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Safe Laboratory Practices
Laboratory Biosafety Level Criteria
Table1: Summary of  Recommended Biosafety  Levels for Infectious Agents
Table1a: Summary of Vertebrate Animal Biosafety Levels (ABSL)
Biological Emergencies
Biosafety Cabinets (BSC's)
Types of BSC's
Comparison of BSC's Characteristics
Selection of a BSC Through Risk Assessment
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Animal Biosafety Level 3 (ABSL3)


Animal Biosafety Level 3 involves practices suitable for work with animals infected with indigenous or exotic agents that present the potential of aerosol transmission and of causing serious or potentially lethal disease.  ABSL-3 builds upon the standard practices, procedures, containment equipment, and facility requirements of ABSL-2.

A. Standard Practices
  1. Aside from the standard policies, procedures, and protocols for emergency situations established by the facility director, appropriate special policies and procedures should be developed as needed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC).

  2. The laboratory or animal facility director limits access to the animal room to the fewest number of individuals possible.  Personnel who must enter the room for program or service purposes when work is in progress are advised of the potential hazard.

  3. An appropriate medical surveillance program is in place.  All personnel receive appropriate immunizations or tests for the agents handled or potentially present (e.g., hepatitis B vaccine, TB skin testing).  When appropriate, a serum surveillance system should be implemented.(9) In general, persons who may be at increased risk of acquiring infection, or for whom infection might have serious consequences, are not allowed in the animal facility unless special procedures can eliminate the extra risk. Assessment should be made by the occupational health physician.

  4. A biosafety manual is prepared or adopted.  Personnel are advised of special hazards, and are required to read and follow instructions on practices and procedures.

  5. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use should be done only in designated areas and are not permitted in animal or procedure rooms.

  6. All procedures are carefully performed to minimize the creation of aerosols or splatters.

  7. Equipment and work surfaces in the room are routinely decontaminated with an effective disinfectant after work with the infectious agent, and especially after overt spills, splashes, or other contamination by infectious materials.

  8. All wastes from the animal room (including animal tissues, carcasses, contaminated bedding, unused feed, sharps, and other refuse animal tissues) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional or local requirements. Incineration is recommended.  The outer surface of the containers is disinfected prior to moving the material (see Special Practices #3 below).

  9. Policies for the safe handling of sharps are instituted.

    1. Needles and syringes or other sharp instruments are restricted in the animal facility for use only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles.

    2. Syringes that re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

    3. Plastic ware should be substituted for glassware whenever possible.

  10. Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility.

  11. A biohazard sign must be posted on the entrance to the animal room whenever infectious agents are present.  The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements for entering the animal room (e.g., the need for immunizations and respirators).

  12. All infectious samples are collected, labeled, transported, and processed in a manner that contains and prevents transmission of the agent(s).

  13. Laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. As necessary, personnel receive updates and/or additional training on procedural or policy changes.  Records of all training provided are maintained.

  14. An insect and rodent control program is in effect.
B. Special Practices
  1. Cages are autoclaved or thoroughly decontaminated before bedding is removed and before they are cleaned and washed. Equipment must be decontaminated according to any local, state, or federal regulations before being packaged for transport or removal from the facility for repair or maintenance.

  2. A spill procedure is developed and posted.  Only personnel properly trained and equipped to work with infectious materials are to clean up spills.  Spills and accidents that result in overt exposures to infectious materials must be immediately reported to the facility director.  Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.

  3. All wastes from the animal room must be autoclaved prior to incineration or other appropriate terminal treatment.

  4. Materials not related to the experiment (e.g., plants, animals) are not permitted in the animal room.
C. Safety Equipment (Primary Barriers)
  1. Uniforms or scrub suits are worn by personnel entering the animal room.  Wrap-around or solid-front gowns should be worn over this clothing. Front-button laboratory coats are unsuitable. The gown must be removed and left in the animal room. Before leaving the animal facility, scrub suits and uniforms are removed and appropriately contained and decontaminated prior to laundering or disposal.

  2. Personal protective equipment used is based on risk assessment determinations.

    1. Personal protective equipment is used for all activities involving manipulations of infectious material or infected animals.

    2. Personnel wear gloves when handling infected animals.  Gloves are removed aseptically and autoclaved with other animal room wastes before disposal.

    3. Appropriate face/eye and respiratory protection (e.g., respirators and face shields) is worn by all personnel entering animal rooms.

    4. Boots, shoe covers, or other protective footwear, and disinfectant foot baths are available and used where indicated.

  3. The risk of infectious aerosols from infected animals or their bedding also can be reduced if animals are housed in containment caging systems, such as open cages placed in inward flow ventilated enclosures (e.g., laminar flow cabinets), solid wall and bottom cages covered with filter bonnets, or other equivalent primary containment systems.

  4. Biological safety cabinets and other physical containment devices are used whenever conducting procedures with a potential for creating aerosols.  These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, or intranasal inoculation of animals.  At BSL-3, all work should be done in a primary barrier; otherwise respirators should be worn by personnel in the room.
D. Facilities (Secondary Barriers)

  1. The animal facility is separated from areas that are open to unrestricted personnel traffic within the building.

  2. Access to the facility is limited by a self-closing and self-locking door. This exterior entry door may be controlled by a key lock, card key, or proximity reader. Entry into the animal room is via a double-door entry which includes a change room and shower(s).  An additional double-door access (air-lock) or double-doored autoclave may be provided for movement of supplies and wastes into and out of the facility, respectively.  Doors to animal rooms open inward and are self-closing.  Doors to cubicles inside an animal room may open outward or slide horizontally or vertically.

  3. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are water resistant.  Penetrations in floors, walls and ceiling surfaces are sealed and openings around ducts and the spaces between doors and frames are capable of being sealed to facilitate decontamination.

  4. A hands-free or automatically operated hand washing sink is provided in each animal room near the exit door.  The sink trap is filled with an appropriate disinfectant after each use.

  5. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas.

  6. Any windows must be resistant to breakage.  Where possible, windows should be sealed. If the animal facility has windows that open, they are fitted with fly screens.

  7. If floor drains are provided, they are always filled with an appropriate disinfectant.

  8. Ventilation should be provided in accordance with criteria from the Guide for Care and Use of Laboratory Animals, latest edition. A ducted exhaust air ventilation system is provided. This system creates directional airflow which draws air into the laboratory from "clean" areas and toward "contaminated" areas. The exhaust air is not recirculated to any other area of the building. Filtration and other treatments of the exhaust air may not be required, but should be considered based on site requirements, and specific agent manipulations and use conditions. The exhaust must be dispersed away from occupied areas and air intakes, or the exhaust must be HEPA-filtered. Personnel must verify that the direction of the airflow (into the animal areas) is proper. It is recommended that a visual monitoring device that indicates and confirms directional inward airflow be provided at the animal room entry. Consideration should be given to installing an HVAC control system to prevent sustained positive pressurization of the animal spaces. Audible alarms should be considered to notify personnel of HVAC system failure.

  9. HEPA-filtered exhaust air from a Class II biological safety cabinet can be recirculated into the animal room if the cabinet is tested and certified at least annually.  When exhaust air from Class II safety cabinets is to be discharged to the outside through the building exhaust air system, the cabinets must be connected in a manner that avoids any interference with the air balance of the cabinets or the building exhaust system (e.g., an air gap between the cabinet exhaust and the exhaust duct).  When Class III biological safety cabinets are used, they should be directly connected to the exhaust system.  If the Class III cabinets are connected to the supply system, it is done in a manner that prevents positive pressurization of the cabinets.

  10. Cages are washed in a cage washer.  The mechanical cage washer has a final rinse temperature of at least 180F.

  11. An autoclave is available which is convenient to the animal rooms where the biohazard is contained.  The autoclave is utilized to decontaminate infectious waste before moving it to other areas of the facility.

  12. If vacuum service (i.e., central or local) is provided, each service connection should be fitted with liquid disinfectant traps and an in-line HEPA filter, placed as near as practicable to each use point or service cock. Filters are installed to permit in-place decontamination and replacement.

  13. Illumination is adequate for all activities, avoiding reflections and glare that could impede vision.

  14. The completed Biosafety Level 3 facility design and operational procedures must be documented.  The facility must be tested for verification that the design and operational parameters have been met prior to operation.  Facilities should be re-verified at least annually against these procedures as modified by operational experience.

  15. Additional environmental protection (e.g., personnel showers, HEPA filtration of exhaust air, containment of other piped services, and the provision of effluent decontamination) should be considered if recommended by the agent summary statement, as determined by risk assessment of the site conditions, or other applicable federal, state, or local regulations.
ABSL1 ABSL2 ABSL3

Reproduced from "Biosafety in Microbiological and Biomedical Laboratories, BMBL 4th Edition" with permission from the Center for Disease Control (CDC).
     
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