Appendix D: Biosafety Level 2 (BSL2) Requirements

Biosafety Level 2 (BSL2) is suitable for experiments involving agents of moderate potential hazard to personnel and the environment.

For example:

• Microorganisms of low biohazard potential, such as those in Risk Group 2 or BSL2.
• Recombinant DNA activity requiring BSL2 physical containment including animal studies that involve the construction of transgenic animals.
• Non-recombinant cell and/or tissue culture systems that require this level of containment.
• Oncogenic viral systems classified as low risk.
• Production activities with Risk Group 1 organisms.

The control of potential biohazards at the BSL2 level is provided by use of standard microbiological practices with the addition of personnel protective equipment (lab coat and gloves).

The following are procedures are used with BSL2 containment requirements. They are based on the recommendation of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) 6th Edition, 2020 and BU policies and procedures.

Standard Microbiological Practices

Access

Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.

Hand washing

Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.

Food

Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.

Eye protection

Personnel who use contact lenses will consult with EHS if required to use eye protection in the lab.

Personal Electronic Devices

Use of personal electronic devices such earbuds and cell phones should not be practiced when working in the lab.

Pipetting

Mouth pipetting is prohibited; mechanical pipetting devices are used.

Sharps

Policies for the safe handling of sharps are instituted.

Splashes & Aerosols

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

Decontamination

Work surfaces are decontaminated upon completion of work, or at the end of the day, and after any spill or splash of viable material with disinfectants that are effective against the agents of concern.

Insect/Rodent Control Program

An insect and rodent control program is in effect.

Special Practices

Access” elem

Access to the laboratory is limited or restricted by the laboratory director when work with infectious agents is in progress. In general, persons who are at increased risk of acquiring infection, or for whom infection may have serious consequences, are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immunosuppressed may be at increased risk of acquiring infections. The laboratory director in consultation with ROHP has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory or animal room.

Policies & Procedures

The Principal Investigator or Laboratory Director establishes policies and procedures whereby only persons who have been advised of the potential hazards and meet specific entry requirements (e.g., immunization) may enter the laboratory.

Biohazard Signs

A biohazard sign must be posted on the entrance to the laboratory when etiologic agents are in use. Appropriate information to be posted includes:

• the agent(s) in use;
• the biosafety level;
• the required immunizations;
• the investigator’s name and telephone number;
• any personal protective equipment that must be worn in the laboratory;
• and any procedures required for exiting the laboratory.

Immunizations

Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).

Manual/SOPs

Biosafety procedures are incorporated into standard operating procedures or in a biosafety manual adopted or prepared specifically for the laboratory by the laboratory director. Personnel are advised of special hazards and are required to read and follow instructions on practices and procedures.

Training

The Principal Investigator or Laboratory Director ensures that laboratory and support personnel receive appropriate training about the potential hazards associated with:

• the work involved;
• the necessary precautions to prevent exposures;
• the exposure evaluation procedures.

Personnel receive annual updates or additional training as necessary for procedural or policy changes.

Sharps

A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels.

Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

Only needle-locking syringes or disposable syringe-needle units (e.g., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.

Syringes that re-sheathe the needle, needleless systems, and other safety devices are used when appropriate.

Broken glassware must not be handled directly by hand, and must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass are decontaminated before disposal according to any local, state, or federal regulations.

Potentially Infectious Material

Cultures, tissues, specimens of body fluids, or potentially infectious wastes are placed in a container with a cover that prevents leakage during collection, handling, processing, storage, transport, or shipping.

Decontamination

Laboratory equipment and work surfaces should be decontaminated with an effective disinfectant:

• on a routine basis;
• after work with infectious materials is finished;
• and especially after overt spills, splashes, or other contamination by infectious materials.

Prior to its removal from the facility, contaminated equipment must be decontaminated according to any local, state, or federal regulations before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, state, or federal regulations.

Spills & Accidents

Spills and accidents that result in overt exposures to infectious materials are immediately reported to the Principal Investigator and Laboratory Director. Medical evaluation, surveillance, and treatment are provided as appropriate, and written records are maintained.

Cleaning

Sinks in the BSL2 area should be made free of clutter and cleaned routinely with appropriate disinfectant such as a 10% bleach solution and flushed down with running water after a few minutes. Water baths and all water reservoirs should be washed periodically with a suitable chemical decontaminant.

Once a month, work spaces should be cleaned and disinfected, as well as other lab areas where clutter accumulates (e.g., storage areas).

The laboratory will set up a routine schedule to perform surface cleaning with appropriate chemical disinfectant of large equipment (such as incubators) as part of laboratory good practices.

Supplies should be rotated and outdated material discarded. Unlabeled material should be eliminated.

Eliminate and clean clutter.

Custodial Services

Only personnel with appropriate authorization may enter a BSL2 facility while BSL2 research activity is in progress.

Animals

Animals not involved in the work being performed are not permitted in the lab.

Safety Equipment (Primary Barriers)

Properly maintained biological safety cabinets, preferably Class II, or other appropriate personal protective equipment or physical containment devices are to be used when:

Procedures that have the potential to create infectious aerosols or splashes are conducted.

These may include:

• centrifuging,
• grinding,
• blending,
• vigorous shaking or mixing,
• sonic disruption,
• opening containers of infectious materials whose internal pressures may be different from ambient pressures,
• inoculating animals intra-nasally,
• harvesting infected tissues from animals or embryonate eggs.

High concentrations or large volumes of infectious agents are used.

Such materials may be centrifuged in the open laboratory with sealed rotor heads or sealed centrifuge safety cups are used, and if these rotors or safety cups are opened only in a biological safety cabinet.

Equipment

Face protection (goggles, mask, face shield, or other splatter guard) is used for anticipated splashes or sprays of infectious or other hazardous materials to the face when the microorganisms must be manipulated outside the BSC.

Protective laboratory coats, gowns, smocks, or uniforms designated for lab use are worn while in the laboratory. This protective clothing is removed and left in the laboratory before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices). All protective clothing is either disposed of in the laboratory or laundered by the institution; it should never be taken home by personnel.

Gloves are worn when hands may contact potentially infectious materials, contaminated surfaces, or equipment. Wearing two pairs of gloves may be appropriate. Gloves are disposed of when overtly contaminated and removed when work with infectious materials is completed or when the integrity of the glove is compromised.

Disposable gloves are not washed, reused, or used for touching “clean” surfaces (keyboards, telephones, etc.). They should not be worn outside the lab. Alternatives to powdered latex gloves should be available. Hands are washed following removal of gloves.

Procedures for Receiving and Inspecting Samples

The PI will designate a responsible person for the purchase of all infectious materials to be used in the BSL2 lab.

Infectious materials will be shipped to the laboratory in accordance with the appropriate Department of Transportation (DOT) and the International Air Transportation Association (IATA) standards for shipping of infectious biological materials.

Upon receipt of the package, it will be placed on a tray covered with absorbent material and opened in the Biological Safety Cabinet prevent any potential exposure to personnel in case the container leaked during transport.

Personnel assigned to open packages will wear lab coat, gloves, and eye protection.

If any containers are found to be damaged, leaking or otherwise contaminated, they will be immediately isolated into a plastic bag along with all packaging materials. The spill will be disinfected and cleaned up. The Principal Investigator, lab director or designee will be notified immediately. The incident will be reported to EHS and as necessary, to other appropriate agencies.

If, after inspection, the samples are intact, they can be placed into labeled secondary containers (unbreakable plastic containers or metal tubes) and then transferred to a storage area.

Only staff who are authorized to do so can remove samples from storage. Removal and use of all such materials must be entered into the logbook.

Unused cultures can be returned to storage after the outer container has been properly disinfected.

Laboratory Facilities (Secondary Barriers)

In a BSL2 lab, the following conditions are to exist:

Doors

Doors that can be locked and secured should be installed for facilities that house restricted areas.

Public

Consideration should be given to locating new laboratories away from public areas.

Sink

Each laboratory contains a sink for handwashing.

Cleaning

The laboratory is designed so that it can be easily cleaned. Carpets and rugs in laboratories are inappropriate.

Bench Tops

Bench tops are impervious to water and resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.

Lab Furniture

Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning. Chairs and other furniture used in laboratory work should be covered with a non-fabric material that can be easily cleaned and decontaminated.

Biological Safety Cabinets

Biological safety cabinets should be installed in such a manner that fluctuations of the room’s air supply and exhaust air do not cause them to operate outside their parameters for containment. Locate BSCs away from doors, windows that can be opened, heavily traveled laboratory areas, and other potentially disruptive equipment to avoid disruption of the BSC’s air flow parameters.

Eyewash Station

An eyewash station is readily available for use.

Lighting

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

Ventilation

There are no specific ventilation requirements. 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. If the laboratory has windows that open to the exterior, they are fitted with fly screens.