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Environment, Health, & Safety Division

4.0    Biosafety Principles and Levels

To determine which controls are required to mitigate hazards and perform work safely, supervisors and work leads must understand and apply the processes and requirements for defining work, identifying hazards, and assessing risks, as discussed in Section 3.0 of this manual. Controls are safeguards employed to contain biological agents or materials and therefore prevent the exposure of workers, other people, or the environment to agents that may harm them.

In biosafety, the term “containment” describes the set of controls, including safe methods, equipment, and facilities needed to protect workers and the environment from biohazardous materials or agents. Controls used for containment in laboratories are described in Biosafety in Microbiological and Biomedical Laboratories (BMBL), Section III, as the “Principles of Biosafety.” These containment controls are listed below and summarized in the next sections:

  • laboratory practices and techniques, 
  • safety and personal protective equipment, and
  • facility design and construction.

The LBNL Biosafety Work Authorization is used to define work, identify hazards, assess risks, and implement any of the containment controls listed above. See Sections 2.0 and 5.1 of this manual and PUB-3000, Section 26.8, for additional information on work authorization documents.

4.1       Laboratory Practices

The first and most important element of control for laboratory containment and research product protection is strict adherence to laboratory biosafety containment practices and good microbiological practice (GMP). Biosafety containment practices include standard microbiological practices and special practices specified by the Centers for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH). GMP is based on widely accepted aseptic practices.

Standard microbiological practices and special practices are administrative controls listed as biosafety level (BL) containment criteria in BMBL and the NIH Guidelines to protect workers and the environment. (See Section 4.4 of this manual for additional information on BL containment categories and criteria.) These practices, along with requirements from other biosafety standards, are used for the safe performance of work documented in the LBNL Biosafety Work Authorization. Standard microbiological practices or special practices for laboratories apply to most LBNL work with biological materials. Standard practices for BL1 and BL2 laboratories address the following topics (see Appendix C and Section 5.0 for more information):

  • access control
  • hand hygiene
  • food and eating
  • pipetting
  • sharps control
  • spill, splash, and aerosol control
  • decontamination of work surfaces, equipment, materials, and spills
  • signage and hazard communication
  • pest management
  • worker training and proficiency
  • occupational health, immunization, and personal health
  • incident reporting, evaluation, and worker treatment
  • biosafety manuals or documents

Good Microbiological Practice (GMP) is also typically needed for containment and good research. GMP is based on aseptic techniques and other good microbiological practices necessary to prevent contamination of the laboratory with the agents being handled and contamination of the work with agents from the environment. See Appendix D for common GMP. Supervisors and work leads are responsible for selecting and instructing workers on the specific GMP needed to conduct the work, or additional safety practices needed for specific agents or procedures.

4.2       Safety and Personal Protective Equipment

Worker exposure to infectious agents may be prevented by the use of standard and activity-specific safety and personal protective equipment (PPE) as primary barriers or controls. The need for additional activity-specific safety equipment or PPE must be determined during risk assessment, and any equipment needed for safety should be included in the Biosafety Work Authorization.

Standard safety equipment and PPE are equipment controls listed as BL containment criteria in BMBL and the NIH Guidelines. They provide primary barriers that prevent worker exposure to infectious agents. See Section 4.4 of this manual for additional information on BL containment categories and criteria. These standard equipment requirements, along with requirements from other biosafety standards, are used and customized for the work to be conducted. They are also summarized in the LBNL Biosafety Work Authorization. Standard safety equipment and PPE are applicable to most work with biological materials at LBNL. The following types of standard equipment and PPE are further discussed in Appendix C and Section 5.0 of this manual:

  • biosafety cabinets
  • PPE
  • other physical containment devices such as centrifuge safety cups

4.3       Facility Design and Construction

Facility design and equipment provide secondary barriers that protect laboratory workers, persons outside the laboratory, the public, and the environment from potentially hazardous materials or agents that may be accidentally released from the laboratory.

Picture   Picture

Standard facilites provide secondary barriers.
Source: HHS CDC Office of Health and Safety.

LBNL designs and operates its facilities where work with biological materials is conducted in accordance with applicable standard facilities criteria. Standard facilities are design features, materials, and equipment incorporated into the laboratory or facility in accordance with BL containment criteria stated in BMBL and the NIH Guidelines (see Section 4.4 for more information).

Standard BL1 and BL2 laboratory facility barriers are sufficient to control most work at LBNL. This is because risks related to most work are associated with direct contact with materials or agents in standard laboratories. Examples of standard facility barriers and equipment are listed below. See Appendix C for standard laboratory facilities criteria that summarize how these barriers must be employed. See Section 5.0 for additional information on each of these topics.

  • doors
  • sinks
  • cleanable surfaces and furnishings
  • window screens
  • ventilation and biosafety cabinets
  • vacuum line filters and traps
  • eyewashes
  • autoclaves

If the risk assessment indicates that there is a risk of exposure to an infectious aerosol, then higher levels of safety equipment and PPE (primary barriers) or multiple secondary facilities barriers are necessary. Multiple secondary facilities barriers are not typically needed at LBNL.

Some standard facility barriers are summarized in the Biosafety Work Authorization. Any additional special facility barriers that are required should also be included in the authorization.

4.4       Biosafety Containment Levels and Criteria

LBNL requires researchers who work with biological materials to implement containment controls in accordance with an established biosafety level (BL). BL is a standard combination of practices and techniques, safety equipment, and facilities to safely contain biohazardous materials or agents to be used in the work, as specified by BMBL or the NIH Guidelines.

Work at LBNL requires routine application of BLs developed for biological laboratories, and occasional application of BLs developed for other types of work such as large-scale recombinant operations. BLs for laboratories are presented in the next section. BLs for large-scale, plant, and animal uses are presented in Section 4.4.2 this manual.

The appropriate BL must be selected once the risk assessment has been completed. The final BL determination should consider all aspects of the work, hazards, and controls. The principal investigator (PI) or supervisor should propose the appropriate BL(s) when submitting the authorization for review. The final BL(s) are determined by the Institutional Biosafety Committee (IBC).

4.4.1    Laboratory Containment Levels

Containment controls for laboratory biosafety are categorized into four BLs. Definitions of each laboratory biosafety level (BL) are provided in Table 8. Work at LBNL is commonly conducted at BL1 or BL2, while work at BL3 or BL4 is not currently conducted. Laboratory work at LBNL must be conducted in accordance with the standard and special work practices, safety equipment, and facility requirements noted in the laboratory BL1 and BL2 criteria listed in Appendix C.


Table 8
Laboratory Biosafety Containment Levels

Present
at LBNL

Biosafety Level (BL)

Biosafety Level Definition

yes

1

BL1 is suitable for work involving agents of unknown or minimal potential hazard to laboratory personnel and the environment,1 or work with defined and characterized strains of viable microorganisms not known to consistently cause disease in healthy adult humans.2 

2

BL2 is suitable for laboratory work involving agents of moderate potential hazard to personnel and the environment.1 Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures, or ingestion of infectious materials.2 

no

3

BL3 is applicable to facilities in which work is conducted with indigenous or exotic agents that may cause serious or potentially lethal disease as a result of exposure by the inhalation route.3 

4

BL4 is required for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, aerosol transmission, or related agents having an unknown transmission risk.3 

Footnotes:

  1. NIH Guidelines
  2. BMBL, fifth edition, Section III
  3. BMBL, fifth edition, Section IV

When developing the Biosafety Work Authorization, the appropriate laboratory BL must be selected after conducting the risk assessment. Typical BLs used for various materials and agents are listed in Sections 4.4.1.1 and 4.4.1.2 of this manual. The final BL(s) are determined by the IBC.

Text Box:4.4.1.1 Laboratory Biosafety Level 1 

BL1 laboratories are not necessarily separated from the general traffic patterns in the building. Work is typically conducted on open benchtops using standard microbiological practices. Special containment equipment or facility design is not required, but may be used as determined by a risk assessment. Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist trained in microbiology or a related science.

Picture 
Laboratory Biosafety Level 1 work with open benchtops and standard microbiological practices. Source: LBNL EH&S.

The BL will be determined as part of the risk assessment. BL1 containment is typically required for laboratory work involving:

  • biological agents that meet the definition of Risk Group (RG) 1 (i.e., agents not associated with disease in healthy adult humans);
  • biological materials not suspected of containing RG2 or higher agents in a quantity or form that may cause human disease (e.g., many soils and nonprimate animal cells);
  • biological agents or materials not characterized by the supplier as RG2 or higher;
  • transgenic or wild-type laboratory animals that have size or growth requirements allowing the use of containment for laboratory animals (e.g., rodents) and are
    • free of zoonotic diseases, and
    • not infected with, implanted with, or containing RG2 or higher agents or materials;
  • laboratory growth of nongreenhouse transgenic plants (see Section 4.4.3 of this manual);
  • biological agents, materials, or animals not typically categorized as RG2 or BL2 (or higher) as detailed Section 4.4.1.2 of this manual.

Text Box:Text Box:4.4.1.2 Laboratory Biosafety Level 2 

BL2 laboratories follow BL1 requirements and additional BL2 requirements such as:

  • Laboratory personnel have specific training in handling any pathogenic agents and are supervised by scientists competent in handling infectious agents and associated procedures.
  • Access to the laboratory is restricted when work is being conducted.
  • All procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets (BSCs) or other physical containment equipment.

The BL will be determined as part of the risk assessment. Laboratory BL2 containment is typically required for laboratory work involving:

  • biological agents categorized as RG2 in the NIH Guidelines or by the supplier;
  • uses of biological agents described as BL2 in BMBL agent summary statements or other BMBL;
  • biological materials that may contain RG2 agents (e.g., sewage);
  • Bloodborne pathogen (BBP) materials (e.g., human blood, human tissues, or human cells);
  • nonhuman primate tissues or cells;
  • viral vectors that are replication defective but still infectious to human cells;
  • laboratory animals (e.g., rodents) infected with, implanted with, or containing RG2 agents or materials (e.g., infected with a human pathogen or containing a xenotransplant of human cells); and
  • tissues or cells potentially containing an RG2 agent (e.g., cells transformed with a RG2 virus).

4.4.2 Additional Containment Categories

Additional types of containment specified in the NIH Guidelines or BMBL may also be applicable to work with biological materials at LBNL. Table 9 below lists these additional containment categories and relevant NIH Guidelines or BMBL section. When selecting a containment level for a type of work listed in Table 9, the supervisor, work lead, and IBC should carefully review this table to determine if the containment category and criteria apply to the planned LBNL work.

Laboratory- or operation-specific authorizations, biosafety manuals, or other documents may be used to document the containment requirements related to the work. If the containment categories or criteria presented in Table 9 are not applicable to the work, the laboratory BLs presented in Section 4.4.1 of this manual are applied.

Text Box:4.4.2.1 Recombinant Large-Scale Containment Levels

Physical containment guidelines from Appendix K of the NIH Guidelines must be used for large-scale research or production activities involving viable organisms containing recombinant DNA molecules. Large scale (BL–Large Scale) is a term used in the NIH Guidelines and the LBNL biosafety policy to describe uses of and containment levels for organisms containing recombinant DNA molecules involving a quantity of culture greater than 10 liters. Note that this quantity category typically means the quantity of a material in a single batch of liquid culture; however, this batch quantity is not defined by NIH and should be used as a guideline to determine the applicability of large-scale containment criteria. Criteria for large-scale containment address the biological hazard associated with organisms containing recombinant DNA only. Large-scale containment criteria must be selected based on the findings of the risk assessment, and then documented in the Biosafety Work Authorization.

Text Box:4.4.2.2 Recombinant Plant Containment Levels

Biosafety containment levels and criteria for recombinant research with plants must be selected based on the findings of the risk assessment, and then documented in the Biosafety Work Authorization. Laboratory or plant biosafety containment levels must be applied to the work as follows:

  • Laboratory BLs and criteria discussed in Section 4.4.1 of this manual and Appendix G of the NIH Guidelines should be used when the research plants are of a size, number, or have growth requirements that allow good containment when using laboratory BLs.
  • Plant BLs (BL-P) must be used when the research plants are of a size, number, or have growth requirements that preclude the use of laboratory BLs. For plant BLs and criteria, see Appendix P (Physical and Biological Containment for Recombinant DNA Research Involving Plants) of the NIH Guidelines.

Table 9
Additional Containment Categories

Containment Category

Standard and Section

Focused Scope of Containment Criteria

large-scale uses of organisms containing recombinant DNA molecules

NIH Guidelines, Appendix K

Physical containment guidelines for large-scale (greater than 10 liters of culture) research or production activities involving viable organisms containing recombinant DNA molecules

recombinant DNA research involving plants

NIH Guidelines, Appendix P

Physical and biological containment conditions and practices suitable to greenhouse operations that conduct experiments involving plants, plant-associated microorganisms, and small animals (e.g., arthropods or nematodes)

recombinant DNA research involving animals

NIH Guidelines, Appendix Q

Containment and confinement practices for research involving whole animals when the animals are of a size or have growth requirements that preclude the use of containment for laboratory animals (i.e., including but not limited to nonhuman primates, cattle, swine, sheep, goats, horses, and poultry) and:

  • The animals’ genomes have been altered by the introduction of recombinant DNA or DNA derived therefrom into the germ line (transgenic animals), or
  • Experiments involving viable recombinant DNA-modified microorganisms have been tested on whole animals.

vertebrate animal BL criteria for indoor research facilities (e.g., vivariums)

BMBL,
Section V

Use of experimentally infected animals housed in indoor research facilities (e.g., vivariums), and the maintenance of laboratory animals that may naturally harbor zoonotic infectious agents

arthropod containment
guidelines

BMBL, Appendix E

Risk assessment and containment for arthropods of public health importance including those that transmit pathogens. Arthropods that only bite, sting, or cause myiasis and infestation are not included. Myiasis is an infestation of tissue by fly larvae, or a disease resulting from such infestation.

 

The plant BLs listed in Appendix P of the NIH Guidelines specify physical and biological containment conditions and practices suitable for conducting greenhouse experiments involving recombinant DNA-containing plants, plant-associated microorganisms, and small animals. Acronyms for plant BLs are BL1-P through BL4-P. The following bullets further clarify terms and applicability of the plant biosafety levels:

  • The term greenhouse refers to a structure with walls, a roof, and a floor designed and used principally for growing plants in a controlled and protected environment. The walls and roof are usually constructed of transparent or translucent material to allow the passage of sunlight for plant growth. The term greenhouse facility includes the actual greenhouse rooms or compartments for growing plants, including all immediately contiguous hallways and head-house areas, and is considered part of the confinement area.
  • The plants covered in Appendix P of the NIH Guidelines include but are not limited to mosses, liverworts, macroscopic algae, and vascular plants including terrestrial crops, forest, and ornamental species.
  • Plant-associated microorganisms include viroids, virusoids, viruses, bacteria, fungi, protozoans, certain small algae, and microorganisms that have a benign or beneficial association with plants, such as certain Rhizobium species and microorganisms known to cause plant diseases. Microorganisms being modified to foster an association with plants are also included.
  • Plant-associated small animals include those arthropods that have an obligate association with plants, are plant pests or plant pollinators, or transmit plant disease agents. They also include other small animals such as nematodes that require the use of plants to test their biological properties. Microorganisms associated with such small animals (e.g., pathogens or symbionts) are also included.

A Practical Guide to Containment developed by Virginia Polytechnic and State University is a good example of how to apply plant BLs in research with greenhouse transgenic plants and microbes.

4.4.2.3 Vertebrate Animal Containment Levels

Biosafety containment levels and criteria for the use or care of vertebrate animals must be selected or developed following the risk assessment and covered in the Biosafety Work Authorization. Laboratory or animal biosafety containment levels must be applied to the work as follows:

  • Text Box:Laboratory biosafety level criteria should be used for laboratory animals such as rodents whose size or growth requirements allow the use of laboratory containment levels specified by the NIH Guidelines. Laboratory BLs and criteria are discussed in Section 4.4.1 of this manual.
  • Text Box:Animal biosafety levels must be applied when 1) recombinant research involves larger animals (e.g., nonhuman primates), 2) animals are infected with human pathogens, or 3) animals may harbor zoonotic agents (see Table 9 for more information). Acronyms for animal biosafety levels are BL1-N through BL4-N. In some cases, animal use, animal care, and hazards at LBNL may not be directly applicable to these established animal biosafety levels and criteria. In these cases, specific criteria that may be applicable may be selected, customized, and incorporated into the Biosafety Work Authorization.

Agent summary statements for zoonotic agents in Section VIII of BMBL also recommend containment levels for laboratory use of zoonotic agents, and for handling animals infected with the agent.

Text Box:

4.4.2.4 Arthropod Containment Levels

Biosafety containment levels and criteria for the use of arthropods must be selected or developed after the risk assessment. Laboratory or arthropod biosafety containment levels must be applied to the work as follows:

  • Laboratory biosafety level criteria should be used for arthropods that do not present risks to humans, plants, or animals (e.g., most research uses of Drosophila spp.).
  • Arthropod containment guidelines discussed in Appendix E of BMBL must be used for arthropods of public health importance including those that transmit pathogens. Arthropods that only bite, sting, or cause myiasis and infestation are not included. Most uses of Drosophila spp. are also excluded from these arthropod containment guidelines. The Appendix E of BMBL references the Arthropod Containment Guidelines published by the American Society of Tropical Medicine and Hygiene.