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Biological Safety Levels 1, 2, 3, & 4

Each biological safety level builds on the previous level, adding constraints and barriers based on the agents or organisms in which the research or work is being conducted. 

Vince McLeod, CIH

Vince McLeod is an American Board of Industrial Hygiene-certified industrial hygienist and the senior industrial hygienist with Ascend Environmental + Health Hygiene LLC in Winter Garden, Florida. He has more...

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Jonathan Klane, M.S.Ed., CIH, CSP, CHMM, CIT

Jonathan Klane, M.S.Ed., CIH, CSP, CHMM, CIT, is senior safety editor for Lab Manager. His EHS and risk career spans more than three decades in various roles as a...

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Biological safety level basics

A very specialized research laboratory that deals with infectious agents is the biosafety lab. Whether performing research or production activities, when working with infectious materials, organisms or perhaps even laboratory animals, the proper degree of protection is of utmost importance. Protection for laboratory personnel, the environment and the local community must be considered and ensured. The protections required by these types of activities are defined as biosafety levels. Biological safety levels are ranked from one to four and are selected based on the agents or organisms on which the research or work is being conducted. Each biological safety level builds on the previous level, adding constraints and barriers. 

The Centers for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH) are our main sources for biological safety information for infectious agents. The publication Biosafety in Microbiological and Biomedical Laboratories is a principal reference and the resource for much of the information presented in this month’s column. As an introduction, we summarize what the different biosafety levels encompass in terms of the typical biological agents used, safe work practices, specialized safety equipment (primary barriers) and facility design (secondary barriers).

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The four biosafety levels were developed to protect against a world of select agents. These agents include bacteria, fungi, parasites, prions, rickettsial agents and viruses, the latter being probably the largest and most important group. In many instances the work or research involves vertebrate animals, everything from mice to cattle. When vertebrates are involved, additional precautions and safety requirements are necessary. Using the most infectious agents also means extensive security measures are in place, not only because of their virulence but also because of their potential for use in bioterrorism.

Biosafety levels

Biosafety Level 1 - Infectious Agents

Level 1

Biological safety level one, the lowest level, applies to work with agents that usually pose a minimal potential threat to laboratory workers and the environment and do not consistently cause disease in healthy adults. Research with these agents is generally performed on standard open laboratory benches without the use of special containment equipment. BSL 1 labs are not usually isolated from the general building. Training on the specific procedures is given to the lab personnel, who are supervised by a trained microbiologist or scientist.

Standard microbiology practices are usually enough to protect laboratory workers and other employees in the building. These include mechanical pipetting only (no mouth pipetting allowed), safe sharps handling, avoidance of splashes or aerosols, and decontamination of all work surfaces when work is complete, e.g., daily. Decontamination of spills is done immediately, and all potentially infectious materials are decontaminated prior to disposal, generally by autoclaving. Standard microbiological practices also require attention to personal hygiene, i.e., hand washing and a prohibition on eating, drinking or smoking in the lab. Normal laboratory personal protective equipment is generally worn, consisting of eye protection, gloves and a lab coat or gown. Biohazard signs are posted and access to the lab is limited whenever infectious agents are present.

Biosafety Level 2: Infectious Agents

Level 2

Biological safety level two would cover work with agents associated with human disease, in other words, pathogenic or infectious organisms posing a moderate hazard. Examples are the equine encephalitis viruses and HIV when performing routine diagnostic procedures or work with clinical specimens. Therefore, because of their potential to cause human disease, great care is used to prevent percutaneous injury (needlesticks, cuts and other breaches of the skin), ingestion and mucous membrane exposures in addition to the standard microbiological practices of BSL 1. Contaminated sharps are handled with extreme caution. Use of disposable syringe-needle units and appropriate puncture-resistant sharps containers is mandatory. Direct handling of broken glassware is prohibited, and decontamination of all sharps prior to disposal is standard practice. The laboratory’s written biosafety manual details any needed immunizations (e.g., hepatitis B vaccine or TB skin testing) and whether serum banking is required for at-risk lab personnel. Access to the lab is more controlled than for BSL 1 facilities. Immunocompromised, immunosuppressed, and other persons with increased risk for infection may be denied admittance at the discretion of the laboratory director.

BSL 2 labs must also provide the next level of barriers, i.e., specialty safety equipment and facilities. Preferably, this is a Class II biosafety cabinet or equivalent containment device for work with agents and an autoclave or other suitable method for decontamination within the lab. A readily available eyewash station is needed. Self-closing lockable doors and biohazard warning signs are also required at all access points.

Biosafety Level 3: Infectious Agents

Level 3

Yellow fever, St. Louis encephalitis and West Nile virus are examples of agents requiring biological safety level 3 practices and containment. Work with these agents is strictly controlled and must be registered with all appropriate government agencies. These are indigenous or exotic agents that may cause serious or lethal disease via aerosol transmission, i.e., simple inhalation of particles or droplets. The pathogenicity and communicability of these agents dictates the next level of protective procedures and barriers. Add to all the BSL 2 practices and equipment even more stringent access control and decontamination of all wastes, including lab clothing before laundering, within the lab facility. Baseline serum samples are collected from all lab and other at-risk personnel as appropriate.

More protective primary barriers are used in BSL 3 laboratories, including solid-front wraparound gowns, scrub suits or coveralls made of materials such as Tyvek® and respirators as necessary. Facility design should incorporate self-closing double-door access separated from general building corridors. The ventilation must provide ducted, directional airflow by drawing air into the lab from clean areas and with no recirculation.

Biosafety Level 4: Infectious Agents

Level 4

Agents requiring biological safety level 4 facilities and practices are extremely dangerous and pose a high risk of life-threatening disease. Examples are the Ebola virus, the Lassa virus, and any agent with unknown risks of pathogenicity and transmission. These facilities provide the maximum protection and containment. To the BSL 3 practices, we add requirements for complete clothing change before entry, a shower on exit and decontamination of all materials prior to leaving the facility.

The BSL 4 laboratory should contain a Class III biological safety cabinet but may use a Class I or II BSC in combination with a positive-pressure, air-supplied full-body suit. Usually, BSL 4 laboratories are in separate buildings or a totally isolated zone with dedicated supply and exhaust ventilation. Exhaust streams are filtered through high-efficiency particulate air (HEPA) filters, depending on the agents used.

We have touched on only the main issues and differences between BSL 1, 2, 3, and 4 laboratories. There are many other concerns and requirements addressed in the CDC manual, such as impervious, easy-to-clean surfaces; insect and rodent control; and total barrier sealing of all wall, floor, and ceiling penetrations. Our goal was to introduce you to the different levels of biological safety practices and facility design considerations. Hopefully, you now have the knowledge to decide whether you should open that door or not.

Biohazardous waste management

Biohazardous wastes are different from chemicals or radioactive waste. Chemicals can react badly, and rad wastes continue to radiate. But our major focus for bio-waste is preventing transmission or infection. So, how can you best control infectious substance waste? Disinfect, decontaminate, sterilize, or incinerate.

Alcohol and bleach are used daily to disinfect surfaces in labs. Other potent chemicals (e.g., aldehydes or ethylene oxide) are available to sterilize bio-wastes. Heat is a common method mostly via autoclaves and similar enclosed systems both in the lab and elsewhere. During the COVID-19 pandemic, vaporized hydrogen peroxide (H2O2) was used to decontaminate N95 respirators in short supply. UV lights are ubiquitous within certain controls. And of course, there’s always incineration, though it comes with environmental woes.

Containers are needed for most bio-wastes including bags, sharps containers, and lines boxes or bins. Often the biggest challenge is managing safe sharps use and disposal (e.g., syringes, needles, razor blades, slides, cover slips, broken glass).

Biological safety training

For biosafety training, you can follow some of the key adult learning principles (ALPs). Make it:

  • Applicable, e.g., just BSL1 and BSL2
  • Learner-focused, e.g., answer their questions and existing expertise
  • Immediate needs, e.g., they need to work with a new pathogen
  • Problem-solving, e.g., how to manage risks of a certain experiment
  • Help set agendas, e.g., suggesting or requesting topics

As applicable to the learners’ context, typical topics are:

  • Biosafety levels 1-4 (including 2+ and 3+)
  • Risk groups
  • Biohazards in lab + being worked with
  • BSCs versus clean benches
  • PPE
  • Universal precautions + bloodborne pathogens (BBPs)
  • Processes
  • Work practices – dos + don’ts.

Lastly, focus on all three KSAs (knowledge, skills, and attitudes). Information helps us understand hazards. Skills allow us to perform necessary actions and hands-on manipulations. But it’s our attitudes that drive our behaviors and help achieve a culture of safety.


  1. Biosafety in Microbiological and Biomedical Laboratories, 5th edition, Centers for Disease Control and Prevention and National Institutes of Health, February 2007.
  2. Biennial Review of the Lists of Select Agents and Toxins, National Select Agent Registry, CDC. Atlanta, GA. 2010.