Chapter 4: Biosafety Levels

West Virginia University adheres to the most current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL) by the U.S. Centers for Disease Control and the NIH Guidelines for Research Involving Recombinant and Synthetic Nucleic Acid Molecules by the National Institutes of Health for establishing biosafety levels and determining appropriate precautions and containment for each biosafety level.

Containment of biohazards is a fundamental objective of a biosafety program. Containment is achieved through a variety of principles including laboratory practices, primary barriers including safety equipment, personal protective equipment (PPE), and secondary barriers such as facility design.

1.     DEFINITIONS:

1.1. Biological Safety Levels (BSL): Designations of laboratories in ascending order based on the degree of risk associated with the laboratory work being conducted and the degree of protection provided to personnel, the environment, and the community.

1.2. Etiologic Agents: Microorganisms and toxins that cause disease in humans and include bacteria, bacterial toxins, viruses, fungi, protozoans, and parasites. These disease-causing microorganisms may also be referred to as infectious agents.

1.3. Risk Group (RG): As defined by the NIH Guidelines for Research Involving Recombinant and Synthetic Nucleic Acid Molecules, Risk Groups are risk-based groups in which etiologic agents are classified into, according to their relative pathogenicity for healthy adult humans. It should be noted that Risk Group and Biological Safety Levels are not always equivalent.

2.     PROCEDURE:

2.1. Risk Groups (RG)

The NIH has classified biological agents into four Risk Groups according to their relative pathogenicity for healthy adult humans:

Risk Group 1 (RG1): RG1 agents are not associated with disease in healthy adults humans. Examples of RG1 agents include Bacillus subtilis, Saccharomyces cerevisiae, and Escherichia coli (K12)

Risk Group 2 (RG2): RG2 agents are associated with human diseases which are rarely serious and for which preventive or therapeutic interventions are often available. Examples of RG2 agents include Staphylococcus aureus, Adenovirusues human(AAV), and Listeria spp.

Risk Group 3 (RG3): RG3 agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available. RG3 agents have high individual risk but low community risk. Examples of RG3 agents include Mycobacterium tuberculosis, Rickettsia rickettsii, and West Nile virus.

Risk Group 4 (RG4): RG4 agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available. RG4 agents have high individual risk and high community risk. Examples of RG4 agents include Ebola virus and Herpesvirus simiae (Herpes B or Monkey B virus).

NOTE: For a full list of agents classified into Risk Groups, visit the following links:

NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules- Appendix B ABSA Risk Group Database

2.2. Universal Biosafety Precaution

It is best biosafety practice to handle all biological material (including tissues, cell culture, and body fluids) which have the potential to be infected as though they are infectious material. Doing this will greatly minimize the risk of exposure.

2.3. Standard Biosafety Lab Practices

The following standard biosafety lab practices apply to all biosafety levels:

• Eating, drinking, storing food or beverages for human consumption, handing contact lenses, and applying cosmetics are prohibited within the laboratory.
• Persons must wash hands after working with potentially infectious materials and before leaving the laboratory.
• Mouth pipetting is prohibited.  Mechanical pipetting devices must be used.
• Cell phones may not be used while wearing gloves. If you must use a cell phone in the lab, remove your gloves and wash your hands prior to touching phone.
• Phones should be disinfected after using in a laboratory environment.
• Procedures should be performed to minimize the creation of splashes and/or aerosols.
• Decontaminate work surfaces after the completion of work and after any spills or splashes with potentially infectious materials.
• Ensure the disinfectant utilized is effective against potential biohazards.
• Decontaminate all cultures and other potentially infectious materials before disposal using an effective method. See Chapter 8: Decontamination, Disinfection, and Spill Response for further guidance regarding effective methods of decontamination.
• A sign incorporating the universal biohazard symbol (Figure 1) must be present at the entrance to the laboratory when infectious agents are present. This is typically BSL2 and higher.
               
Figure 1. Universal Biohazard Symbol


Sharps Policy
• All sharps, regardless of being utilized for biohazards or not, must be disposed of in an approved sharps disposal container.
• Needles must never be recapped.
• An approved, puncture-resistant sharp disposal container must be conveniently placed within a reasonable distance to where the sharp is utilized.
• Non-disposable sharps must be placed in a hard-walled container for transport.
• Broken glassware must not be handled directly. Utilize a brush and dust pan, tongs, or forceps to clean broken glassware.

2.4. Biological Safety Level 1 (BSL1)

Biological Safety Level 1 is suitable for work involving agents not known to consistently cause disease in immunocompetent adult humans. BSL1 agents present minimal potential hazard to laboratory personnel and the environment.

Special Practices for BSL1

• No special practices are required at BSL1.

Safety Equipment for BSL1

Containment and Primary Barriers

• Special containment devices, such as Biological Safety Cabinets (BSC) are not typically required at BSL1.

Personal Protective Equipment (PPE)

• Lab coats, gowns, or uniforms are recommended to prevent contamination of personal clothing.
• Safety glasses are recommended to be worn in the laboratory at all times. Safety glasses are required to be worn when conducting procedures that have the potential to create splashes of biohazards or other hazardous material.
• Gloves must be worn to protect hands from exposure to biohazards.
• Gloves should be changed when contamination or when the integrity is compromised.
• Remove gloves and wash hands when work with biohazards has been completed and before leaving laboratory.

Laboratory Facilities for BSL1

• Laboratories should have doors for access control.
• Laboratories must have a sink for hand washing.
• Laboratories should be designed to be easily cleaned.
• Non-porous floors and surfaces
• Carpet and rugs are not appropriate
• Lab benches must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
• Chairs must be made of a non-porous material that can be easily cleaned and decontaminated.

2.5. Biological Safety Level 2 (BSL2)

Biological Safety Level 2 is suitable for work involving agents that pose moderate hazards to personnel and the environment. BSL2 builds upon the aforementioned requirements of BSL1.

Special Practices for BSL2

• The laboratory is responsible for signage and notifying all persons entering laboratory of the potential hazards.
• The laboratory supervisor must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BSL2 agents.
• The laboratory supervisor must ensure that laboratory personnel complete mandatory biosafety training, as well as any specialized training determined to be necessary for the laboratory.
• See Chapter 3: Training for further guidance related to required training.
• Potentially infectious materials must be placed in a durable, leak proof secondary container during storage and transport within a facility.
• Laboratory equipment should be regularly decontaminated, as well as, after spills or other potential contamination.
• Equipment must be decontaminated before repair, maintenance, or removal from the laboratory. See  Chapter 8: Decontamination, Disinfection, and Spill Response for further guidance.
• Spills involving infectious material must be contained, decontaminated, and cleaned up by staff whom are properly trained and equipped to work with infectious material. See Chapter 8: Decontamination, Disinfection, and Spill Response for further guidance.
• Incidents resulting in exposure or potential exposure to infectious materials must be immediate evaluated and treated according to procedures described in Chapter 6: Occupational Health and Exposure Response.
• Animals and plants not associated with the work being performed must not be permitted in the laboratory.
• Cellphone or other personal electronic device use should be limited to when not actively working with biohazards.
• Gloves must be removed before handling cell phones or other personal electronic devices.

Safety Equipment for BSL2

Containment and Primary Barriers

• All procedures involving high concentrations or large volumes of infectious agents must be conducted within a Biological Safety Cabinet (BSC) or other approved physical containment devices. Biological Safety Cabinets must also be used for the manipulation of infectious materials that may generate an aerosol.
• Such procedures include, but are not limited to: pipetting, centrifuging, grinding, blending, shaking, sonicating, vortexing, opening containers of infectious materials, inoculating animals intranasally, and harvesting infected tissue from animals or eggs.

Personal Protective Equipment (PPE)

• Lab coats must be worn when working with hazardous materials and must remain in the laboratory. They may be reusable cloth lab coats or disposable.
• Lab coats must be removed before leaving the laboratory area.
• Refer to Chapter 10: Personal Protective Equipment for guidance on laundering reusable lab coats.
• Safety glasses must be worn when working in a BSL2 laboratory.
• Eye and face protection such as goggles, mask, or face shield must be utilized if manipulating or handling infectious material or microorganisms outside the BSC.
• It is not required if you are transporting closed containers of infectious material between storage and the BSC.
• Gloves must be worn to protect hands from working with infectious materials and other biohazards. Glove selection should be based on appropriate risk assessment.
• Nitrile gloves are the preferred choice given their wide range of protection.
• Gloves must be changed when contaminated, glove integrity is compromised, or when otherwise necessary.
• Gloves must be removed, followed by hand washing, when working with infectious material and other biohazards is complete and before leaving the laboratory.
• Gloves must not be washed and reused.

Laboratory Facilities for BSL2

• Laboratory doors should be self-closing and have locks.
• Laboratories must have a sink for hand washing and maintain a supply of soap and paper towels.
• Laboratories should be designed to be easily cleaned.
• Non-porous floors and surfaces
• Carpet and rugs are not appropriate
• Lab benches must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
• Chairs must be made of a non-porous material that can be easily cleaned and decontaminated.
• Biological Safety Cabinets must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations.
• Vacuum lines should be protected with in-line HEPA filters.
• Eye wash station must be readily available in the laboratory.

2.6. Biological Safety Level 2+ (BSL2+)

Although not officially recognized as a biosafety level, BSL2+, also referred to as BSL2 with enhancements, is utilized when work is conducted in a BSL2 laboratory with biohazards that may pose a higher risk than standard BLS2 agents. There is no standard list of BSL2+ agents; however, the need for BSL2+ practices are determined through risk assessment. An example of a BSL2+ agent is Lentiviral vectors when utilized with oncogenes.

BSL2+ implement aspects of biosafety practices and procedures typically found at BSL3 within a BSL2 laboratory setting.

In addition to all practices and procedures utilized for BSL2 work, the following must be adhered to:

• The door to the lab must be kept closed while performing experiments.
• A BSL2+ procedure door hang tag (Figure 2) must be placed on the door.
• Two (2) pairs of gloves must be worn. (The outer layer must be discarded into the biohazard waste inside the biosafety cabinet.)
• A solid-front disposable lab-coat needs to be worn over a regular lab-coat.
• All work must be performed inside a biosafety cabinet, and sealed centrifuge rotors must be opened and loaded and unloaded with samples inside the biosafety cabinet.
• In case of an accidental needle stick or exposure, Occupational Medicine will need to be contacted to start prophylaxis treatment within 2 hours for the treatment to be successful.

2.7. Biological Safety Level 3 (BSL3)

BSL3 is designated where work is performed with indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route. BSL3 work must be performed within a specially designated, high containment area. BSL3 principles build on those applicable for BSL2.

General principles and practices of BSL3 are discussed within this section. Specific SOPs related to WVU’s High Containment Area (HCA) are available to authorized users of the HCA. The aforementioned SOPs provide detailed procedures and policies related to the use of the WVU HCA.

Special Practices for BSL3

• All persons entering the laboratory must be advised of the potential hazards and meet specific entry/exit requirements.
• Lab personnel must be provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the lab.
• The laboratory supervisory must ensure that laboratory personnel complete mandatory biosafety training, as well as any specialized training determined to be necessary for the laboratory.
• West Virginia University Occupational Medicine department is responsible for conducting applicable medial surveillance.
• The Biosafety Office must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BSL3 agents.
• Potentially infectious material must be placed in a durable, leak-proof container during collection, handling, processing, storage, or transport.
• Lab equipment should be routinely decontaminated, as well, as after spills, splashes, or other potential contamination.
• Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material.
• Equipment must be decontaminated before repair, maintenance, or removal from the laboratory.
• Incidents resulting in exposure or potential exposure to infections materials must be immediate evaluated and treated according to procedures described in Chapter 6: Occupational Health and Exposure Response.
• Animals and plants not associated with the work being performed must not be permitted in the laboratory.

Safety Equipment for BSL3

Containment and Primary Barriers

• All procedures involving the manipulation of infectious materials must be conducted within a class 2, at minimum, Biological Safety Cabinet (BSC) or other physical containment device.
• Centrifuges must be equipped with aerosol tight rotors, which are only opened inside of a BSC.
• If an aerosol tight rotor is not available, the centrifuge must be operated within a BSC.

Personal Protective Equipment (PPE)

• Lab workers are not permitted to wear personal clothing, shoes, or jewelry in the BSL3 High Containment Area (HCA).
• Prior to entering the HCA, lab workers must change into facility captured scrubs and shoes.
• Depending on the procedure being performed, either a solid-front gown or Tyvek suit is required to be worn.
• Eye, face, and respiratory protection is required for entry into the HCA. A Powered, Air-Purifying Respirator (PAPR) must be worn at all times within the HCA.
• Gloves must be worn to protect hands from working with infections materials and other biohazards. Glove selection should be based on appropriate risk assessment.
• Double gloves are required for use in the HCA.
• Nitrile gloves are the preferred choice given their wide range of protection.
• Gloves must be changed when contaminated, glove integrity is compromised, or when otherwise necessary.
• Outer gloves must be removed when working with infectious material and other biohazards is complete and before leaving the laboratory.
• Gloves must not be washed and reused.

Laboratory Facilities for BSL3

• Laboratory doors should be self-closing and have locks.
• The laboratory must be separated from areas that are open to unrestricted traffic.
• Access to the lab is through two self-closing doors, separated by an anteroom.
• The lab must be designed with sustained, directional airflow from “clean” areas towards “potentially contaminated” areas.
• Under failure conditions, the airflow must not be reversed.
• Lab personnel must be able to verify directional airflow at all times via visual monitoring devices.
• Audible alarms should be considered to notify personnel of airflow disruption.
• The building must exhaust air through a HEPA filter and not re-circulate to other areas of the building.
• Laboratories must have a hands-free sink for hand washing and maintain a supply of soap and paper towels.
• The sink should be located near the exit door.
• Each separate room must have a hand washing sink.
• Laboratories should be designed to be easily cleaned.
• Seams, floors, walls, and ceiling surfaces should be sealed.
• Non-porous floors and surfaces.
• Carpet and rugs are not permitted.
• Floors must be slip resistant, impervious to liquids, and resistant to chemicals.
• Lab benches must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
• Chairs must be made of a non-porous material that can be easily cleaned and decontaminated.
• Biological Safety Cabinets must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations.
• Vacuum lines must be protected with in-line HEPA filters.
• Eye wash stations must be readily available in the laboratory.

2.8. Biological Safety Level 4 (BSL4)

• Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease that is frequently fatal.
• West Virginia University does not operate any laboratories at BSL4.
• If you would like to learn more about principles and practices of BSL 4, please refer to the CDC Biosafety in Microbiological and Biomedical Laboratories (BMBL), page 45 for more information.

3.     REFERENCES:

• CDC Biosafety in Microbiological and Biomedical Laboratories (BMBL)
• NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules
• Duane, E. G. (2013). A Practical Guide to Implementing a BSL-2+ Biosafety Program in a Research Laboratory. Applied Biosafety, 18(1), 30–36 https://doi.org/10.1177/153567601301800105

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Version History:

Revision Control Table
Version	Changes	Revised by
1.0	Initial version	Matt Stinoski
2.0 (7/2/24)	Updated website links, fixed typos and wording	Josh Parenti