Biosafety level 1

Biosafety level 1 (BSL-1) is suitable for work with well-characterized agents which do not cause disease in healthy humans. In general, these agents should pose minimal potential hazard to laboratory personnel and the environment.^[21] At this level, precautions are limited relative to other levels. Laboratory personnel must wash their hands upon entering and exiting the lab. Research with these agents may be performed on standard open laboratory benches without the use of special containment equipment. However, eating and drinking are generally prohibited in laboratory areas.^[21] Potentially infectious material must be decontaminated before disposal, either by adding a chemical such as bleach or isopropanol or by packaging for decontamination elsewhere.^[21] Personal protective equipment is only required for circumstances where personnel might be exposed to hazardous material.^[21] BSL-1 laboratories must have a door which can be closed to limit access to the lab. However, it is not necessary for BSL-1 labs to be isolated from the general building.^[22]

This level of biosafety is appropriate for work with several kinds of microorganisms including non-pathogenic strains of Escherichia coli and Staphylococcus, Bacillus subtilis, Saccharomyces cerevisiae and other organisms not suspected to contribute to human disease.^[23] Due to the relative ease and safety of maintaining a BSL-1 laboratory, these are the types of laboratories generally used as teaching spaces for high schools and colleges.^[22]

Biosafety level 2

At this level, all precautions used at Biosafety level 1 are followed, and some additional precautions are taken. BSL-2 differs from BSL-1 in that:

• "laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists."^[24][25]
• Access to the laboratory is limited when work is being conducted.
• Certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.^[21]
• Extreme precautions are taken with contaminated sharp items.

Biosafety level 2 is suitable for work involving agents of moderate potential hazard to personnel and the environment.^[22] This includes various microbes that cause mild disease to humans, or are difficult to contract via aerosol in a lab setting.^[26] Examples of pathogens classified as "Risk Group 2" in the United States include hepatitis A, B, and C viruses, human immunodeficiency virus (HIV), pathogenic strains of Escherichia coli and Staphylococcus, Salmonella, Plasmodium falciparum, and Toxoplasma gondii.^[26][27] Notably, the European Union departs from the United States and classifies HIV and hepatitis B – G as Risk Group 3 agents best handled at BSL-3.^[28]

Prions, the infectious agents that transmit prion diseases such as vCJD, are typically handled under Biosafety Level 2 or higher.^[24] This is due to the lack of any evidence of aerosol transmission and relatively higher infective dose of prion diseases, though some circumstances (such as handling animal-infective prions in a facility which cares for vulnerable animals) would require BSL-3 conditions.^[24]

Biosafety level 3

Biosafety level 3 is appropriate for work involving microbes which can cause serious and potentially lethal disease via the inhalation route.^[21] This type of work can be done in clinical, diagnostic, teaching, research, or production facilities.^[22] Here, the precautions undertaken in BSL-1 and BSL-2 labs are followed, as well as additional measures including:

• A laboratory-specific biosafety manual must be drafted which details how the laboratory will operate in compliance with all safety requirements.^[21]
• All laboratory personnel are provided medical surveillance and offered relevant immunizations (where available) to reduce the risk of an accidental or unnoticed infection.^[21]
• All procedures involving infectious material must be done within a biological safety cabinet.^[21]
• Laboratory personnel must wear solid-front protective clothing (i.e. gowns that tie in the back). This cannot be worn outside of the laboratory and must be discarded or decontaminated after each use.^[21]

In addition, the facility which houses the BSL-3 laboratory must have certain features to ensure appropriate containment. The entrance to the laboratory must be separated from areas of the building with unrestricted traffic flow.^[21] Additionally, the laboratory must be behind two sets of self-closing doors (to reduce the risk of aerosols escaping).^[22] The construction of the laboratory is such that it can be easily cleaned. Carpets are not permitted, and any seams in the floors, walls, and ceilings are sealed to allow for easy cleaning and decontamination.^[21] Additionally, windows must be sealed, and a ventilation system installed which forces air to flow from the "clean" areas of the lab to the areas where infectious agents are handled.^[21] Air from the laboratory must be filtered before it can be recirculated.^[21]

A 2015 study by USA Today journalists identified more than 200 lab sites in the U.S. that were accredited biosafety levels 3 or 4.^[29] The Proceedings of a Workshop on "Developing Norms for the Provision of Biological Laboratories in Low-Resource Contexts" provides a list of BSL-3 laboratories in those countries.^[30]

Biosafety level 3 is commonly used for research and diagnostic work involving various microbes which can be transmitted by aerosols and/or cause severe disease. These include Francisella tularensis, Mycobacterium tuberculosis, Chlamydia psittaci, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, SARS-CoV-1, MERS-CoV, Coxiella burnetii, Rift Valley fever virus, Rickettsia rickettsii, several species of Brucella, chikungunya, yellow fever virus, West Nile virus, Yersinia pestis,^[27] and SARS-CoV-2.^[31]

Biosafety level 4

Biosafety level 4 (BSL-4) is the highest level of biosafety precautions, and is appropriate for work with agents that could easily be aerosol-transmitted within the laboratory and cause severe to fatal disease in humans for which there are no available vaccines or treatments. BSL-4 laboratories are generally set up to be either cabinet laboratories or protective-suit laboratories. In cabinet laboratories, all work must be done within a class III biosafety cabinet. Materials leaving the cabinet must be decontaminated by passing through an autoclave or a tank of disinfectant. The cabinets themselves are required to have seamless edges to allow for easy cleaning. Additionally, the cabinet and all materials within must be free of sharp edges to reduce the risk of damage to the gloves. In a protective-suit laboratory, all work must be done in a class II biosafety cabinet by personnel wearing a positive pressure suit. To exit the BSL-4 laboratory, personnel must pass through a chemical shower for decontamination, then a room for removing the positive-pressure suit, followed by a personal shower. Entry into the BSL-4 laboratory is restricted to trained and authorized individuals, and all persons entering and exiting the laboratory must be recorded.^[21]

As with BSL-3 laboratories, BSL-4 laboratories must be separated from areas that receive unrestricted traffic. Additionally, airflow is tightly controlled to ensure that air always flows from "clean" areas of the lab to areas where work with infectious agents is being performed. The entrance to the BSL-4 lab must also employ airlocks to minimize the possibility that aerosols from the lab could be removed from the lab. All laboratory waste, including filtered air, water, and trash must also be decontaminated before it can leave the facility.^[21]

Biosafety level 4 laboratories are used for diagnostic work and research on easily transmitted pathogens which can cause fatal disease. These include a number of viruses known to cause viral hemorrhagic fever such as Marburg virus, Ebola virus, Lassa virus, and Crimean-Congo hemorrhagic fever. Other pathogens handled at BSL-4 include Hendra virus, Nipah virus, and some flaviviruses. Additionally, poorly characterized pathogens which appear closely related to dangerous pathogens are often handled at this level until sufficient data are obtained either to confirm continued work at this level, or to permit working with them at a lower level.^[27] This level is also used for work with Variola virus, the causative agent of smallpox, though this work is only performed at the Centers for Disease Control and Prevention in Atlanta, United States, and the State Research Center of Virology and Biotechnology in Koltsovo, Russia.^[32]

• Regular inspection of positive-pressure suits to locate any leaks^[33]
• SPECT machine at BSL-4 imaging facility that separates subjects with pathogens from the machines^[1]
• The circular containment tube separates the patient table in the "hot" zone (pathogen present) from the "cold" zone around this MRI machine.
• Air pressure resistant (APR) door to separate the hot and cold zones
• Working inside a BSL-4 lab with air hoses providing positive air pressure
• Inside a Class III biological safety cabinet with an aerosol control platform
• Effluent decontamination system of a BSL-4 lab of NIAID