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An Overview of Biosafety Levels

Biosafety levels refer to both physical and administrative barriers that combine to protect personnel and the environment from microbial agents. Proper management of biosafety levels is an important component of a laboratory information management system

Many scientists take the designated biosafety levels of their labs for granted. In reality, the biosafety of a lab is something that needs to be proactively maintained by complying with applicable regulations and policies. Here, an overview is presented on what the different biosafety levels mean, and what the facilities and work practices required for each level contribute to protecting personnel from infection, and the environment from accidental releases of infectious agents or genetically modified organisms. 

What are biosafety levels?

In the United States, biological research laboratories are generally designated a biosafety level of either 1, 2, 3, or 4 (BSL-1, BSL-2, BSL-3, BSL-4). In Canada, the labs are designated by containment level, or pathogen protection level in Europe. For the sake of simplicity, this article will focus on the U.S. Centers for Disease Control and Prevention (CDC) designated BSLs-1 through 4.


What dictates biosafety levels for research?

Biological safety levels , as described in the CDC’s Biosafety in Microbiology and Biomedical Laboratories (BMBL) are determined based on a combination of the facility design and capabilities, engineering controls (i.e. chemical fume hoods or biosafety cabinets that physically separate laboratorians from a hazard), administrative controls (or standard operating procedures), and personal protective equipment (PPE). When implemented in agent- and task-appropriate combinations, this is what makes up a BSL.

The appropriate level of controls are determined based on a risk assessment performed by the lab director in collaboration with biosafety professionals. This risk assessment will factor in the biological agents being studied, the type of manipulation of the organism being performed, the amount of an agent being produced, the procedures that will be performed, the route and risk of transmission along with the potential consequences of personnel and environmental exposure to the agent, and any preventative or prophylactic measures that may be available in the event of an exposure. 

Microbial agents are categorized by the CDC and National Institutes of Health (NIH), or World Health Organization (WHO) based on risk groups (RG1-4), which generally correlate with biosafety levels of the labs in which they are studied. A RG1 organism is defined  by the CDC as a microbe that is not known to be pathogenic to humans, or by the WHO as an agent that is unlikely to cause disease in humans or animals. 


biosafety levels

Biosafety levels are defined by how much risk is involved in working with particular pathogens (©2020 by Troy Sutton) 

The next group, RG2, consists of agents that are capable of causing disease in healthy human adults that are typically not serious. As per the WHO, these agents are capable of causing mild disease in humans or animals, and pose a low hazard risk to laboratory workers, livestock, and the environment. 

Risk group 3 agents would be capable of causing serious disease in healthy humans, but for which there are therapeutic interventions available (CDC) or those that can cause serious disease in humans or animals, but are not readily transmissible (WHO). 

Finally, RG4 agents are those that likely cause fatal human diseases in healthy adults, and for which therapeutic or prophylactic interventions are not available (CDC). The WHO defines RG4 agents as an agent that causes potentially lethal diseases in humans and animals, that is readily transmissible, either directly or indirectly, and for which therapeutic or preventative measures are not readily available (high individual, high community risk).  

Basics of Biolab Design

Best practices for laboratory design are spelled out by the NIH in the Design Requirements Manual. Very briefly, the different biosafety levels have a number of features common to all laboratories. This is due to the fact that the control measures are additive. For example, a BSL-2 lab will have all control measures from a BSL-1 lab, but with additional requirements. One such example is the requirement for non-porous surfaces. This allows for effective cleaning in the event of a biological or chemical spill. The non-porous surface requirement extends to lab benches, walls, floors, and chairs. One question that often comes up is the acceptability of wood surfaces (i.e. cabinets). In short, as long as the wood is sealed with paint or a lacquer type coating, it would be considered non-porous and meets the standards. 

For BSL-1 labs, while air handling should be dedicated to the lab, it is often multiple pass, meaning that the air from the lab is re-circulated, and may be shared with other rooms, such as office spaces. Conversely, in BSL-2 labs, BSL-3 laboratories, and BSL-4 laboratories, it is required that air handling systems be dedicated to lab facilities, and it must be set up as a single-pass system. This means that outdoor air is brought into the system, is conditioned, circulated through the lab, and is exhausted to the exterior of the building. In combination with a negative air balance relative to public hallways (air flows into the lab), this ensures that any pathogens are contained within the laboratory, minimizing the risk of release. 

Another common requirement is that a hand washing sink be present, for obvious reasons. Many laboratorians seem to believe that handwashing is only necessary after removing gloves or leaving the lab at the end of a work session. However, the CDC requires at BSL-2 and above, that hands are washed before exiting a lab, regardless of whether or not anything was touched. The mere act of entering a lab necessitates hand washing. 

Finally, as containment level increases from BSL-1 to 4, security requirements compound significantly. While BSL-1 requires a minimal level of security, such as authorization from the laboratory director, BSL-2 labs have the additional requirement of restricting access from public areas. This is typically accomplished by maintaining locked doors on the labs. As this is a very large subject, each BSL will be discussed in dedicated posts in the near future. 

Personal Protective Equipment

As is the case for facility design, PPE requirements compound with increasing biosafety level as well. In a BSL-1 lab, gloves are required, and the use of lab coats and eye protection are strongly encouraged. However, these both are mandatory in BSL-2 labs. For work being conducted at BSL-3, respiratory protection is also required. 


Face shields are part of the personal protective equipment of laboratory workers handling coronaviruses.

This can be accomplished through the use of an N-95 filtering facepiece respirator, for which the user is required to successfully pass either a qualitative or quantitative fit test to ensure a tight seal and adequate protection. However, many working in BSL-3 labs opt for powered air purifying respirators, or PAPRs. These are hoods worn by the user that are connected to a belt-mounted fan with a filter. This creates a positive pressure system that prevents access of pathogenic microbes to the user’s face and head, providing a high level of protection. These units, while noisy, tend to be more comfortable as they do not add to the effort required for respiration. On the extreme end, BSL-4 labs require the use of full-body positive pressure suits to completely isolate the worker from the agents they are handling. 


Worker in a BSL-3 laboratory. 

Additional Containment Levels and Facilities

Although the 4 biosafety levels introduced above apply to standard laboratories, the containment requirements and practices are not always practical or feasible. Thus, additional and specific biosafety levels have been created. 

For animal research, there are 4 biosafety levels (ABSL-1 through 4). These levels not only dictate PPE and engineering controls to be used by the researchers, but they also outline housing, bedding and waste handling, and often wellness check requirements based on the microbial agent being studied. While the CDC focuses primarily on risks to humans, an organism being studied at BSL-1 could conceivably require ABSL-2 or even ABSL-3 containment if the agent poses a risk to the animal colonies. Similarly, the U.S. Department of Agriculture has a designated BSL-3Ag level of containment. This is required for higher consequence pathogens of livestock, primarily those that are exotic, also referred to as foreign animal diseases. In this situation, where an animal cannot be handled in a biological safety cabinet or isolation cage, stringent facility design, PPE requirements, and SOP’s would be implemented to ensure that the agents are not released to the environment.

Similar to the animal containment levels described above, there is also a series of insect, or arthropod containment levels (ACL-1 through 4). In these facilities, particularly at ACL-1, insects frequently escape their primary containment vessels (i.e. housing flasks). To combat this potential release, traps must be placed strategically throughout the room to capture any escaped insects. In ACL-3 labs, researchers may study arboviral agents, such as Dengue virus or Zika virus in their natural mosquito host. 


Aedes aegypti mosquito, the vector of several viral diseases

However, ACL-3 facilities may also be required for handling and studying exotic mosquito species that would be capable of surviving in the local environment should they escape containment.

For research involving plants and plant pathogens, BSL-1P through 4P designations are put in place. These typically refer to greenhouses or growth chambers where the plants may be housed. Not only are these containment levels put into place for plant pathogens, but also for transgenic plants and noxious weeds; essentially plants that would have significant impacts on the local ecosystem or agriculture should they be released and become established. A detailed explanation of these facilities can be found in the Practical Guide to Plant Containment.

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