Emergency Preparedness Lab Safety Training Fire Prevention Construction Safety Exposure Assessment Environmental
EHS Home Biosafety Manual Part 2

Biosafety Manual

1992; Revised 1999
  1. POLICY
    1. Purpose
    2. Scope
    3. Responsibilities
  2. GUIDELINES
    1. General Introduction
    2. General Procedures to Minimize Exposure
    3. Classification of Agents
    4. Biosafety Levels (BSL)
      Summary Table
      BSL 1
      BSL 2
      BSL 3
      BSL 4
      Experimentally or Naturally Infected Vertebrate Animals
    5. Working With Human Tissues
    6. Cell Culture
    7. Recombinant DNA Research
    8. Gene Therapy
    9. Transgenics
    10. Selection of Biological Safety Cabinets
      Types of Cabinets
    11. Use of Biological Safety Cabinets
      Certification
    12. Personnel Exposure Control Plans / Procedures
    13. Training
    14. Personal Protective Equipment
    15. Record Keeping
    16. Disinfection and Sterilization
    17. Spill Procedures
    18. Disposal Procedures
    19. Shipment
  3. REFERENCES

  4. Appendix 1 Points of Contact
  5. Appendix 2 Bloodborne Pathogen Exposure Decision tree
  6. Appendix 3 BBP Exposure Incident Report
  7. Appendix 4 Hepatitis B Vaccination Declination
I. POLICY
  1. Purpose
    The purpose of this manual is to specify controls and safe handling practices for microorganisms (viruses, bacteria, fungi, rickettsia, mycoplasma, protozoans, multicellular parasites, and prions), toxins, recombinant DNA molecules, human blood or tissues, and animal cell cultures.

  2. Scope
    This written program applies to all University of Utah research performed at the medical center, main campus or at off campus facilities. Students are covered as well as part and full time employees.

  3. Responsibilities
    1. Institutional Biosafety Committee (IBC) shall:
      1. Meet as required for review of recombinant DNA (r-DNA) research in accordance with National Institutes of Health (NIH) guidelines.
      2. Review this Biosafety Manual and the current inventory of r-DNA activity. In the event of any significant violations or accidents, they shall report the incident to the NIH Office of Recombinant DNA Activities.
      3. Determine the necessity for health surveillance and prophylaxis for research projects.
    2. Environmental Health and Safety shall:
      1. Provide consultation and technical information on the safe handling of biological agents and toxins.
      2. Be responsible for updating the University Biosafety Manual.
      3. Coordinate and provide oversight for the annual certification of biological safety cabinets by an outside contractor.
      4. Review and recommend purchases of biological safety cabinets and other related safety equipment.
      5. Advise in the disinfection of facilities and equipment.
      6. Assist in the development of safety and exposure control plans and training programs.
    3. Principal Investigators and/or Laboratory Supervisors shall:
      1. Receive approval from the IBC prior to conducting r-DNA research.
      2. Register potentially infectious agents with Environmental Health and Safety.
      3. Maintain and annually review laboratory specific standard operating procedures (e.g., bloodborne pathogen exposure control plan).
      4. Ensure their research laboratory staff and students are trained on the contents of this program and follow its requirements.
      5. Survey laboratories for compliance with standards and policies regarding safe handling and use of biological agents and toxins.
      6. Enforce compliance with the approved standards and policies of the University.
      7. Encourage employees to report any changes in their health status.
      8. As applicable, advise the r-DNA committee, Institutional Review Board for Research with Human Subjects (IRB), Institutional Animal Care and Use Committee (IACUC), and EHS of any significant changes in approved protocol involving use of biological agents and/or toxins.
      9. Comply with shipping requirements for biohazardous substances and toxins.
    4. Researchers, Technicians and Students will:
      1. Adhere to the established policies, SOP's, and guidelines for biological safety as trained.
      2. Inform immediate supervisor of any unsafe practice or conditions in the work area.
      3. Report any change in health status to the supervisor if there is a possibility it may be work related.
      4. Report all biological spills and incidents to the supervisor.
II. GUIDELINES

  1. General Introduction
    1. Eating, drinking, smoking, or storage of foods must not be permitted in the laboratory.
    2. Personnel must wash their hands after handling infectious material, removal of gloves, and before leaving the laboratory.
    3. Control the biohazard area.
      1. Keep laboratory doors and windows closed while work is in progress.
      2. Post a warning sign, such as the universal biohazard symbol, when infectious material is present in the area. This warning sign should identify the agent and indicate the requirements for entry.
      3. Limit access to the laboratory during procedures involving biohazardous agents.

  2. General Procedures to Minimize Exposure
    1. Aerosols
      1. Aerosols refers to liquid droplets or solid particulates dispersed in air. Aerosols are too small a particle to see by the unaided eye and remain suspended for a period of time. The production of aerosols while handling infectious agents accounts for the greatest source of laboratory-acquired infections.
      2. Generation of aerosols may be caused in the use of centrifuges, blenders, shakers, magnetic stirrers, sonicators, pipettes, vortex mixers, syringes and needles, freeze-dried samples, vacuum sealed samples, mortar and pestles, culture tubes, inoculating loops, and separatory funnels.
      3. Control of aerosols
        1. Perform activities in a biological safety cabinet; or chemical fume hood when appropriate.
        2. Keep tubes stoppered when vortexing or centrifuging.
        3. Allow aerosols to settle prior to opening centrifuge, blender, or mixed tubes.
        4. Place cloth soaked with disinfectant over work surface to deactivate possible spills or droplets of biohazardous agents. Soaked gauze can be wrapped around ampoules while breaking, needles while being removed from a vial, or stoppers being removed from tubes.
        5. Slowly reconstitute or dilute contents of an ampoule.
        6. Mix solutions by discharging the secondary fluid down the side of the container or as close as possible to the surface of the primary solution.
        7. Allow inoculating needle to cool before touching biological specimens.
    2. Pipetting
      1. Never mouth pipette.
      2. No infectious mixture should be prepared by bubbling air through the liquid with the pipette.
      3. No infectious materials should be forcibly discharged from pipettes.
    3. Syringes and needles
      1. Avoid the use of syringes and needles if possible. Use the needle-locking type or a disposable syringe needle unit.
      2. Needles should not be re-sheathed,bent, broken or removed from disposable syringes. Needles and syringes should be discarded in biosafety labeled sharps containers for later autoclaving. Do not discard needles into disinfectant pans containing pipettes or other glassware; they must be sorted from the syringes and needles.

  3. Classification of Agents on the Basis of Hazard

    These agents as listed by the Centers for Disease Control and Prevention (CDC) are those biological agents known to infect humans as well as selected animal agents that may pose theoretical risks if inoculated into humans. Included are lists of representative genera and species known to be pathogenic; mutated, recombined, and non-pathogenic species and strains are not considered. Non-infectious life cycle stages of parasites are excluded. This is a list of the more commonly encountered agents and is not meant to be all inclusive. They are divided into Risk Groups which correspond to the equivalent Biosafety Level. Another source of information to assist in hazard determination are Material Safety Data Sheets provided by the Health Canada Laboratory Centre for Disease Control.

    1. Risk Group 1 (RG1) Agents

      2. RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents include asporogenic Bacillus subtilis or Bacillus licheniformis, Escherichia coli-K12, adeno-associated virus types 1 through 4, and recombinant AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus.

      Those agents not listed in Risk Groups (RG's) 2, 3 and 4 are not automatically or implicitly classified in RG1; a risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed.

    2. Risk Group 2 (RG2) Agents

      RG2 agents are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available.

      1. Bacterial Agents including Chlamydia
        - Acinetobacter baumannii (formerly Acinetobacter calcoaceticus)
        --Actinobacillus
        --Actinomyces pyogenes         (formerly Corynebacterium pyogenes )
        --Aeromonas hydrophila
        --Amycolata autotrophica
        --Archanobacterium haemolyticum         (formerly Corynebacterium haemolyticum )
        --Arizona hinshawii - all serotypes
        --Bacillus anthracis
        --Bartonella henselae, B. quintana, B. vinsonii
        --Bordetella including B. pertussis
        --Borrelia recurrentis, B. burgdorferi
        --Burkholderia         (formerly Pseudomonas species) except those listed in RG3)
        --Campylobacter coli, C. fetus, C. jejuni
        --Chlamydia psittaci , C. trachomatis , C. pneumoniae
        --Clostridium botulinum , Cl. chauvoei, Cl. haemolyticum, Cl. histolyticum, Cl. novyi, Cl. septicum, Cl. tetani
        --Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale
        --Dermatophilus congolensis
        --Edwardsiella tarda
        --Erysipelothrix rhusiopathiae
        --Escherichia coli         - all enteropathogenic, enterotoxigenic, enteroinvasive and strains bearing K1 antigen, including E. coli O157:H7
        --Haemophilus ducreyi, H. influenzae
        --Helicobacter pylori
        --Klebsiella         - all species except K. oxytoca (RG1)
        --Legionella including L. pneumophila
        --Leptospira interrogans         - all serotypes
        -- Listeria
        --Moraxella
        --Mycobacterium        except those listed in Appendix B-III-A (RG3)) including M.avium complex, M. asiaticum, M. bovis BCG vaccine strain, M. chelonei, M. fortuitum, M. kansasii, M. leprae, M. malmoense, M. marinum, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi
        --Mycoplasma ,         except M. mycoides and M. agalactiae which are restricted animal pathogens
        --Neisseria gonorrhoeae , N. meningitidis
        --Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. transvalensis
        --Rhodococcus equi
        --Salmonella         including S. arizonae, S. cholerasuis, S. enteritidis, S. gallinarum-pullorum, S. meleagridis, S. paratyphi, A, B, C, S. typhi , S. typhimurium
        --Shigella         including S. boydii, S. dysenteriae, type 1, S. flexneri, S. sonnei
        --Sphaerophorus necrophorus
        --Staphylococcus aureus
        --Streptobacillus moniliformis
        --Streptococcus including S. pneumoniae, S. pyogenes
        --Treponema pallidum, T. carateum
        --Vibrio cholerae, V. parahemolyticus, V. vulnificus
        --Yersinia enterocolitica
      2. Fungal Agents
        --Blastomyces dermatitidis
        --Cladosporium bantianum, C. (Xylohypha) trichoides
        --Cryptococcus neoformans
        --Dactylaria galopava (Ochroconis gallopavum)
        --Epidermophyton
        --Exophiala (Wangiella) dermatitidis
        --Fonsecaea pedrosoi
        --Microsporum
        --Paracoccidioides braziliensis
        --Penicillium marneffei
        --Sporothrix schenckii
        --Trichophyton
      3. Parasitic Agents
        --Ancylostoma human hookworms including A. duodenale, A. ceylanicum
        --Ascaris         including Ascaris lumbricoides suum
        --Babesia         including B. divergens, B. microti
        --Brugia filaria worms         including B. malayi, B. timori
        --Coccidia
        --Cryptosporidium including C. parvum
        --Cysticercus cellulosae (hydatid cyst, larva of T. solium )
        --Echinococcus including E. granulosis, E. multilocularis, E. vogeli
        --Entamoeba histolytica
        --Enterobius
        --Fasciola including F. gigantica, F. hepatica
        --Giardia including G. lamblia
        --Heterophyes
        --Hymenolepis including H. diminuta, H. nana
        --Isospora
        --Leishmania including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana, L. peruvania, L. tropica
        --Loa loa filaria worms
        --Microsporidium
        --Naegleria fowleri
        --Necator human hookworms including N. americanus
        --Onchoerca filaria worms including, O. volvulus
        --Plasmodium including simian species, P. cynomologi, P. falciparum, P. malariae, P. ovale, P. vivax
        --Sarcocystis including S. sui hominis
        --Schistosoma including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi
        --Strongyloides including S. stercoralis
        --Taenia solium
        --Toxocara including T. canis
        --Toxoplasma including T. gondii
        --Trichinella spiralis
        --Trypanosoma including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cruzi
        --Wuchereria bancrofti filaria worms
      4. Viruses
        Adenoviruses, human - all types
        Alphaviruses (Togaviruses) - Group A Arboviruses
        --Eastern equine encephalomyelitis virus
        --Venezuelan equine encephalomyelitis vaccine strain TC-83
        --Western equine encephalomyelitis virus
        Arenaviruses
        --Lymphocytic choriomeningitis virus (non-neurotropic strains)
        --Tacaribe virus complex
        --Other viruses as listed in the reference source
        Bunyaviruses
        --Bunyamwera virus
        --Rift Valley fever virus vaccine strain MP-12
        --Other viruses as listed in the reference source
        Calciviruses
        Coronaviruses
        Flaviviruses (Togaviruses) - Group B Arboviruses
        --Dengue virus serotypes 1, 2, 3, and 4
        --Yellow fever virus vaccine strain 17D
        --Other viruses as listed in the reference source
        Hepatitis A, B, C, D, and E viruses
        Herpesviruses - except Herpesvirus simiae (Monkey B virus) (see RG4 -Viral Agents )
        --Cytomegalovirus
        --Epstein Barr virus
        -- Herpes simplex types 1 and 2
        -- Herpes zoster
        --Human herpesvirus types 6 and 7
        Orthomyxoviruses
        --Influenza viruses types A, B, and C
        --Other tick-borne orthomyxoviruses as listed in the reference source
        Papovaviruses
        --All human papilloma viruses
        Paramyxoviruses
        --Newcastle disease virus
        --Measles virus
        --Mumps virus
        --Parainfluenza viruses types 1, 2, 3, and 4
        --Respiratory syncytial virus
        Parvoviruses
        --Human parvovirus (B19)
        Picornaviruses
        --Coxsackie viruses types A and B
        --Echoviruses - all types
        --Polioviruses - all types, wild and attenuated
        Prions
        --Transmissible spongioform encephalopathies (TME) agents (Creutzfeldt-Jacob disease and kuru agents)
        Retroviruses
        --Human immunodeficiency virus (HIV) types 1 and 2
        --Human T cell lymphotropic virus (HTLV) types 1 and 2
        --Simian immunodeficiency virus (SIV)
        Rhabdoviruses
        --Vesicular stomatitis virus

    3. Risk Group 3 (RG3) Agents
      RG3 agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available.

      1. Bacterial Agents Including Rickettsia
        --Bartonella
        --Brucella including B. abortus, B. canis , B. suis
        --Burkholderia (Pseudomonas) mallei, B. pseudomallei
        --Coxiella burnetii
        --Francisella tularensis
        --Mycobacterium bovis (except BCG strain), M. tuberculosis
        --Pasteurella multocida type B -"buffalo" and other virulent strains
        --Rickettsia akari, R. australis, R. canada, R. conorii, R. prowazekii, R. rickettsii, R, siberica, R. tsutsugamushi, R. typhi (R. mooseri)
        --Yersinia pestis

      2. Fungal Agents
        --Coccidioides immitis (sporulating cultures; contaminated soil)
        --Histoplasma capsulatum, H. capsulatum var.. duboisii

      3. Parasitic Agents
        None
      4. Viruses and Prions
        Alphaviruses (Togaviruses) - Group A Arboviruses
        --Semliki Forest virus
        --St. Louis encephalitis virus
        --Venezuelan equine encephalomyelitis virus (except the vaccine strain TC-83, see RG2)
        --Other viruses as listed in the reference source
        Arenaviruses
        --Flexal
        --Lymphocytic choriomeningitis virus (LCM) (neurotropic strains)
        Bunyaviruses
        --Hantaviruses including Hantaan virus
        --Rift Valley fever virus
        Flaviviruses (Togaviruses) - Group B Arboviruses
        --Japanese encephalitis virus
        --Yellow fever virus
        --Other viruses as listed in the reference source
        Poxviruses
        --Monkeypox virus
        Prions
        --Transmissible spongioform encephalopathies (TME) agents (Creutzfeldt-Jacob disease and kuru agents)(see the reference source)
        Retroviruses
        --Human immunodeficiency virus (HIV) types 1 and 2
        --Human T cell lymphotropic virus (HTLV) types 1 and 2
        --Simian immunodeficiency virus (SIV)
        Rhabdoviruses
        --Vesicular stomatitis virus

    4. Risk Group 4 (RG4) Agents
      RG4 agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available. The University of Utah does not have containment facilities that support BSL4 research.

      1. Bacterial Agents
        None
      2. Fungal Agents
        None
      3. Parasitic Agents
        None
      4. Viral Agents
        Arenaviruses
        --Guanarito virus
        --Lassa virus
        --Junin virus
        --Machupo virus
        --Sabia
        Bunyaviruses (Nairovirus)
        --Crimean-Congo hemorrhagic fever virus
        Filoviruses
        --Ebola virus
        --Marburg virus
        Flaviruses (Togaviruses) - Group B Arboviruses
        --Tick-borne encephalitis virus complex including Absetterov, Central European encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer encephalitis viruses
        Herpesviruses (alpha)
        --Herpesvirus simiae (Herpes B or Monkey B virus)
        Paramyxoviruses
        --Equine morbillivirus
        Hemorrhagic fever agents and viruses as yet undefined

    5. Animal Viral Etiologic Agents in Common Use
      The following list of animal etiologic agents is appended to the list of human etiologic agents. None of these agents is associated with disease in healthy adult humans; they are commonly used in laboratory experimental work.
      A containment level appropriate for RG1 human agents is recommended for their use. For agents that are infectious to human cells, e.g., amphotropic and xenotropic strains of murine leukemia virus, a containment level appropriate for RG2 human agents is recommended.
      Baculoviruses
      Herpesviruses
      --Herpesvirus ateles
      --Herpesvirus saimiri
      --Marek's disease virus
      --Murine cytomegalovirus
      Papovaviruses
      --Bovine papilloma virus
      --Polyoma virus
      --Shope papilloma virus
      --Simian virus 40 (SV40)
      Retroviruses
      --Avian leukosis virus
      --Avian sarcoma virus
      --Bovine leukemia virus
      --Feline leukemia virus
      --Feline sarcoma virus
      --Gibbon leukemia virus
      --Mason-Pfizer monkey virus
      --Mouse mammary tumor virus
      --Murine leukemia virus
      --Murine sarcoma virus
      --Rat leukemia virus
    6. Murine Retroviral Vectors
      Murine retroviral vectors to be used for human transfer experiments (less than 10 liters) that contain less than 50% of their respective parental viral genome and that have been demonstrated to be free of detectable replication competent retrovirus can be maintained, handled, and administered, under BL1 containment.
    1. Biosafety Levels (BSL)
      The four biosafety levels correspond directly to the four risk groups of microorganisms listed in Section C. The agents of minimal hazard are in Class or Biosafety Level (BSL) 1, with the more dangerous microorganisms in class or BSL4. The following descriptions were taken from Biosafety in Microbiological and Biomedical Laboratories.


      TABLE 1. Summary of recommended Biosafety Levels for Infectious Agents
      Level Practices and Techniques Safety Equipment Facilities
      1
      		 Standard Microbiological practices None: primary containment provided adherence to laboratory practices during open bench operations. Basic
      2
      		Level 1 practices plus: Laboratory coats; decontamination of all infectious wastes; limited access; protective gloves and biohazard warning signs as indicated. Partial Containment equipment (i.e., Class I or II Biological Safety Cabinets) used to conduct mechanical manipulative procedures that have high aerosol potential that may increase the risk of exposure to personnel. Basic
      3
      		 Level 2 practices plus: special laboratory clothing; controlled access. Partial containment used for all manipulations of infectious material. Containment
      4
      		 Level 3 practices plus: entrance through change room where street clothing is removed and laboratory clothing is put on; shower on exit; all wastes are decontaminated on exit from the facility. Maximum containment equipment (i.e., Class III biological safety cabinet or partial containment equipment in combination with full-body, air-supplied, positive-pressure personnel suit) used for all procedures and activities. Maximum Containment


      1. Biosafety Level 1 (Risk Group 1 Agents)
        Biosafety Level 1 is suitable for work involving agents of known or of minimal potential hazard to laboratory personnel and the environment. The laboratory is not separated from the general traffic patterns in the building. Work is generally conducted on open bench tops. Special containment equipment is not required or generally used. Laboratory personnel 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. The following standard and special practices, safety equipment, and facilities apply to agents assigned to Biosafety Level 1:
        1. Standard Microbiological Practices
          1. Access to the laboratory is limited or restricted at the discretion of the Principal Investigator/Supervisor when experiments are in progress.
          2. Work surfaces are decontaminated once a day and after any spill of viable material.
          3. All contaminated liquid or solid wastes are decontaminated before disposal.
          4. Technical pipetting devices are used; mouth pipetting is prohibited.
          5. Eating, drinking, smoking, and applying cosmetics are not permitted in the work area. Food may be stored in cabinets or refrigerators should be located outside of the work area.
          6. Persons wash their hands after they handle viable materials and animals and before leaving the laboratory.
          7. All procedures are performed carefully to minimize the creation of aerosols.
          8. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.
        2. Special Practices
          1. Contaminated materials that are to be decontaminated at a site away from the laboratory are placed in a durable leak-proof container which is closed before removed from the laboratory.
          2. An insect and rodent control program is in effect.
        3. Containment Equipment
          Special containment equipment is generally not required for manipulation of agents assigned to BSL 1.
        4. Laboratories Facilities
          1. The laboratory is designed so that it can be easily cleaned.
          2. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
          3. Laboratory furniture is sturdy. Spaces between benches, cabinets. And equipment are accessible for cleaning.
          4. Each laboratory contains a sink for hand washing.
          5. If the laboratory has windows that open, they are fitted with fly screens.
      2. Biosafety Level 2 (Risk Group 2 Agents)
        Biosafety Level 2 is similar to Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs in that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists, (2) access to the laboratory is limited when work is being conducted, and (3) certain procedures in which infectious aerosols are created are conducted in biological safety cabinets or other physical containment equipment. The following standard and special practices safety equipment, and facilities apply to agents assigned to BSL 2.
        1. Standard Microbiological Practices
          1. Access to the laboratory is limited or restricted by the Principal Investigator/Supervisor when work with infectious agents is in progress.
          2. Work surfaces are decontaminated at least once a day and after any spill of viable material.
          3. All infectious liquid or solids wastes are decontaminated before disposal.
          4. Mechanical pipetting devices are used; mouth pipetting is prohibited.
          5. Eating, drinking, smoking, and applying cosmetics are not permitted in the work area. Food may be stored in cabinets or refrigerators designated and used for this purpose only. Food storage cabinets or refrigerators should be located outside of the work area.
          6. Persons wash their hands after handling infectious materials and animals when they leave the laboratory.
          7. All procedures are performed carefully to minimize the creation of aerosols.
        2. Special Practices
          1. Contaminated materials that are to be decontaminated at a site away from the laboratory are placed in a durable leak-proof container which is closed before being removed from the laboratory.
          2. The Principal Investigator/Supervisor limits access to the laboratory. In general, persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. The director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.
          3. The Principal Investigator/Supervisor establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet any specific entry requirements (e.g., immunization) enter the laboratory or animal rooms.
          4. When the infectious agent(s) in use in the laboratory require special provisions for entry (e.g., vaccination), a hazard warning sign, incorporating the universal biohazard symbol, is posted on the access door to the laboratory work area. The hazard warning sign identifies the infectious agent, lists the name and telephone number of the Principal Investigator/Supervisor or other responsible person(s) for entering the laboratory.
          5. An insect and rodent control program is in effect.
          6. Laboratory coats, gowns, smocks,or uniforms are worn wile in the laboratory. Before leaving the laboratory for non-laboratory area (e.g., cafeteria, library, administrative offices), this protective clothing is removed and left in the laboratory or covered with a clean coat not used in the laboratory.
          7. Animals not involved in the work being performed are not permitted in the laboratory.
          8. Special are is taken to avoid skin contamination with infectious materials; gloves should be worn when handling infected animals and when the skin contact with infectious materials is unavoidable.
          9. All wastes from laboratories and animal rooms are appropriately decontaminated before disposal.
          10. Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for the injection or aspiration of infectious fluids. Extreme caution should be used when handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. Needles should not be bent, sheared, replaced in the sheath or guard or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse.
          11. Spills and accidents which result in overt exposures to infectious materials are immediately reported to the Principal Investigator/Supervisor. Medical evaluation, surveillance and treatment are provided as appropriate and written records are maintained.
          12. When appropriate, considering the agent(s) handled, baseline serum samples for laboratory and other at-risk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the facility.
          13. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read instructions on practices and procedures to follow them.
        3. Containment Equipment
          Biological safety cabinets(Class 1 or 2) or other appropriate personal protective or physical containment devices are used whenever:
          1. Procedures with a high potential for creating infectious aerosols are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intra-nasally, and harvesting infected tissues from animals or eggs.
          2. High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed heads or centrifuge safety cups are used and if they are opened only in a biological safety cabinet.
        4. Laboratory Facilities
          1. The laboratory is designed so that it can easily be cleaned.
          2. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
          3. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.
          4. Each laboratory contains a sink for hand washing.
          5. If the laboratory has windows that open, they are fitted with fly screens.
          6. An autoclave for decontaminating infectious laboratory wastes is available.
      3. Biosafety Level 3 (Risk Group 3 Agents)
        Biosafety Level 3 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 as a result of exposure by the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the manipulation of infectious material are conducted within biological safety cabinets or other physical containment devices of by personnel wearing appropriate personal protective clothing and devices. The laboratory has special engineering and design features. It is recognized, however, that many existing facilities may not have all the facility safeguards recommended for BSL 3 (e.g., access zone, sealed penetrations, and directional airflow,etc.). In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in laboratories where facility features satisfy BSL 2 recommendations provided the recommended "Standard Microbiological Practices," "Special Practices," and "Containment Equipment" for BSL3 are rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made only by the Principal Investigator.

        The following standard and special safety practices, equipment, and facilities apply to agents assigned to BSL3:

        1. Standard Microbiological Practices
          1. Work surfaces are decontaminated at least once a day and after any spill of viable material.
          2. All infectious liquid or solid wastes are decontaminated before disposal.
          3. Mechanical pipetting devices are used; mouth pipetting is prohibited.
          4. Eating, drinking, smoking, storing food, and applying cosmetics are not permitted in the work area.
          5. Persons wash their hands after handling infectious materials and animals and when they leave the laboratory.
          6. All procedures are performed carefully and accurately.
        2. Special Practices
          1. Laboratory doors are kept closed when experiments are in progress.
          2. Contaminated materials that are to be decontaminated at a site away from the laboratory are placed in a durable, leak-proof container which is closed before being removed from the laboratory.
          3. The Principal Investigator/Supervisor controls access to the laboratory and restricts access to persons whose presence is required for program or support purposes. Persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. The director has the final responsibility for assessing each circumstance and determinating who may enter or work in the laboratory.
          4. The Principal Investigator/Supervisor establishes policies and procedures whereby only persons who have been advised of the potential biohazard, who meet any specific entry requirements (e.g., immunization), and who comply with all entry and exit procedures enter the laboratory or animal rooms.
          5. When infectious materials or infected animals are present in the laboratory or containment module, a hazard warning sign, incorporating the universal biohazard symbol, is posted on all laboratory and animal room access doors. The hazard warning sign identifies the agent, lists the name and telephone number of the Principal Investigator/Supervisor or other responsible person(s), and indicates any special requirements for entering the laboratory, such as the need for immunizations, respirators, or other personal protective measures.
          6. All activities involving infectious materials are conducted in biological safety cabinets or other physical containment devices within the containment module. No work in open vessels is conducted on the open bench.
          7. The work surfaces of biological safety cabinets and other containment equipment are decontaminated when work with infectious materials is finished. Plastic-baked paper toweling used on non-perforated work surfaces within biological safety cabinets facilitates clean-up.
          8. An insect or rodent control program is in effect.
          9. Laboratory clothing that protects street clothing (e.g., solid front or wrap-around gowns, scrubs, suits, coveralls) is worn in the laboratory. Laboratory clothing is not worn outside the laboratory, and it is decontaminated before being laundered.
          10. Special care is taken to avoid skin contamination with infectious materials; gloves should be worn when handling infected animals and when skin contact with infectious materials is unavoidable.
          11. Molded surgical masks or respirators are worn in rooms containing infected animals.
          12. Animals and plants not related to the work being conducted are not permitted in the laboratory.
          13. All wastes from laboratories and animal rooms are appropriately decontaminated before disposal.
          14. Vacuum lines are protected with high efficiency particulate air (HEPA)filters and liquid disinfectant traps.
          15. Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for the infection or aspiration of infectious fluids. Extreme caution should be used when handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. Needles should not be bent, sheared, replaced in the sheet or guard or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse.
          16. Spills and accidents which result in overt or potential exposures to infectious materials are immediately reported to the Principal Investigator/Supervisor. Appropriate medical evaluation, surveillance, and treatment are provided and written records are maintained.
          17. Baseline serum samples for all laboratory and other at-risk personnel should be collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory.
          18. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read instructions on practices and procedures and to follow them.
        3. Containment Equipment
          Biological safety cabinets (Class 1, 2, or 3) or other appropriate combinations of personal protective or physical containment devices (e.g., special protective clothing, masks, gloves, respirators, centrifuge safety cups, sealed centrifuge rotors, and containment caging for animals) are used for all activities with infectious materials which pose a threat of aerosol exposure. These include: manipulation of cultures and of those clinical or environmental materials which may be a source of infectious aerosols; the aerosol challenge of experimental animals; harvesting of tissues or fluids from infected animals and embryonated eggs, and necropsy of infected animals.
        4. Laboratory Facilities
          1. The laboratory is separated from areas which are open to unrestricted traffic flow within the building. Passage through two sets of doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. Physical separation of the high containment laboratory from access corridors or other laboratories of activities may also be provided by a double-doored clothes change room (showers may be included), airlock, or other access facility which requires passage through two sets of doors before entering the laboratory.
          2. The interior surfaces of walls, floors, and ceilings are water resistant so that they can be easily cleaned. Penetrations in these surfaces are sealed or capable of being sealed to facilitate decontaminating the area.
          3. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
          4. Laboratory furniture is sturdy and spaces between benches, cabinets, and equipment are accessible for cleaning.
          5. Each laboratory contains a sink for hand washing. The sink is foot, elbow, or automatically operated and is located near the laboratory exit door.
          6. Windows in the laboratory are closed and sealed.
          7. Access doors to the laboratory or containment module are self-closing.
          8. An autoclave for decontaminating laboratory wastes is available, preferably within the laboratory.
          9. A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air into the laboratory through the entry areas. The exhaust air is not recirculated to any other area of the building, is discharged to the outside, and is dispersed away from occupied areas and air intakes. Personnel must verify that the direction of the airflow (into the laboratory) is proper. The exhaust air from the laboratory room can be discharged to the outside without being filtered or otherwise treated.
          10. The HEPA-filtered exhaust air from Class 1 or Class 2 biological safety cabinets is discharged directly to the outside or through the building exhaust system. Exhaust air from Class 1 or 2 biological safety cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every twelve months. If the HEPA-filtered exhaust air system, it is connected to this system in a manner (e.g., thimble unit connection) that avoids any interference with the air balance or the cabinets or building exhaust system.

      4. Biosafety Level 4 (Risk Group 4 Agents)
        Biosafety Level 4 is required for work with dangerous and exotic agents which pose a high individual risk of life-threatening disease. Members of the laboratory staff 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 competent scientists who are trained and experienced in working with these agents. Access to the laboratory is strictly controlled by the Principal Investigator/Supervisor. 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.

        The University of Utah does not have containment facilities that support BSL4 research.

      Table 2. Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally Infected Vertebrate Animals are Used.
      Level Practices and Techniques Safety Equipment Facilities
      1
      		 Standard animal care and management practices. None Basic
      2
      		 Laboratory coats; decontamination of all infectious wastes and of animal cages prior to washing; limited access; protective gloves and hazard warning signs as indicated. Partial containment equipment and/or personal protective devices used for activities and manipulations of agents or infected animals that produce aerosols. Basic
      3
      		 Level 2 practices plus: special laboratory clothing; controlled access. Partial containment and/or personal protective devices used for all activities and manipulations of all agents or infected animals. Containment
      4
      		 Level 3 practices plus: entrance through clothes change room where street clothing is removed and laboratory clothing is put on; shower on exit; all wastes are decontaminated before removal from the facility. Maximum containment equipment (i.e., Class 3 biological safety cabinet or partial containment equipment in combination with full-body, air-supplied positive-pressure personnel suit) used for all procedures and activities. Maximum Containment

    2. Working With Human Tissues
      Biosafety Level 2 practices and procedures must be followed when handling human blood, blood products, body fluids and tissues because of the infectious agents they may contain. This is consistent with the concept known as "Universal Precautions". The OSHA Bloodborne Pathogen Standard requires limiting exposure to blood and other potentially infectious materials since any exposure could result in transmission of bloodborne pathogens which could lead to disease or death. A site specific (laboratory, clinic, etc.) Exposure Control Plan must be developed and made readily available to all at-risk employees. The primary goal is to prevent transmission of Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), and other bloodborne pathogens. HBV vaccination is available at the Wasatch Clinic to all occupationally at-risk employees as well as students. Under no circumstances shall anyone work with cells derived from themselves or from first degree relatives since the host immune systems may not provide adequate protection.

      Training on safe handling of human blood, blood products, body fluids and tissues is mandatory. EHS coordinates periodic train-the-trainer sessions for Principal Investigators and Supervisors. The most recent schedule is posted on the EHS home page. An employee with no prior experience in handling human pathogens must be trained in the laboratory prior to handling infectious materials. Participation in work involving infectious agents will be allowed only after proficiency has been demonstrated to the satisfaction of the Principal Investigator or Laboratory Supervisor.

    3. Cell Culture
      The following must be handled at BSL2 or higher containment level:
      1. All cell lines of human/primate origin
      2. Any cell lines derived from lymphoid or tumor tissue
      3. All cell lines exposed to or transformed by any oncogenic virus
      4. All cell lines exposed to or transformed by amphotropic packaging systems
      5. All clinical material (e.g., samples of human tissues and fluids obtained after surgical resection or autopsy)
      6. All cell lines new to the laboratory (until proven to be free of all adventitious agents)
      7. All mycoplasma-containing cell lines
        The cell line must be classified at the same level as that recommended for the agent when cell cultures are known to contain an etiologic agent, an oncogenic virus or amphotropic packaging system.

    4. Recombinant DNA Research
      Recombinant DNA molecules are defined as either: (1) molecules that are constructed outside living cells by joining natural or syntehtic DNA segments to DNA molecules that can replicate in a living cell, or (2)DNA molecules that result from the replication of a moelcule described in (1).
      Synthetic DNA segments which are likely to yield a potentially harmful polynucleotide or polypeptide (e.g., a toxin or a pharmacologically active agent) are considered as equivalent to their natural DNA counterpart. If the synthetic DNA segment is not expressed in vivo as a biologically active polynucleotide or polypeptide product, it is exempt from the NIH Guidelines. Genomic DNA of plants and bacteria that have acquired a transposable element, even if the latter was donated from a recombinant vector no longer present, are not subject to the NIH Guidelines unless the transposon itself contains recombinant DNA.
      1. Federal Guidelines and Registering Experimental Protocols
        All research conducted at the University of Utah involving recombinant DNA molecules must meet current NIH guidelines. Some experimental protocols must be approved by the IBC and in special instances by a committee at the NIH or USDA as well. The principal investigator is responsible for determining the status of his/her experiments and filing the proper documents if review is required.
      2. Emergency Plans
        The NIH Guidelines instruct to prepare a set of emergency plans covering accidental spills and resulting personnel contamination for work involving rDNA. Section Q of this document constitutes a basic spill plan.

        Research that is carried out at physical containment level BL2 or higher requires the principal investigator prepare or adopt a biosafety manual. This manual may serve as the basis for preparing a more specific document.


    5. Human Gene Therapy
      All protocols involving the generation of r-DNA for human gene therapy must be approved by the IBC prior to submission to outside agencies and the initiation of experimentation. Prior approval by the IRB is required before commencing gene therapy in humans.

    6. Transgenics
      Investigators who create transgenic animals must complete a Biological Materials Registration Form and submit it to the IBC for approval. In addition, the protocol must receive approval from the IACUC.

      Experiments to genetically engineer plants by rDNA methods may require approval from the IBC. The NIH guidelines provide specific plant biosafety containment recommendations to prevent release of transgenic plant materials to the environment. Protocol must be registered with the IBC.

    7. Selection of Biological Safety Cabinets
      Biological Safety Cabinets serve as an effective primary barrier against biological or infectious agents by surrounding the immediate work area. It is the ideal complement to, not replacement for, careful work practice.

      The cabinets are equipped with High Efficiency Particulate Air (HEPA) filters which have 99.97% efficiency against 0.3 micron particles. HEPA filters offer no protection against volatiles, such as ether, alcohol, etc.

      Selection of the correct biologicalsafety cabinet is based on the classification of the agent, the associated biosafety level for the particular agent, and chemicals which will be used in the research.

      Table 3. Types of Cabinets
      CABINET OPERATIONS AND USES
      Horizontal Laminar Flow or Clean Bench Filtered air flow across the work surface toward te operator, providing a protection for the product, but not the worker. Do not use the infectious materials, toxic chemicals, sensitizing agents, or radionuclides.
      Class 1 Only the exhaust air is filtered, therefore protection is provided to the user and to the environment, but not to the experiment. The operator's hands and arms may be exposed to hazardous materials inside the cabinet. This cabinet may be used with low to moderate risk biological agents.
      Class 2 These have vertical laminar air flow with HEPA filtered supply and exhaust air. They protect the worker, the product, and the environment. For use with low to moderate risk biological agents.
      Class 2, Type A Recirculated 70% of the air inside the cabinet. Exhausts 30% into room after filtration. 75 fpm average face velocity. Do not use with volatile radionuclides or toxic chemicals.
      Class 2, Type B1 Recirculated 30% of the air inside the cabinet and exhausts the rest to the outside of the building. Maintains 100 fpm average velocity. Contaminated ducts are under negative pressure. May be used with minute amounts of volatiles.
      Class 2, B2 Referred to as Total Exhaust. No recirculation. 100% exhausted outside after filtration. Maintains 100 fpm average face velocity. All contaminated ducts are under negative pressure. Suitable where volatile toxic chemicals and radionuclides are required.
      Class 3, or Glove box Is gas-tight and maintained under negative air pressure. Used to work with highly infectious, carcinogenic, or hazardous materials. All operations are conducted through rubber gloves attached to entry portals.

Click here for the second half of the Biosafety Manual:
           Biosafety Manual Part K through References