory coats, gowns or uniforms is strongly recommended.
  2. Special Practices
    1. Contaminated materials that are to be decontaminated at a site away from the laboratory shall be placed in a durable container that is sealed before leaving the lab. 
    2. An insect and rodent control program is in effect. 
  3. Containment Equipment
    1. Special containment equipment is generally not required for manipulations of agents assigned to BL 1.
  4. Laboratory Facilities 
    1. The laboratory should be designed so that it is easily cleaned. 
    2. Bench tops should be impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. 
    3. Each laboratory should contain a hand-washing sink. 
    4. If the laboratory has windows that open, they should be fitted with screens. 
    5. An autoclave for the decontamination of infectious laboratory wastes should be available in the same building as the laboratory. (See Appendix 9)

Biosafety Level 2

(BL2) is similar to BL 1 and is suitable for experiments involving agents of moderate potential hazard to personnel and the environment. It differs in that laboratory personnel have specific training in handling pathogenic agents, access to the laboratory is limited when experiments are being conducted and procedures involving large volumes or high concentrations are conducted in BSC’s or other physical containment equipment. BL 2 practices, equipment and facilities are those which are applicable to clinical, diagnostic, teaching and other facilities working with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. [The Hepatitis agents (Hepatitis A, Hepatitis B, Hepatitis non A-non B), the Salmonellae, and Toxoplasma spp, are representative of micro-organisms assigned to this containment level.] Primary hazards to personnel working with these agents relate to accidental auto-inoculation or ingestion of infectious materials. Procedures with high aerosol potential may predictably and significantly increase the risk of exposure and must be conducted in primary containment equipment or devices. 

  1. Standard Microbiological Practices
    1. Laboratory doors are kept closed when experiments are in progress. 
    2. Work surfaces are decontaminated at least daily and after each spill of viable material. (See Section IV for a list of common disinfectants) 
    3. All contaminated liquids or solid waste are decontaminated before being disposed of or otherwise handled. (See Section VI) 
    4. Mechanical Pipetting devices are used; mouth pipetting is prohibited. 
    5. Eating, drinking, smoking, storing food and applying cosmetics are not permitted in the work area. 
    6. Persons must wash their hands after handling infectious materials or animals and before they leave the lab. 
    7. All procedures are conducted carefully to minimize the creation of aerosols. 
    8. Laboratory coats, gowns or uniforms must be worn in the laboratory, but must NOT be worn in nonlaboratory areas when soiled or contaminated. 
  2. Special Practices 
    1. Contaminated materials that are to be decontaminated at a site away from the laboratory shall be placed in durable, leakproof containers which are sealed before being removed from the laboratory. 
    2. An insect and rodent control program is in effect.
    3. The laboratory supervisor will ensure that only persons who have been advised of the potential hazard and met any specific entry requirements (e.g. immunization) may enter the laboratory or animal rooms.
    4. When infectious materials or infected animals are present in the laboratory or animal rooms, a hazard warning sign, incorporating the universal biohazard symbol, is posted on all laboratory and animal room access doors and on other items (i.e. equipment, containers, materials) as appropriate to indicate the presence of viable infectious agents. The hazard warning sign should identify the agent, list the name of the laboratory supervisor or other responsible person(s) and indicate any special requirements for entering the area (immunization, respirators, etc.). 
    5. Access to the Laboratory is limited by the laboratory supervisor when experiments are being conducted. 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. Persons at increased risk may include children, pregnant women, and individuals who are immunodeficient or immunosuppressed. The lab manager or lab supervisor has the final responsibility for assessing each individual circumstance and determining who may enter or work in the laboratory. 
    6. Animals not involved in the experiment being performed are not permitted in the lab. 
    7. The use of hypodermic needles and syringes is restricted to gavage, parental injection, and aspiration of fluids from laboratory animals and diaphragm vaccine bottles. Hypodermic needles and syringes are not used as a substitute for automatic pipetting devices in the manipulation of infectious fluids. Serial dilutions of infectious agents should not be done in diaphragm bottles with needles and syringes because of the hazards of autoinoculation and of aerosol exposure. Cannulas should be used instead of sharp needles whenever possible. 
    8. If activities of lesser biohazard potential are conducted in the laboratory concurrently with activities requiring BL 2, all activities will be conducted at BL 2. 
    9. Gloves should be worn for all procedures requiring the handling of infectious materials or infected animals.
    10. All spills, accidents, and overt or potential exposures to infectious materials must be immediately reported to the laboratory supervisor. A written record must be prepared and maintained. Appropriate medical evaluation, surveillance, and treatment must be provided. (See Sections VII and VIII) k. Safety or operational instructions which identify known and potential hazards and which specify practices and procedures to minimize or eliminate such risks should be prepared or adopted as necessary. Personnel must be advised of special hazards and are required to read and follow standard practices and procedures. 
  3. Containment Equipment

    Biological safety cabinets (Class I, II, or III) (See Appendix 6) or other appropriate personal protective or physical containment devices are used whenever: 

    1. Procedures with a high potential for creating aerosols are conducted. These may include centrifugation, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient, intranasal inoculation of animals, and harvesting infected tissue from animals or eggs.
    2. High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed heads or centrifuge safety cups are used and if they are opened only in a biological safety cabinet. 4. Laboratory Facilities a. The laboratory should be designed so that it is easily cleaned. b. Bench tops should be impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. The use of plastic-backed absorbent toweling over the work surface is recommended. 
    3. Each laboratory should contain a hand-washing sink.
    4. If the laboratory has windows that open, they should be fitted with screens. 
    5. An autoclave for the decontamination of infectious laboratory wastes should be available in the same building as the laboratory. (See Appendix 9) Biosafety Level 3 (BL3) is suitable for experiments involving agents of high potential risk to personnel and the environment. 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. Access to the laboratory is controlled by the lab supervisor. The laboratory has special engineering and design features and physical containment equipment and devices. All procedures involving the manipulation of infectious material are conducted within biological safety cabinets or other containment devices or by personnel wearing appropriate personal protective clothing. Level three practices, safety equipment and facilities are those that are applicable to clinical, diagnostic, teaching, research or production facilities working with indigenous or exotic agents that may cause serious and potentially lethal infections. (Mycobacterium Tuberculosis, St. Louis Encephalitis Virus, and Coxiella burnetti are representative of microorganisms assigned to this level.) Primary hazards to personnel working with these agents relate to auto-inoculation, ingestion, and exposure to infectious aerosols. 

Biosafety Level 3 

  1. Microbiological Practices
    1. Laboratory doors are kept closed when experiments are in progress.
    2. Work surfaces are decontaminated at least daily and after each spill of viable material. (See Section IV) 
    3. All contaminated liquids or solid wastes are decontaminated before being disposed of or otherwise handled. (See Section VI)
    4. Mechanical pipetting devices are used; mouth pipetting is prohibited. 
    5. Eating, drinking, smoking, storing food, and applying cosmetics are not permitted in the work area.
    6. Persons must wash their hands after handling viable materials or animals and before they leave the laboratory.
    7. All procedures are conducted carefully to minimize the creation of aerosols.
    8. Laboratory clothing that protects street clothing (i.e. solid front or wrap-around gowns, scrub suits, coveralls, etc.) is worn in the laboratory. FRONT-BUTTON LABORATORY COATS ARE UNSUITABLE. Laboratory clothing is not to be worn outside of the laboratory and is decontaminated before being laundered. 
  2. Special Practices
    1. All contaminated materials shall be decontaminated within the generating laboratory. 
    2. An insect and rodent control program is in effect. 
    3. The laboratory supervisor will ensure that only persons who have been advised of the potential biohazard, meet any specific entry requirements (e.g. immunization) and comply with all entry and exit procedures may enter the laboratory or animal room. 
    4. When infectious materials or infected animals are present in the laboratory or animal rooms, a hazard warning sign incorporating the universal biohazard symbol is posted on all laboratory and animal room access doors and on other items (i.e. equipment, containers, materials) as appropriate to indicate the presence of viable infectious agents. The hazard warning sign should identify the agent, list the name of the laboratory supervisor or other responsible person(s) and indicate any special conditions of entry into the area (immunizations, respirators, etc.).
    5. Access to the laboratory is controlled by the laboratory supervisor and is restricted to persons whose presence is required for program or support needs. Persons who are at increased risk if acquiring an infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. Persons at increased risk may include children, pregnant women, and individuals who are immunodeficient or immunosuppressed. The lab supervisor has the final responsibility for assessing each individual circumstance and determining who may enter or work in the laboratory. 
    6. Animals and plants not related to the experiment being conducted are not permitted in the laboratory.
    7. The use of hypodermic needles is restricted to gavage, parenteral injection, and aspiration of fluids from lab animals and diaphragm vaccine bottles. Hypodermic syringes are not used as a substitute for automatic pipetting devices in the manipulation of infectious fluids. Serial dilutions of infectious agents should not be done in diaphragm bottles with syringes due to the potential for autoinoculation and aerosol exposure.
    8. If activities of lesser biohazard potential are conducted in the laboratory concurrently with activities requiring BL 3, all work will be conducted at BL 3.
    9. Gloves are worn when handling infectious materials or animals. Gloves should be removed aseptically and autoclaved with other laboratory wastes before being disposed. 
    10. All spills, accidents and overt or potential exposures to infectious materials must be immediately reported to the laboratory supervisor. A written report must be prepared and maintained. Appropriate medical evaluation, surveillance and treatment must be provided.
    11. Safety or operational instructions which identify known and potential hazards and which specify practices and procedures to minimize or eliminate such risks should be prepared or adopted. Personnel should be advised of special hazards and must read and follow required practices and procedures. 
    12. Molded surgical masks or respirators are worn in rooms containing infected animals. 
    13. m. All activities involving infectious materials are conducted in biological safety cabinets or other physical containment devices. No work in open vessels is conducted on the open bench. 
    14. The work surfaces of biological safety cabinets and other containment equipment are decontaminated when an experiment is finished. The use of plastic-backed paper toweling on non-perforated work surfaces within biological safety cabinets facilitates clean up following the completion of activities. 
    15. Baseline serum samples should be collected and stored for all laboratory and other at-risk personnel. Additional serum specimens must be collected in cases of known or suspected exposure.
  3. Biosafety Equipment
    Biological safety cabinets or other physical containment equipment for devices are used for all procedures and manipulations involving infectious material. 
  4. Laboratory Facilities
    1. The surfaces of walls, floors and ceilings are water-resistant and can be easily cleaned. Openings in these surfaces are sealed or capable of being sealed to facilitate decontaminating the area. 
    2. Bench tops should be impervious to water and resistant to acids, alkalis, organic solvents and moderate heat. 
    3. A foot or elbow-operated hand-washing sink is provided near each laboratory exit door. 
    4. Windows in the laboratory are closed and sealed. 
    5. An autoclave for decontamination of laboratory wastes is available within the laboratory. Infectious wastes which must be removed to another area in the same building for decontamination must be held and transported in a covered, leakproof container. (See Appendix 9) 
    6. The laboratory is separated from areas which are open to unrestricted traffic flow within the building. Separation is provided by either a double-door change room and shower or an airlock or other access facility which requires passage through two sets of doors to enter the laboratory. Access to the laboratory area is designed to prevent entrance of free-living arthropods. 
    7. Access doors to the laboratory are self closing and locking.
    8. An exhaust air ventilation system is provided. This system creates directional airflow that draws air into the laboratory through the entry area. The building exhaust system can be used for this purpose if the exhaust air is not recirculated to any other area of the building. Personnel must verify that proper directional airflow (into the laboratory) is achieved. However, air within the laboratory can be recirculated. The exhaust air from the laboratory is discharged directly to the outside or through the building exhaust system so that it is dispersed away from occupied buildings and air intakes. The exhaust air from the laboratory that does not come from the biological safety cabinet can be discharged to the outside without being treated.
    9. In laboratories which have supply air systems, the supply air and exhaust air system are interlocked to assure inward airflow at all times 
    10. The HEPA-filtered exhaust air from Class I or Class II biological safety cabinets should be discharged directly to the outside or through the building exhaust system. Air may be recirculated within the laboratory only after it has been filtered through tested and certified cabinet exhaust HEPA filters. Exhaust air from Class III biological safety cabinets is to be discharged to the outside through a building exhaust air system. It is recommended that these cabinets be connected to this system to avoid any interference with the air balance of the cabinet or building exhaust system.
    11. Biological safety cabinets must be certified annually.

E. Animal Specifications

These guidelines presuppose that laboratory animal facilities, operational practices, and quality of animal care meet applicable standards and regulations and that appropriate species selection has been made for animal experiments (e.g. Guide for the Care and Use of Laboratory Animals, HEW Publication No. (NIH) 78-23, Rev. 1978, and Laboratory Animal Welfare Regulations, 9 CFR, Subchapter A, Parts 1, 2 and 3).

Ideally, facilities for laboratory animals used for studies of infectious or noninfectious disease should be physically separated from other activities such as animal production and quarantine, clinical laboratories and especially from facilities that provide patient care. Animal facilities should be designed and constructed to facilitate cleaning and housekeeping. A "clean corridor/dirty corridor" layout is very useful in reducing cross-contamination. Floor drains should be installed in animal facilities only on the basis of clearly defined needs. If floor drains are installed, the drain trap should always contain water. This section describes three combinations of practices, safety equipment and facilities for experiments on animals infected with agents which produce or may produce human infection. These combinations provide increasing levels of protection to personnel and to the environment and are recommended as minimal standards for activities involving infected laboratory mammals. 

Animal Biosafety Level 1

  1. Standard Practices
    1. Doors to animal rooms are self-closing and are kept closed when experiments are in progress. 
    2. Work surfaces are decontaminated following use or spills of viable materials. (See Section IV) 
    3. Eating, drinking, smoking, and storing food are not permitted in animal rooms. 
    4. Personnel shall wash their hands after handling viable cultures and animals and before leaving the animal room.
    5. All procedures are carefully conducted to minimize the creation of aerosols. 
    6. An insect and rodent control program is in effect. 
  2. Special Practices
    1. Bedding materials from cages used for animals infected with agents transmissible to humans are decontaminated--preferably by autoclaving--before being discarded. Cages used for animals infected with agents transmissible to humans are washed and/or rinsed with water, heated to at least 180°F, for at least 20 minutes.
    2. The wearing of laboratory coats, gowns or uniforms in the animal room is recommended. It is further recommended that laboratory coats worn in the animal room not be worn in other areas. 
  3. Biosafety Equipment
    Special containment equipment is generally not required for animals infected with agents assigned to BL 1. 
  4. Animal Facilities
    1. The animal facility should be designed and constructed to facilitate cleaning and housekeeping. 
    2. A hand washing sink is available in the animal facility. 
    3. If the animal facility has windows that open, they shall be fitted with screens. 
    4. The animal facility shall be provided with inward directional airflow and that exhaust air be discharged to the outside without being recirculated to other rooms.

Animal Biosafety Level 2

  1. Standard Practices
    1. Doors to animal rooms are self-closing and are kept closed when experiments are in progress. 
    2. Work surfaces are decontaminated following use or spills of viable materials. (See Section IV) 
    3. Eating, drinking, smoking, and storing of food are not permitted in animal rooms. 
    4. Personnel shall wash their hands after handling viable cultures and animals and before leaving the animal room. 
    5. All procedures are carefully conducted to minimize the creation of aerosols. 
    6. An insect and rodent control program is in effect. 
  2. Special Practices 
    1. Cages shall be autoclaved before bedding is removed and before they are cleaned and washed. 
    2. Laboratory coats, gowns or uniforms shall be worn in the animal room but must not be worn elsewhere. 
    3. Surgical-type masks shall be worn by all personnel entering animal rooms housing nonhuman primates. 
    4. Access to the animal room is restricted by the laboratory or animal facility supervisor to personnel who have been advised of the potential hazard and who need to enter on an approved basis when experiments are in progress. In general, persons who may be at increased risk of acquiring infection or for whom infection might be unusually hazardous are not allowed in the animal room. Persons at increased risk may include children, pregnant women, and individuals who are immunodeficient or immunosuppressed. The lab supervisor has the final responsibility for assessing individual circumstances and determining who may enter or work in the animal room. 
    5. The laboratory supervisor will assure that only persons who have been advised of the potential hazard and meet any specific requirements (e.g. immunization) may enter the animal room. 
    6. Hazard warning signs, incorporating the universal biohazard warning symbol are posted on access doors to animal rooms when materials containing or animals infected with agents assigned to BL 2 or higher are present. The hazard warning sign should identify agent(s) in use, list the name of the laboratory supervisor or other responsible person(s) and indicate any special conditions for entry into the animal room (e.g., immunization, respirators). 
    7. Gloves are worn by personnel handling animals when the hazard of contact infection exists. Forceps should be used when handling and inoculating small laboratory animals to further reduce exposures of personnel to bites, scratches or unnecessary contact with infected animals. 
    8. All waste from the animal rooms are appropriately decontaminated-- preferably by autoclaving-before being disposed of. Infected animal carcasses are autoclaved before being disposed of in sealed, leakproof containers. (See Section VI)
    9. The use of hypodermic needles and syringes is restricted to gavage, parenteral injection or aspiration of fluids from laboratory animals and diaphragm bottles. Serial dilutions of infectious agents should not be done in diaphragm bottles with needles and syringes because of the hazards of autoinoculation and of aerosol exposure. Canulas should be used instead of sharp needles whenever possible.
    10. If floor drains are provided, the drain trap is always filled with water.
  3. Containment Equipment
    Biological safety cabinets, other physical containment devices and/or personal protective devices (e.g. respirators, face shields) are used whenever procedures with a high potential for creating aerosols are conducted. These may include necropsy of infected animals, harvesting of infected tissues or fluids from animals or eggs, intranasal inoculation of animals and manipulations of high concentrations or large volumes of infectious materials. (See Appendix 6) 
  4. Animal Facilities
    1. The animal facility shall be designed and constructed to facilitate cleaning and housekeeping. 
    2. A hand washing sink shall be available in the room where infected animals are housed. 
    3. If the animal facility has windows that open, they shall be fitted with screens.
    4. The animal facility shall be provided with inward directional airflow and that exhaust air be discharged to the outside without being recirculated to other rooms.
    5. An autoclave to decontaminate infectious waste is available in the same building with the animal facility. (See Appendix 9) 

Animal Biosafety Level 3

  1. Standard Practices
    1. Doors to animal rooms are self-closing and self-locking and are kept closed when experiments are in progress. 
    2. Work surfaces are decontaminated following use or spills of viable materials. (See Section IV) 
    3. Eating, drinking smoking, and storing of food are not permitted in the animal room.
    4. Personnel shall wash their hands after handling viable cultures and animals and before leaving the laboratory. 
    5. All procedures are carefully conducted to minimize the creation of aerosols.
    6. An insect and rodent control program is in effect. 
  2. Special Practices
    1. Cages are autoclaved before bedding is removed and before they are cleaned and washed. 
    2. Warp-around or solid-front gowns or uniforms are worn by personnel entering the animal room. Front-button laboratory coats are unsuitable. Protective gowns must remain in the animal room and must be decontaminated before being laundered. 
    3. Surgical-type masks or other respiratory protection devices are worn by personnel entering rooms housing animals infected with agents assigned to BL 3.
    4. Access to the animal room is restricted by the supervisor or other responsible person to personnel who have been advised of the potential hazard and who need to enter on a program or service basis when experiments are in progress In general, persons who may be at increased risk of acquiring infection or for whom infection might be unusually hazardous are not allowed in the animal room. Persons at increased risk may include children, pregnant women, and individuals who are immunodeficient or immunosuppressed. The lab supervisor or other responsible person has the final responsibility for assessing individual circumstances and determining who may enter or work in the animal room.
    5. The laboratory supervisor or other responsible person will assure that only persons who have been advised of the potential hazard and meet any specific requirements (e.g. immunization) may enter the animal room.
    6. Hazard warning signs, incorporating the universal biohazard warning symbol are posted on access doors to animal rooms when materials containing, or animals infected with, agents assigned to BL 2 or higher are present. The hazard warning sign should identify the agent(s) in use, list the name of the animal room supervisor or other responsible person (s) and indicate any special conditions of entry into the animal room (e.g. immunizations, respirators ). 
    7. Gloves are worn by personnel when handling infectious agents and animals. Gloves should be removed aseptically and autoclaved with other animal room wastes before being disposed of or reused.
    8. All wastes from the animal room are autoclaved before being disposed of. All animal carcasses are disposed of as biohazardous waste. 
    9. The use of hypodermic needles and syringes is restricted to gavage, parenteral injection or aspiration of fluids from laboratory animals and diaphragm bottles. Serial dilutions of infected agents should not be done in diaphragm bottles with needles and syringes because of the hazards of autoinoculation and of aerosol exposure. Cannulas should be used instead of sharp needles whenever possible. 
    10. If floor drains are provided, the drain trap is always filled with water. 
    11. If vacuum lines are provided, they shall be protected with HEPA filters and liquid traps. (See Appendix 6) l. Boots, shoe covers or other protective footwear and disinfectant foot baths are provided and used when indicated. 
  3. Biosafety Equipment 
    1. Personal protective clothing and equipment or other physical containment devices are used for all procedures and manipulations of infectious materials or infected animals.
    2. Infected laboratory animals are housed in partial containment caging systems such as open cages place in ventilated enclosures, solid wall and bottom cages covered by filter bonnets or other equivalent primary containment systems. 
  4. Animal Facilities
    1. The animal facility should be designed and constructed to facilitate cleaning and housekeeping and should be separated from areas which are open to unrestricted personnel traffic within the building. Separation shall be provided by an airlock or other access device which requires passage through two sets of doors to gain access to the animal room by a double door change room and shower.
    2. A foot- or elbow-operated hand washing sink is provided near each animal room exit door. 
    3. Windows in the animal room are closed and sealed.
    4. The animal room is provided with a ventilation system which creates an inward directional flow of air and assures that exhaust air is discharged directly to the outside or through the building exhaust system without being recirculated to any other area of the facility. Air within the animal room may be recirculated.
    5. An autoclave for decontamination of wastes is available within the animal room or animal facility. Materials to be autoclaved outside the animal room are transported in a covered leakproof container. (See Appendix 9) 
    6. The surface, of walls, floors and ceilings are water resistant and easily cleaned. Openings in these surfaces are sealed or capable of being sealed to facilitate fumigation or space decontamination.
    7. In animal facilities which have supply air systems, the supply air and exhaust air systems are electrically or mechanically interlocked to assure inward air-flow at all times.      
    8. The exhaust air from Class I or Class II biological safety cabinets can only be recirculated within the animal room after appropriate filtration through tested and certified cabinet exhaust HEPA filters. i. Biological safety cabinets must be certified yearly (See Appendix 6). 

F. Recommended Biosafety Levels for Infectious Agents and Infected Animals

The selection of an appropriate biosafety level for work with a particular agent is dependent upon a number of factors. 

The most important of these include: the virulence, pathogenicity, biological stability, and communicability of the agent; the nature or functions of the laboratory; the quantity and concentration of the agent; the endemicity of the agent; and the availability of effective vaccines or therapeutic measures.

If a combination of increasingly stringent primary and secondary containment procedures and facilities are used, laboratory studies and manipulations can be safely conducted on agents that are correspondingly more hazardous. 

In general, the biosafety level used for activities using infectious agents or infected animals should be commensurate with that required for the agent of highest virulence known or likely to be encountered in the course of contemplated work. 

For example: all diagnostic sera of human origin should be considered potentially infectious for hepatitis and handled under conditions which reasonably preclude cutaneous, oral, and parenteral exposure to personnel: sputa should be considered as potentially infectious for tuberculosis and in addition should be handled under conditions which reasonably preclude the generation of aerosols. If in the course of diagnostic or other laboratory examinations there is evidence that the materials being studied contain only an agent of higher or lower risk than expected, the biosafety level should be raised or lowered accordingly. In selecting the appropriate biosafety level, the laboratory manager and supervisor should determine the nature of the specific laboratory activity in which the infectious agent will be used.

Occasions will arise when the laboratory supervisor should select a biosafety level higher than that recommended in these guidelines. For example, a higher biosafety level may be indicated by the unique nature of the proposed activity (e.g. the need for special containment of experimentally generated aerosols during inhalation studies) or by the proximity of the laboratory to areas of special concern (e.g. a laboratory located near patient care areas). Additionally, if the procedures involve large quantities or highly concentrated preparations of infectious agents or manipulations which are likely to produce aerosols, a higher biosafety level may be recommended. The American Committee on Arthropod-Borne Viruses (ACAV) registered 424 arboviruses as of December 31, 1979. The ACAV Subcommittee on Arbovirus Laboratory Safety (SALS) publication Laboratory Safety for Arboviruses and Certain Other Viruses of Vertebrates (in press ) has assigned these 424 agents to biosafety levels 2-4 based on risk assessments. Those 94 agents assigned to biosafety levels 3-4 and seven arboviruses which are indigenous or are commonly used in laboratories and assigned to biosafety level 2 are included in Section A. The remaining 330 arboviruses registered with ACAV as of December 31, 1979 are assigned to biosafety level 2 and are listed in Section B. Arboviruses not included on the December 31, 1979 registry will be individually assessed by SALS and subsequently assigned to an appropriate biosafety level.