Biological Safety Levels

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A. Risk Groups

Biological agents can be categorized in Risk Groups (RG) based on their relative risk. Risk groups are based on the following factors:
•   Pathogenicity of the organism
•   Mode of transmission and host range
•   Availability of effective preventive measures, such as vaccines
•   Availability of effective treatment, including antibiotics and antiviral medication.
  • RG1: are not associated with disease in healthy adult humans or animals
    Examples: E.coli K12 Host-Vector Systems, Adeno-associated virus, B. subtilis
  • RG2: associated with disease which is rarely serious and for which preventative or therapeutics is often available
    Examples: S. aureus, Hepatitis B virus, Cryptococcus neoformans
  • RG3: associated with serious or lethal human disease for which preventative or therapeutics may be available
    Examples:  Mycobacterium tuberculosis, Yellow Fever Virus
  • RG4: associated with lethal human disease for which preventative or therapeutics are not readily available
    Examples: Ebola virus, Herpesvirus simaie

B. Biological Safety Levels

While Risk Groups only take into account the organism or virus, Biosafety Levels prescribe procedures, equipment and facilities and levels of containment required to provide protection to the worker, occupants of building, the environment and local community.

Laboratory Practice and Technique

The most important element of containment is strict adherence to standard microbiological practices and techniques. Persons working with infectious agents or infected materials must be aware of potential hazards, and must be trained and proficient in the practices and techniques required for handling such material safely. The PI or laboratory supervisor is responsible for providing or arranging for appropriate training of personnel.

Safety Equipment (Primary Barriers)

Safety equipment includes items such as biological safety cabinets and personal protective equipment designed to eliminate or minimize exposures to hazardous biological materials. The biological safety cabinet (BSC) is the principal device used to provide containment of infectious splashes or aerosols generated by many microbiological procedures.

Safety equipment may also include items for personal protection such as personal protective clothing, respirators, face shields, safety glasses or goggles. In some situations, personal protective clothing may form the primary barrier between personnel and the infectious materials.

Facility Design (Secondary Barriers)

The design of a facility is important in providing a barrier to protect those working inside and outside the laboratory and to protect people or animals in the community from infectious agents which may be accidentally released from the laboratory. Facilities must be commensurate with the laboratory's function and the recommended biosafety level for the agent being manipulated.

The secondary barrier(s) needed will depend on the risk of transmission of specific agents.

Biosafety Level 1 (BSL-1)

BSL-1 applies to the basic level of containment and essentially represents good microbiological practice with no special primary or secondary barriers required. It is appropriate for work with defined and characterized strains of viable microorganisms not known to consistently cause disease in healthy adult humans. This includes organisms such as Bacillus subtilis, Vibrio harveyi, Saccharomyces cerevisiae or laboratory strains of E. coli.

Laboratory Design

  • A dedicated handwashing sink is available in the lab.
  • Doors can be locked to prevent access.
  • All surfaces, including floors, chairs and benchtops are easily cleanable.
  • Benchtops are impervious to water and resistant to heat, organic solvents, acids, alkalis and other chemicals.
  • Windows in labs that open to the exterior are fitted with screens.

Laboratory Procedures

  • Eating, drinking, handling contact lenses, applying cosmetics and storing food for human consumption is not permitted in laboratory areas. Food must be stored outside of the laboratory area in refrigerators designated for this purpose.
  • Wear appropriate PPE, including disposable gloves and lab coat. Add eye protection if a splash hazard exists.
  • Wash hands in a dedicated sink after working with potentially hazardous materials and before leaving the laboratory.
  • Never mouth pipette; use mechanical pipetting devices.
  • Limit access by janitorial and maintenance staff to lab spaces when handling BSL 1 materials.

Personal Protective Equipment

  • Wear disposable exam gloves, preferably nitrile, to protect hands from exposure to hazardous materials.
  • Change gloves when contaminated or the integrity is compromised.
  • Never wash or reuse disposable gloves.
  • Wear cotton laboratory coats, to prevent contamination of clothing. Do not launder lab coats at home.


  • Biosafety cabinets and containment equipment are not required for manipulation of BSL 1 materials.

Biosafety Level 2 (BSL-2)

BSL-2 is appropriate for work involving agents that pose moderate hazards to personnel and the environment and where vaccines or post-exposure treatment is available.

Many of the viral agents used in campus research, such as adenovirus, cytomegalovirus, and other herpes viruses fall within the BSL-2 level of work. Microorganisms assigned to this containment level include salmonella spp., V. cholerae and S. aureus.

In addition to BSL-1 conditions, this level of work also requires the following:

Laboratory Design

  • Airflow is directional, single pass.
  • Lab work area is separate from public and eating areas
  • Access is restricted to authorized personnel only

Laboratory Procedures

  • Conduct procedures with a potential to create aerosols in a biosafety cabinet (BSC).
  • Decontaminate work surfaces with an appropriate disinfectant at completion of tasks and after any spill or splash.


  • Lab staff and students who work in BSL-2 labs must complete Introduction to Biosafety training. Additionally, upon joining a lab, staff should be oriented to:
  • Procedures to follow after a significant exposure
  • PI-approved use of sharps when working with infectious agents
  • Review of personal hygiene procedures (hand hygiene, permissible areas for eating/drinking)
  • Tasks that should be conducted in a biosafety cabinet
  • Proper use of a biosafety cabinet.
  • Operation and use of laboratory equipment and decontamination methods
  • Medical waste handling
  • Biological material spill procedures


  • Work in a biosafety cabinet when performing tasks that can create aerosols.
  • Autoclaves are available and should be used to decontaminate all potentially infectious waste.
  • Eyewashes are available within a 10 second walk from location of research.
  • Safety centrifuge cups/safety blenders are designed to prevent aerosols from being released during centrifugation or homogenization of infectious materials.

Personal Protective Equipment

  • Gloves, disposable nitrile
  • Cotton lab coat
  • Face shield
  • Safety glasses or safety goggles
  • Under certain circumstances, additional PPE may be required:

    • Sleeve covers
    • Shoe covers
    • Disposable gowns
    • Respirators (Certain respirators are designed to prevent inhalation of aerosols.  EHS staff must conduct a risk assessment and all persons required to wear a respiratory must pass a medical exam and participate in training prior to use of a respirator.)
    • If a BSC cannot be used, PPE provides the primary barrier to prevent exposure to infectious materials.


  • Entrance to a lab where BSL-2 materials are handled must be posted with the international biohazard symbol.  Request room door signs from EHS, Biosafety.
  • Equipment that is used in conjunction with biohazardous materials must be labeled with the universal biohazard symbol.

BSL3 and BSL4

BSL3 and BSL4 apply to work with exotic agents of increasingly greater potential for causing serious human illness or death. No work at the BSL3 or 4 level is currently being done, and facilities that would meet the requirements of these biosafety levels are not available at Princeton.

A good summary of requirements at each laboratory biosafety level can be found at