Guide to Biosafety Levels 1,2,3, and 4 in Laboratory Settings


biosafety levels for laboratory settings guide

Occupational hazards are present in various work settings. In laboratories, scientists, researchers, and other personnel are commonly exposed to biological hazards. If not handled accordingly and properly, contact with biohazards could result in health problems and environmental degradation. Considering this, it is crucial to understand and learn how to identify these biological hazards to ensure workplace safety and regulatory compliance.

Biosafety levels are categories designed to make hazard identification in the workplace easier. This guide aims to provide an in-depth look at the classifications of biosafety levels, how these hazards are categorized and outline the precautionary measures needed to handle biohazards safely.


What is a Biological Hazard?

What is a bio hazard

A biological hazard, or biohazard, encompasses any organism or byproduct that poses a threat to the health of living organisms, primarily humans. This category includes not only microorganisms but also biological materials that can impact human health indirectly through environmental degradation or contamination.

Laboratories and other workplaces that handle biological materials are exposed to a wide array of bio hazards, each with specific characteristics and associated risks:

  • Bacteria - These are single-celled organisms that can rapidly multiply under suitable conditions. While many bacteria are harmless or beneficial, pathogenic bacteria can cause diseases such as tuberculosis, salmonellosis, and anthrax.
  • Viruses - Much smaller than bacteria, viruses require a living host to multiply. They can cause diseases ranging from the common cold to more severe illnesses like Ebola or COVID-19.
  • Fungi - Including yeasts and molds, fungi can be pathogenic to humans, particularly those with weakened immune systems. They are known to cause infections like ringworm or more serious conditions such as histoplasmosis.
  • Parasites - These are organisms that live on or in a host organism, causing damage or disease. Examples include protozoa like Plasmodium, which causes malaria, and helminths like tapeworms.
  • Prions - Unlike other biohazards, prions are infectious agents composed entirely of protein. They cause rare but fatal neurodegenerative diseases by inducing abnormal folding of normal cellular proteins, particularly in the brain.
  • Biologically Active Substances - This category includes toxins, allergens, and bioactive compounds that can cause health problems upon exposure. Examples are the botulinum toxin produced by Clostridium botulinum and mycotoxins produced by certain molds.

Risks Associated with Biological Hazards

The risks associated with biological hazards depend on a number of factors that characterize the nature of the hazard. This includes the pathogenicity of the organisms, their mode of transmission, the infectious dose, and more.

Pathogenicity refers to the inherent ability of a biological agent to cause disease. Agents are categorized based on their pathogenic potential, which dictates the level of containment required. The higher the level of pathogenicity, the more stringent measures needed to be taken to contain the spread of the biohazard.

Another critical factor in biohazard risk assessment is the mode of transmission of the hazardous agent. Generally, transmission can occur through direct contact, airborne spread, vector-borne transmission, or ingestion. This factor does not only influence the severity of the risk but also the safety measures required against it.

The infectious dose, or the amount of a pathogen required to cause infection, also influences risk severity. Some agents, like the tuberculosis bacterium, require a very small number of organisms to infect a host, while others may require larger amounts to pose a significant risk.

With that in mind, here’s an overview of the most common hazards and risks associated with handling and working with potentially hazardous biological materials:

  • Infectious Diseases: Direct contact with or inhalation of infectious agents can lead to diseases that vary in severity from mild to life-threatening.
  • Toxic Effects: Exposure to toxins produced by biohazards can lead to poisoning, with symptoms ranging from mild irritation to severe organ damage or death.
  • Allergic Reactions: Sensitivity to specific biohazards can result in allergic reactions, which may include symptoms like itching, swelling, respiratory distress, and in severe cases, anaphylaxis.
  • Oncogenic Effects: Some viruses are known to cause cancer after prolonged exposure or chronic infections. For example, human papillomavirus (HPV) is linked to cervical cancer.
  • Environmental Contamination: Improper handling or disposal of biohazards can lead to environmental contamination, which not only affects the ecosystem but can also pose a public health risk if pathogens enter water supplies or food chains.


What Are Biosafety Levels?

Biosafety Levels (BSLs) are a series of protections, rooted in international standards, designed to protect laboratory personnel, as well as the surrounding environment and community, from potentially infectious microbes. These levels are classified from BSL-1 to BSL-4, with each level increasing in safety measures based on the microorganisms' ability to infect and cause illness in a healthy adult human, the severity of the disease, and the nature of the work being conducted.

Biosafety levels consist of several key components, each of which is carefully picked out to protect workers from various biological hazards. The BSL components include standard microbiological practices to serve the foundation of an effective laboratory safety program.

Aside from this, the BSL also lists the appropriate safety equipment depending on the hazard present. Safety tools and equipment, such as personal protective equipment and biological safety cabinets, are critical first-line physical barriers against biohazard exposure. As a secondary barrier, the BSL classification also includes facility design into consideration when creating a safe laboratory facility effectively.

Biosafety Risk Assessment Guide

Conducting a biosafety risk assessment is a critical step in managing biological hazards in the laboratory. It allows employees to gain a comprehensive understanding of their work environment and gauge the level of risk they are in and the appropriate safety and control measures needed in case of emergency. Here’s how to do it:

  1. Identify the Hazards: Begin by determining which biological agents are present in the laboratory and understanding their infectious potential and modes of transmission.
  2. Evaluate the Risks: Assess the likelihood and severity of exposure to these hazards, considering factors such as the volume of work, the concentration of agents, and the susceptibility of the workforce.
  3. Implement Control Measures: Based on the risk level, select and implement appropriate control measures, including engineering controls, administrative controls, and personal protective equipment (PPE).
  4. Review and Update Controls: Regularly review the effectiveness of control measures and update them as necessary, especially when new agents are introduced or when procedures change.

Importance of Biosafety Classifications in Laboratory Safety

Biosafety classifications serve as a foundational framework in the establishment and maintenance of safe laboratory practices. They are an integral part of a broader strategy to ensure health, safety, and environmental protection, facilitating scientific progress while safeguarding against potential risks:

Protecting Laboratory Personnel

The primary objective of biosafety classifications is to protect individuals working within the laboratory from exposure to harmful biological agents. By classifying labs according to the types of biohazards they handle, it ensures that personnel are equipped with the necessary knowledge and tools to work safely. Each increase in biosafety level mandates more stringent safety measures, tailored to the potential dangers posed by the pathogens being studied or utilized.

Preventing Cross-Contamination

Proper biosafety measures prevent cross-contamination between different experiments, as well as within the broader environment. This is essential in preventing the accidental creation of more dangerous strains of microorganisms through genetic recombination or other laboratory errors. Controlled environments help maintain the integrity of scientific data by ensuring that experimental results are due to intended variables rather than environmental contaminants.

Facilitating Compliance and Standardization

Biosafety classifications help standardize safety protocols across different laboratories and institutions worldwide. This standardization is crucial for compliance with national and international safety regulations. It simplifies the enforcement of safety standards and makes it easier for institutions to adopt best practices, as they can reference universally recognized guidelines.

Aiding in Risk Assessment and Management

Biosafety levels provide a clear basis for risk assessment by categorizing microorganisms based on their infectivity, pathogenicity, and the potential hazard they pose to both humans and the environment. This categorization aids laboratory managers in implementing appropriate risk management strategies, including the selection of proper safety equipment, the design of laboratory layouts, and the determination of workflow processes that minimize exposure risks.

Supporting Emergency Preparedness and Response

By adhering to specific biosafety levels, laboratories are better prepared to handle emergencies, such as spills or accidental releases of pathogens. Preparedness plans, including response actions and communication strategies, can be tailored to the specific risks associated with different biosafety levels, ensuring rapid and effective responses that minimize harm and contain potential outbreaks.

Ensuring Public and Environmental Health

Ultimately, the rigorous application of biosafety classifications protects not just laboratory workers but also the general public and the environment. By preventing the escape of dangerous pathogens, these classifications help avoid potentially catastrophic public health crises and environmental damage. This is particularly important in the context of global health, where the accidental release of a pathogen could lead to widespread disease outbreaks.


4 Biosafety Levels

The Centers for Disease Control and Prevention (CDC) along with the National Institutes of Health (NIH) categorize biosafety practices into four levels, each designed to provide appropriate containment and safety measures based on the risk of infection, severity of disease, and transmissibility of the biological agents being handled.

Biosafety Level 1 (BSL-1)

This classification is the lowest tier in the hierarchy of biosafety containment levels and applies to agents that do not typically cause disease in healthy humans and present minimal potential hazards to laboratory workers and the environment. It is suited for work involving well-characterized agents that are not considered to pose a threat of infection when exposed to humans.

  • Laboratory Practices - Requires basic microbiological techniques such as proper handwashing, avoiding eating or drinking in the lab, and safely managing sharps. Personnel must be trained in handling the specific microorganisms used and in the procedures conducted within the lab.
  • Safety Equipment - Unlike higher biosafety levels, BSL-1 does not require the use of biosafety cabinets or other containment equipment for routine manipulations. Basic PPE such as gloves, lab coats, and eye protection is used to prevent skin contact with hazardous materials, protect against splashes, and reduce the likelihood of contamination.
  • Facility Requirements - BSL-1 labs do not require specialized facilities. They can be part of a typical laboratory setting without the need for special containment structures. Biological waste should be decontaminated by autoclave or chemical disinfection before disposal according to institutional and environmental safety regulations.

Biosafety Level 2 (BSL-2)

BSL-2 builds on BSL-1, designed for work involving agents that pose moderate hazards to personnel and the environment. These agents can lead to human disease but generally have effective treatments and pose a limited risk of spread. BSL-2 labs are commonly found in clinical and diagnostic environments, as well as in research institutions where moderate-risk infectious agents are studied.

  • Laboratory Practices - Laboratory access is restricted when work is being conducted. Appropriate biohazard warning signs indicating the organisms in use and the required precautions are posted at the laboratory entrance. Procedures are in place for addressing spills, exposures, or other accidental releases of biohazards, including immediate medical evaluation and treatment.
  • Safety Equipment - Requirement for the use of personal protective equipment (PPE) such as gloves, lab coats, and eye protection to prevent skin and mucous membrane exposure, splashes, or other contact with infectious agents. For procedures that have the potential to create infectious aerosols or splashes, work must be performed within a Class II biological safety cabinet or other equivalent containment equipment.
  • Facility Requirements - Laboratories are designed to contain spills with doors that close automatically and a sink for handwashing and an eyewash station must be available near the laboratory workspace. Furthermore, an autoclave for sterilizing waste and other materials should be available within the building or in a nearby facility to decontaminate microbial cultures and other potentially infectious materials before disposal.

Biosafety Level 3 (BSL-3)

BSL-3 involves handling microbes that are either indigenous or exotic, and that can cause serious respiratory transmission diseases. These pathogens often require special precautions due to the risk of airborne transmission and the potentially severe consequences of infection. BSL-3 laboratories are important in studying pathogens that pose a significant risk of airborne transmission, necessitating stringent controls to protect both the laboratory personnel and the public.

  • Laboratory Practices - Laboratory access is controlled, and all personnel must be registered and trained specifically for BSL-3 conditions. Aside from that procedures that involve handling infectious aerosols or large volumes of infectious agents are conducted under more stringent physical and operational conditions to prevent exposure along with a detailed emergency response protocol.
  • Safety Equipment - PPE at BSL-3 includes respirators, face shields, and gowns, which are used in addition to gloves and lab coats. Personnel must change clothing before entering and shower upon exiting the laboratory. Also, all manipulations of potentially infectious materials take place within BSCs or other enclosed equipment to prevent exposure to infectious aerosols.
  • Facility Requirements - BSL-3 laboratories are physically separated from other laboratory areas and it is maintained at negative pressure relative to surrounding areas to ensure that any aerosols containing pathogens are contained within the lab. All possible exits are sealed to prevent airborne transmission. Autoclaves or other validated decontamination methods are available within the containment zone for sterilizing waste materials.

Biosafety Level 4 (BSL-4)

BSL-4 is the highest level of biological safety and is appropriate for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening diseases for which there are no available vaccines or treatments. BSL-4 labs are vital for global health security as they provide the infrastructure for studying and developing interventions against some of the world’s most deadly diseases. Research conducted in these facilities is crucial for vaccine development, therapeutic research, and understanding the transmission mechanisms of high-risk pathogens.

  • Laboratory Practices - Access to BSL-4 labs is strictly controlled, and limited to personnel who have specific authorization and training. Furthermore, all procedures involving the manipulation of infectious materials are conducted within a Class III biological safety cabinet or by wearing a full-body, air-supplied, positive-pressure suit.
  • Safety Equipment - Requirement to wear full-body suits equipped with a dedicated air supply to prevent contamination from the external environment. Also, personnel must shower and undergo a full body decontamination process both before entering and upon leaving the laboratory.
  • Facility Requirements - BSL-4 laboratories are typically isolated from other buildings or are in a clearly demarcated and highly controlled area within a larger building. The facility is designed to be completely sealed off from the outside environment. It also features negative pressure to prevent accidental leaks. All materials exiting the laboratory, including all waste, must be autoclaved or otherwise decontaminated before removal from the facility.

Biosafety Levels Chart

The biosafety chart is a tool used to visualize the different biosafety levels and summarize the escalation in safety measures and facility requirements as the biosafety levels increase, reflecting the higher risks associated with more dangerous pathogens.

biosafety level chart

Standards and Regulations on Biosafety

Several key standards and regulations govern biosafety protocols in laboratory settings. These help ensure that biological hazards are properly managed to protect both laboratory personnel and the public. Here is a summary of the standards and regulations on bio and laboratory safety:
Body Standard/Regulation Description
OSHA Bloodborne Pathogens Standard (29 CFR 1910.1030) Protects workers from health hazards presented by bloodborne pathogens. Requires exposure control plans and proper PPE usage.
OSHA General Duty Clause (Section 5(a)(1) of the OSH Act) Requires employers to provide a safe workplace by removing recognized hazards, including biological hazards, that can cause serious harm or death.
CDC & NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL) A comprehensive manual detailing biosafety levels, practices, equipment, and facility requirements. Widely adopted as the primary guide for biosafety in labs.
NIH NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules Guidelines for safe handling of genetically engineered organisms and viruses, to prevent misuse and ensure safety in genetic research.
EPA Resource Conservation and Recovery Act (RCRA) Governs the disposal of biohazardous and other hazardous wastes, ensuring environmentally safe management and disposal practices.


Laboratory Safety Strategies Against Biological Hazards

Ensuring safety in laboratories dealing with biological hazards requires a comprehensive approach that incorporates multiple control measures. These measures are designed to protect laboratory personnel, the environment, and the public from potential exposure to harmful biological agents.

Control Measures

Implementing effective control measures against biohazards involves a combination of engineering controls, administrative controls, and personal protective equipment (PPE). Engineering controls aim to remove the hazard at the source, before it comes into contact with the worker. These are the most preferred and effective in the hierarchy of controls.

On the other hand, Administrative controls involve changing how or when people work. These controls are designed to minimize the exposure to hazards through policies and procedures. Lastly, PPE serves as the last line of defense to protect individuals from exposure to hazardous agents. It is used in conjunction with other control measures to ensure comprehensive protection.

Here is a brief overview of the key control measures used for laboratory safety:

  • Biological Safety Cabinets (BSCs): Enclosures that prevent airborne contaminants from escaping into the laboratory environment. They are essential for work with toxic vapors or particulate matter that might be infectious.
  • HVAC Systems with HEPA Filters: High-efficiency particulate air (HEPA) filters in ventilation systems ensure that any air exiting the lab is free of infectious agents.
  • Autoclaves: Used for sterilizing equipment and deactivating biohazards before disposal.
  • Pass-Through Boxes: Sealed containers that allow materials to be passed into and out of containment areas without direct exposure.
  • Training Programs: Regular training on the proper handling of biohazards and response procedures for potential exposure or spills.
  • Access Restrictions: Limiting laboratory access to trained and authorized personnel only.
  • Standard Operating Procedures (SOPs): Detailed procedures for safely working with biohazards, which include specific instructions on using, storing, and disposing of biological agents safely.
  • Emergency Response Plans: Protocols that outline steps to be taken in the event of an accidental spill or exposure, including communication strategies and remediation measures.
  • Gloves: Protect hands from exposure to hazardous materials and prevent the transfer of contaminants.
  • Gowns or Lab Coats: Provide body protection from spills and splashes of infectious or toxic substances.
  • Respirators or Masks: Essential for preventing inhalation of infectious aerosols, particularly in BSL-3 and BSL-4 settings.
  • Face Shields or Goggles: Protect the eyes and face from splashes or sprays of hazardous substances.

By integrating these engineering, administrative, and personal protective measures, laboratories can create a safer environment that minimizes the risks associated with biological hazards. However, the successful implementation of these control measures requires regular maintenance, compliance monitoring, and continuous improvement to maintain the highest level of biosafety.

Response to Biohazard Exposure

In case of biohazard exposure, it is essential to act promptly and effectively to minimize health risks and contain potential contamination.

Immediate Action

Much like any emergency, one of the first steps is to contain the area as quickly as possible. This will prevent further exposure and prevent the spread of the biohazard. Determine the extent of the exposure, including the type of biohazard, the amount, and the route of exposure. Immediately inform laboratory supervisors and safety officers. Quick communication is essential for effective response and to initiate emergency protocols.


In case of hazard contact or exposure, it is particularly important to wash the affected area with soap and water thoroughly for at least 15 minutes. Avoid scrubbing harshly, which can cause breaks in the skin and further entry of the biohazard. If the eyes are exposed, rinse them at an eyewash station for at least 15 minutes, keeping eyelids open to ensure thorough cleansing.

Medical Evaluation

Arrange for immediate medical evaluation, even if the exposure seems minor. Some symptoms from biohazard exposure can be delayed. Provide the healthcare provider with detailed information about the biohazard, the circumstances of the exposure, and any safety data sheets (SDS) available.

Report and Review

Record all details pertaining to the exposure incident for future reference and to comply with health and safety regulations. To prevent repeating any mistakes, analyze the incident to identify any shortcomings in current safety protocols and to improve future response strategies.


FAQ on Biosafety Levels

What is the principle of biosafety levels?

The principle of biosafety levels (BSLs) is to provide a series of containment levels that correspond to the increasing risk of exposure to infectious agents. Each level defines specific laboratory practices, safety equipment, and facility design requirements to safely handle pathogens and protect laboratory personnel and the environment.

What is the difference between biosafety and biosecurity?

Biosafety involves the implementation of effective laboratory practices, safety equipment, and facility designs to protect people and the environment from exposure to infectious or hazardous biological agents. Biosecurity refers to the protection, control, and accountability measures implemented to prevent the loss, theft, misuse, diversion, or intentional release of pathogens and toxins.

What does biohazard mean in laboratory?

In a laboratory setting, a biohazard refers to any biological agent (such as a virus, bacterium, prion, or fungus) or condition that has the potential to adversely affect human health. Laboratories label biohazards to signify the presence of these risk factors and to ensure that strict containment measures are adhered to.

What are the biohazard diseases?

Biohazard diseases are illnesses that can be caused by exposure to biological hazards. These include a wide range of infections such as tuberculosis, HIV/AIDS, hepatitis, influenza, Ebola, and many other bacterial, viral, fungal, or prion diseases that pose significant health risks.

What is the treatment of biohazard?

The treatment of biohazard exposures depends on the type of biohazard involved. Generally, it includes immediate measures such as decontamination, medical evaluation, and possibly specific medical treatments like antibiotics for bacterial exposures or antivirals for viral exposures. Preventative measures, such as vaccinations or prophylactic treatments, may also be administered depending on the risk assessment and the nature of the biohazard.

The material provided in this article is for general information purposes only. It is not intended to replace professional/legal advice or substitute government regulations, industry standards, or other requirements specific to any business/activity. While we made sure to provide accurate and reliable information, we make no representation that the details or sources are up-to-date, complete or remain available. Readers should consult with an industrial safety expert, qualified professional, or attorney for any specific concerns and questions.


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Author: Herbert Post

Born in the Philadelphia area and raised in Houston by a family who was predominately employed in heavy manufacturing. Herb took a liking to factory processes and later safety compliance where he has spent the last 13 years facilitating best practices and teaching updated regulations. He is married with two children and a St Bernard named Jose. Herb is a self-described compliance geek. When he isn’t studying safety reports and regulatory interpretations he enjoys racquetball and watching his favorite football team, the Dallas Cowboys.