Biological Safety Manual - Chapter 04: Laboratory Biosafety Level Criteria

Title

Biological Safety Manual - Chapter 04: Laboratory Biosafety Level Criteria

Introduction

Biosafety Levels are safety controls that include standard microbiological practices, special practices, safety equipment, and laboratory facility design. These elements apply to activities involving infectious microorganisms, toxins, and laboratory animals. The four levels are organized in ascending order by the degree of protection provided to personnel, the environment, and the community. Special practices address any unique risks associated with the handling of agents requiring increasing levels of containment. Appropriate safety equipment and laboratory facilities enhance worker and environmental protection.

Prior to project initiation, a risk assessment should be performed to identify the specific hazards associated with the planned work. When necessary, labs should append the standard recommendations for each BSL (summarized in Table 1) to ensure any anticipated risks are properly addressed. The risk assessment process is discussed in Chapter 02 of the Biological Safety Manual. Each lab is expected to implement risk mitigation strategies to preserve worker safety, and should work with Environmental Health and Safety (EHS), the Institutional Biosafety Committee (IBC), or other applicable institutional committee(s) to resolve any questions that arise during the risk assessment.

Table of Contents

1. Laboratory Safety Levels
   1. Biosafety Level 1
   2. Biosafety Level 2
   3. Biosafety Level 3
2. Table 1: Summary of Recommended Biosafety Levels for Infectious Agents
3. Section I: Designating Biological Safety Level 2 at UNC
   1. Forms, Documents and Training
   2. Laboratory Facilities
   3. Safety Equipment
   4. Special Practices
4. Appendix A: Biological Safety Level 2 Checklist
5. Appendix B: Cryogenic Preservation of Biological Materials
   1. Safety Practices
   2. Emergency Exposures/Spills

Laboratory Biosafety Levels

Biosafety Levels cover BSL-1 through BSL-4. Since UNC-Chapel Hill does not have any BSL-4 facilities, only BSL-1 through BSL-3 will be discussed below.

Biosafety Level 1

Biosafety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel and the environment. BSL-1 laboratories are not necessarily separated from the general traffic patterns in the building. Work is typically conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is not required but may be used as determined by appropriate risk assessment. Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist with training in microbiology or a related science.

The following standard practices, safety equipment, and facility requirements apply to BSL-1:

A. Standard Microbiological Practices

1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory and worker safety.
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.
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.
   1. 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.
   2. 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. Long hair is restrained so that it cannot contact hands, specimens, containers, or equipment.
6. A sign is posted at the entrance to the laboratory when infectious materials are present. Posted information includes:
   1. the laboratory’s Biosafety Level;
   2. the supervisor’s or other responsible personnel’s name and telephone number;
   3. PPE requirements;
   4. general occupational health requirements (e.g., immunizations, respiratory protection);
   5. required procedures for entering and exiting the laboratory;
   6. Agent information (in accordance with the institutional policy).
7. Gloves comprise an essential part of PPE
   1. Gloves are worn to protect hands from exposure to hazardous materials
   2. Glove selection is based on an appropriate risk assessment.
   3. Gloves are not worn outside the laboratory.
   4. Gloves should be changed when contaminated, glove integrity is compromised, or when otherwise necessary.
   5. Do not wash or reuse disposable gloves.
   6. 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. Persons must wash their hands after working with potentially hazardous materials and before leaving the laboratory.
10. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area.
11. Mouth pipetting is prohibited; mechanical pipetting devices must be used.
12. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed, implemented, and followed; policies are consistent with applicable state, federal, and local requirements. Whenever practical, laboratory supervisors should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions must always be taken with sharp items. These include:
    1. Plasticware is substituted for glassware whenever possible.
    2. 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.
       1. Uncapping of needles is performed in such a manner to reduce the potential for recoil causing an accidental needlestick.
       2. Needles are not bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
       3. 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).
       4. 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
    3. Non disposable sharps must be placed in a hard walled container for transport to a processing area for decontamination, preferably by autoclaving.
    4. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plastic ware should be substituted for glassware whenever possible.
13. Perform all procedures to minimize the creation of splashes and/or aerosols.
14. 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.
15. Decontaminate all cultures, stocks, and other potentially infectious materials before disposal using an effective method, consistent with applicable institutional, local, and state requirements.. Depending on where the decontamination will be performed, the following methods should be used prior to transport:
    1. Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leak-proof container and secured for transport. For infectious materials, the outer surface of the container is disinfected prior to moving materials and the transport container has a universal biohazard label.
    2. Materials to be removed from the facility for decontamination must be packed in accordance with applicable local, state, and federal regulations.
16. An effective integrated pest management program is required. See Chapter 15 of the Biological Safety Manual.
17. Animals and plants not associated with the work being performed are not permitted in the laboratory.

B. Special Practices

None required.

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

1. Special containment devices or equipment, such as biosafety cabinets (BSCs), are not generally required.
2. Protective laboratory coats, gowns, or uniforms are worn to prevent contamination of personal clothing.
3. Protective eyewear is worn by personnel when conducting procedures that have the potential to create splashes of microorganisms or other hazardous materials. Eyes protection and face protection are disposed of with other contaminated laboratory waste or decontaminated after use. Persons who wear contact lenses in laboratories should also wear eye protection.
4. In circumstances where research animals are present in the laboratory, the risk assessment considers appropriate eye, face, and respiratory protection, as well as potential animal allergens

D. Laboratory Facilities (Secondary Barriers)

1. Laboratories have doors for access control.
2. Laboratories have a sink for hand washing.
3. The laboratory should be designed so that it can be easily cleaned.
   1. Carpets and rugs in laboratories are not appropriate.
   2. Spaces between benches, cabinets, and equipment are accessible for cleaning.
4. Laboratory furniture must be capable of supporting anticipated loads and uses.
   1. Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
   2. Chairs used in laboratory work must be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant.
5. Laboratories windows that open to the exterior should be fitted with screens.
6. Illumination is adequate for all activities and avoids reflections and glare that could impede vision.

Biosafety Level 2

Biosafety Level 2 (BSL-2) builds upon BSL-1. BSL-2 is suitable for work with agents associated with human disease and pose moderate hazards to personnel and the environment. BSL-2 differs from BSL-1 in that:

1. laboratory personnel have specific training in handling pathogenic agents and are supervised by scientists competent in handling infectious agents and associated procedures;
2. access to the laboratory is restricted when work is being conducted; and
3. all procedures in which infectious aerosols or splashes may be created are conducted in BSCs or other physical containment equipment.

The following standard and special practices, safety equipment, and facility requirements apply to BSL-2:

A. Standard Microbiological Practices

1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory and worker safety.
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.
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.
   1. 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.
   2. 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. Long hair is restrained so that it cannot contact hands, specimens, containers, or equipment.
6. A sign incorporating the universal biohazard symbol is posted at the entrance to the laboratory when infectious materials are present. Posted information includes:
   1. the laboratory’s Biosafety Level;
   2. the supervisor’s or other responsible personnel’s name and telephone number;
   3. PPE requirements;
   4. General occupational health requirements (e.g., immunizations, respiratory protection);
   5. Required procedures for entering and exiting the laboratory;
   6. Agent information (in accordance with the institutional policy).
7. Gloves comprise an essential part of PPE.
   1. Gloves are worn to protect hands from exposure to hazardous materials
   2. Glove selection is based on an appropriate risk assessment.
   3. Gloves are not worn outside the laboratory.
   4. Gloves should be changed when contaminated, glove integrity is compromised, or when otherwise necessary.
   5. Do not wash or reuse disposable gloves.
   6. 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. Persons must wash their hands after working with potentially hazardous materials and before leaving the laboratory.
10. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area .
11. Mouth pipetting is prohibited; mechanical pipetting devices must be used.
12. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed, implemented, and followed; policies are consistent with applicable state, federal, and local requirements. Whenever practical, laboratory supervisors should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions imust always be taken with sharp items. These include:
    1. Plasticware is substituted for glassware whenever possible.
    2. 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.
       1. Uncapping of needles is performed in such a manner to reduce the potential for recoil causing an accidental needlestick.
       2. Needles are not bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
       3. 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).
       4. 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.
    3. Non disposable sharps must be placed in a hard walled container for transport to a processing area for decontamination, preferably by autoclaving.
    4. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plastic ware should be substituted for glassware whenever possible.
13. Perform all procedures to minimize the creation of splashes and/or aerosols.
14. 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.
15. Decontaminate all cultures, stocks, and other potentially infectious materials before disposal using an effective method, consistent with applicable institutional, local, and state requirements.. Depending on where the decontamination will be performed, the following methods should be used prior to transport:
    1. Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leak-proof container and secured for transport. For infectious materials, the outer surface of the container is disinfected prior to moving materials and the transport container has a universal biohazard label.
    2. Materials to be removed from the facility for decontamination must be packed in accordance with applicable local, state, and federal regulations.
16. An effective integrated pest management program is required. See Chapter 15 of the Biological Safety Manual.
17. Animals and plants not associated with the work being performed are not permitted in the laboratory.

B. Special Practices

1. Access to the laboratory is controlled when work is being conducted.
2. The laboratory supervisor is responsible for ensuring that laboratory personnel demonstrate proficiency in standard microbiological practices and techniques for working with agents requiring BSL-2 containment.
3. Laboratory personnel must be provided medical surveillance , as appropriate, and offered available immunizations for agents handled or potentially present in the laboratory.
4. Properly maintained BSCs or other physical containment devices are used, when possible, whenever:
   1. Procedures with a potential for creating infectious aerosols or splashes are conducted. These include pipetting, centrifuging, grinding, blending, shaking, mixing, sonicating, opening containers of infectious materials, inoculating animals intranasally, and harvesting infected tissues from animals or eggs.
   2. High concentrations or large volumes of infectious agents are used.Such materials may be centrifuged in the open laboratory using sealed rotors or centrifuge safety cups with loading and unloading of the rotors and centrifuge safety cups in the BSC or another containment device.
   3. If it is not possible to perform a procedure within a BSC or other physical containment device, a combination of appropriate personal protective equipment (PPE) and administrative controls are used, based on a risk assessment.
5. Laboratory equipment is decontaminated routinely; after spills, splashes or other potential contamination; and before repair, maintenance, or removal from the laboratory.
6. A method for decontaminating all laboratory waste is available (e.g.,autoclave, chemical disinfection, incineration, or other validated decontamination method).
7. Incidents that may result in exposure to infectious materials are immediately evaluated per institutional policies. All such incidents are reported to the laboratory supervisor and any other personnel designated by the institution. Appropriate records are maintained.

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

1. Protective laboratory coats, gowns, or uniforms designated for laboratory use are worn while working with hazardous materials and removed before leaving for non-laboratory areas (e.g., cafeteria, library, and administrative offices). Protective clothing is disposed of appropriately or deposited for laundering by the institution. Laboratory clothing is not taken home.
2. Eye protection and face protection (e.g., safety glasses, goggles, mask, face shield or other splatter guard) are used for manipulations or activities that may result in splashes or sprays of infectious or other hazardous materials. Eye protection and face protection are disposed of with other contaminated laboratory waste or decontaminated after use.
3. The risk assessment considers whether respiratory protection is needed for the work with hazardous materials. If needed, relevant staff are enrolled in a properly constituted respiratory protection program.
4. In circumstances where research animals are present in the laboratory, the risk assessment considers appropriate eye, face, and respiratory protection, as well as potential animal allergens.

D. Laboratory Facilities (Secondary Barriers)

1. Laboratory doors should be self-closing and have locks in accordance with the institutional policies.
2. Laboratories must have a sink for hand washing. The sink may be manually, hands-free, or automatically operated. It should be located near the exit door.
3. An eyewash station is readily available in the laboratory.
4. The laboratory should be designed so that it can be easily cleaned and decontaminated.
   1. Carpets and rugs in laboratories are not appropriate.
   2. Spaces between benches, cabinets, and equipment are accessible for cleaning.
5. Laboratory furniture must be capable of supporting anticipated loads and uses.
   1. Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
   2. Chairs used in laboratory work must be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant.
6. Laboratory windows that open to the exterior are not recommended. However, if a laboratory does have windows that open to the exterior, they must be fitted with screens.
7. Illumination is adequate for all activities and avoids reflections and glare that could impede vision.
8. Vacuum lines in use are protected with liquid disinfectant traps and in-line HEPA filters or their equivalent. Filters are replaced, as needed, or are on a replacement schedule determined by a risk assessment.
9. There are no specific requirements for ventilation systems. However, the planning of new facilities considers mechanical ventilation systems that provide an inward flow of air without recirculation to spaces outside of the laboratory.
10. BSCs and other primary containment barrier systems are installed and operated in a manner to ensure their effectiveness.
    1. 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.
    2. 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). 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.
    3. BSCs are certified at least annually to ensure correct performance.

To designate your lab as a BSL-2 space follow the procedure and checklist in Chapter 04, Section 1 of the Biological Safety Manual.

Biosafety Level 3

Biosafety Level 3 (BSL-3) is suitable for work with indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route exposure. Laboratory personnel must receive specific training in handling pathogenic and potentially lethal agents, and they are supervised by scientists competent in handling infectious agents and associated procedures.

A BSL-3 laboratory has special engineering and design features.

The following standard and special safety practices, safety equipment, and facility specifications are recommended for BSL-3:

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.
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.
   1. 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.
   2. 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, specimens, containers, or equipment.
7. Gloves comprise an essential part of PPE.
   1. Gloves are worn to protect hands from exposure to hazardous materials.
   2. Glove selection is based on an appropriate risk assessment.
   3. Gloves are not worn outside the laboratory.
   4. Gloves should be changed when contaminated, glove integrity is compromised, or when otherwise necessary.
   5. Do not wash or reuse disposable gloves.
   6. 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. Persons wash their hands after working with potentially hazardous materials and before leaving the laboratory.
10. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area .
11. Mouth pipetting is prohibited; mechanical pipetting devices must be used.
12. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed, implemented, and followed; policies are consistent with applicable state, federal, and local requirements. Whenever practical, laboratory supervisors should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions must always be taken with sharp items. These include:
    1. Plasticware is substituted for glassware whenever possible.
    2. 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.
       1. Uncapping of needles is performed in such a manner to reduce the potential for recoil causing an accidental needlestick.
       2. Needles are not bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
       3. 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).
       4. 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.
    3. Non disposable sharps must be placed in a hard walled container for transport to a processing area for decontamination, preferably by autoclaving.
    4. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plastic ware should be substituted for glassware whenever possible.
13. Perform all procedures to minimize the creation of splashes and/or aerosols.
14. 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.
15. Decontaminate all cultures, stocks, and other potentially infectious materials before disposal using an effective method, consistent with applicable institutional, local, and state requirements.. Depending on where the decontamination will be performed, the following methods should be used prior to transport:
    1. Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leak-proof container and secured for transport. For infectious materials, the outer surface of the container is disinfected prior to moving materials and the transport container has a universal biohazard label.
    2. Materials to be removed from the facility for decontamination must be packed in accordance with applicable local, state, and federal regulations.
16. An effective integrated pest management program is required. See Chapter 15 of the Biological Safety Manual.
17. 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 laboratory areas is required for scientific or support purposes are authorized to enter.
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.
3. The laboratory supervisor is responsible for ensuring that laboratory personnel demonstrate proficiency in standard microbiological practices and techniques for working with agents requiring BSL-3 containment.
4. A system is established for reporting and documenting near misses, laboratory accidents, exposures, unanticipated absences 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-3 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 laboratory. Once removed, the primary container is opened within a BSC in BSL-3 containment unless a validated inactivation method is used. See Appendix K. The inactivation method is documented in-house with viability testing data to support the method.
7. All procedures involving the manipulation of infectious materials are conducted within a BSC or other physical containment device, when possible. No work with open vessels is conducted on the bench. If it is not possible to perform a procedure within a BSC or other physical containment device, a combination of PPE and other administrative and/or engineering controls, such as centrifuge safety cups or sealed rotors, are used, based on a risk assessment. Loading and unloading of the rotors and centrifuge safety cups take place in the BSC or another containment device.
8. Laboratory equipment is routinely decontaminated after spills, splashes, or other potential contamination, and before repair, maintenance, or removal from the laboratory.
   1. 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.
9. A method for decontaminating all laboratory waste is available in the facility, preferably within the laboratory (e.g., autoclave, chemical disinfection, or other validated decontamination method).
10. Decontamination of the entire laboratory is considered when there has been gross contamination of the space, significant changes in laboratory usage, major renovations, or maintenance shutdowns. Selection of the appropriate materials and methods used to decontaminate the laboratory is based on a risk assessment.
11. Decontamination processes are verified on a routine basis.

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

1. Laboratory workers wear protective clothing with a solid-front, such as tie-back or wrap-around gowns, scrub suits, or coveralls. Protective clothing is not worn outside of the laboratory. Reusable clothing is decontaminated before being laundered. Clothing is changed when contaminated.
2. Based on work being performed, additional PPE may be required.
   1. Eye protection and face protection (e.g., safety glasses, goggles, mask, face shield or other splash guard) are used for manipulations or activities that may result in splashes or sprays of infectious or other hazardous materials. Eye protection and face protection are disposed of with other contaminated laboratory waste or decontaminated after use.
   2. Two pairs of gloves are worn when appropriate.
   3. Respiratory protection is considered. Staff wearing respiratory protection are enrolled in a properly constituted respiratory protection program.
   4. Shoe covers are considered.
3. In circumstances where research animals are present in the laboratory, the risk assessment considers appropriate eye, face, and respiratory protection, as well as potential animal allergens.

D. Laboratory Facilities (Secondary Barriers)

1. The laboratory is separated from areas that are open to unrestricted traffic flow within the building.
   1. Laboratory access is restricted. Laboratory doors are lockable in accordance with institutional policies. Access to the laboratory is through two consecutive self-closing doors. A clothing change room and/or an anteroom may be included in the passageway between the two self-closing doors.
2. Laboratories have a sink for handwashing. The sink is hands-free or automatically operated and should be located near the exit door. If a laboratory suite is segregated into different zones, a sink is also available for handwashing in each zone.
3. An eyewash station is readily available in the laboratory.
4. The laboratory is designed, constructed, and maintained to facilitate cleaning, decontamination, and housekeeping.
   1. Carpets and rugs are not permitted.
   2. Spaces between benches, cabinets, and equipment are accessible for cleaning.
   3. Seams, floors, walls, and ceiling surfaces are sealed. Spaces around doors and ventilation openings are capable of being sealed to facilitate space decontamination.
   4. Floors are slip-resistant, impervious to liquids, and resistant to chemicals. Flooring is seamless, sealed, or poured with integral cove bases
   5. Walls and ceilings are constructed to produce a sealed smooth finish that can be easily cleaned and decontaminated.
5. Laboratory furniture can support anticipated loads and uses.
   1. Benchtops are impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
   2. Chairs used in laboratory work are covered with a non-porous material that can be easily cleaned and decontaminated with an appropriate disinfectant.
6. All windows in the laboratory are sealed.
7. Illumination is adequate for all activities and avoids reflections and glare that could impede vision.
8. Vacuum lines in use are protected with liquid disinfectant traps and in-line HEPA filters or their equivalent. See Appendix A, Figure 11. Filters are replaced, as needed, or are on a replacement schedule determined by a risk assessment. Vacuum lines not protected as described are capped. The placement of an additional HEPA filter immediately prior to a central vacuum pump is considered.
9. A ducted mechanical air ventilation system is required. This system provides sustained directional airflow by drawing air into the laboratory from “clean” areas toward “potentially contaminated” areas. The laboratory is designed such that under failure conditions the airflow will not be reversed at the containment barrier.
   1. A visual monitoring device that confirms directional airflow is provided at the laboratory entry. Audible alarms to notify personnel of airflow disruption are considered.
   2. The laboratory exhaust air is not re-circulated to any other area in the building.
   3. The laboratory exhaust air is dispersed away from occupied areas and from building air intake locations or the exhaust air is HEPA filtered.
10. BSCs and other primary containment barrier systems are installed and operated in a manner to ensure their effectiveness.
    1. 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.
    2. 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). 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.
    3. BSCs are certified at least annually to ensure correct performance
    4. Class III BSCs are provided supply air in such a manner that prevents positive pressurization of the cabinet or the room.
11. Equipment that may produce infectious aerosols is used within primary barrier devices that exhaust air through HEPA filtration or other equivalent technology before being discharged into the laboratory. These HEPA filters are tested annually and replaced as needed.
12. Facility is constructed to allow decontamination of the entire laboratory when there has been gross contamination of the space, significant changes in usage, major renovations, or maintenance shutdowns. Selection of the appropriate materials and methods used to decontaminate the laboratory is based on the risk assessment.
    1. Facility design consideration is given to means of decontaminating large pieces of equipment before removal from the laboratory.
13. Enhanced environmental and personal protection may be necessary based on risk assessment and applicable local, state, or federal regulations. These laboratory enhancements may include one or more of the following: an anteroom for clean storage of equipment and supplies with dress-in, shower-out capabilities; gas-tight dampers to facilitate laboratory isolation; final HEPA filtration of the laboratory exhaust air; laboratory effluent decontamination; containment of other piped services; or advanced access control devices, such as biometrics.
14. When present, HEPA filter housings have gas-tight isolation dampers, decontamination ports, and/or bag-in/bag-out (with appropriate decontamination procedures) capability. All HEPA filters are located as near as practicable to the laboratory to minimize the length of potentially contaminated ductwork. The HEPA filter housings allow for leak testing of each filter and assembly. The filters and housings are certified at least annually.
15. The BSL-3 facility design, operational parameters, and procedures are verified and documented prior to operation. Facilities are tested annually or after significant modification to ensure operational parameters are met. Verification criteria are modified as necessary by operational experience.
16. Appropriate communication systems are provided between the laboratory and the outside (e.g., voice, fax, and computer). Provisions for emergency communication and emergency access or egress are developed and implemented.

Table 1 - Summary of Recommended Biosafety Levels for Infectious Agents

BSL-1

Agents

• Well-characterized agents not known to consistently cause disease in immunocompetent adult humans and present minimal potential hazard to laboratory personnel and the environment.

Practices

• Standard microbiological practices.

Primary Barriers and Safety Equipment

• No primary barriers required;
• Protective laboratory clothing; and
• Protective face, eyewear, as needed.

Facilities (Secondary Barriers)

• Laboratory doors;
• Sink for handwashing;
• Laboratory bench;
• Windows fitted with screens; and
• Adequate lighting.

BSL-2

Agents

• Agents associated with human disease and pose moderate hazards to personnel and the environment.

Practices

• BSL-1 practice plus:
  1. Limited access;
  2. Occupational medical services including medical evaluation, surveillance, and treatment, as appropriate;
  3. All procedures that may generate an aerosol or splash conducted in a BSC;
  4. Biohazard warning signs; and
  5. Decontamination process needed for laboratory equipment.

Primary Barriers and Safety Equipment

• BSCs or other primary containment device used for manipulations of agents that may cause splashes or aerosols;
• Protective laboratory clothing; and
• Other PPE, including respiratory protection, as needed.

Facilities (Secondary Barriers)

• Self-closing doors;
• Sink located near exit; and
• Windows sealed or fitted with screens.

BSL-3

Agents

• Indigenous or exotic agents;
• May cause serious or potentially lethal disease through the inhalation route of exposure.

Practices

• BSL-2 practice plus:
  1. Controlled access;
  2. Decontamination of all waste;
  3. Decontamination of laboratory clothing before laundering;
  4. Viable material removed from laboratory in primary and secondary containers;
  5. Viable material opened only in BSL-3 or ABSL-3 laboratories; and
  6. All procedures with infectious materials performed in a BSC.

Primary Barriers and Safety Equipment

• BSCs for all procedures with viable agents;
• Solid front gowns, scrubs, or coveralls;
• Two pairs of gloves, when appropriate; and
• Appropriate protective eyewear, respiratory protection, as needed.

Facilities (Secondary Barriers)

• Ventilation system with negative airflow into laboratory; and
• Autoclave available, preferably in laboratory.

BSL-4

UNC-Chapel Hill does not have BSL-4 facilities.

Section I: Designating Biological Safety Level 2 at UNC-Chapel Hill

1. Forms, Documents, and Training

Lab Safety Plan & Schedule F (Biological Hazards)

The first step in designating BSL-2 space at UNC-Chapel Hill is notifying EHS of your desire to do so. This is done by submitting an up-to-date copy of your Laboratory Safety Plan (LSP).

You can access all current LSP documents on the EHS website.

The Lab Safety Plan can be daunting; to complete the requirements for each section properly, refer to Chapter 02 of the UNC-Chapel Hill Laboratory Safety Manual.

For BSL-2 consideration, the Schedule F (Biological Hazards) of the Lab Safety Plan is completed and submitted to EHS for approval. You must update the Lab Safety Plan with EHS annually.

Schedule G (Recombinant DNA)

Recombinant DNA research on campus probably falls into one of the three following groups that may require registration with the UNC-Chapel Hill Institutional Biosafety Committee (IBC):

• Exempt Experiments;
• Experiments Requiring Prior Approval; or
• Experiments Requiring IBC Notice Simultaneous with Initiation.

Examples of each, forms, and other information is available on the EHS "Recombinant DNA" webpage.

Annual In-House Training

All biological hazards listed on the Schedule F will be reviewed annually with all laboratory workers including those handling and/or treating biohazard waste. The annual in-house training form (or an equivalent) will be used to document this training and kept in the lab safety binder. These requirements are reviewed with new staff and the Schedule F is reviewed annually. Lab workers can view all lab members' training requirements on the "EHS Compliance Portal" webpage.

Bloodborne Pathogens Requirements (Including Human Cell Lines)

All workers exposed to human source materials will complete Bloodborne Pathogens (BBP) training annually. OSHA defines workers as exposed to BBP if they work with human blood or other potentially infectious material (including human cell lines-continuous or primary.)

Complete the online self-study BBP training. You may want to forward this link along to any lab workers who will require this training.

All workers to which the OSHA Bloodborne Pathogen standard applies should have, or officially decline, the Hepatitis B vaccination series at the University Employee Occupational Health Clinic.

For more information about the risk of human tissue and the Bloodborne Pathogen Standard please refer to the EHS "Bloodborne Pathogens" webpage.

View the CDC's "Hepatitis B Vaccination of Adults" webpage for more information about the Hepatitis B vaccination series.

2. Laboratory Facilities

Proper Doors

Laboratory doors are self-closing and have locks in accordance with the institutional policies. The Principal Investigator is ultimately responsible for the control of, and access to, laboratories where Risk Group 2 agents are stored or manipulated.

You must keep laboratory doors closed while experiments are in progress. This practice not only protects persons who might otherwise enter the laboratory, it reduces interruptions to laboratory staff that could lead to accidents. Biological safety cabinets and laboratory hoods work best, and offer the most worker protection, when the doors to the laboratory are closed. For general information regarding access control of UNC-Chapel Hill labs, review Chapter 06 of the UNC-Chapel Hill Biological Safety Manual.

Entryway signs

BSL-2 entryway signs are posted for lab spaces in which large volumes or high concentrations of Risk Group 2 agents are present. Posting entryway signs for all other BSL-2 lab space is also required (e.g. for areas where human blood or other potentially infectious material including human cell lines is present).

EHS has supplied an approved BSL-2 door placard for the designated BSL-2 space. The Principal Investigator has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.

You may submit a request for a new entrance sign on the EHS "Lab Entrance Sign" webpage.

Hand Washing Sink

The lab space designated at BSL-2 has a sink available for hand washing. The sink may be manual, hands-free, or automatically operated and should have soap and disposable paper towels readily available at all times for washing hands at the sink. Ideally, the sink is located near the exit door.

Easily Cleanable

The laboratory should be designed so that it can be easily cleaned and decontaminated. This can be difficult in older buildings that were designed without present day biosafety precautions in mind. Carpets and rugs are not permitted in laboratories. Check areas for worn and damaged bench tops or flooring that may harbor microbes in the event of a spill.

Speak with your department's business manager and/or submit an online service request to have Facilities Services repair an area.

Proper Bench Tops

Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals. Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between benches, cabinets, and equipment should be accessible for cleaning.

Speak with your department’s business manager and/or submit an online service request to have Facilities Services repair an area.

Proper Chairs at Biological Safety Cabinets

Chairs used at the biological safety cabinet must be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant. Cloth covered chairs should not be used at the biosafety cabinet. The chairs must be capable of supporting anticipated loads and uses. This practice should be applied to chairs at lab benches too.

Proper Windows

Laboratory windows that open to the exterior are not recommended. However, if a laboratory does have windows that open to the exterior, they must be fitted with screens.

Speak with your department’s business manager and/or submit an online service request to have Facilities Services fit or repair openings to the exterior of the building.

Biological Safety Cabinet Location

You must consult with EHS regarding the placement and use of your biological safety cabinet. Contact us at biosafety@ehs.unc.edu to arrange an appointment.

Biological safety cabinets (BSC), (aka “tissue culture hoods”) must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs should be located away from doors, windows that can be opened, heavily traveled laboratory areas, and other possible airflow disruptions.

If a BSC has passed certification in place with the vents running, deflectors are not technically necessary. However, EHS highly recommends their installation. The BSC’s air curtain is delicate and seasonal airflow variations in laboratory buildings can be significant enough to disrupt its protective barrier.

Speak with your department’s business manager and/or submit an online service request to have Facilities Services install deflectors at room air vents.

In-line HEPA Filters

Vacuum lines located at biosafety cabinets (aka: “tissue culture hoods”) must have protection via an absorbent or liquid disinfectant trap and a High Efficiency Particulate Air (HEPA) filter, or its equivalent to prevent contamination of the vacuum system. You must replace filters as needed. This practice should also apply to aspirating liquid at the BSL-2 benchtop.

You may order in-line HEPA filters on the Fisher Scientific website; search for part # 09-744-75 or # 09-744-76.

Eyewash Station

To be compliant with BSL-2 standards at UNC-Chapel Hill, eyewash stations must be plumbed units that meet the ANSI Standard Z358.1-2004. Personal eye flush squeeze bottles do not meet ANSI requirements, because they cannot deliver the required minimum flow rate and duration. EHS discourages the presence of these bottles, particularly in BSL-2 labs because they have a limited shelf life, are prone to contamination, and are ineffective at dual-eye or eye-face irrigation.

Facilities Services can install a deck-mounted unit at an existing sink (complete an online service request). EHS recommends the following unit to compensate for the distance and obstructions to the nearest safety shower from some labs: Fisher Scientific deck mount eye wash #S47711.

For more information about eyewash facilities in UNC-Chapel Hill labs, see Chapter 03 of the UNC-Chapel Hill Laboratory Safety Manual.

Emergency Shower

A shower facility, other than emergency drench hoses, must be located in the building. To compensate for the distance and obstructions to the nearest safety shower from some designated BSL-2 space, EHS recommends the following unit: Fisher Scientific deck mount eye wash #S47711.

Proper Ventilation

There are no specific requirements on ventilation systems in BSL-2 labs at UNC-Chapel Hill. However, planning of new facilities should consider mechanical ventilation systems that provide an inward flow of air without recirculation to spaces outside of the laboratory. Chapter 07.III. (Facility Requirements) of the UNC-Chapel Hill Laboratory Safety Manual indicates the following:

"Exhaust ventilation systems are designed to maintain an inflow of air from the corridor into the work area. The exhaust air from the work area must discharge directly to the outdoors, and clear of occupied buildings and air intakes. Exhaust air from the work area must not recirculate. The exhaust air from glove boxes must filter through high-efficiency particulate air (HEPA) and charcoal filters. EHS will determine the need for and type of treatment for other primary containment equipment. Exhaust air treatment systems that remove toxic chemicals from the exhaust air by collection mechanism such as filtration or absorption must operate in a manner that permits maintenance, to avoid direct contact with the collection medium. All exhaust air from primary containment equipment must discharge directly to the outdoors and disperse clear of occupied buildings and intakes. Exhaust systems for highly toxic substances must contain engineered fail-safe mechanisms to prevent loss of containment due to utility outages."

Biological Safety Cabinet Exhaust

Because most biological safety cabinets (BSC) at UNC-Chapel Hill re-circulate HEPA-filtered exhaust air into the laboratory environment, the cabinet should be tested and certified at least annually and operated according to manufacturer’s recommendations. Never use hazardous chemicals in these cabinets - the vapors bypass the HEPA (particulate) filters and enter your breathing zone.

Some BSCs on campus may be connected to the laboratory exhaust system by either a thimble (canopy) connection or a direct (hard) connection. Provisions to assure proper safety cabinet performance and air system operation should be verified annually. Only minute amounts of hazardous chemicals should be used in an exhausted cabinet. More information about biological safety cabinets is available in the UNC-Chapel Hill Biological Safety Manual.

Autoclave

In UNC-Chapel Hill laboratories, an autoclave must be accessible to decontaminate all biohazard waste before disposal. This varies from the UNC Hospitals requirement for biohazard waste because UNC Hospitals incinerates all biohazard waste. If an autoclave is not accessible to your lab, contact EHS.

Refer to the EHS "Biohazard Waste Management" webpage for waste decontamination requirements.

Chemical Disinfection of Liquid Microbiological Waste

Refer to the “Liquids” section of the Biohazard Waste Disposal Chart. If your liquid waste was used for propagating microbes/viral vectors/toxins AND you are unable to autoclave your liquid biohazard waste, you will need to make application to the North Carolina Medical Waste Division to dispose of this chemically disinfected liquid microbiological waste down the drain. Refer to the EHS "Chemical Treatment of Liquid Microbiological Waste" webpage for more information and to access the "Request for Approval to Chemical Treat Liquid Microbiological Waste" form.

3. Safety Equipment

Biohazard Labels

Laboratory equipment used for BSL-2 containment is posted with the universal biohazard warning symbol (to communicate hazard to maintenance workers, visitors, etc.). This symbol is used to identify the actual or potential presence of a biological hazard on or in freezers, incubators, centrifuges, biological safety cabinets, etc. which are used with agents listed on Schedule F of the Laboratory Safety Plan. Biohazard warning labels may be printed on a color printer from the EHS "Safety Labels and Signage" webpage, or they may be ordered from the Fisher Scientific website - search for part # 18-999-934.

Biological Safety Cabinet Maintenance

Because most biological safety cabinets (BSC) (aka “tissue culture hoods”) on campus re- circulate HEPA filtered exhaust air into the laboratory environment, the cabinet should be tested and certified at least annually and operated according to manufacturer’s recommendations. Never use hazardous chemicals in these cabinets - the vapors bypass the HEPA filter to enter your breathing zone.

Some BSCs on campus can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or a direct (hard) connection. Provisions to assure proper safety cabinet performance and air system operation should be verified annually. Only minute amounts of hazardous chemicals should be used in an exhausted cabinet.

 All testing, certification, and decontamination services for research and clinical laboratories will be provided by Precision Air Technology, Inc.

Precision Air Technology, Inc.
 P.O. Box 46449
 Raleigh, NC 27620

Phone: (919) 212-1300

Email (preferred): info@precisionairtechnology.com 
Fax: (801) 740-3346

Please provide the following information to Precision Air for service requests:

• Contact Name;
• Contact phone number, email;
• Building and room number of equipment;
• Building and office room number;
• Equipment manufacturer model;
• Equipment manufacturer serial number; and
• Brief description of the service request.

Biological Safety Cabinet Use

Biological safety cabinets (BSC) (aka "tissue culture hoods") must be used whenever procedures with a potential for creating infectious aerosols or splashes are conducted. These may include pipetting, centrifuging, grinding, blending, shaking, mixing, sonicating, opening containers of infectious materials, inoculating animals intra-nasally, and harvesting infected tissues from animals or eggs.

BSCs must be used whenever procedures with high concentrations or large volumes of infectious agents are conducted. Such materials should only be centrifuged in the open laboratory when sealed rotor heads or centrifuge safety cups are used.

Centrifuge safety Precautions

Many activities associated with centrifuges may create significant amounts of infectious aerosol, including:

• filling centrifuge tubes;
• removing plugs or caps from tubes after centrifugation;
• removing supernatant;
• re-suspending sedimented pellets;
• breakage of tubes during centrifugation; and
• centrifugation itself.

Follow these steps to prevent the generation of aerosols in centrifuges:

1. Routinely inspect the centrifuge to ensure there is no leakage.
2. Do not overfill centrifuge tubes.
3. Wipe the outside of the tubes with an appropriate disinfectant after they are filled and sealed.
4. Centrifuge inside a biological safety cabinet. If a biological safety cabinet is not available, internal aerosol containment devices (e.g., sealed canisters, safety cups or buckets with covers, heat sealed tubes or sealed rotors) should be used.
5. Remove aerosol containment devices and open them in a biological safety cabinet. If the biological safety cabinet is in use, a minimum of 10 minutes settling time should be allowed before opening.

Lab Coats

Protective laboratory coats, gowns, smocks, or uniforms designated for laboratory use are worn while working in designated BSL-2 space. You must remove protective clothing before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices). You must appropriately dispose of protective clothing or deposit the protective clothing for laundering by the institution. (Please see the EHS "Personal Protective Equipment References" webpage for more information.) You must not take laboratory clothing home.

Chapter 05.III. of the UNC-Chapel Hill Laboratory Safety Manual has more information regarding laboratory clothing, protective apparel, and laundering at UNC-Chapel Hill.

Eye & Face Protection

Eye and face protection (goggles, mask, face shield, or other splatter guard) is used for anticipated splashes or sprays of infectious or other hazardous materials when the microorganisms must be handled outside the BSC or containment device. Eye and face protection is disposed of with other contaminated laboratory waste or decontaminated before reuse. Persons who wear contact lenses in laboratories also wear eye protection.

Chapter 05.12 of the Environment, Health & Safety manual details UNC-Chapel Hill’s laboratory eye and face protection policy.

Gloves

Special care is taken to avoid skin contamination at BSL-2. Gloves are worn to protect hands when handling experimental animals and when skin contact with the agent is unavoidable. Glove selection is based on an appropriate risk assessment. Alternatives to latex gloves are available. Gloves are not worn outside the laboratory. Chapter 05.II. of the UNC-Chapel Hill Laboratory Safety Manual details requirements for the use of gloves in laboratories. In addition, BSL-2 laboratory workers should:

1. Change gloves when contaminated, integrity has been compromised, or when otherwise necessary. Wear two pairs of gloves when appropriate.
2. Remove gloves and wash hands when work with hazardous materials has been completed and before leaving the laboratory.
3. Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated laboratory waste. You must rigorously follow hand washing protocols.

Respiratory Protection with Infected Animals

Eye, face, and respiratory protection is used in rooms containing infected animals as determined by the risk assessment and EHS.

See Chapter 14 of the UNC-Chapel Hill Laboratory Safety Manual for safe handling of laboratory animals.

Sharps Precautions

Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from lab animals and diaphragm bottles. Only needle-locking syringes or disposable syringe- needle units (i.e., needle is integral to the syringe) are used at BSL-2. Use extreme caution when handling needles and syringes to avoid auto inoculation and the generation of aerosols during use and disposal. Do not bend or shear the needle, replace the needle in the needle sheath or guard, or remove the needle from the syringe after you use the needle.

You must promptly place needles and syringes into appropriately labeled plastic sharps containers. Red containers are clearly marked as “Biohazardous Sharps” and autoclaved (remember to mark an “X” with autoclave tape directly over the biohazard warning label) prior to disposal or, if the sharps are not biohazardous, white or clear plastic containers are labeled “Nonhazardous Sharps”.

When ordering the plastic sharps containers online from the Fisher Scientific website, search for part # 1482664B and/or 14830124.

Labels for these containers can be obtained from the EHS "Safety Labels and Signage" webpage.

For other biohazardous waste collection and disposal methods, a Disposal Chart is available on the EHS "Biohazard Waste Management" webpage.

4. Special practices

Entry/Exit Requirements

All persons entering the designated BSL-2 area are advised of the potential hazards and they meet specific entry/exit requirements when manipulations involving materials from the Lab Safety Plan’s Schedule F (Biological Hazards) are taking place. These requirements are reviewed with new staff and the Schedule F is reviewed annually as part of the lab’s annual in-house training.

You must remove lab coats, gowns, smocks, and gloves before exiting the BSL-2 laboratory for non-laboratory areas, Leave the lab coats, gowns, smocks, and gloves in the laboratory. All workers wash their hands after they de-glove and prior to exiting the lab after they handle materials involving viable material.

Medical Surveillance/Serum Samples

Each laboratory must establish policies and procedures describing the collection and storage of serum samples from at-risk personnel as appropriate. If applicable, this will be listed on the Lab Safety Plan’s Schedule F (Biological Hazards) under “Medical Surveillance.” For more information, contact EHS Biological Safety or the University Employee Occupational Health Clinic. Procedures for animal surveillance are discussed in Chapter 14 of the UNC-Chapel Hill Laboratory Safety Manual.

Immunizations

Laboratory personnel are provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory. If applicable, this should be listed on the Lab Safety Plan’s Schedule F Biological Hazards) under “Medical Surveillance.”

If your group is working with a known pathogen for which there is an effective vaccine, the vaccine should be made available to all workers. For more information, contact EHS biological safety or the University Employee Occupational Health Clinic.

Anyone in the lab working with (including treating waste) human blood or other potentially infectious material (including human cell lines, established, or primary) is required by the OSHA Bloodborne Pathogen standard to have, seek, or officially decline the Hepatitis B vaccination series. For questions regarding this and other vaccination series, please contact the University Employee Occupational Health Clinic. View the CDC's "Hepatitis B Vaccination of Adults" webpage for more information about the Hepatitis B vaccination series.

For more information about the risk of human tissue and the Bloodborne Pathogen Standard please refer to the EHS "Bloodborne Pathogens" webpage.

Chapter 14 of the UNC-Chapel Hill Laboratory Safety Manual discusses procedures for animal bites and immunizations.

Biosafety Manual

The UNC-Chapel Hill Biosafety Manual and Exposure Control Plan has been adopted as laboratory policy and the Schedule F (Biological Hazards) is reviewed with each worker at least annually during annual in-house training. A paper copy of the Biosafety Manual and Schedule F must be available and accessible to workers at all times and documentation of the annual in-house training is available in the lab safety binder.

The latest copy of the UNC-Chapel Hill Laboratory Exposure Control Plan is available on the EHS "Bloodborne Pathogens" webpage. Print the General Version and keep in your safety binder.

Worker Proficiency

The Principal Investigator has the final responsibility for determining who may enter or work in the BSL-2 space and for advising persons of the potential hazard and entry requirements (e.g. immunization) for entry. The Principal Investigator must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BSL-2 agents. At a minimum, this includes training in aseptic techniques and in the biology of the organisms used in the experiment so that the potential biohazards can be understood and appreciated. Laboratory personnel must perform all procedures carefully to minimize the creation of aerosols.

Eating, drinking, smoking, and applying cosmetics are not permitted in the work area. Food will be stored and consumed in designated areas used for this purpose only. ("Food Use Safety Labels" are available on the EHS "Safety Labels and Signage" webpage.) These requirements are reviewed with new staff and the Schedule F is reviewed annually as part of the lab’s annual in-house training.

For more information about animal handling technical proficiency, see Chapter 14 of the UNC-Chapel Hill Laboratory Safety Manual.

Proper Containerization

Place potentially infectious materials in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility. Use a leak-proof box, preferably equipped with a gasket seal lid, for transport of potentially infectious materials from one location to another on campus. This is particularly important when moving samples from patient care areas in UNC Hospitals to the lab. Containers such as igloo coolers or Rubbermaid containers will suffice provided they have enough absorbent material placed inside and a biohazard warning label on the outermost container. ("Biohazard" labels are available on the EHS "Safety Labels and Signage" webpage.)

Routine Decontamination

You must decontaminate laboratory equipment and work surfaces once a day and after any spill of viable material. The appropriate disinfectant and recommended contact time is listed on the Lab Safety Plan’s Schedule F (Biological Hazards) under “Safety Precautions.” Decontaminate equipment prior to repair, maintenance, or removal of the equipment from the laboratory.

The lab has determined how and where the decontamination of all cultures, stocks, and other potentially infectious materials will be performed before disposal. This is listed on your Lab Safety Plan’s Schedule F (Biological Hazards) under “Safety Precautions.”

Refer to the EHS "Biohazard Waste Management" webpage for waste decontamination requirements.

Spill Kit & Clean-Up

Workers are properly trained and equipped to contain, decontaminate, and clean up spills involving infectious material. The emergency plan that describes the procedures to be followed if an accident contaminates personnel or the environment is listed on the Lab Safety Plan’s Schedule F (Biological Hazards) under #8 "Emergency Procedures." At a minimum, bleach is provided in the lab space and plenty of paper towels are available for spill clean-up purposes.

Refer to the EHS "Biohazard Waste Management" webpage for waste decontamination requirements.

Reporting Exposure Incidents

Incidents that may result in overt exposures to materials handled at BSL-2 will be immediately evaluated and treated according to procedures described in this manual. All such incidents must be reported to the laboratory supervisor, the University Employee Occupational Health Clinic, and the Institutional Biosafety Committee.

Pets and House Plants Prohibited in Laboratories

Animals and plants not associated with the work being performed are not permitted in the laboratory. This is also consistent with the UNC-Chapel Hill Environment, Health & Safety Manual, Chapter 02.07: Animals on Campus.

Aerosol Generation Precautions

You must conduct all procedures involving the manipulation of infectious materials that may generate an aerosol within a biological safety cabinet (aka "tissue culture hood") or other physical containment device.

Appendix A: Biological Safety Level 2 Checklist

Use the Biosafety Level 2 checklist for guidance when designating new BSL‐2 lab space at UNC-Chapel Hill. This checklist is designed to answer common/general questions during the set‐up process. EHS requires that you register your lab space. You can do this by completing an online Laboratory Safety Plan. Feel free to consult the online Biological Safety Manual or contact EHS for guidance.

Please see attached Laboratory checklist for designating Biological Safety Level 2 (BSL-2) at UNC-Chapel Hill.

Appendix B: Cryogenic Preservation of Biological Materials

Liquid nitrogen dewars are commonly utilized for cryogenic preservation of biological materials.

However, liquid nitrogen exhibits a boiling point of -195.8° C and expands over 600 fold when brought to room temperature. Cryogenic preservation vials or “cryovials” stored in the liquid phase of liquid nitrogen can rupture upon warming if liquid nitrogen has infiltrated them, resulting in an explosion hazard. Pieces of the cryovial may be propelled towards personnel resulting in physical injury, particularly to the eyes, and exposure to the cryovial’s contents. For this reason, cryogenic storage of hazardous materials including infectious agents and biological toxins is strongly discouraged. Contact the Biological Safety Officer for guidance.

Safety Practices

Exposure to liquid nitrogen may damage living tissue. Personnel must be properly attired including the use of long pants or skirts and closed toed shoes. Additional required Personal Protective Equipment (PPE) includes lab coats, disposable gloves, thermal outer gloves and eye/face protection such as safety glasses and/or a face shield.

Utilize screw capped cryogenic preservation vials with rubber O rings designed specifically for cryogenic preservation. Ensure caps are tightly screwed on. Store cryovials in the gaseous phase above the liquid nitrogen to avoid infiltration. Ensure laboratory personnel are aware of the explosion hazard to avoid overfilling liquid nitrogen dewars to the point of submerging cryovials.

When removing cryovials from the dewar, immediately place them in a sealed and leak-proof container to contain a possible explosion. Example secondary containers include 50 mL conical tubes or Tupperware containers. Rubber ice buckets do not constitute an appropriate secondary container. If desired, ice or dry ice can be placed in the secondary container or the secondary container can be placed in a larger container with ice or dry ice. The explosion hazard will no longer exist moments after bringing the cryovial to room temperature.

Work in a properly ventilated area when replenishing liquid nitrogen dewars to avoid an asphyxiation hazard. Patiently watch over the dewar to avoid overflow. Spilled liquid nitrogen can damage living tissue as well as facilities and equipment.

Emergency Exposures/Spills

Immediate medical treatment is required if a cryogenic liquid is spilled and leads to skin or eye exposure. Contact the University Employee Occupational Health Clinic (919-966-9119) during work hours for immediate medical care or call 911. Large spills of cryogenic liquids can lead to an oxygen-deficient environment. Personnel should immediately evacuate the area and call 911.

Back to Chapter Three

Proceed to Chapter Five