Chapter 4: Biosafety Principles

Containment

Laboratory biosafety practices are based on the principle of containment of biological agents to prevent exposure to laboratory workers and the outside environment. Primary containment protects the laboratory workers and the immediate laboratory environment from exposure to biological agents. Primary containment is achieved through good microbiological technique and the use of safety equipment and personal protective equipment. Secondary containment protects the environment outside the laboratory, and is provided by facility design and operational procedures.

Laboratory Practice and Technique

The use of good microbiological technique is the most important element of containment. Personnel working with biological agents must be aware of hazards, and must be trained to safely handle and dispose of these materials. Although we are all responsible for our own safety, the Principal Investigator is responsible for ensuring that persons working in their laboratory are adequately trained.

This Biosafety Manual has been developed to provide general policies and procedures when working with biological agents at the University of Nevada, Reno. Each individual laboratory must supplement this manual with laboratory specific policies; procedures and training that will minimize the specific risks present in the laboratory.

Safety Equipment

Safety equipment includes biological safety equipment, safety centrifuge cups, and other engineered controls designed to minimize exposure to biological agents. Biological safety cabinets (BSCs) are the most important safety equipment for protection of personnel and the laboratory environment, and most BSCs also provide product protection. Safety equipment is most effective at minimizing exposure when workers are trained on the proper use of such equipment, and the equipment is regularly inspected and maintained.

Personal Protective Equipment

Personal protective equipment includes safety eyewear, lab coats, gloves, and other protective equipment, and is used to supplement the containment provided by laboratory practices and safety equipment. Personal protective equipment is considered the least desirable containment method because its failure results in direct exposure of personnel to the biological agent.

Facility Design

Facility design features include physical separation of laboratories from public access, specially designed ventilation systems (to prevent airborne biological agents from migrating outside the laboratory), and autoclaves. These design features protect personnel working outside the immediate laboratory, as well the outside environment.

Biosafety Levels

The CDC/NIH has developed four biosafety levels that describe laboratory practices and techniques, safety equipment, and facility design features recommended for work with specific infectious organisms. Descriptions of the 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) 5th Edition. The recommended biosafety level for an organism represents conditions under which the agent can normally be handled safely; however, specific circumstances may dictate that the recommended conditions be raised or lowered. As outlined in the BMBL, the four biosafety levels are summarized below:

Biosafety Levels and Information for Handling Agents
Biosafety Level Agents Practices Safety Equipment Facilities
1 Not known to cause disease in healthy adults. Standard Microbiological Practices None required Open bench top, sink required
2 Associated with human disease, hazard: auto-inoculation, ingestion, mucous membrane exposure BSL-1 practice plus:
  • Limited access
  • Biohazard warning signs
  • ­Sharps precautions
  • Biosafety manual

Primary barriers:

Class I or II BSCs or other containment used for manipulations of agents that cause splashes or aerosols of infectious materials; PPE: lab coats; gloves; eye/face protection as needed

BSL-1 plus: Autoclave available
3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences

BSL-2 practice plus:

  • ­Controlled access
  • Decontamination of all waste
  • Decontamination of lab clothing before laundering
  • Consider medical surveillance program

Primary barriers:

Class I or II BSCs or other physical containment devices used for all manipulations of agents; PPE: protective lab clothing; gloves; respiratory protection as needed

BSL-2 plus:

  • Physical separation from access corridors
  • ­Self-closing, double door access
  • ­Exhausted air not recirculated
  • ­Directional airflow into laboratory
4 Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agent with unknown risk of transmission

BSL-3 practices plus:

  • ­Clothing change before entering
  • ­Shower on exit
  • ­All material decontaminated on exit from facility

Primary barriers:

All procedures conducted in Class III BSCs or Class I or Class II BSCs in combination with full-body, air-supplied, positive pressure personnel suit

BSL-3 plus:

  • Separate building or isolated zone
  • ­Dedicated supply/exhaust, vacuum, and decon systems
  • Other requirements outlined in BMBL

Consult the BMBL for a more complete description of the four biosafety levels, as well as recommended biosafety levels for specific organisms.

In addition to the four biosafety levels described above, there are also four biosafety levels for work with infectious agents in vertebrate animals and plants. For a complete description of the animal and plant biosafety levels, consult the BMBL.

Routes of Transmission

Skin and Mucous Membrane Contact

Low energy procedures such as decanting of liquids, pipetting, removal of screw caps, vortex mixing, streaking agar plates, and inoculation of animals, can result in the generation of infectious droplets, as well as result in direct contact with infectious material. Eye contact is also a route of exposure and safety eyewear must be worn as needed to prevent sprays or splashes to the eyes.

Ingestion

Mouth pipetting presents the highest risk for ingestion of infectious material. Splashing of material into the mouth, and indirect oral exposure through touching the mouth with contaminated hands, and eating and drinking in the lab can also result in ingestion of infectious material.

Percutaneous Inoculation

Use of syringes and needles are considered the greatest risk of exposure through inoculation. Inoculation can also occur as a result of cuts and scratches from contaminated items, and animal bites.

Inhalation

Many procedures have the potential for generation of respirable aerosols, including: sonication, centrifugation, “blowing out” of pipettes, heating inoculating loops, and changing litter in animal cages.