Biosafety Levels 1, 2, 3 & 4: What’s the Difference?

Biosafety levels (BSL-1 through BSL-4) are a set of containment classifications defined by the Centers for Disease Control and Prevention (CDC) that govern how laboratories handle biological agents. Each level introduces new requirements, covering personnel practices, protective equipment, and facility design, in addition to the previous level’s requirements. The higher the level, the more dangerous the agents involved and the more stringent the controls required. Autoclaves play a critical role in decontamination at every level, with requirements that escalate significantly at BSL-3.

Key Takeaways:

• The CDC defines four biosafety levels. Each one builds on the one before it, adding layers of containment.
• BSL assignment is determined by a biological risk assessment, not by the agent alone.
• Autoclaves are required for waste decontamination at BSL-1 and above, with specialized pass-through configurations required at BSL-3.
• BSL-3 and BSL-4 labs are federally regulated and require registration with the CDC and/or the United States Department of Agriculture (USDA).
• Two additional classifications — Animal Biosafety Levels (ABSL) and Agricultural Biosafety Levels (BSL-Ag) — address research settings that fall outside the standard four-level framework.

What Are Biosafety Levels?

Biological safety levels — often abbreviated to biosafety levels or BSL — are a series of protections that apply to biological laboratory settings, including autoclave-related activities. Biosafety levels are individual safeguards designed to protect laboratory personnel, as well as the surrounding environment and community.

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These levels, which are ranked from one to four, are designated based on the biohazards (such as agents or organisms) laboratory personnel encounter in the lab. For example, a basic lab setting that specializes in the research of non-lethal agents that pose minimal threat to lab workers and the environment would generally be considered BSL-1, which is the lowest biosafety level. By way of comparison, a research laboratory that specializes in handling potentially deadly infectious agents, such as the Ebola virus, would be designated as a BSL-4 lab — the highest and most stringent biosafety level.

It’s also important to keep in mind that while sometimes used interchangeably, biohazard levels are distinct from biosafety levels. To clarify, biohazard levels are used to classify agents based on the risk they pose to human life. BSLs, on the other hand, focus on the precautions that labs need to take to protect staff from these biohazards. This means that lab personnel can expect to encounter level one biohazards in a BSL-1 environment, level two biohazards in a BSL-2 setting, and so on.

How Are Biosafety Levels Defined?

The CDC sets biosafety levels to indicate what specific controls a laboratory must have in place for the containment of microbes and biological agents. As of 2026, the most up-to-date information can be found in the Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. Each biosafety level builds upon the previous level, thereby creating layer upon layer of constraints and barriers.

Each biosafety level — BSL-1 through BSL-4 — is defined based on the following:

• Risks related to containment
• Severity of infection
• Transmissibility
• Nature of the work conducted within the lab
• Origin of the microbe
• Agent in question
• Route of exposure

More specifically, when determining BSLs for different agents and microbes, the CDC recommends performing a biological assessment:

1. Identify the agent’s hazardous characteristics: Assess the agent’s ability to cause disease, severity, transmission routes, infectious dose, stability, and host range.
2. Identify laboratory procedure hazards: Determine the risks presented by handling techniques, equipment use, aerosol generation, and exposure routes, such as skin contact, ingestion, inhalation, and needlesticks.
3. Determine required biosafety level and controls: Select the appropriate BSL based on risk assessment, factoring in safety precautions, facility safeguards, and regulatory requirements.
4. Select additional risk mitigation measures: Identify supplemental controls beyond the baseline BSL requirements indicated by the risk assessment.
5. Evaluate the risk: Review the overall risk profile with relevant stakeholders, including the principal investigator, institution biosafety professionals, and biosafety committees.

Biosafety levels dictate the types of work practices allowed to take place in a lab setting; they also heavily influence the overall design of a facility and the type of specialized safety equipment in it.

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Biosafety Level 1 (BSL-1)

The lowest of the four biosafety levels, biosafety level 1 (BSL-1) applies to laboratory settings in which personnel work with low-risk microbes that pose little to no threat of infection in healthy adults. Because of this, BSL-1 labs typically do not need to be isolated from surrounding facilities.

Types of Agents Studied

Some examples of the microbes handled in biosafety level-1 labs include:

• Bacillus subtilis (a non-pathogenic soil bacterium commonly used in research)
• Nonpathogenic strains of E. coli
• Saccharomyces cerevisiae (baker’s yeast)

Commonly Used Equipment

As the lowest biosafety level, BSL-1 labs don’t require the use of specialized equipment to carry out research, which is typically conducted on benches. Surfaces should be easy to clean and able to tolerate standard laboratory chemicals.

Safety Protocols

Safety protocols for biosafety level 1 labs — which require only basic microbial practices — include:

• Mechanical pipetting (no mouth pipetting allowed)
• Safe sharps handling
• Avoidance of splashes or aerosols
• Daily decontamination of all work surfaces when work is complete
• Regular handwashing
• Prohibition of food, drink, and smoking materials
• The use of personal protective equipment (PPE), such as goggles, gloves, and a lab coat or gown
• Biohazard signs

BSL-1 labs also require immediate decontamination after spills. Infectious materials should also be decontaminated prior to disposal, generally through the use of an autoclave.

Biosafety Level 2 (BSL-2)

Biosafety level 2 (BSL-2) covers all laboratories that work with agents associated with human diseases — that is, pathogenic or infectious organisms — that pose a moderate health hazard if inhaled, ingested, or exposed to the skin.

BSL-2 labs must follow the same standard microbial practices as BSL-1 labs while also implementing additional precautions to address the higher risk associated with the agents they study. Personnel working in biosafety level 2 laboratories are expected to take even greater care to prevent injuries, such as cuts and other breakage to the skin, as well as ingestion and mucous membrane exposures.

Types of Agents Studied

BSL-2 labs might study the following:

• Dengue
• Equine encephalitis viruses
• Hepatitis A, B, and C
• HIV
• Lyme disease
• Measles
• Mumps
• Salmonella
• Staphylococcus aureus (staph infections)
• Varicella zoster virus (chicken pox)

Commonly Used Equipment

Given the nature of the agents present in BSL-2 labs, workspaces must have access to equipment capable of decontaminating waste, such as an autoclave (or another approved decontamination method). All procedures that could cause infection from aerosols or splashes must be performed within a biological safety cabinet.

Safety Protocols

In addition to the safety protocols established for BSL-1 labs, BSL-2 labs are subject to the following safety controls:

• The use of PPE, including lab coats, gloves, eye protection, and — in some cases — face shields
• Decontamination of infectious materials prior to disposal, generally through the use of an autoclave
• Self-closing, lockable doors
• Access to a sink and eyewash station
• Biohazard warning signs

Access to a biosafety level 2 lab is far more restrictive than to a biosafety level 1 lab. Outside personnel, or those with an increased risk of contamination, are often restricted from entering the area while work is underway.

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Biosafety Level 3 (BSL-3)

Once again building on the two prior biosafety levels, a biosafety level 3 (BSL-3) laboratory typically conducts research into or work on microbes that are either indigenous or exotic and can cause serious or potentially lethal disease through inhalation.

Types of Agents Studied

Common examples of microbes found in BSL-3 labs include:

• Anthrax
• SARS-CoV-2 (COVID-19)
• Hantavirus
• Malaria
• Mycobacterium tuberculosis
• Rift Valley fever
• Rocky Mountain spotted fever
• West Nile virus
• Yellow fever

Commonly Used Equipment

To ensure the safety of laboratory staff, biosafety cabinets, incinerators, autoclaves, and a sustained directional airflow system — to draw air into the laboratory from clean, non-laboratory areas toward potentially contaminated areas — must be present.

The BMBL specifies that BSL-3 labs should have an autoclave available within the laboratory space itself, preferably a pass-through (double door) configuration. Pass-through units are built into the containment wall with one door opening to the contained lab and the other opening to the non-contained corridor, allowing sterilized waste to exit the lab without compromising containment.

Safety Protocols

Microbes found within biosafety level 3 settings are so serious that work is often strictly controlled and registered through the appropriate government agencies. Laboratory personnel are also under medical surveillance and may require immunizations for the microbes they work with. BSL-3 and BSL-4 laboratories that handle CDC/USDA-designated select agents are subject to federal inspection and must maintain ongoing biosafety and biosecurity programs as required by the Select Agent Regulations (42 CFR Part 73 and 9 CFR Part 121).

Common safety controls within a BSL-3 lab include:

• The use of PPE, including goggles and gloves; respirators may also be required
• The use of solid-front wraparound gowns, scrub suits, and/or coveralls is often required
• Access to a hands-free sink and eyewash station available near the exit
• Self-closing set of locking doors with access away from general building corridors

Access to a BSL-3 laboratory is restricted and controlled at all times.

Biosafety Level 4 (BSL-4)

Biosafety level 4 (BSL-4) labs are rare; however, a small number exist in the U.S. and around the world. As the highest level of biological safety, BSL-4 labs work with highly dangerous and exotic microbes. Infections caused by these types of microbes are often fatal and come without treatment or vaccines.

Types of Agents Studied

Agents present in BSL-4 labs might include:

• Bolivian hemorrhagic fever (or other tropical hemorrhagic fevers)
• Ebola virus
• Lassa fever
• Marburg virus

Commonly Used Equipment

Since biosafety level 4 labs deal with the most dangerous agents, they are required to house advanced equipment that enables researchers to safely carry out their studies. All BSL-4 environments must provide access to class III biosafety cabinets and be equipped with dedicated air supply and exhaust air (plus vacuum lines and decontamination systems). Liquid effluents from all lab areas, including sinks, biosafety cabinets, and autoclave chambers, must be decontaminated by heat treatment before being released from the facility.

Safety Protocols

In addition to biosafety level 3 considerations, biosafety level 4 laboratories must follow these safety protocols:

• Personnel must change clothing before entering the facility and shower upon exiting
• All materials must be decontaminated before leaving the facility
• Personnel must wear the PPE from lower BSL levels, as well as a full-body, air-supplied, positive pressure suit

BSL-4 labs are extremely isolated, often located in an isolated and restricted zone of a building or in a separate building entirely. BSL-4 labs also feature a dedicated supply of exhaust air, as well as vacuum lines and decontamination systems.

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Additional Biosafety Levels

In addition to the biosafety levels previously discussed, specialized biosafety classifications exist for certain types of research. Two key examples are Animal Biosafety Levels (ABSL) and Agricultural Biosafety Levels (BSL-Ag), which are designed to address the unique risks associated with animal and agricultural research.

Animal Biosafety Levels

ABSL classifications are used in research involving animals that carry or are infected with potentially hazardous biological agents. These levels — ABSL-1 through ABSL-4 — align with traditional BSLs, but include additional containment measures to protect researchers, prevent cross-contamination, and ensure animal welfare.

• ABSL-1: Used for research involving animals that carry well-characterized agents that aren’t known to cause disease in humans.
• ABSL-2: Research involves animals infected with agents that pose moderate health hazards to humans, such as certain viruses, bacteria, and parasites.
• ABSL-3: Labs work with animals infected with agents that can cause serious or potentially lethal disease in humans as well as agents that spread through aerosol transmission, such as Mycobacterium tuberculosis.
• ABSL-4: Denotes a laboratory environment where researchers work with animals infected with highly dangerous pathogens that cause life-threatening disease, pose a high risk of aerosol transmission, or have an unknown transmission risk.

Agricultural Biosafety Levels

BSL-Ag is a specialized biosafety designation used for facilities handling pathogens that pose a risk to agricultural animals, plants, or ecosystems. Unlike standard BSLs, which focus on protecting human health, BSL-Ag is designed to prevent the spread of infectious agents that could devastate livestock, crops, or wildlife.

Unlike ABSL, which has four levels, BSL-Ag does not prescribe to a tiered system. Instead, BSL-Ag facilities incorporate stringent containment measures tailored to the specific agricultural pathogens being studied. These measures often meet, or exceed, those of BSL-3 labs due to the economic and ecological consequences of an outbreak.

BSL-Ag labs are characterized by the following features:

• Primary containment: Enclosures designed to prevent the escape of pathogens via air, water, or direct contact
• Secondary containment: Dedicated ventilation systems with HEPA filtration, airtight doors, and controlled airflows to prevent external contamination
• Personnel safety: Researchers are required to wear specialized PPE and follow strict decontamination procedures before exiting
• Environmental protection: Waste management and sterilization protocols ensure that no contaminated materials leave the facility

What Are the Differences Between Biosafety Levels?

Knowing the difference between biosafety levels and their corresponding safety requirements is imperative for anyone working with microbes in a lab setting; readers can use the chart below as a quick reference guide.

Biosafety Level	BSL-1	BSL-2	BSL-3	BSL-4	ABSL	BSL-Ag
Description	– No containment – Defined Organisms – Unlikely to cause disease	– Containment – Moderate Risk – Disease of varying severity	– High Containment – Aerosol Transmission – Serious/Potentially lethal disease	– Max Containment – “Exotic”, High-Risk Agents – Life-threatening disease	– Aligns with the traditional BSL levels – Includes additional containment measures	– Designation for facilities handling pathogens posing a risk to agricultural animals, plants, or ecosystems
Sample Organisms	Bacillus subtilis, nonpathogenic strains of E. coli, Saccharomyces cerevisiae	Dengue; equine encephalitis viruses; Hepatitis A, B, and C; HIV; lyme disease; measles; mumps; salmonella; Staphylococcus aureus; Varicella zoster virus	Anthrax, COVID-19, hantavirus, malaria, Mycobacterium tuberculosis, Rift Valley fever, Rocky Mountain spotted fever, West Nile virus, Yellow fever	Bolivian hemorrhagic fever (or other tropical hemorrhagic fevers), Ebola virus, Lassa fever, Marburg virus	Adeno-associated virus, Staphylococcus aureus, Mycobacterium tuberculosis, Ebola virus	Avian influenza virus, classical swine fever virus, eastern equine encephalitis virus, foot and mouth disease
Pathogen Type	Agents present minimal potential hazard to personnel and the environment	Agents associated with human disease and post moderate hazards to personnel and the environment	Indigenous or exotic agents, agents that present a potential for aerosol transmission, and agents causing serious or potentially lethal disease	Dangerous and exotic agents that post a high risk of aerosol-transmitted laboratory infections and life-threatening disease	ABSL-1 through ABSL-4 follow the same pathogen type pathway as BSL-1 through BSL-4	Designed to prevent the spread of infectious agents that could devastate livestock, crops, or wildlife
Commonly Used Equipment	Doesn’t require the use of specialized equipment. Research is typically conducted on benches	Must include access to biological safety cabinets and decontamination equipment, such as incinerators and autoclaves	Biological safety cabinets, incinerators, autoclaves, and a sustained directional airflow system must be present	Must provide access to class III biosafety cabinets and be equipped with dedicated air supply and exhaust air (plus vacuum lines and decontamination systems). Liquid effluents from lab must be decontaminated by heat treatment before release	In addition to the equipment used in BSL labs, ABSL facilities regularly use animal restraint devices, cages, and cage washers.	Equipment used is similar in nature to that of BSL-3 and BSL-4 labs
Autoclave Requirements	None	None	Pass-thru autoclave with bioseal required in laboratory room	Pass-thru autoclave with bioseal required in laboratory room	Autoclave requirements typically align with the corresponding BSL level	Double-door autoclaves engineered with bioseals

Which Biosafety Level Does Your Lab Need?

BSL designation isn’t self-selected, but rather determined through a formal risk assessment process involving a principal investigator, institutional biosafety officer, and institutional biosafety committee. That said, you can use the following general guidance to orient your planning:

• If your lab works with well-characterized, non-pathogenic agents in teaching or basic research settings, your work likely falls under BSL-1. Standard laboratory infrastructure will suffice, and you are not required to have an in-room autoclave (though one should be accessible for waste decontamination).
• If your lab handles common human pathogens, you likely need BSL-2 designation. You’ll need an accessible autoclave (though not necessarily in-room), a biological safety cabinet, self-closing lockable doors, and restricted access.
• If your lab works with aerosol-transmissible agents capable of causing serious or lethal disease, BSL-3 is required. Plan for a pass-through autoclave built into the containment wall, a directional airflow ventilation system, and federal registration if select agents are involved.
• If your lab handles exotic, life-threatening pathogens with no known treatment, BSL-4 applies. These facilities require years of specialized planning, design, and construction.

Labs in the planning or construction phase should determine autoclave placement and configuration during facility design, not after. At BSL-3 or higher, retrofitting a pass-through autoclave into an existing wall is far more complex and costly than accounting for it in the design from the start.

What Makes BSL-3 & BSL-4 Autoclaves Different?

A standard floor-standing or countertop autoclave is generally adequate for waste decontamination in BSL-1 and BSL-2 facilities. At BSL-3 and BSL-4, however, the requirements are substantially different.

BSL-3 Autoclave Requirements

The most important distinction at BSL-3 is the bioseal. BSL-3 autoclaves must be hermetically sealed into the containment wall using a continuously welded stainless-steel frame and durable gasket. This bioseal ensures that the negative air pressure maintained in a BSL-3 lab, which is required to prevent the outward movement of air and pathogens, is not compromised by the autoclave installation. All electrical conduit and plumbing penetrations through the frame must be fully sealed.

A pass-through configuration is the other key requirement. Materials loaded into the autoclave from inside the containment lab exit through a separate door on the non-contained side after sterilization is complete. Door interlock systems prevent both doors from being open simultaneously, maintaining the integrity of the containment barrier at all times.

Additional BSL-3 autoclave requirements include:

• HEPA-filtered exhaust venting: Exhaust air from the autoclave must pass through HEPA filtration before being discharged, preventing any aerosolized pathogens from escaping during the sterilization cycle.
• Effluent decontamination: Liquid condensate from BSL-3 autoclaves must be treated as infectious waste and decontaminated before leaving the containment zone.
• Cycle validation and biological indicators: Per BSL-3 verification requirements, autoclaves must be tested regularly using biological indicators and/or Bowie-Dick tests. Documentation must confirm that the autoclave achieves validated time-temperature parameters for every cycle. Annual preventative maintenance, including door seal inspection, valve testing, and vent filter replacement or testing, is required.

When specifying an autoclave for a BSL-3 facility, verify that the manufacturer offers a true bioseal (not an “air differentiated seal” or “vermin seal,” neither of which meet biocontainment requirements), and confirm that door interlock behavior in the event of a power failure maintains containment rather than defaulting to an open position.

BSL-4 Autoclave Requirements

BSL-4 facilities carry forward all BSL-3 requirements and add a layer of stringency that reflects the extreme risk profile of the agents involved.

Pass-through autoclaves are required in BSL-4 facilities. Additionally, any gas discharged from the autoclave chamber must pass through HEPA filters or be otherwise decontaminated, and autoclave decontamination processes must be designed so that no unfiltered air or steam that’s been exposed to infectious materials can be released to the environment. This is a more absolute requirement than at BSL-3, where the emphasis is on best practice and risk-based engineering.

Effluent decontamination at BSL-4 is also more comprehensive in scope. All liquid waste from the entire facility must be decontaminated through heat treatment before discharge to the sanitary sewer. This process must be validated both physically and biologically, with biological validation performed at least annually, and carefully documented.

BSL-4 autoclaves are also subject to the following compliance requirements per the Federal Select Agent Program:

• Annual verification that system operational parameters (volume, pressure, temperature settings) remain consistent with biologically validated conditions
• Annual certification testing of all associated HEPA filters, including operating vent, pressure relief vent, and chamber effluent/vent filters
• Annual verification that failure detection and emergency communication systems function correctly
• Annual confirmation that appropriate filter media (HEPA or PTFE) is maintained

For additional biosafety level safety tips, we encourage you to download our free biosafety level checklist and microbe guide. Consolidated Sterilizer Systems also manufactures sterilizers for BSL-1, BSL-2, and BSL-3 laboratories — contact us today to learn more about any of our laboratory autoclaves.

Frequently Asked Questions

Q: What is the difference between a biosafety level and a biohazard level?

A: The difference between a biosafety level and a biohazard level is one of classification versus practice. Biohazard levels classify agents by the risk they pose to human health. Biosafety levels define the containment controls that a laboratory must implement when working with those agents. The two systems work together — BSL-1 labs work with Risk Group 1 agents, BSL-2 labs with Risk Group 2 agents, and so on.

Q: What are the four biosafety levels?

A: The four biosafety levels established by the Centers for Disease Control and Prevention (CDC) are:

• BSL-1, the lowest level, which covers work with well-characterized agents that post minimal risk to healthy adults
• BSL-2, which applies to labs working with agents that cause moderate human disease, such as HIV or hepatitis
• BSL-3, which covers work with agents that can cause serious or lethal disease through inhalation, such as Mycobacterium tuberculosis
• BSL-4, the highest level, which is reserved for work with exotic, often fatal pathogens for which no vaccines or treatments exist

Each level builds on the one before it, adding progressively stricter requirements for protective equipment, laboratory design, and waste decontamination.

Q: What is BSL-1 vs. BSL-2?

A: The difference between BSL-1 and BSL2 is the risk level of the agents being studied and the corresponding containment requirements. BSL-1 applies to work with agents not known to cause disease in healthy adults and requires only basic laboratory practices, personal protective equipment, and accessible handwashing facilities.

BSL-2 applies to work with agents that can cause moderate human disease and adds requirements including restricted access during active work, a biological safety cabinet for aerosol-generating activities, self-closing lockable doors, and an accessible autoclave. Personnel in BSL-2 labs also receive specialized training in handling pathogenic agents.

Q: Is there a biosafety level 5?

A: There is no biosafety level 5. The CDC’s framework, as defined in the Biosafety in Microbiological and Biomedical Laboratories (BMBL), formally recognizes four biosafety levels, with BSL-4 representing the maximum level of containment currently defined for biological laboratory work.