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Biosafety Levels for Labs

Laboratories using microorganisms for teaching or research purposes are assigned a Biosafety Level (1–4), dependent on the potential for risks to human health of those working in the lab space. 

At UW Oshkosh there are no laboratories with a Biosafety Level over a 2.

Basic Biological Safety – Principles and Practices

What are Biosafety Levels?

Risk Group 1 – Biosafety Level 1 (BL1)

Agents at this level are those which are well characterized and which are not known to consistently cause disease in healthy adults humans or animals.

  • E. coli K-12 (non-pathogenic lab strain)
  • Saccharomyces cerevisiae
  • Bacillus subtillis
  • Candida albicans

RG1 agents are so numerous they are not specifically listed in Appendix B of the NIH Guidelines.

Recombinant RG1 agents can be modified so that they can infect humans and can be used to introduce foreign genes. Increased precautions may be appropriate for some recombinant RG1 experiments.

 

Risk Group 2 – Biosafety Level 2 (BL2)

Agents assigned to this containment level include a broad spectrum of indigenous moderate risk agents for which vaccinations or therapies are generally available. They are transmissible to humans through accidental ingestion or entry through broken skin or contact with the mucous membranes.

  • Hepatitis A, B, C, D
  • Salmonella spp.
  • Adenovirus
  • Staphylococcus aureus

BSL-2 is required at a minimum for manipulating viable human derived materials and is recommended level for animal diagnostic laboratory operations.

 

Risk Group 3 – Biosafety Level 3 (BL3)

Agents assigned to this level are associated with disease which may cause serious or lethal infection and for which preventive or therapeutic interventions may be available.

Aerosol is a common route of transmission for most RG3 agents (HIV is an exception). Transmission by the aerosol route increases the risk potential for these pathogens because aerosol-transmitted pathogens are more difficult to contain.

  • Francisella tularensis
  • Mycobacterium tuberculosis complex species (including M. bovis)
  • Human immunodeficiency virus (HIV)

Features of BL-3

Personnel must typically wear respiratory protection and demonstrate proficiency in lab techniques that ensure personnel protection. Specific facility features designed to protect the surrounding environment are also required.


Risk Group 4 – Biosafety Level 4 (BL4)

Agents assigned to this BSL are dangerous and exotic with a high individual risk of life-threatening disease and which may be transmitted via the aerosol route. Vaccinations or therapies are not available for these agents.

  • Ebola virus
  • Herpes B virus (Cercopithecine)
  • Lassa fever virus

Features of BL – 4

Personnel perform work in full-body positive pressure protective gear or in Class III glove boxes. A separate facility specifically designed to protect the surrounding environment are required as well.

 

 

Practices and Procedures – The Second Element of BL Selection

As already mentioned, the nature of the agent is the starting point for BL selection. Procedures to be carried out could be considered the final selection criteria.

There are a number of procedures that can increase the risk for personnel as well as environmental contamination potential. These include:

  • Work with large quantities of samples
  • Work with an agent involving live animals
  • Work with concentrated cultures
  • Work with large single volumes (i.e. greater than 1 liter) of biologically active materials.

Under these conditions, an increase of the biosafety level assignment or enhancement of practices and techniques may be warranted. These decisions should be made by the primary investigator before work involving infectious agents is undertaken. This process is part of the Biological Risk Assessment.

 

Biosafety Level 1 – Good Microbiological Practices

All lab operations involving viable biological agents must be conducted at BL1. Most campus laboratories are BL1.

Facilities features are minimal at this level but do include:

  • Doors for access control
  • A sink for hand washing
  • Bench tops that are impervious so they can be disinfected
  • Sturdy lab furniture
  • Spaces accessible for cleaning
  • Fly screens if the lab has window that open

The core of BL1 and the foundation of all biosafety levels are Standard Microbiological Practices. In the following section the standard microbiological practices for BL1 through BL3 will be discussed.

 

  • BL1 – Standard Microbiological Practices
  1. Access to the lab is limited or restricted at the discretion of the lab director when experiments to work with cultures and specimens are in progress. This practice can ensure that distractions are kept to a minimum.
  2. Persons wash their hands after handling viable materials, after removing gloves, and before leaving the lab. This practice is not only necessary for good hygiene; it will also minimize any potential for contamination to remove from one lab to another.
  3. Eating, drinking, smoking, handling or applying cosmetics, and storing food for human use are not permitted in the work area. Food is stored outside of the work area in an area designated for that purpose. All hand creams, lip balms and other lotions should not be stored or applied in the open lab environment where container surfaces can become contaminated by activities in the lab.
  4. Mouth pipetting is prohibited. Use mechanical pipetting devices. Mouth pipetting presents an opportunity for accidental ingestions and should not be performed.
  5. Policies for safe handling of sharps are implemented. Safe sharps procedures are discussed later in this module.
  6. All procedures are performed carefully to minimize the creation of aerosols or splashes. Perform your procedures in the safest, cleanest manner you can. Use only the quantity of material you need and do dry runs of new procedures to refine and learn the process before you begin work with viable materials, etc.
  7. Work surfaces are decontaminated at least once a day and after any spill of viable material. The basic procedures for proper cleaning and disinfection are also presented later in this module.
  8. All cultures, stocks and other regulated wastes are decontaminated by an approved decontamination method. Biohazardous waste classification, segregation, storage, treatment and disposal will be discussed later in this module.

 

  • Biosafety Level 1: Primary Barriers

There are no special containment devices or equipment like biosafety cabinets required at this level.

The following recommendations are made regarding personnel protective equipment:

  • Lab coats are recommended to prevent contamination of street clothes.
  • Fluid-resistant gloves are recommended, especially if you have broken skin or rashes on your hands. Nitrile and/or vinyl gloves should be available for use in the lab.

Protective eyewear should be worn when conducting procedures that involve manipulation of viable biological materials or other hazardous materials. Minimally, safety glasses should be worn. Where procedures are likely to generate a splash, splash goggles should be worn.


Biosafety Level 2 Laboratories

Biosafety level 2 work requires attention to aerosol minimization, limiting handling of viable biological materials, and safe sharps procedures. Therefore, facilities where BL2 work is to be carried out must meet the following requirements (in addition to the BL1 Facility requirements):

  • Doors are lockable
  • Lab is easily cleaned and no carpets are rugs are permitted
  • Chairs and other lab furniture used in lab are covered with non-fabric materials that can be easily decontaminated.
  • Biosafety cabinets are properly installed and located.
  • An eyewash station is readily available
  • Lab illumination is adequate for all activities, avoiding reflections and glare that could impede vision.
  • While there are no specific ventilation requirements, new facilities should be designed so that the ventilation systems provide an inward flow of air without recirculation to spaces outside the lab.

 

As previously mentioned, the standard microbiological practices are the basis of biosafety containment practices at all levels.

At BL2, the following additional special practices are required.

  • Regarding restricted access, persons who are at increased risk for acquiring infection or for whom infection may have serious consequences are not permitted in the lab.
  • Policies and procedures are in place to ensure that only personnel who have been advised of the biohazard present and meet the vaccination requirements (if applicable) are permitted in the lab.
  • A biohazard sign must be posted on the entrance to the lab when etiologic agents are in use. The sign must include the biosafety level, the agents in use, the primary investigator’s name and contact numbers, required PPE and exit procedures. The Biosafety program will provide signs for BL2 labs to ensure compliance with current requirements.
  • When appropriate, considering the agents handled, baseline serum samples for lab or other at-risk personnel are collected and stored. Serum banking is not generally practiced at UWO.
  • Biosafety procedures are incorporated into standard operating procedures or in a biosafety manual adopted or prepared specifically for the lab. Personnel are advised or special hazards and are required to read and follow instructions on practices and procedures.

 

 

  • Use of Sharps in BSL2 Labs

Adequate precautions must be always taken with any contaminated sharp items. Sharps handling is discussed later in this module, but at BL2 some specific practices are emphasized and should be followed. These include:

  • Sharps use restricted in the lab and used only when there is no alternative.
  • Glassware should be replaced with plastic-ware whenever possible.
  • Needle-locking or disposable syringe-needle units are used for injection or aspiration of infectious materials.
  • Non-disposable sharps are placed in hard-walled containers for reprocessing.
  • Broken glassware is not handled directly by hand. Use a broom and dustpan to pick up broken pieces.
  • Use sharp devices with safety features.

 

 

“Safer Sharps”

A “safer sharp” is one that has a safety mechanism built into the design that allows the use to enclose or retract the sharp end without recapping or otherwise manipulating the device. Examples include:

  • Disposable scalpels with shields
  • Needles that are equipped with a shield
  • Blood collection systems that retract the needle with a spring

History shows that the bulk of sharps accidents occur from recapping or other actions involved with the disposal process. The design of a safer sharp is to minimize that exposure risk. OSHA regulations now require that “safer sharps” be used in human health care and clinical lab settings.

 

BL2 – Standard Microbiological Practices

  • Cultures, tissues, specimens of body fluids, or potentially infectious wastes are placed in a container with a cover that prevents leakage during collection, handling, processing, storage transport or shipping.
  • Lab equipment and work surfaces should be decontaminated with an effective disinfectant on a routine basis, at the conclusion of procedures with potentially infectious materials, and whenever a spill or splash occurs. Contaminated equipment must be decontaminated before it is sent for repair or maintenance.
  • Spills and accidents that result in exposure to potentially infectious materials (including viable clinical specimens or cell materials) must be immediately reported to the primary investigator. Proper incident response and reporting is critical to ensure that you receive the best possible care in the event that the exposure results in infection.
  • Animals not involved in the work being performed are not permitted in the lab. The lab is no place for pets.


Avoid Aerosols 

Doing aerosol-generating activities with biohazardous materials is a major concern. It is important to note that pathogens not normally transmitted by the aerosol route can be an aerosol hazard when you perform procedures that generate aerosols.

Some common activities that generate aerosols include:

  • Vortexing
  • Popping tube caps
  • Pipetting, pouring
  • Homogenization
  • Loading and injecting syringes
  • Centrifugation
  • Changing bedding of infected animals
  • Blending

If possible do aerosol-generating activities in containment such as a Biosafety Cabinet (BSC). When aerosol-generating activities need to be done outside containment, use appropriate safety practices.

 

BL2 Primary Barriers

  • Using a Biological Safety Cabinet (BSC) to Minimize Your Exposure Risk

 

A biological safety cabinet should be used whenever:

  • Conducting procedures with a potential for creating aerosols or splashes
  • Using high concentrations of infectious agents
  • Using large volumes of infectious agents

A biosafety cabinet is equipped with high efficiency particulate air (HEPA) filters that are capable of capturing particulates. This feature protects not only the user but the materials inside the cabinet as well. However, this is only possible when the cabinet is used properly.

 

  • Proper Use of A Biological Safety Cabinet
    • Turn it on – check the gauge
    • Maintain a constant air curtain
    • Establish a “Clean to Dirty” work pattern
    • Avoid clutter; keep grille area clear
    • Disinfect working surface and interior
    • Ensure that BSC is certified annually
  • Biosafety Cabinets – No Substitute for a Chemical Fume Hood

 

Biosafety cabinets are designed to capture particulates ONLY. They do not capture vapors. Additionally, whether a BSC is exhausted to the outdoors or to the room, most BSCs will recirculate the air within the cabinet.

Hazardous chemicals, especially volatiles, should not be used in a BSC. The cabinet will simply recirculate and concentrate these vapors and increase your chemical exposure and the risk for an explosion.

  • Flames in the BSC: Not Recommended

 

When used in conjunction with flammables (i.e., ethanol) in the BSC, flames are a significant fire hazard and must not be used. Flames are also disruptive to the airflow, which is critical for the proper function of the BSC, and they can damage the HEPA filters.

Alternatives to flame sterilization would include the use of pre-sterilized equipment and materials or flameless incinerating equipment.

 

  • Ultraviolet (UV) Lights: A Possible Burn Hazard

 

Some biosafety cabinets are equipped with UV lights. These lights (usually tubes that are purplish-black in color) are intended to be germicical but cannot be used as a stand-alone means of disinfection under most circumstances. They are a much greater burn/exposure hazard to personnel working in the immediate environment than an effective means of disinfection.

If your BSC is equipped with a UV light, know where the “ON/OFF” switch is and how it functions. Do not work in the BSC with the UV light on and do not work in close proximity (in the same culture room) to a BSC where the light is on.

 

BL2 – Personnel Protective Equipment (PPE) 

Remember that at biosafety level 2, the biological materials that you work with are assumed to be a biological exposure risk through:

  • Accidental ingestion
  • Contact with the mucous membranes (primarily through splashes to the eyes nose, or mouth)
  • Entry through broken skin (either through cuts with contaminated objects, contact with broken skin and possibly through prolonged contact with skin that appears to be undamaged)

Therefore, you must use PPE that is designed to effectively block the route of exposure that your procedures are likely to create.

PPE selection will vary depending on the nature of the work that you will be doing and the materials encountered. Even so, we will review the principles of PPE selection in this section.

 

PPE: Body Protection at BL-2

In lab applications and animal applications where anticipated contamination is minimal (i.e. work with diagnostic specimens, small rodents or birds), a lab coat is the minimum body protection that must be worn. In some research applications, disposable fluid-resistant coveralls may be warranted based on the risk assessment.


PPE: Eye & Face Protection at BL-2

Eye protection requirements at BL2 are essentially the same as discussed for BL1. Safety glasses are required at a minimum for all lab applications where work with chemical or biological materials is underway. Where procedures are likely to create a splash or spray of biological materials, goggles must be worn unless this will hinder your ability to safely execute the procedure.

Face shields should be considered to further protect against contamination of your face that can lead to accidental ingestion or mucous membrane exposure. However, a face shield is not a substitute for eye protection and must be worn in conjunction with eye protection.

 

PPE: Gloves at BL2

Glove use at BL2 is required whenever work with viable biological material is underway. Gloves must be changed when overtly contaminated or when the integrity of the glove is compromised. Dispose of gloves at BL2 as biohazardous waste.

 

Disposable gloves can degrade quickly. If your gloves are "sticky" or are discolored in the cuffs or fingertips, degradation has probably already started. Always inspect your gloves thoroughly before you begin working to minimize your exposure risk.

 

 

Biosafety Level 3: Facility Features

Biosafety Level 3 facilities and procedures are typically required for diagnostic or research work with agents that may cause serious or potentially lethal disease as a result of exposure by the inhalation route. With this said, the safety features of most common university labs will not meet BL3 containment requirements. In addition the facility features required for BL2, lab environments operating at BL3 must have the following features:

  • Access to the lab is restricted. The lab is separated from areas that are open to unrestricted flow within the building.
  • Passage through a series of two self-closing doors into lab from the access corridor is standard. (This creates a buffer zone that prevents air from drifting out of the lab directly into the corridor).
  • A ducted exhaust air ventilation system is provided that draws air from the "cleanest" area to the "dirtiest" area. Exhaust air is not recirculated to other parts of the building, and must be dispersed away from occupied areas and air intakes.

 

Note: There are currently no BL3 facilities on the UWO campus.

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