Biosafety Levels (BSL-1, BSL-2, BSL-3 and BSL-4)

Biosafety is the prevention of risk to human health and safety, and the conservation of the environment and the pathogen, as a result of the use for research and commerce of infectious or genetically modified organisms.

• Biosafety is an important concept in microbiology as bio-related research activities may involve manipulation of microbial, animal, or plant cells which might potentially be pathogenic.
• The risks associated with the laboratory activities occur either from the samples or the procedural requirements.
• Application to standard microbiological techniques and employing facilities suitable for the risk level of the pathogen helps to protect the researcher from laboratory-acquired infections.

The biosafety levels are thus designed to identify various protective measures that are to be taken in a laboratory setting to protect the researchers, the environment, and the microorganisms.

• These levels are defined by the Central for Disease Control and Prevention (CDC), where each of these levels is outlined with specific practices and safety requirements.
• Biosafety level designations are based on the combination of the design features, equipment, practices, and procedures required while working with agents from the various risk groups.
• The allocation of a pathogenic agent to a biosafety level for laboratory work must be based on the risk assessment.
• Such assessments take the risk group as well as other factors into consideration while establishing the appropriate biosafety level. The biosafety levels, thus, might differ from one region to another.
• As per the CDC, biosafety levels are of four types depending on the risk associated with the microorganism and the facilities available. The levels of containment range from the biosafety level 1 (BSL-1), which is the lowest to the level 4 (BSL-4), which is the highest.

Biosafety Levels

1. Biosafety Level -1 (BSL-1)

• Biosafety Level 1 is the level appropriate for work involving well-characterized agents not known to consistently cause disease in immune-competent adult humans and cause a minimal potential hazard to the laboratory personnel and the environment.
• Biosafety level 1 is the lowest safety level, and the precautions required for the level are thus limited and not as extensive.
• These laboratories provide general space in which work is done with viable agents that are not associated with disease in healthy adults.

Requirements

• The BSL-1 laboratories are not necessarily separated from the general traffic in the building.
• Most of the work is typically conducted on open bench tops using general microbiological practices.
• Unique laboratory design or containment equipment are not required but may be used depending on the risk assessment.
• Laboratory personnel must be provided with specific training in the procedures to be conducted in the laboratory, which is then supervised by a scientist with training in microbiology or related sciences.

The following are the standard practices, safety equipment, and facility requirements required in BSL-1:

Standard microbiological practices

• The laboratory supervisor should implement the policies regarding the access control to the laboratory.
• Laboratory personnel must wash their hands after working with potentially hazardous materials and before leaving the laboratory.
• Activitttiiies like eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food are not be permitted in laboratory areas.
• Mouth pipetting is prohibited; mechanical pipetting devices must be employed.
• All procedures to be conducted in the laboratory should be performed while avoiding the creation of splashes and aerosols.
• The work surface like the benchtops should be disinfected after work and after any spill of potentially hazardous biological material.
• The supervisor must ensure that all the laboratory personnel acquires appropriate training and necessary precautions while performing their tasks.

Safety practices

• There are no safety specific safety practices required for BSL-1.

Safety equipment

• Special containment devices like the Bio-safety Cabinets are not required for BSL-1.
• In order to prevent the contamination of personal clothing, protective laboratory coats, gowns, or uniforms are recommended.
• While conducting tests with a high possibility of aerosol formation, protective eyewear can be used.

Uses

• Biosafety Level-1 is commonly used while performing tests on microbial agents that are not known to cause diseases in immune-compromised individuals.
• These laboratories include the laboratories used for teaching purposes in colleges and training centers.

Organisms

• The common organisms that require Biosafety Level-1 containment include less hazardous organisms like Agrobacterium radiobacter, Aspergillus niger, Bacillus thuringiensis, Escherichia coli strain K12, Lactobacillus acidophilus, Micrococcus leuteus, Neurospora crassa, Pseudomonas fluorescens, Serratia marcescens.
• However, the requirement of the biosafety level might differ depending on the risk assessment of the pathogen.

2. Biosafety Level-2 (BSL-2)

• Biosafety level-2 laboratories are the laboratories that are used for the tasks involving microbial agents of moderate potential hazards to the laboratory personnel, the environment, and the agent.
• However, the infectious agents or the toxins might pose a moderate danger if accidentally inhaled, swallowed, or exposed to the skin.
• The precautions associated with biosafety level-2 are comparatively more extensive than BSL-1, but BSL-1 and BSL-2 laboratories are generally considered as basic laboratories.

Requirements

• BSL-2 laboratories like BSL-1 laboratories are not necessarily separated from the general traffic patterns in the building.
• However, access into the laboratory is limited while BSL-2 experiments are in progress.
• The annual inspection of the laboratories is also an important part of the BSL-2 requirements. These might include changing the filters or replacement of some devices.
• The work is mostly conducted on sterilized bench tops except for some processes that might form aerosols. The latter is conducted in safety cabinets.
• The precautions to be followed in BSL-2 include all the precautions of the BSL-1 and some additional precautions.

Standard microbiological practices

• All the laboratory personnel must wash their hands after using viable microorganisms and before leaving the laboratory.
• Eating, drinking, smoking, and handling contact lenses in the laboratory are strictly prohibited.
• Mechanical pipetting should be done instead of mouth pipetting.
• All contaminated cultures, glassware, plastic ware, and biologically contaminated waste must be treated as bio-hazards and thus, autoclaved.
• Work surfaces must be decontaminated with disinfectant at the end of the day or after any spills or splashes.
• Used hypodermic syringes and needles, Pasteur pipettes, razor blades, contaminated broken glass, and blood vials are treated as medical waste and discarded in puncture-resistant sharps disposal containers.

Safety Practices

• People with increased risk of acquiring infections like the immune-compromised and pregnant individuals should not be allowed to enter the BSL-2 laboratories while the laboratories are at work.
• An annual review of the BSL-2 manual should be done to update the guidelines.
• Documented policies and procedures should be established that limit the entrance to individuals who know of the potential hazards and are appropriately trained.
• A biohazard symbol is placed on pieces of equipment where biohazardous materials are used or stored.

Safety Equipment

• Protective coats are to be worn while entering the laboratory and then removed and kept in the laboratory post work.
• The laboratory design should be made such that it can be easily cleaned and decontaminated with minimum nooks and corners.
• The laboratory doors should be closed whenever work with hazardous biomaterials is conducted.
• An autoclave must be available.

Uses

• Biosafety level-2 laboratories are mostly used for routine analysis and culture of moderately hazardous agents.
• Besides, some of the laboratories used for teaching and training purposes are also BSL-2 laboratories.

Organisms

• The organisms that require BSL-2 laboratories include the pathogenic strains of E. coli, Staphylococcus, Salmonella, Plasmodium falciparum, Toxoplasma, and Herpes Simples Viruses.
• The allocation of organisms to the laboratories, however, might differ depending on the risk assessment.

Video- Understanding BioSafety Levels By Microbiology Mantra.

3. Biosafety Level-3 (BSL-3)

• Biosafety level 3 (BSL-3) is the level where work is performed with agents that may cause severe or potentially lethal disease through inhalation or aerosol formation, to the personnel, and may even contaminate the environment.
• The tasks performed in the BSL-3 laboratories involve indigenous or exotic agents where the potential for infection by aerosols is high, and the disease may have lethal consequences.
• Autoinoculation and ingestion present primary hazards to personnel working with these agents at this level.
• Working in such laboratories require laboratory personnel with specific training in handling pathogenic and potentially lethal agents, along with supervisors scientists competent in handling infectious agents and associated procedures.

Requirements

• Biosafety Level 3 containment laboratories for animals and research are the most challenging containment level facilities to design and operate.
• These laboratories should be certified for use before initial operation and subsequently on an annual schedule or after a program change, renovation, or replacement of system components that may affect the operating environment of the laboratory.
• BSL-3 laboratories are also called the containment laboratory as they require containment equipment to protect the personnel, the microbial agent, and the environment.
• The requirements for BSL-3 include all the requirements of the BSL-1 and BSL-2 laboratories, along with some additional design features and special equipment.

Standard Microbiological Practices

• The entry to the BSL-3 laboratories is limited to individuals with appropriate training in handling BSL-3 organisms, all of whom are selected by the laboratory supervisor.
• Besides the general procedures and laboratory practices, the supervisor also formulates additional policies to limit the entry to the laboratory.
• All the procedures to be conducted in the BSL-3 must be conducted within a biosafety cabinet to prevent the exposure of the aerosols to the laboratory personnel.
• Personnel working in the laboratory must wear personal protective equipment before entering the laboratory and then remove them before leaving.
• The work surfaces and sinks should be decontaminated once every work shift or after any spills or splashes.
• The BSL-3 laboratories should be separated from the general traffic in a building to limit entry into the laboratories at all times.

Safety Practices

• The doors of the BSL-3 laboratories are closed at all times with appropriate BSL-3 signs outside the suite, along with a universal biohazard sign and emergency contact information.
• Laboratory personnel must have medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory.
• Each institution should consider the collection and storage of serum samples from at-risk personnel.
• A laboratory-specific biosafety manual, which is available and accessible to all, must be prepared and adopted as a policy.
• The laboratory supervisor must check for the demonstration of proficiency in standard and special microbiological practices by all laboratory personnel before working with BSL-3 agents.
• Potentially hazardous materials must be placed in a durable, leak-proof container or vial during collection, processing, storage, or transport within a facility.
• All laboratory equipment should be routinely decontaminated after work or after any spills or splashes.
• The laboratory biosafety manual must define procedures t be adopted in the case of exposure to infectious materials, and these should be treated accordingly.
• No work in the BSL-3 laboratories should be conducted on an open bench or an open vessel. All the activities involving the infectious agents must be conducted within Biosafety cabinets or other physical containment devices.

Safety Equipment

• Biosafety cabinets are to be used for the manipulation of all infectious agents.
• Individual protection gears like personal protective equipment, coats, gloves, and respiratory protection should be worn while entering the laboratories and then removed before leaving.
• The air flowing in the laboratory shouldn’t be recirculated to any area of the laboratory and should be HEPA-filtered prior to being discharged to the outside.
• The filters, manuals, equipment, vacuum pipes, autoclaves, etc. should be revised and reviewed annually.

Uses

• BSL-3 laboratories are used for clinical, diagnostic, teaching, research, or production facilities.
• These laboratories are used for the handling and manipulation of highly infectious agents that prose direct severe effects on the health of the personnel.
• These are used for the studies regarding the effects of infectious agents and various toxins and their effects.

Organisms

• The pathogens that require BSL-3 laboratories include HIV, H1N1 flu, Yersinia pestis, Mycobacterium tuberculosis, SARS, Rabies Virus, West Nile Virus, Ricketts, etc.
• The placement of the organisms in different Biosafety levels, however, might defer and should also be determined after risk assessment.

4. Biosafety Level 4 (BSL-4)

• Biosafety level 4 is the highest level that is employed while working with dangerous infectious agents that present a high individual as well as environmental risk in the form of life-threatening disease, aerosol transmission, or unknown risk of transmission.
• The BSL-4 laboratories are often used while handling and manipulating Risk Group 4 pathogens that are extremely dangerous, with no known vaccines or therapies, and require extreme precautions during work.
• The BSL-4 laboratories are of two types; cabinet laboratory where all the work is performed in a Class III biosafety cabinet or similar physical containment with very carefully formulated precautions and suit laboratory where all the laboratory personnel are required to wear full-body, air-supplied suits protective gears in the form of PPEs.

Requirements

• The requirements of BSL-4 laboratories are extensive with specific laboratory design, training procedures, and highly protective equipment and personal gears.
• These laboratories should be certified for use before initial operation and subsequently on an annual schedule or after a program change, renovation, or replacement of system components that may affect the operating environment of the laboratory.
• BSL-4 laboratories are also termed the maximum containment laboratories as they have secondary barriers to prevent hazardous materials from escaping into the environment.
• The BSL-4 laboratories should follow the requirements of all BSL-1, BSL-2, and BSL-3, along with additional specific precautions.

Standard microbiological practices

• No work conducted within the BSL-4 should be done on an open bench or an open vessel.
• The work stations, equipment, and sinks should be sterilized post work.
• The laboratory personnel should be in protective gear that might include full-body PPEs, gloves, masks, and coats.
• The doors of the laboratories should be closed at all times with the laboratory placed away from the general traffic in the building.
• Activities like drinking, eating, mouth pipetting should be avoided at all costs.
• Only people that are trained in handling the BSL-4 organisms and the equipment in the laboratory should be allowed into the laboratory.

Safety Practices

• Viable or intact biological materials to be removed from the Class III cabinet in a BSL-4 are transferred in a nonbreakable, sealed primary container with a nonbreakable, sealed secondary container.
• No materials, except the biological materials that are to remain in a viable or intact state, are removed from the BSL-4 laboratory unless they have been autoclaved or decontaminated before they leave the facility.
• Only individuals whose presence in the facility is required for microbiological processes or support purposes are authorized to enter. Individuals that are at increased risk of acquiring an infection or for whom infection may be unusually hazardous are not allowed in the laboratory.
• Personnel can enter and leave the facility only after the clothing change and through the shower rooms.
• When the BSL-4 laboratory is at work or when infectious materials or infected animals are present in the laboratory, a hazard warning sign, along with the universal biohazard symbol, is placed on all access doors.
• A system is set up for reporting laboratory accidents, exposures, and the medical surveillance of potential laboratory-associated illnesses.

Safety equipment

• A Class III biological safety cabinet or Class I or II biological safety cabinets used in conjunction with one-piece personnel suits ventilated by a life support system are to be present in a BSL-4 while conducting all procedures within the facility.
• Walls, floors, and ceilings of the laboratories must form a sealed internal shell which facilitates fumigation and is animal and insect-proof.
• A double-doored autoclave is placed for decontaminating materials passing out of the facility.
• The exhaust air from the facility is filtered through HEPA filters before being discharged to the outside so as to prevent its entry into occupied buildings and air intakes.

Uses

• BSL-4 laboratories are used for diagnostic and research work on easily transmitted pathogens, causing fatal diseases.
• These laboratories are used for new and unknown pathogenic microbes, for which no vaccines or therapies are available.
• They are also used for clinical and production facilities that require highly sophisticated techniques and advanced processes.

Organisms

• The BSL-4 level pathogens include the risk group IV organisms like Ebola virus, SARS-CoV-2, Central European Encephalitis virus, Hemorrhagic viruses, etc.

References and Further Readings

1. National Research Council (US) Committee on Hazardous Biological Substances in the Laboratory. Biosafety In The Laboratory: Prudent Practices for the Handling and Disposal of Infectious Materials. Washington (DC): National Academies Press (US); 1989. Appendix A, Biosafety in Microbiological and Biomedical Laboratories.Available from: https://www.ncbi.nlm.nih.gov/books/NBK218631/
2. Mourya, D. T., Yadav, P. D., Majumdar, T. D., Chauhan, D. S., & Katoch, V. M. (2014). Establishment of Biosafety Level-3 (BSL-3) laboratory: important criteria to consider while designing, constructing, commissioning & operating the facility in Indian setting. The Indian journal of medical research, 140(2), 171–183.
3. Janosko, K., Holbrook, M. R., Adams, R., Barr, J., Bollinger, L., Newton, J. T., Ntiforo, C., Coe, L., Wada, J., Pusl, D., Jahrling, P. B., Kuhn, J. H., & Lackemeyer, M. G. (2016). Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures. Journal of visualized experiments: JoVE, (116), 52317. https://doi.org/10.3791/52317
4. Standard Microbiological Practices for Biosafety Level 1 Laboratories at the University of Tennessee-Knoxville, Institute of Agriculture and Graduate School of Medicine. January, 2013.
5. Biosafety Manual. Boston University Research Support.

Sources

• 3% – https://www.moh.gov.sg/docs/librariesprovider7/useful-info-and-guidelines-documents/bsl3_certificationrequirements_final.pdf
• 2% – https://www.slideshare.net/amjadkhanafridi4all/biosafety-level-3
• 1% – https://www.who.int/csr/resources/publications/biosafety/en/Biosafety7.pdf
• 1% – https://www.who.int/csr/disease/coronavirus_infections/Biosafety_InterimRecommendations_NovelCoronavirus_19Feb13.pdf
• 1% – https://www.umt.edu/research/compliance/IBC/safetymanual.php
• 1% – https://www.phe.gov/s3/BioriskManagement/biosafety/Pages/Biosafety-Levels.aspx
• 1% – https://www.phe.gov/s3/BioriskManagement/biocontainment/Pages/BSL-Requirements.aspx
• 1% – https://www.nap.edu/read/1197/chapter/9
• 1% – https://www.nafahq.org/biosafety-level-3-bsl-3-facilities/
• 1% – https://www.kent.edu/compliance/biohazard-classes-agents-pdf
• 1% – https://www.csuohio.edu/sites/default/files/BSL%201.pdf
• 1% – https://www.cmich.edu/office_provost/ORGS/Lab_Safety/Biological_Safety/Documents/SectionIV-LaboratoryBiosafetyLevelCriteria.pdf
• 1% – https://www.cdc.gov/selectagent/resources/Checklist-NIH-BL4.pdf
• 1% – https://sites.google.com/site/bioterrorbible/BIO-LABS/Biosafety-Level-4
• 1% – https://quizlet.com/11896718/biohazard-flash-cards/
• 1% – https://ehrs.upenn.edu/health-safety/biosafety/special-containment
• 1% – http://www.saflii.org/za/legis/consol_reg/rrttromlataihmasohp1430/
• 1% – http://www.bio.iitb.ac.in/bsbe_safety.pdf
• 1% – http://www.adph.org/ALPHTN/assets/072308_weirich.pdf