Chapter 4: Biosafety Principles
The risk of exposure to biological agents in a research environment depends on a number of factors (e.g., the agent, its virulence, subject’s susceptibility, route of transmission, etc.). In general, the biosafety procedures followed are designed to prevent such exposures by containing the agents being handled and using the appropriate types of PPE. To properly design the containment, it is important to recognize the potential routes of transmission for the given agent.
Routes of Transmission
Skin and Mucous Membrane Contact
Procedures, such as decanting of liquids, pipetting, opening of screw caps, vortexing, streaking agar plates, and inoculation of animals, can potentially result in the generation of splatters of infectious droplets, as well as direct contact with infectious material. Direct contact to the mucous membranes of the eyes, nose, and mouth is considered a route of exposure. Appropriate eye and face protection should be used when performing work that generate splashes.
Ingestion
Performing mouth pipetting is prohibited in the laboratory. It presents a risks of ingesting infectious material. Touching the face and mouth when working can cause an oral exposure to the infectious material being handled. Storage of food or drinks and their consumption, and wearing of contact lenses in the laboratory is prohibited.
Laboratory personnel should refrain from the use of personal electronic mobile devices such as cell phones, headphones, and earbuds when working in the laboratory. They can cause distractions and could potentially become contaminated.
Percutaneous Inoculation
Use of syringes and needles presents the greatest risk of exposure through inoculation. Accidental inoculation can also occur as a result of cuts and scratches from contaminated items including syringes used for animal inoculations, as well as animal bites.
Inhalation
Certain procedures have the potential for generation of respirable aerosols, including sonication, centrifugation, “blowing out” of pipettes, heating inoculating loops, and changing bedding from the cages of infected animals. Aerosol refers to a droplet of 5 microns or less in diameter that can potentially be suspended in air for some amount of time. Aerosols may contain infectious particles.
Containment
The term “containment” is used to describe safe methods for handling infectious agents in the laboratory environment. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other people, and the outside environment to potentially hazardous agents. The four elements of containment include administrative controls, work practices, personal protective equipment, and facility design.
Primary Containment
The protection of personnel and the immediate laboratory environment from exposure to infectious agents is provided by good microbiological techniques, use of appropriate PPE, and the use of appropriate safety equipment, such as the Biological Safety Cabinets (BSC).
Secondary Containment
Protecting the laboratory’s external environment from exposure to infectious materials is accomplished by a combination of the facility design and safe operational practices. The risk evaluation of the work to be done with a specific agent will determine the appropriate combination of these elements.
Safety Equipment
Safety equipment includes biological safety equipment, sealed containers, safety centrifuge cups, down-draft tables, and other engineered controls designed to minimize exposure to biological agents. Biological safety cabinets are among the most important safety equipment for protection of personnel and the laboratory environment, and most also provide product protection. Safety equipment is most effective at minimizing exposure when workers are trained in the proper use of such equipment and the equipment is regularly inspected and maintained.
Biological Safety Cabinets (BSC)
Proper use of a BSC provides a high level of containment that protects the operator from exposure while providing some protection from contamination of the material being handled within the work environment.
Because of its importance in providing containment and safety protections in the laboratory, a BSC is considered one of the most critical piece of safety equipment in the biological laboratories. When used appropriately, BSCs are designed to capture aerosols that may be generated during work with biological materials. The BSC is equipped with a high efficiency particulate air (HEPA) filtration that filters the air in BSC before being recirculated or exhausted. There are three types of BSCs (Class I, II, and III) used in laboratories. Open-fronted Class I and Class II BSCs are containment devices that provide a primary barrier offering significant levels of protection to laboratory personnel and to the environment when used in combination with good laboratory technique.
The Class I BSC is suitable for work involving low-to-moderate risk agents where there is a need for containment, but not for product protection. It provides protection to personnel and the environment from contaminants within the cabinet but does not protect the work within the cabinet from “dirty” room air.
The Class II BSC protects the material being manipulated inside the cabinet (e.g., cell cultures, microbiological stocks) from external contamination. It meets requirements to protect personnel, the environment, and the product. The two basic types of Class II BSCs are Type A and Type B. The primary difference between the two types may be found in the ratio of air that is exhausted or recirculated and the manner in which exhaust air is removed from the work area.
The Class III BSC is typically used to work with highly infectious microbiological agents including those classified as RISK Group IV or agents requiring a BSL4 laboratory. It provides the maximum protection to personnel, the environment, and the product. Another type of device that is typically referred to as a “laminar flow” or a “clean bench” can sometimes be seen used in the lab. It is important to note that this device must not be utilized for work with biohazardous or chemically hazardous agents. These units provide product protection by ensuring the product is exposed only to clean HEPA-filtered air. They do not provide protection to personnel working or the environment.
PIs are responsible for ensuring the proper maintenance of lab equipment. BSCs used as primary barriers must be certified annually by a qualified vendor. Contact the Biosafety Officer or the EHS for information about testing and certification vendors or other BSC-related information.
Proper operation and maintenance of a BSC requires knowledge of how the system operates, as well as training and experience in effective techniques for working within the cabinet volume without compromising its functions. Additional details concerning the design and use of BSCs are provided in Appendix C.
Two specialized forms of quality control are strongly recommended for all BSCs and are required for cabinets used to contain Risk Group 2 or higher agents:
- At least daily, or each time the BSC is operated, the operator or user should observe the magnahelic gauge and note its relative position. Magnahelic gauges measure the pressure drop across the outlet HEPA filter and are important indicators of filter integrity and loading. The gauge will typically indicate the same measurement over a long period of time. A significant change in the reading over a short period of time may indicate an issue including clogging or a leaking HEPA filter. In such cases, the BSC should not be used until the problem is identified and resolved. If the BSC located within a laboratory does not have a magnahelic gauge, users must understand the operation of the airflow monitor, controls, and alarm settings.
- The BSC must be tested and recertified annually or after it is moved to a different location. Testing and recertification is performed by a contractor certified to test the BSC. EHS can provide information on qualified vendors. The certification ensures that the BSC is meeting its operating specifications and providing maximum protection. In addition, certifiers provide service and preventive maintenance for BSC and can often forecast expensive requirements like HEPA filter replacements, allowing PIs to budget for the event.
- If BSC recertification is required, the recertification must be completed before the current certification expires. If the certification lapses, the BSC may not be used for BSL2 or higher procedures until it is recertified. The lab will report the lapsed recertification to EHS immediately. EHS will inform the PI and lab workers not to use the BSC and affix an “OUT OF SERVICE” notice and assist the lab to get the BSC recertified. Unless a good reason exists for more frequent certification, a one-year certificate life is appropriate. The certificate will generally expire on the last day of the month in which the certification was performed, one year later (for example, a certificate issued on June 2, 2007 will expire on June 30, 2008).
Personal Protective Equipment (PPE)
PPE is designed to protect the wearer against chemical, biological, radiological, or mechanical irritants. Different types and levels of PPE may be required for individuals, depending upon each person’s role within a laboratory, the materials handled, and the work being performed.
In order to ensure that all laboratory faculty, staff, students, affiliates and visitors are sufficiently protected from the hazards present in the workplace, the Personal Protection Equipment in Laboratories Policy has established the minimum PPE requirements, based on best laboratory practices.
Facility Design
The design of a facility is important in providing a barrier to protect people working inside and outside the laboratory, as well as to protect people or animals in the community from infectious agents that may be accidentally released from the laboratory. Facility design must be commensurate with the laboratory’s function and the recommended biosafety level for the agent being used or stored.
The recommended secondary barrier(s) will depend on the risk of transmission of specific agents. For example, the exposure risks for most laboratory work in BSL1 and BSL2 facilities will be direct contact with the agents or inadvertent contact exposures through contaminated work environments. Secondary barriers in these laboratories may include separation of the laboratory work area from public access; availability of decontamination equipment (e.g., autoclave*); and handwashing facilities. In BSL3 facilities, additional safeguards, such as directional airflow airlock-controlled entry and exiting, a shower used for personnel to shower out may be required.
As the risk for aerosol transmission increases, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Such design features could include specialized ventilation systems to ensure directional airflow; air treatment systems to decontaminate or remove agents from exhaust air; controlled access zones; an airlock at the laboratory entrance; or separate buildings or modules for physical isolation of the laboratory building itself.
Note: It is IBC policy that autoclaves used to sterilize biohazardous materials be validated quarterly using a sporulation test and that validation records be kept (see Appendix D). Unless sterilization of the waste is otherwise specified by the IBC, solid biohazardous waste should be directly disposed into red bags as medical waste without autoclaving.
Biosafety Levels
Four biosafety levels (BSLs) represent combinations of laboratory practices and techniques, safety equipment, and laboratory facilities. Each combination is specifically appropriate for the operations performed and the documented or suspected routes of transmission of the infectious agents, as well as for the laboratory function or activity. The recommended biosafety level for an organism represents the conditions under which the agent can be ordinarily handled safely.
NIH’s Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules classifies “human etiologic agents” on the basis of their relative pathogenicity. Agents are categorized into four risk groups (RG).
As a general rule for selecting the appropriate laboratory containment, the biosafety safety level (BSL) selected should correspond with the highest risk group (RG) category of the organisms involved. For example, work with vaccinia virus which is a Risk Group 2 (RG2) agent, should be conducted at BSL2 or higher and Mycobacterium tuberculosis which is a Risk Group 3 (RG3) should be conducted at BSL3.
Descriptions of biosafety levels, as well as assigned biosafety levels for specific organisms, are contained in the CDC/NIH document, Biosafety in Microbiological and Biomedical Laboratories (BMBL) 6th edition. The BMBL outlines four biosafety levels, summarized below:
Description of Biosafety Levels
Note: Consult the BMBL 6th edition for a more complete description of the four biosafety levels, as well as recommended biosafety levels for specific organisms.
Description of Animal Biosafety Levels
In addition to the four biosafety levels described above, there are also four biosafety levels for work with infectious agents in vertebrate animals, referred to as the Animal Biosafety Level (ABSL).
| ABSL | Agents | Practices | Primary Barriers and Safety Equipment | Facilities (Secondary Barriers) |
| 1 | Not known to consistently cause disease in healthy human adults; RG1 | Standard animal care, use, and management practices, including appropriate medical surveillance programs. | As required under the Program of Veterinary Care and Use of Animals at BU for normal care, use, and handling of each species.
PPE: laboratory coats and gloves; eye, face protection, as needed | Standard animal facility, remote housing facility, or laboratory No recirculation of exhaust air Directional air flow recommended Hand washing sink recommended |
| 2 | Agents associated with human disease; Hazard: percutaneous injury, ingestion, mucous membrane exposure RG2 | ABSL1 practices plus: Limited access Biohazard warning signs Sharps precautions Biosafety manual Decontamination of all infectious wastes and of animal cages prior to washing | ABSL1 equipment plus primary barriers: containment equipment appropriate for animal species.
PPE: laboratory coats, gloves, face, eye and respiratory protection as needed. | ABSL1 facility plus: Autoclave available Hand washing sink available in the animal room. Mechanical cage washer recommended Negative airflow into animal and procedure rooms recommended |
| 3 | Indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposure; RG3 | ABSL2 practices plus: Controlled access Decontamination of clothing before laundering Cages decontaminated before bedding removed Disinfectant foot bath as needed | ABSL2 equipment plus: Containment equipment for housing animals and cage dumping activities Class I, II or III BSCs available for manipulative procedures (inoculation, necropsy) that may create infectious aerosols. PPE: appropriate respiratory protection | ABSL2 facility plus: Physical separation from access corridors Self-closing, double-door access Sealed penetrations Sealed windows Autoclave available in facility Entry through ante-room or airlock Negative airflow into animal and procedure rooms Hand washing sink near exit of animal or procedure room |
| 4 | Dangerous/exotic agents which post high risk of aerosol transmitted laboratory infections that are frequently fatal, for which there are no vaccines or treatment. Agent with a close or identical antigenic relationship to an agent requiring BSL4 until data are available to redesignate the level. Related agents with unknown risk of transmission life-threatening disease; RG4 | ABSL3 practices plus: Entrance through change room where personal clothing is removed and laboratory clothing is put on; shower upon exiting All wastes are decontaminated before removal from the facility | ABSL3 equipment plus: Maximum containment equipment (e.g., Class III BSC or partial containment equipment in combination with full body, air-supplied, positive-pressure personnel suit) used for all procedures and activities | ABSL3 facility plus: Separate building or isolated zone Dedicated supply and exhaust, vacuum, and decontamination systems Other requirements outlined in the text |
Note: Consult the BMBL 6th edition for a more complete description of the four Animal biosafety levels, as well as recommended containment requirements for biosafety specific type of animals.
Chapter 5: Laboratory Biosafety Practices