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Biological Safety Levels

A biosafety level is the level of the biocontainment precautions required to isolate dangerous biological agents in an enclosed facility. The levels of containment range from the lowest biosafety level 1 to the highest at level 4. In the United States, the Centers for Disease Control and Prevention (CDC) have specified these levels. In the European Union, the same biosafety levels are defined in a directive. Sabanci University is following the same directive in accordance with Turkish biological safety regulation.

The term "containment" is used in describing safe methods for managing infectious materials in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory users, other people, and the outside environment to potentially hazardous agents.

Biocontainment can be classified by the relative danger to the surrounding environment as biological safety levels (BSL). As of 2006, there are four safety levels. These are called BSL1 through BSL4.

Biosafety Level 1

This level is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory users and the environment (CDC, 1997).  It includes several kinds of bacteria and viruses including canine hepatitis, Escherichia coli, varicella (chicken pox), as well as some cell cultures and non-infectious bacteria. At this level precautions against the biohazardous materials in question are minimal, most likely involving gloves and some sort of facial protection. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open benchtops using standard microbiological practices. Usually, contaminated materials are left in open (but separately indicated) rubbish receptacles. Decontamination procedures for this level are similar in most respects to modern precautions against everyday microorganisms (i.e., washing one's hands with anti-bacterial soap, washing all exposed surfaces of the lab with disinfectants, etc.). In a lab environment all materials used for cell and/or bacteria cultures are decontaminated via autoclave.  Laboratory users have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science.

Biosafety Level 2

This level is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting, such as C. diff, hepatitis A, B, and C, influenza A, Lyme disease, dengue fever, Salmonella, mumps, Bacillus subtilis, measles, HIV, scrapie, MRSA, VRSA, etc. Genetically modified organisms have also been classified as level 2 organisms, even if they pose no direct threat to humans. This designation is used to limit the release of modified organisms into the environment. Approval by the FDA is required to release these organisms. An example is genetically modified food crops. BSL-2 differs from BSL-1 in that:

  • Laboratory users have specific training in handling pathogenic agents and are directed by scientists with advanced training;
  • Access to the laboratory is limited when work is being conducted;
  • Extreme precautions are taken with contaminated sharp items; and
  • Certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.

Biosafety Level 3

This level is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease after inhalation.  It includes various bacteria and viruses that can cause severe to fatal disease in humans, but for which vaccines or other treatment exist, such as Mycobacterium tuberculosis, Bacillus anthracis, West Nile virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Hendra virus, SARS corona virus, Salmonella typhi, Coxiella burnetii, Rift Valley fever virus, Rickettsia rickettsia, and yellow fever virus.

Laboratory users have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents. This is considered a neutral or warm zone.

All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features.

It is recognized, however, that some existing facilities may not have all the facility features recommended for Biosafety Level 3 (i.e., double-door access zone and sealed penetrations). In this circumstance, an acceptable level of safety for the conduct of routine procedures, (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, susceptibility testing, etc.), may be achieved in a biosafety level 2 facility, providing:

  • The filtered exhaust air from the laboratory room is discharged to the outdoors,
  • The ventilation to the laboratory is balanced to provide directional airflow into the room,
  • Access to the laboratory is restricted when work is in progress, and
  • The recommended standard microbiological practices, special practices, and safety equipment for biosafety level 3 are rigorously followed.

Biosafety Level 4

This level is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections, agents which cause severe to fatal disease in humans for which vaccines or other treatments are not available, such as Bolivian and Argentine haemorrhagic fevers, Marburg virus, Ebola virus, Lassa fever, Crimean-Congo haemorrhagic fever, and other various haemorrhagic diseases. When dealing with biological hazards at this level, the use of a Hazmat suit and a self-contained oxygen supply is mandatory. The entrance and exit of a Level 4 biolab will contain multiple showers, a vacuum room, an ultraviolet light room, and other safety precautions designed to destroy all traces of the biohazard. Multiple airlocks are employed and are electronically secured to prevent both doors opening at the same time. All air and water service going to and coming from a biosafety level 4 lab will undergo similar decontamination procedures to eliminate the possibility of an accidental release.

Agents with a close or identical antigenic relationship to Biosafety Level 4 agents are handled at this level until sufficient data is obtained either to confirm continued work at this level, or to work with them at a lower level.

Laboratory users have specific and thorough training in handling extremely hazardous infectious agents and they understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics. They are supervised by qualified scientists who are trained and experienced in working with these agents. Access to the laboratory is strictly controlled by the laboratory director.

The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. A specific facility operations manual is prepared or adopted. Building protocols for preventing contamination often use negatively pressurized facilities, which, if compromised, would severely inhibit the containment of an outbreak of aerosol pathogens.

Within work areas of the facility, all activities are confined to Class III biological safety cabinets, or Class II biological safety cabinets used with one-piece positive pressure personnel suits ventilated by a life support system. The Biosafety Level 4 laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment. The laboratory is kept at negative air pressure, so that air flows into the room if the barrier is penetrated or breached. Furthermore, an airlock is used during personnel entry and exit. Table 7.1. summarizes the biosafety levels.


Table 7.1 Biosafety levels

Biosafety Level





Infectious Agents

Unlikely to cause disease in healthy workers or animals


Low individual and community risk

Can cause human or animal disease but unlikely to be a serious hazard


Moderate individual risk, limited community risk


Effective treatments available

Cause serious human or animal disease but not ordinarily spread by casual contact


High individual risk, low community risk


Cause very serious human or animal disease, often untreatable and transmitted


High individual risk, high community risk

Examples of infectious agents in this risk level


E. coli, California encephalitis viruses, many influenza viruses

Anthrax, Q fever, tuberculosis, Hantaviruses, human immuno-deficiency viruses

Ebola viruses, Herpes B virus (Monkey virus), foot and mouth disease


Standard well-designed experimental animal and laboratory facilities

Level 1 plus: Separate laboratory, room surfaces impervious and readily cleaned, biohazard sign

Level 2 plus: Controlled access double door entry and body shower, air pressure must be negative at all times, no recirculation, HEPA filtration, backup power

Specialized, secure, completely self-contained unit with specialized ventilation, fully monitored; air lock entry and exit,

Safety Equipment

Handwashing facilities, laboratory coats

Level 1 plus: autoclave, HEPA filtered class I or II biological safety cabinet, personal protective equipment

Level 2 plus: Autoclave, HEPA filtered class II biological safety cabinet, personal protective equipment to include solid front laboratory clothing, head covers, dedicated footwear, and gloves, appropriate respiratory protection

Class III biological safety cabinets, positive pressure ventilated suits


Basic safe laboratory practices

Use of personal protective equipment laboratory coat worn only in the laboratory, gloves, decontamination

Users fully trained, written protocols; showers, wastes disposed of as contaminated, use of biological safety cabinets, personal protective devices

Access only to certified staff, rigorous sterilization / decontamination procedures

Adopted from Canadian Council on Animal Care.

Risk Assessment

For typical laboratory operations, biosafety level classifications are convenient.  Based on laboratory specific conditions, the Responsible Faculty Member or LS/LSS is in charge of implementing more (or less) strict practices.  Risk assessment decisions count for the following:

  • Pathogenicity - the ability of an organism to cause disease.
  • Virulence - the severity of disease.
  • Transmission route - parenteral, ingestion, mucous membrane exposure, or inhalation. Organisms such as M. tuberculosis require more strict control than organisms that are transmitted via direct contact, e.g., HBV.
  • Agent stability - survival in environment or otherwise prolonged viability (spore formation).
  • Infectious dose - the dose required to cause infection in humans or animals (ID 50 refers to the dose needed to infect 50 % of the exposed population).
  • Antibiotic resistance.
  • The use of recombinant DNA - any of the above risk factors and modifications should be taken into consideration.

All of the above factors are inherent to a particular microbe; external factors to be considered in a risk assessment include:

  • Titer/volume of material used - titer may increase several orders of magnitude compared to levels in clinical samples, upon culturing.
  • Availability of effective treatment or vaccine.
  • Nature of activities - e.g., potential for splashes, volume used, skills and training level of the users.
  • Health status of the lab user - such as immune status, pregnancy, vaccination status.

Tissue Cultures And Cell Lines

Cell lines obtained from commercial sources may become contaminated with adventitious agents while used in the laboratory.  The extent of screening varies among providers and while most test for bacteria, mycoplasma, and fungi, they do not routinely include testing for viruses other than those categorized as “Bloodborne Pathogens”.

Cell cultures known to contain an infectious agent or oncogenic virus should be manipulated at the Biosafety Level appropriate for the agent, usually BSL-2. 

For activities with materials not known to contain infectious agents, the following hazard classification applies:

BSL-1 is appropriate for well-established lines of cells of sub-primate origin if they do not harbour a primate virus and are free of bacteria, fungi, and mycoplasma.  However, working with these materials at BSL-2 is recommended because of the additional degree of protection from contamination provided by BSL-2 practices, particularly the use of a Biological Safety Cabinet. 

BSL-2 is appropriate for activities with: all primate cell lines, even well established ones, all cells derived from primate lymphoid or tumor tissues; all primate tissue; all human clinical material; cultured cells new to the laboratory until proven contaminant-free; and, cells exposed to or transformed by a primate oncogenic virus.

These activities and the use of any cells purposely infected with or suspected of harbouring agents defined as bloodborne pathogens are covered by the Bloodborne Pathogens Standard (in accordance with CDC).  Laboratories using human cell strains (non‑transformed cells) propagated from primary explants must also comply with the Standard because they are considered “unfixed human tissue” which is covered by the regulation.