Biosafety Levels: A Complete Guide from BSL-1 to BSL-4 | Safety Protocols & Lab Requirements

Table of Contents

• Introduction to Biosafety Level
• 1. Biosafety Level -1 (BSL-1)
• 2. Biosafety Level-2 (BSL-2)
• 3. Biosafety Level-3 (BSL-3)
• 4. Biosafety Level 4 (BSL-4)
• References

Introduction to Biosafety Level

• Biosafety refers to the prevention of risk to human health and safety, conservation of the environment, and the pathogen itself due to the use of infectious or genetically modified organisms in research and commerce.
• It is a crucial concept in microbiology, as bio-related research often involves manipulation of microbial, animal, or plant cells that may be potentially pathogenic.
• Risks in laboratory activities may arise either from the biological samples being handled or from the specific procedural requirements involved.
• The application of standard microbiological techniques and the use of facilities appropriate to the pathogen's risk level help protect researchers from laboratory-acquired infections.
• Biosafety levels are created to outline the protective measures needed in a lab setting to safeguard researchers, the environment, and the microorganisms involved.
• These levels are defined by the Centers for Disease Control and Prevention (CDC), with each level specifying detailed practices and safety requirements.
• Biosafety level designations are based on a combination of design features, equipment, practices, and procedures required when working with agents from different risk groups.
• The assignment of a pathogenic agent to a particular biosafety level must be based on a thorough risk assessment.
• Such risk assessments consider the risk group of the agent along with other relevant factors to determine the appropriate biosafety level.
• Biosafety levels may vary across different regions due to varying local assessments and regulations.
• According to the CDC, there are four biosafety levels, ranging from biosafety level 1 (BSL-1), which represents the lowest risk, to biosafety level 4 (BSL-4), which represents the highest level of containment and risk.

1. Biosafety Level -1 (BSL-1)

• Biosafety Level 1 (BSL-1) is suitable for work involving well-characterized agents that are not known to consistently cause disease in immune-competent adult humans and pose minimal potential hazard to laboratory personnel and the environment.
• It represents the lowest level of biosafety, and therefore the precautions required are limited and not extensive.
• Laboratories operating at BSL-1 provide general workspace for handling viable agents that are not associated with disease in healthy adults.

Requirements

• BSL-1 laboratories are not necessarily separated from general building traffic and can be located in unrestricted areas.
• Most laboratory work is performed on open bench tops using standard microbiological practices.
• Specialized laboratory design or containment equipment is not required at BSL-1, though they may be used based on the outcomes of a risk assessment.
• Laboratory personnel must receive specific training in the procedures to be performed and must be supervised by a scientist trained in microbiology or a related discipline.

The following elements are required in BSL-1 laboratories: 

Standard microbiological practices

• The laboratory supervisor is responsible for implementing policies related to access control to the laboratory.
• Laboratory personnel must wash their hands after handling potentially hazardous materials and before exiting the laboratory.
• Activities such as eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food are strictly prohibited in laboratory areas.
• Mouth pipetting is not allowed; mechanical pipetting devices must be used.
• All laboratory procedures should be carried out in a manner that minimizes the creation of splashes and aerosols.
• Work surfaces, including benchtops, must be disinfected after completing work and immediately after any spill involving potentially hazardous biological material.
• The supervisor is responsible for ensuring that all laboratory personnel receive appropriate training and follow necessary safety precautions during their tasks.

Safety practices

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

Safety equipment

• Special containment devices such as Biosafety Cabinets are not required for BSL-1 laboratories.
• To prevent contamination of personal clothing, it is recommended to wear protective laboratory coats, gowns, or uniforms.
• When performing procedures with a high potential for aerosol generation, the use of protective eyewear is advised.

Uses

• Biosafety Level 1 is commonly used for handling microbial agents that are not known to cause disease in immune-compromised individuals.
• BSL-1 laboratories are often found in educational settings, including teaching laboratories in colleges and training centers.

Organisms

• Common organisms that require Biosafety Level-1 containment include less hazardous organisms such as Agrobacterium radiobacter, Aspergillus niger, Bacillus thuringiensis, Escherichia coli strain K12, Lactobacillus acidophilus, Micrococcus luteus, Neurospora crassa, Pseudomonas fluorescens, and Serratia marcescens.
• However, the required biosafety level may vary depending on the specific risk assessment of the pathogen.

2. Biosafety Level-2 (BSL-2)

• Biosafety Level-2 (BSL-2) laboratories are designed for tasks involving microbial agents that pose moderate potential hazards to laboratory personnel, the environment, and the agent.
• These agents or toxins may present a moderate danger if accidentally inhaled, swallowed, or exposed to the skin.
• The precautions required for BSL-2 are more extensive than those for BSL-1, but both BSL-1 and BSL-2 laboratories are generally considered basic laboratories.

Requirements

• BSL-2 laboratories, like BSL-1 laboratories, are not necessarily separated from general building traffic patterns.
• However, access to the laboratory is restricted while BSL-2 experiments are ongoing.
• Annual inspections of BSL-2 laboratories are required, which may include tasks like changing filters or replacing certain devices.
• Most work is conducted on sterilized bench tops, except for procedures that may generate aerosols, which should be performed in safety cabinets.
• The precautions required for BSL-2 include all the precautions of BSL-1, along with additional safety measures.

Standard microbiological practices

• All laboratory personnel must wash their hands after handling viable microorganisms and before leaving the laboratory.
• Eating, drinking, smoking, and handling contact lenses are strictly prohibited in the laboratory.
• Mechanical pipetting must be used instead of mouth pipetting.
• All contaminated cultures, glassware, plasticware, and biologically contaminated waste must be treated as biohazards and 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 must be treated as medical waste and disposed of in puncture-resistant sharps disposal containers.

Safety Practices

• Individuals at increased risk of infections, such as immune-compromised or pregnant individuals, should not be allowed to enter BSL-2 laboratories while experiments are in progress.
• An annual review of the BSL-2 manual should be conducted to update safety guidelines.
• Documented policies and procedures must be established to limit access to individuals who are aware of the potential hazards and have received appropriate training.
• A biohazard symbol should be placed on equipment where biohazardous materials are used or stored.

Safety Equipment

• Protective coats must be worn while entering the laboratory and should be removed and kept in the laboratory after work.
• The laboratory design should allow for easy cleaning and decontamination, with minimal nooks and corners.
• Laboratory doors must be kept closed while working with hazardous biomaterials.
• An autoclave must be available for sterilization purposes.

Uses

• BSL-2 laboratories are primarily used for routine analysis and culture of moderately hazardous agents.
• Some laboratories used for teaching and training purposes are also classified as BSL-2.

Organisms

• Organisms that require BSL-2 laboratories include pathogenic strains of E. coli, Staphylococcus, Salmonella, Plasmodium falciparum, Toxoplasma, and Herpes Simplex Viruses.
• The allocation of organisms to BSL-2 laboratories may vary based on the results of a risk assessment.

3. Biosafety Level-3 (BSL-3)

• Biosafety Level 3 (BSL-3) is used for work with agents that can cause severe or potentially lethal diseases through inhalation or aerosol formation, posing significant risks to personnel and the environment.
• Tasks in BSL-3 laboratories involve indigenous or exotic agents where aerosol transmission is highly likely, and the disease may have fatal consequences.
• Autoinoculation and ingestion are the primary hazards to personnel working with these agents at this level.
• Working in BSL-3 laboratories requires laboratory personnel to have specific training in handling pathogenic and potentially lethal agents, along with supervision by scientists who are competent in managing infectious agents and related procedures.

Requirements

• BSL-3 containment laboratories, especially those used for animal research, are the most challenging facilities to design and operate.
• These laboratories must be certified for use before initial operation and subsequently on an annual basis, or after any program change, renovation, or replacement of system components that may affect the operating environment.
• BSL-3 laboratories are also referred to as containment laboratories, as they require specialized containment equipment to protect personnel, the microbial agent, and the environment.
• The requirements for BSL-3 laboratories include all the requirements of BSL-1 and BSL-2 laboratories, along with additional design features and specialized equipment.

Standard Microbiological Practices

• Entry to BSL-3 laboratories is restricted to individuals with appropriate training in handling BSL-3 organisms, all of whom are selected by the laboratory supervisor.
• In addition to general procedures and laboratory practices, the supervisor formulates additional policies to limit entry to the laboratory.
• All procedures in BSL-3 laboratories must be conducted within a biosafety cabinet to prevent exposure to aerosols.
• Personnel working in the laboratory must wear personal protective equipment (PPE) before entering and remove it before leaving.
• Work surfaces and sinks must be decontaminated after each work shift or following any spills or splashes.
• BSL-3 laboratories should be separated from general building traffic to restrict access and ensure safety at all times.

Safety Practices

• The doors of BSL-3 laboratories must remain closed at all times, with appropriate BSL-3 signs displayed outside the suite, including a universal biohazard sign and emergency contact information.
• Laboratory personnel must undergo medical surveillance and be offered appropriate immunizations for agents handled or potentially present in the laboratory.
• Institutions should consider collecting and storing serum samples from at-risk personnel.
• A laboratory-specific biosafety manual must be prepared, adopted as policy, and made accessible to all laboratory personnel.
• The laboratory supervisor must ensure that all personnel demonstrate proficiency in standard and special microbiological practices before working with BSL-3 agents.
• Potentially hazardous materials must be placed in durable, leak-proof containers or vials during collection, processing, storage, or transport within the facility.
• Laboratory equipment should be routinely decontaminated after work or after any spills or splashes.
• The laboratory biosafety manual must define procedures for exposure to infectious materials, and these should be treated accordingly.
• No work in BSL-3 laboratories should be conducted on an open bench or open vessel; all activities involving infectious agents must be conducted within biosafety cabinets or other physical containment devices.

Safety Equipment

• Biosafety cabinets must be used for the manipulation of all infectious agents in BSL-3 laboratories.
• Individual protective gear, such as personal protective equipment (PPE), coats, gloves, and respiratory protection, should be worn before entering the laboratory and removed before leaving.
• Air flowing in the laboratory must not be recirculated; it should be HEPA-filtered before being discharged to the outside.
• Filters, manuals, equipment, vacuum pipes, autoclaves, and other systems should be revised and reviewed annually.

Uses

• BSL-3 laboratories are used for clinical, diagnostic, teaching, research, and production purposes.
• These laboratories handle and manipulate highly infectious agents that pose direct severe health risks to personnel.
• They are used for studies on the effects of infectious agents and various toxins and their impact on health.

Organisms

• Pathogens that require BSL-3 laboratories include HIV, H1N1 flu, Yersinia pestis, Mycobacterium tuberculosis, SARS, Rabies Virus, West Nile Virus, Rickettsia, and others.
• The classification of organisms into different biosafety levels may vary and should be determined after conducting a thorough risk assessment.

4. Biosafety Level 4 (BSL-4)

• Biosafety Level 4 (BSL-4) is the highest level of containment used for working with highly dangerous infectious agents that pose life-threatening risks to individuals and the environment, including potential aerosol transmission or unknown transmission risks.
• BSL-4 laboratories are used for handling Risk Group 4 pathogens, which are extremely dangerous, have no known vaccines or therapies, and require stringent precautions during handling.

There are two types of BSL-4 laboratories:

1. Cabinet laboratory: All work is performed in a Class III biosafety cabinet or similar physical containment with strict precautions.
2. Suit laboratory: Laboratory personnel are required to wear full-body, air-supplied protective suits (PPE) during all work.

Requirements

The requirements for Biosafety Level 4 (BSL-4) laboratories are rigorous and involve extensive laboratory design, training protocols, and highly protective equipment. Key aspects include:

• Certification: BSL-4 laboratories must be certified for use before the initial operation, with annual certifications or after any significant changes or renovations that might affect the containment systems.
• Maximum Containment: BSL-4 labs are often referred to as maximum containment laboratories due to their additional secondary barriers designed to prevent hazardous materials from escaping into the environment.
• Design and Equipment: These labs require specialized designs, with strict control over air flow, waste disposal, and containment measures to prevent accidental release of hazardous agents.
• Comprehensive Precautions: In addition to the requirements for BSL-1, BSL-2, and BSL-3 labs, BSL-4 labs must adhere to extra safety measures, ensuring complete containment and minimal risk.

Standard microbiological practices

The standard microbiological practices for Biosafety Level 4 (BSL-4) laboratories include:

• No Open Work: All work in a BSL-4 lab must be done within a biosafety cabinet or other contained systems to prevent any exposure or aerosol formation.
• Sterilization: Workstations, equipment, and sinks must be thoroughly sterilized after every session to avoid contamination or accidental release of hazardous materials.
• Personal Protective Equipment (PPE): Personnel must wear full-body PPE, including gloves, masks, and coats, to prevent exposure to dangerous agents.
• Access Control: The laboratory doors must be closed at all times, and the laboratory should be isolated from general traffic in the building to prevent accidental exposure.
• Strict Prohibition of Risky Activities: Activities like drinking, eating, and mouth pipetting are strictly prohibited.
• Restricted Access: Only individuals who are specially trained in handling BSL-4 organisms and equipped to use the specific lab equipment should be allowed to enter the laboratory.

Safety Practices

Safety practices for Biosafety Level 4 (BSL-4) laboratories include:

• Handling and Transfer of Biological Materials: Viable or intact biological materials must be transferred in sealed, nonbreakable primary containers, which are then placed in nonbreakable, sealed secondary containers to prevent any leakage or contamination.
• Decontamination Before Removal: No materials, except for biological agents that need to remain intact or viable, can be removed from the BSL-4 lab unless they have been thoroughly autoclaved or otherwise decontaminated to eliminate any risk of infection.
• Restricted Access: Only individuals whose presence is necessary for microbiological processes or support activities are allowed entry. Personnel who are at increased risk of infection, or those for whom the infection might be particularly hazardous, are strictly prohibited from entering the laboratory.
• Clothing and Entry Procedures: All personnel must change into lab-specific clothing before entering and exit through shower rooms to prevent contamination from leaving the lab.
• Warning Signs: During active work or when infectious agents or animals are present in the lab, hazard warning signs with the universal biohazard symbol must be posted on all access doors.
• Accident and Exposure Reporting: A system should be in place for reporting any laboratory accidents or exposures. In addition, medical surveillance should be conducted to monitor potential illnesses related to laboratory work.

Safety equipment

In addition to the aforementioned safety practices, the required safety equipment for a Biosafety Level 4 (BSL-4) laboratory includes:

• Biological Safety Cabinets: A Class III biological safety cabinet must be used for handling all procedures within the BSL-4 lab. Alternatively, Class I or II biological safety cabinets may be used in conjunction with one-piece personnel suits that are ventilated by a life support system to ensure the safety of laboratory personnel.
• Sealed Laboratory Design: The walls, floors, and ceilings of the laboratory must be constructed to form a sealed internal shell. This shell allows for fumigation to sterilize the facility and also ensures that the space is impervious to animals and insects, minimizing the risk of contamination.
• Double-Door Autoclave: A double-door autoclave is required for decontaminating materials as they exit the facility, ensuring that no hazardous materials leave the lab without proper sterilization.
• Exhaust Air Filtration: All exhaust air from the BSL-4 facility must pass through HEPA filters before being discharged outdoors. This ensures that potentially contaminated air does not enter any occupied buildings or air intakes, maintaining a high level of environmental safety.

Uses

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

Organisms

BSL-4 level pathogens include risk group IV organisms such as:

• Ebola virus
• SARS-CoV-2
• Central European Encephalitis virus
• Hemorrhagic viruses
• Other highly dangerous pathogens

References

• 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/
• Mourya, D. T., Yadav, P. D., Majumdar, T. D., Chauhan, D. S., & Katoch, V. M. (2014). Establishment of Biosafety Level-3 (BSL-3) Laboratory: Key Considerations for Designing, Constructing, Commissioning, and Operating the Facility in the Indian Setting. The Indian Journal of Medical Research, 140(2), 171–183.
• 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
• Standard Microbiological Practices for Biosafety Level 1 Laboratories at the University of Tennessee-Knoxville, Institute of Agriculture and Graduate School of Medicine, January 2013.
• Biosafety Manual. Boston University Research Support.