BIOSAFETY IN MICROBIOLOGICAL AND BIOMEDICAL LABORATORIES

6th Edition

CDC & NIH

Revised June 2020

Biosafety Level 4

Biosafety Level 4 (BSL-4) is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening diseases that are frequently fatal, agents for which there are no vaccines or treatments, or work with a related agent with unknown risk of transmission. Agents with a close or identical antigenic relationship to agents requiring BSL-4 containment are handled at this level until sufficient data are obtained to re-designate the level. Laboratory staff receive specific and thorough training in handling extremely hazardous infectious agents. Laboratory staff understand the primary and secondary containment functions of standard and special practices, containment equipment, and laboratory design characteristics. All laboratory staff and supervisors are competent in handling agents and procedures requiring BSL-4 containment. The laboratory supervisor controls access to the laboratory in accordance with institutional policies.

There are two models for BSL-4 laboratories:

1. Cabinet Laboratory: manipulation of agents is performed in a Class III BSC;
2. Suit Laboratory: personnel wear a positive-pressure supplied-air protective suit.

BSL-4 cabinet and suit laboratories have special engineering and design features to prevent microorganisms from dissemination into the environment. The following standard and special practices, safety equipment, and facility specifications are necessary for BSL-4.

A. Standard Microbiological Practices

1. The laboratory supervisor enforces the institutional policies that control safety in and access to the laboratory.
2. The laboratory supervisor ensures that laboratory personnel receive appropriate training regarding their duties, potential hazards, manipulations of infectious agents, necessary precautions to minimize exposures, and hazard/exposure evaluation procedures (e.g., physical hazards, splashes, aerosolization) and that appropriate records are maintained. Personnel receive annual updates and additional training when equipment, procedures, or policies change. All persons entering the facility are advised of the potential hazards, are instructed on the appropriate safeguards, and read and follow instructions on practices and procedures. An institutional policy regarding visitor training, occupational health requirements, and safety communication is considered.
3. Personal health status may affect an individual’s susceptibility to infection and ability to receive available immunizations or prophylactic interventions. Therefore, all personnel, and particularly those of reproductive age and/or those having conditions that may predispose them to increased risk for infection (e.g., organ transplant, medical immunosuppressive agents), are provided information regarding immune competence and susceptibility to infectious agents. Individuals having such conditions are encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance. See Section VII.
4. A safety manual specific to the facility is prepared or adopted in consultation with the facility director and appropriate safety professionals. The safety manual is available, accessible, and periodically reviewed and updated as necessary.a. The safety manual contains sufficient information to describe the biosafety and containment procedures for the organisms and biological materials in use, appropriate agent-specific decontamination methods, and the work performed.b. The safety manual contains or references protocols for emergency situations, including exposures, medical emergencies, facility malfunctions, and other potential emergencies. Training in emergency response procedures is provided to emergency response personnel and other responsible staff according to institutional policies.
5. A sign incorporating the universal biohazard symbol is posted at the entrance to the laboratory when infectious materials are present. Posted information includes: the laboratory’s Biosafety Level, the supervisor’s or other responsible personnel’s name and telephone number, PPE requirements, general occupational health requirements (e.g., immunizations, respiratory protection), and required procedures for entering and exiting the laboratory. Agent information is posted in accordance with the institutional policy.
6. Long hair is restrained so that it cannot contact hands, specimen, containers, or equipment.
7. Gloves are worn to protect hands from exposure to hazardous materials.
   
   a. Glove selection is based on an appropriate risk assessment.
   
   b. Inner gloves are not worn outside the laboratory.
   
   c. Change inner gloves when contaminated, glove integrity is compromised, or when otherwise necessary.
   
   d. Do not wash or reuse disposable gloves, and dispose of used gloves with other contaminated laboratory waste.
8. Gloves and other PPE are removed in a manner that minimizes personal contamination and transfer of infectious materials outside of the areas where infectious materials and/or animals are housed or manipulated.
9. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption are not permitted in laboratory areas. Food is stored outside the laboratory area.
10. Mouth pipetting is prohibited. Mechanical pipetting devices are used.
11. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware are developed, implemented, and followed; policies are consistent with applicable state, federal, and local requirements. Whenever practical, laboratory supervisors adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions are always taken with sharp items. These include:
    a. Plasticware is substituted for glassware whenever possible.
    
    b. Use of needles and syringes or other sharp instruments is limited in the laboratory and is restricted to situations where there is no alternative (e.g., parenteral injection, blood collection, or aspiration of fluids from laboratory animals or diaphragm bottles). Active or passive needle-based safety devices are to be used whenever possible.
    
    i. Uncapping of needles is performed in such a manner to reduce the potential for recoil causing an accidental needlestick.
    
    ii. Needles are not bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
    
    iii. If absolutely necessary to remove a needle from a syringe (e.g., to prevent lysing blood cells) or recap a needle (e.g., loading syringes in one room and injecting animals in another), a hands-free device or comparable safety procedure must be used (e.g., a needle remover on a sharps container, the use of forceps to hold the cap when recapping a needle).
    
    iv. Used, disposable needles and syringes are carefully placed in puncture-resistant containers used for sharps disposal immediately after use. The sharps disposal container is located as close to the point of use as possible.
    
    c. Non-disposable sharps are placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving
    
    d. Broken glassware is not handled directly. Instead, it is removed using a brush and dustpan, tongs, or forceps.
    
12. Perform all procedures to minimize the creation of splashes and/or aerosols.
    
13. Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant. Spills involving infectious materials are contained, decontaminated, and cleaned up by staff who are properly trained and equipped to work with infectious material. A spill procedure is developed and posted within the laboratory.
14. Decontaminate all cultures, stocks, and other potentially infectious materials before disposal using an effective method, consistent with applicable institutional, local, and state requirements. A method for decontaminating all laboratory wastes is available in the laboratory (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method). See B. Special Practices, #7 in the following subsection for additional details.
15. An effective integrated pest management program is implemented. See Appendix G.
16. Animals and plants not associated with the work being performed are not permitted in the laboratory.

B. Special Practices

1. All persons entering the laboratory are advised of the potential hazards and meet specific entry/exit requirements in accordance with institutional policies. Only persons whose presence in the facility or individual laboratory rooms is required for scientific or support purposes are authorized to enter. Additional training/security requirements may be required prior to gaining independent access to BSL-4 laboratories.
2. All persons who enter operational laboratory areas are provided information on signs and symptoms of disease and receive occupational medical services including medical evaluation, surveillance, and treatment, as appropriate, and offered available immunizations for agents handled or potentially present in the laboratory.a. An essential adjunct to such an occupational medical services system is the availability of a facility for the isolation and medical care of personnel with potential or known Laboratory-associated infections.
3. Laboratory personnel and support staff are trained and approved to work in the facility. The laboratory supervisor is responsible for ensuring that, prior to working independently with agents requiring BSL-4 containment, laboratory personnel demonstrate high proficiency in standard and special microbiological practices and techniques for working with agents requiring BSL-4 containment. Personnel are required to read and follow instructions on practices, and procedural changes are addressed as part of the protocol review.
4. A system is established for reporting and documenting near misses, laboratory accidents, exposures, unanticipated absence due to potential Laboratory-associated infection, and for the medical surveillance of potential laboratory-associated illnesses.
5. Incidents that result in exposure to infectious materials are immediately evaluated per institutional policy. All such incidents are reported to the laboratory supervisor, institutional management, and appropriate safety, compliance, and security personnel according to institutional policy. Appropriate records are maintained.
6. Biological materials that require BSL-4 containment are placed in a durable, leak-proof sealed primary container and then enclosed in a non-breakable, sealed secondary container prior to removal from the BSL-4 facility by authorized personnel. These materials are transferred through a disinfectant dunk tank, fumigation chamber, or decontamination shower for receipt by authorized personnel. Once removed, the primary container is not to be opened outside BSL-4 containment unless a validated inactivation method is used (e.g., gamma irradiation). The inactivation method is documented in-house with viability testing data to support the method.
7. All waste is decontaminated by a verified method prior to removal from the laboratory.
8. Equipment is routinely decontaminated and is decontaminated after spills, splashes, or other potential contamination and before repair, maintenance, or removal from the laboratory. Equipment or material that might be damaged by high temperatures or steam is decontaminated using an effective and verified method, such as a gaseous or vapor method, in an airlock or chamber designed for this purpose.
9. A logbook, or other means of documenting the date and time of all persons entering and leaving the laboratory, is maintained.
10. An inventory system for agents stored within the laboratory is in place.
11. While the laboratory is operational, personnel enter and exit the laboratory through the clothing change and shower rooms except during emergencies. All personal clothing and jewelry (except eyeglasses) are removed in the outer clothing change room. All persons entering the laboratory use laboratory clothing, including undergarments, pants, shirts, socks, jumpsuits, shoes, and gloves, as appropriate. All persons leaving the laboratory take a personal body shower. Used laboratory clothing and other waste, including gloves, are not removed from the inner change room through the personal shower. These items are treated as contaminated materials and decontaminated before laundering or disposal.
12. After the laboratory has been completely decontaminated by verification of a validated method and all infectious agents are secured, necessary staff may enter and exit without following the clothing change and shower requirements described above.
13. Daily inspections of essential containment and life support systems are completed and documented before laboratory work is initiated to ensure that the laboratory is operating according to established parameters.
14. Only necessary equipment and supplies are stored inside the laboratory. All equipment and supplies taken inside the laboratory are decontaminated before removal from the laboratory.
    
    a. Supplies and materials that are not brought into the laboratory through the change room are brought in through a dunk tank, previously decontaminated double-door autoclave, fumigation chamber, or airlock. After securing the outer doors, personnel within the laboratory retrieve the materials by opening the interior doors of the autoclave, fumigation chamber, or airlock. The inner door is secured after materials are brought into the facility. The outer door of the autoclave or fumigation chamber is not opened until the autoclave, fumigation chamber, or airlock has been operated through a successful decontamination cycle.

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

Cabinet Laboratory

1. All procedures involving the manipulation of infectious materials are conducted
   within a Class III BSC.
2. A Class III BSC 

a. Double-door, pass-through autoclave for decontaminating materials passing out of the Class III BSC(s). The autoclave doors are interlocked so that only one door can be opened at any time and are automatically controlled so that the outside door to the autoclave can only be opened after a successful decontamination cycle has been completed.

b. A pass-through dunk tank, fumigation chamber, or equivalent decontamination method so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the cabinet. Containment between the cabinet and the surrounding laboratory is maintained at all times.

c. A HEPA filter on the supply air intake and two HEPA filters in series on the exhaust outlet of the unit. Supply air is provided in such a manner that prevents positive pressurization of the cabinet. There are gas-tight dampers on the supply and exhaust ducts of the cabinet to permit gas or vapor decontamination of the unit. Ports for injection of test medium are present on all HEPA filter housings.

d. An interior constructed with smooth finishes that can be easily cleaned and decontaminated. All sharp edges on cabinet finishes are eliminated to reduce the potential for cuts and tears of gloves. Equipment to be placed in the Class III BSC is also free of sharp edges or other surfaces that may damage or puncture the cabinet gloves.

1. Gloves that are inspected for damage prior to use and changed if necessary. Gloves are replaced annually during cabinet recertification.
2. The cabinet is designed to permit maintenance and repairs of cabinet mechanical systems (e.g., refrigeration, incubators, centrifuges) to be performed from the exterior of the cabinet whenever possible.
3. Manipulation of high concentrations or large volumes of infectious agents within the Class III BSC is performed using physical containment devices inside the cabinet whenever practical. Such materials are centrifuged inside the cabinet using sealed rotors or centrifuge safety cups.
4. The interior of the Class III BSC and all contaminated plenums, fans, and filters are decontaminated using a validated gaseous or vapor method when there have been significant changes in cabinet usage, before major renovations or maintenance shutdowns, and in other situations, as determined by risk assessment. Success of the decontamination is verified before accessing the interior spaces of the cabinet.
5. The Class III BSC is certified at least annually.
6. For Class III BSCs directly connected via a double-door, pass-through to a BSL-4 suit laboratory, materials may be placed into and removed from the Class III BSC via the suit laboratory.
7. Workers in the laboratory wear protective laboratory clothing with a solid front, such as tie-back or wrap-around gowns, scrubs, or coveralls. Shoe coverings are considered based on a risk assessment. a. Upon exit, all protective clothing is removed in the inner change room before showering. b. Prescription eyeglasses are decontaminated before removal through the personal body shower.
8. Disposable gloves are worn underneath cabinet gloves to protect the worker from exposure should a break or tear occur in a cabinet glove.

Suit Laboratory

1. All procedures involving the manipulation of infectious materials are conducted within a Biological Safety Cabinet (BSC) or other physical containment devices. No work with open vessels is conducted on the bench.
   • Equipment that may produce aerosols is used within primary barrier devices that exhaust air through HEPA filtration before being discharged into the laboratory or facility exhaust system. These HEPA filters are tested annually and replaced as needed.
   • Materials centrifuged in the laboratory use sealed rotors or centrifuge safety cups. Loading and unloading of the rotors and centrifuge safety cups take place in the BSC or another containment device.
2. All procedures are conducted by personnel wearing a one-piece, positive-pressure supplied-air suit.
   • All persons don laboratory clothing, such as scrubs, before entering the room used for donning positive-pressure suits.
   • Procedures are in place to control and verify the operation of the one-piece positive-pressure supplied-air suit, including gloves, before each use.
   • Decontamination of outer suit gloves is performed during the course of normal laboratory operations to remove gross contamination and minimize further contamination of the laboratory.
   • Inner disposable gloves are worn to protect the laboratorian should a break or tear in the outer suit gloves occur. Disposable inner gloves are not worn outside the inner change area.
   • Upon exit from the chemical shower, inner gloves and all laboratory clothing are removed and discarded or collected for autoclaving before laundering prior to entering the personal shower.
   • Prescription eyeglasses are decontaminated before removal through the personal body shower.
3. A chemical shower is provided to decontaminate the surface of the positive-pressure suit before the worker leaves the laboratory. In the event of an emergency exit or failure of the chemical shower system, a method for decontaminating positive-pressure suits, such as a gravity-fed supply of chemical disinfectant, is provided.
4. An automatically activated emergency power source is provided at a minimum for the laboratory exhaust system, alarms, lighting, entry and exit controls, BSCs, and door gaskets.
   • Monitoring and control systems for air supply, exhaust, life support, alarms, entry and exit controls, and security systems are on an uninterrupted power supply (UPS).
5. A double-door autoclave, dunk tank, or fumigation chamber is provided at the containment barrier for the passage of materials, supplies, or equipment in or out of the laboratory.
6. Hands-free sinks inside the suit laboratory are placed near procedure areas.
7. An eyewash station for use during maintenance is readily available in the laboratory area.
8. Walls, floors, and ceilings of the laboratory are constructed to form a sealed internal shell to facilitate fumigation and prohibit animal and insect intrusion. The internal surfaces of this shell are resistant to liquids and chemicals used for cleaning and decontamination of the area. Floors are monolithic, sealed, and coved.
   • All penetrations in the internal shell of the laboratory, suit storage room, and the inner change room are sealed.

D. Laboratory Facilities (Secondary Barriers)

Cabinet Laboratory

1. The BSL-4 cabinet facility may be located in a separate building or a clearly demarcated and isolated zone within a building.
   • Facility access is restricted. Laboratory doors are lockable.
   • Exit from the laboratory is by sequential passage through an inner (i.e., dirty) changing area, a personal shower, and an outer (i.e., clean) change room upon exiting the cabinet laboratory.
2. An automatically activated emergency power source is provided, at a minimum, for the laboratory exhaust system, alarms, lighting, entry and exit controls, BSCs, and door gaskets.
   • Monitoring and control systems for air supply, exhaust, life support, alarms, entry and exit controls, and security systems are on an uninterrupted power supply (UPS).
3. A double-door autoclave, dunk tank, fumigation chamber, or ventilated airlock is provided at the containment barrier for the passage of materials, supplies, or equipment.
4. A hands-free sink is provided near the door of the cabinet laboratory(ies) and the inner change room. A sink is provided in the outer change room.
5. An eyewash station is readily available in the laboratory.
6. Walls, floors, and ceilings of the cabinet laboratory are constructed to form a sealed internal shell to facilitate fumigation and prohibit animal and insect intrusion. The internal surfaces of this shell are resistant to liquids and chemicals used for cleaning and decontamination of the area. Floors are monolithic, sealed, and coved.
   • All penetrations in the internal shell of the cabinet laboratory and inner change room are sealed.
   • Openings around doors into the cabinet laboratory and inner change room are minimized and capable of being sealed to facilitate decontamination.
7. Services and plumbing that penetrate the cabinet laboratory walls, floors, or ceiling are installed to ensure that no backflow from the laboratory occurs. These penetrations are fitted with two (in series) backflow prevention devices. Consideration is given to locating these devices outside of containment. Atmospheric venting systems are provided with two HEPA filters in series and are sealed up to the second filter.
8. Furniture is minimized, of simple construction, and capable of supporting anticipated loads and uses.
   • Spaces between benches, cabinets, and equipment are accessible for cleaning and decontamination.
   • Benchtops are impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
   • Chairs used in laboratory work are covered with a non-porous material that can be easily cleaned and decontaminated.
9. Windows are break-resistant and sealed.
10. Illumination is adequate for all activities and avoids reflections and glare that could impede vision.
11. If Class II BSCs or other primary containment barrier systems are needed in the cabinet laboratory, they are installed and operated in a manner to ensure their effectiveness.
    • BSCs are installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs are located away from doors, heavily traveled laboratory areas, and other possible airflow disruptions.
    • BSCs can be connected to the laboratory exhaust system by either a canopy connection (Class IIA only) or directly exhausted to the outside through a hard connection (Class IIB, IIC, or III). Cabinet exhaust air passes through two HEPA filters, including the HEPA in the BSC, prior to release outside. Class IIA or IIC BSC exhaust can be safely recirculated back into the laboratory environment if no volatile toxic chemicals are used in the cabinet.
    • BSCs are certified at least annually to ensure correct performance, or as specified in Appendix A, Part 7.
12. Central vacuum systems are discouraged. If there is a central vacuum system, it does not serve areas outside the cabinet. Two in-line HEPA filters are placed near each use point and overflow collection is provided while in use. Filters are installed to permit in-place decontamination and replacement.
13. A dedicated, non-recirculating ventilation system is provided. Only cabinet laboratories with the same HVAC requirements (i.e., other BSL-4 cabinet laboratories, ABSL-4 cabinet facilities) may share ventilation systems if gas-tight dampers and HEPA filters isolate each individual laboratory system.
    • The supply and exhaust components of the ventilation system are designed to maintain the laboratory at negative pressure to surrounding areas and provide differential pressure or directional airflow, as appropriate, between adjacent areas within the laboratory.
    • Redundant supply fans are recommended. Redundant exhaust fans are required. Supply and exhaust fans are interlocked to prevent positive pressurization of the cabinet laboratory.
    • The ventilation system is monitored and alarmed to indicate malfunction or deviation from design parameters. A visual monitoring device is installed outside of containment so proper differential pressures within the laboratory may be verified prior to entry and during regular checklist procedures. Visual monitoring is also in place within containment.
    • Supply air to and exhaust air from the cabinet laboratory, inner change room, and fumigation/decontamination chambers pass through a HEPA filter. The air exhaust discharge is located away from occupied spaces and building air intakes.
    • All HEPA filters are located as near as practicable to the laboratory to minimize the length of potentially contaminated ductwork. All HEPA filters are tested and certified annually.
    • The HEPA filter housings are designed to allow for in situ decontamination and verification of the validated decontamination process prior to removal. The design of the HEPA filter housing has gas-tight isolation dampers, decontamination ports, and the ability to individually scan each filter in the assembly for leaks

14. Pass-through dunk tanks, fumigation chambers, or equivalent decontamination methods are provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the laboratory. Access to the exit side of the pass-through is limited to those with authorized access to the BSL-4 laboratory and with specific clearance, if required.
15. Liquid effluents from cabinet laboratory sinks, floor drains, autoclave chambers, and other sources within the cabinet laboratory are decontaminated by a proven method, preferably heat treatment, before being discharged to the sanitary sewer.

• Decontamination of all liquid effluents is documented. The decontamination process for liquid effluents is validated physically and biologically. Biological validation is performed at least annually or more often, if required by institutional policy.
• Effluents from personal body showers and toilets may be discharged to the sanitary sewer without treatment.

16. A double-door, pass-through autoclave is provided for decontaminating materials passing out of the cabinet laboratory. Autoclaves that open outside of the laboratory are sealed to the wall through which the autoclave passes. This bioseal is durable, airtight, and capable of expansion and contraction. Positioning the bioseal so that the equipment can be accessed and maintained from outside the laboratory is strongly recommended. The autoclave doors are interlocked so that only one can be opened at any time and are automatically controlled so that the outside door to the autoclave can only be opened after the decontamination cycle has been completed.

• Gas discharge from the autoclave chamber is HEPA-filtered or decontaminated. Autoclave decontamination processes are designed so that unfiltered air or steam exposed to infectious material cannot be released to the environment.

17. The facility design parameters and operational procedures are documented. The facility is tested to verify that the design and operational parameters have been met prior to operation. Facilities are also re-tested annually or after significant modification to ensure operational parameters are maintained. Verification criteria are modified, as necessary, by operational experience.
18. Appropriate communication systems are provided between the laboratory and the outside (e.g., voice, fax, video, and computer). Provisions for emergency communication and emergency access or egress are developed and implemented.

D. Laboratory Facilities (Secondary Barriers) for Suit Laboratory

1. The BSL-4 suit facility may be located in a separate building or a clearly demarcated and isolated zone within a building.
   • Facility access is restricted. Laboratory doors are lockable.
   • Entry into the laboratory is through an airlock fitted with airtight doors.
   • Exit from the laboratory is by sequential passage through the chemical shower, inner (i.e., dirty) change room, personal shower, and outer (i.e., clean) changing area.
2. Personnel who enter this area wear a positive-pressure suit supplied with HEPA-filtered breathing air. The breathing air systems have redundant compressors, failure alarms, and emergency back-up capable of supporting all workers within the laboratory to allow the personnel to safely exit the laboratory.
3. A chemical shower is provided to decontaminate the surface of the positive-pressure suit before the worker leaves the laboratory. In the event of an emergency exit or failure of the chemical shower system, a method for decontaminating positive-pressure suits, such as a gravity-fed supply of chemical disinfectant, is provided.
4. An automatically activated emergency power source is provided at a minimum for the laboratory exhaust system, alarms, lighting, entry and exit controls, BSCs, and door gaskets.
   • Monitoring and control systems for air supply, exhaust, life support, alarms, entry and exit controls, and security systems are on an uninterrupted power supply (UPS).
5. A double-door autoclave, dunk tank, or fumigation chamber is provided at the containment barrier for the passage of materials, supplies, or equipment in or out of the laboratory.
6. Hands-free sinks inside the suit laboratory are placed near procedure areas.
7. An eyewash station for use during maintenance is readily available in the laboratory area.

8. Walls, floors, and ceilings of the laboratory are constructed to form a sealed internal shell to facilitate fumigation and prohibit animal and insect intrusion. The internal surfaces of this shell are resistant to liquids and chemicals used for cleaning and decontamination of the area. Floors are monolithic, sealed, and coved.
   • All penetrations in the internal shell of the laboratory, suit storage room, and the inner change room are sealed.
9. Services and plumbing that penetrate the laboratory walls, floors, or ceiling are installed to ensure that no backflow from the laboratory occurs. Breathing air systems are exempt from this provision. These penetrations are fitted with two (in series) backflow prevention devices. Consideration is given to locating these devices outside of containment. Atmospheric venting systems are provided with two HEPA filters in series and are sealed up to the second filter.
10. Decontamination of the entire laboratory is performed using a validated gaseous or vapor method when there have been significant changes in usage, before major renovations or maintenance shutdowns, and in other situations, as determined by risk assessment. Decontamination is verified prior to any change in the status of the laboratory.
11. Furniture is minimized, of simple construction, and capable of supporting anticipated loads and uses.
    • Spaces between benches, cabinets, and equipment are accessible for cleaning, decontamination, and unencumbered movement of personnel.
    • Benchtops are impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
    • Chairs used in laboratory work are covered with a non-porous material that can be easily cleaned and decontaminated.
    • Sharp edges and corners are avoided.
12. Windows are break-resistant and sealed.
13. Illumination is adequate for all activities and avoids reflections and glare that could impede vision.
14. BSCs and other primary containment barrier systems are installed and operated in a manner to ensure their effectiveness. See Appendix A.
    • BSCs are installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs are located away from doors, windows that can be opened, heavily traveled laboratory areas, and other possible airflow disruptions.
    • BSCs can be connected to the laboratory exhaust system by either a canopy connection (Class IIA only) or directly exhausted to the outside through a hard connection (Class IIB, IIC, or III), which contains a HEPA filter.
    • Class IIA or IIC BSC exhaust can be safely recirculated back into the laboratory environment if no volatile toxic chemicals are used in the cabinet.
    • BSCs are certified at least annually to ensure correct performance, or as specified in Appendix A, Part 7.
    • Class III BSCs are provided supply air in such a manner that prevents positive pressurization of the cabinet or the room.
15. Central vacuum systems are discouraged. If there is a central vacuum system, it does not serve areas outside the laboratory. Two in-line HEPA filters are placed near each use point and overflow collection is provided while in use. Filters are installed to permit in-place decontamination and replacement. Consideration is made to the provision of two HEPA filters in series as close to the vacuum pump as possible.
16. A dedicated, non-recirculating ventilation system is provided. Only laboratories or facilities with the same HVAC requirements (i.e., other BSL-4 laboratories, ABSL-4, ABSL-3Ag, ABSL-4Ag facilities) may share ventilation systems if gas-tight dampers and HEPA filters isolate each individual laboratory system.
    • The ventilation system is designed to maintain the laboratory at negative pressure to surrounding areas and provide differential pressure or directional airflow as appropriate between adjacent areas within the laboratory.
    • Redundant supply fans are recommended. Redundant exhaust fans are required. Supply and exhaust fans are interlocked to prevent positive pressurization of the laboratory.
    • The ventilation system is monitored and alarmed to indicate malfunction or deviation from design parameters. A visual monitoring device is installed outside of containment so proper differential pressures within the laboratory may be verified prior to entry and during regular checklist procedures. Visual monitoring is also in place within containment.
    • Supply air to the laboratory, including the decontamination shower, passes through a HEPA filter. All exhaust air from the suit laboratory, decontamination shower, and fumigation or decontamination chambers passes through two HEPA filters, in series, before discharge to the outside. The exhaust air discharge is located away from occupied spaces and air intakes.
    • All HEPA filters are located as near as practicable to the laboratory to minimize the length of potentially contaminated ductwork. All HEPA filters are tested and certified annually.
    • The HEPA filter housings are designed to allow for in situ decontamination of the filter and verification of the validated process prior to removal. The design of the HEPA filter housing has gas-tight isolation dampers, decontamination ports, and the ability to individually scan each filter in the assembly for leaks.

17. Pass-through dunk tanks, fumigation chambers, or equivalent decontamination methods are provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the laboratory. Access to the exit side of the pass-through is limited to those individuals authorized to be in the facility and provided appropriate clearance if required.
18. Liquid effluents from chemical showers, sinks, floor drains, autoclave chambers, and other sources within the laboratory are decontaminated by a proven method, preferably heat treatment, before being discharged to the sanitary sewer.

• Decontamination of all liquid effluents is documented. The decontamination process for liquid effluents is validated physically and biologically. Biological validation is performed at least annually or more often if required by institutional policy.
• Effluents from personal body showers and toilets may be discharged to the sanitary sewer without treatment.

19. A double-door, pass-through autoclave(s) is provided for decontaminating materials passing out of the laboratory. Autoclaves that open outside of the laboratory are sealed to the wall through which the autoclave passes. This bioseal is durable, airtight, and capable of expansion and contraction. Positioning the bioseal so that the equipment can be accessed and maintained from outside the laboratory is strongly recommended. The autoclave doors are interlocked so that only one can be opened at any time and be automatically controlled so that the outside door to the autoclave can only be opened after a successful decontamination cycle has been completed.

• Gas discharge from the autoclave chamber is HEPA-filtered or is decontaminated. Autoclave decontamination processes are designed so that unfiltered air or steam exposed to infectious material cannot be released to the environment.

20. The facility design parameters and operational procedures are documented. The facility is tested to verify that the design and operational parameters have been met prior to operation. Facilities are also re-tested annually or after significant modification to ensure operational parameters are maintained. Verification criteria are modified, as necessary, by operational experience.
21. Appropriate communication systems are provided between the laboratory and the outside (e.g., voice, fax, video, and computer). Provisions for emergency communication and emergency access or egress are developed and implemented.