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					Biosafety Training

         Glenn McLean
         University Biosafety Officer
         Research and Biological Safety Office
         Environmental Health and Safety Department
         206-221-7770

         Phone:   206.543.7278
         Email:   glennm3@u.washington.edu
                          Why Biosafety?

1) Minimize Risk to lab Personnel and the Environment

   By 1978 Pike/Sulkin identified 4,079 laboratory-associated infections (LAI), 168
    deaths occurring between 1930-1978.
   Potential release to the environment - wild plants/animals, agriculture, livestock

2) It’s Required
   UW Administrative Policy Statement 12.3
   Washington State Bloodborne Pathogen Regulations
   Biosafety in Microbiological and Biomedical Laboratories
   NIH recombinant DNA guidelines


                                                                                     2
UW Administrative Policy Statement
               12.3

     Research projects involving biological hazards and
  recombinant DNA must be reviewed by the Institutional
                Biosafety Committee (IBC)




                                                          3
Washington State Bloodborne Pathogen Regulations
                  WAC 296-823

   Applies to all those working with HIV, Hepatitis B and C,
       human blood, tissue, body fluid with visible blood, cells
   Requires initial and annual training for the duration of work:
    http://www.ehs.washington.edu/psotrain/corsdesc.shtm
   A lab specific Exposure Control Plan must be in place:
    http://www.ehs.washington.edu/forms/rbs/researchlaboratorysupplementalform.doc
   Must be offered Hepatitis B vaccination within 10 days of
    starting work



                                                                                     4
NIH/CDC Biosafety in Microbiological
 and Biomedical Laboratories (BMBL)


         Introduced in 1984
         Now in its 5th Edition (2007)

         Must be followed for all research at the
         University of Washington regardless of
         funding source


                                                    5
                              BMBL
 General risk assessment information
 Description of Biosafety Levels 1-4
    (facility, equipment, practices)
 Agent Specific information
 Additional information regarding biosafety cabinets,
  decontamination, transportation, biosecurity, toxins,
  training, and more....


                                                          6
NIH Recombinant DNA Guidelines
Must be followed for all recombinant DNA research at the
  University of Washington regardless of funding source

 Identifies types of experiments covered and the level of
  oversight required (Section III)
 Roles and Responsibilities for NIH, UW, PI, IBC, BSO
  (Section IV)
 Classification of biological agents on the basis of hazard
  (virus, bacteria, parasites, fungus - Appendix B)
 Description of Biosafety Levels 1-4 (Appendix G,P,Q)

                                                               7
                 PI Responsibilities
    Submission
 Determine level of review required
 Submit information to the Institutional Biosafety
      Committee (IBC) for review and approval


Research Project hazard Assessment (RPHA) form:

   Required for new projects and every 3 years thereafter


                                                             8
                                                                                                 48.3.1/PI File



DATE:          November 13, 2008

TO:            John Doe Researcher, MD, PhD, DVM

FROM:          Glenn Mclean
               University Biosafety Officer

BUA:           1000-001-999 (Significant Change)                       IACUC Protocol#: 9999-01

SUBJECT:       (Project Title)

On behalf of the Institutional Biosafety Committee I have reviewed your Research Project Hazard Assessment for
biohazards. Based upon the information you provided I have approved research with the following agents if
conducted at the following biosafety levels and in the locations given:

                   Agent (none animal use)                   BSL Required               Building / Room
1. Human tissue, blood, body fluids, cells
2. Non-human primate tissue, blood, body fluids, cells              2                      xxy / S123
3. Lentiviral vectors, non-HIV pseudotyped, rep. deficient
4. Standard recombinant DNA techniques
                                                                    1                xxy / S123, S124, S130
      (e.g. non pathogenic E. coli)
                     Agent (use in mice)                    ABSL Required               Building / Room
5. Human cells transduced with agent #3                             2                 ABC vivarium / room
6. siRNA                                                            1                 ABC vivarium / room
                      Agent (use in rats)                   ABSL Required               Building / Room
7. Human cell lines (e.g. pluripotent stem cells from
                                                                    2                 XYZ vivarium / room
      human fibroblasts)
                                        Additional Requirements / Notes
 A) This revision adds the following:


                           EH&S USE ONLY – DO NOT WRITE IN THIS BOX
                                    Biological Safety Officer Approval
                                              Glenn Mclean
              Signature                             Name                                    Date

                                                                                                                  9
         PI Responsibilities - continued
    Prior to Initiation
   Instruct and train lab staff on:
       Lab specific biosafety practices
       spill / exposure procedures
       shipping requirements
   Inform lab staff of precautionary medical practices that are
    advised (e.g. vaccinations, etc.)



                                                                   10
       PI Responsibilities - continued
    While research is on-going
   Supervise to ensure the required safety practices are in use
   Investigate and report any significant problems, violations,
    accidents, or exposures (rbso@u.washington.edu , OARS )
   Remain in communication with the IBC to keep your
    biological use approval current (e.g. change in location, new
    agents). Submit new information to rbso@u.washington.edu .
   Insure no project is modified prior to IBC approval


                                                                    11
          Biological Safety Levels 1 – 4
                  recDNA guidelines: Appendix G
                       BMBL: Section IV



                 What do the levels describe?

   Standard microbiological practices
   Special practices (e.g. decontamination procedures, vaccinations)
   Safety equipment (e.g. containment devices, PPE)
   Lab facilities (e.g. benches, chairs, sinks)


                                                                        12
                      Biosafety Level 1

                      Well-characterized agents
Do not consistently cause disease in immunocompetent adult humans
Minimal potential hazard to laboratory personnel and the environment.



   E. coli-K12, Bacillus subtilis, canine hepatitis virus, murine cell lines




                                                                           13
                Biosafety Level 2

                  Well-characterized agents
    Moderate hazard to personnel and the environment.
Preventative and therapeutic interventions are often available



Measles virus, Human cells, Toxoplasma, Adenovirus, Lentiviral
                vectors (non-HIV pseudotyped)



                                                                 14
                  Biosafety Level 3
                  Indigenous or exotic agents
May cause serious or potentially lethal disease through inhalation
  Preventative and therapeutic interventions may be available

   BSL-2 plus:                   Additional training
                                 Lab specific Biosafety Manual
                                 Protective equipment (inhalation)
                                 Engineering and design features
                                 Increased oversight by BSO


        Anthrax, SARS, TB, St. Louis encephalitis virus
                                                                     15
                  Biosafety Level 4
                  Dangerous and exotic agents
        High individual risk of life-threatening disease
                     Aerosol transmission
Preventative / therapeutic interventions are not usually available



             None at the University of Washington


   Marburg Virus, Congo-Crimean hemorrhagic fever, Ebola
                                                                 16
What Equipment /Practices are needed?




                                    17
                          BSL-1 Practices
                     (Std. microbiological Practices)
   Access to the lab is limited      Fly screens recommended if
   Sink for handwashing when          windows are operable
    leaving lab                       Decontaminate at least daily, or
   Eating, drinking, smoking,         after spill
    handling contact lenses,          Bench tops are impervious to
    applying cosmetics, storing        water, resistant to heat /solvents /
    food is prohibited                 acids / alkalis
   No mouth pipetting                no carpets / rugs
   Lab coats recommended             No fabric furniture or chairs
   Minimize aerosols                 Surfaces accessible for cleaning

                                                                              18
Fabric Chair to be discarded
                               19
              BSL-1 Practices continued

                   Policy For Handling Sharps
Don’t   bend, break, recap, or remove needles from disposable syringes
Develop    and implement specific policies for safe handling
Adopt improved engineering and work practice controls that
   reduce the risk of injuries
Use   a red puncture resistant sharps container for disposal
Do    not handle broken glassware directly by hand.
Use   plastic labware when possible


                                                                          20
BSL-2 Containment and Practices

         BSL-1 plus additional:

         1)   Administrative controls
         2)   Safety equipment
         3)   Safe practices




                                        21
Administrative Controls

   Training
   Biohazard Signs

   UW Biosafety Manual




                          22
                 Training

   General Biosafety training (e.g. this class)
   Lab specific hands-on training by supervisor or PI




                                                         23
            Biohazard Warning Signs

Equipment
 (e.g. centrifuge, freezer, incubator)
Biohazard waste bags / containers
Transporters / Shippers




                                         24
             Biohazard Door Signs
        restrict access when work is in-process



-Name of the infectious agent

-Special entry requirements
   (minimum of lab coat when
   work is in-process)

-Name, phone # of PI or other
  responsible person




                                http://www.ehs.washington.edu/ftproot/biosign.doc
                                                                                    25
UW Biosafety Manual
 (must be available in the lab)




              Project Review Process
              Biohazard Control Procedures
              Occupational Health Program
              Bloodborne Pathogen Program




                                             26
      Safety Equipment

   Personal Protective Equipment (PPE)
   Biological Safety Cabinets
   Centrifuge Safety Cups / Sealed rotors
   Autoclaves




                                             27
  Personal Protective Equipment (PPE)


                                    Lab Coat
                                    Gloves
                                    Additional PPE to be
                                    determined by risk assessment




Note: Minimum of lab coat is required in work area when procedures are
                        in-process at BSL-2
                                                                         28
     Shields / Barriers




- Make sure PPE covers your street cloths -   29
Biological Safety Cabinet (BSC)




Aerosol generating activities at BSL-2 must be performed
         in a BSC or other containment device              30
Biological Safety Cabinet (BSC)

- Personnel Protection
- Product Protection
- Environmental Protection




                              31
        Biological Safety Cabinets
High Efficiency Particulate Air (HEPA) filters




        Not for use with heavy chemicals
           degrades sealant / gaskets            32
      Biosafety Cabinet Certification
       All BSCs on campus must be annually certified

                Test integrity of HEPA filters
                Test airflow rates




For more information:
http://www.ehs.washington.edu/fsobiocab/index.shtm

(Note: Paperwork required for purchasing new or relocating a BSC)
                                                                    33
   Protect the front air curtain, keep items off the grill
   Store equipment outside of the BSC
   Decontaminate in / Decontaminate out
                                                              34
   Use of flammable gas is restricted
       Avoid use of open flames
In-Line HEPA filters are recommended
             Chemical Fume Hood




Personnel protection, but no product or environmental protection
                                                                   36
              Laminar Flow Cabinet




Product Protection only. No Personnel or environmental protection   37
Centrifuge Safety Cups                             Sealed Rotors




   Required when centrifuging biological agents at BSL-2 outside of a
                           biosafety cabinet
                                                                        38
         Centrifuging Infectious Agents:

   Load /unload inside the BSC
   Insure rotor is properly balanced
   Know the speed ratings for rotor and tubes
   If unattended, post a warning sign with contact information
   Have an emergency plan in place, many lab associated
    infections have been linked to centrifuge use



                                                                  39
                               - If small enough -

                                Place in BSC
                         Use of Safety cups / sealed
                          rotors are not required.


option for older units that are not equipped with safety features
                                                                    40
                 Autoclaves
http://www.ehs.washington.edu/rbsbiosafe/autoclave.shtm




  All users must adhere to proper operating and
             monitoring procedures.
                     Autoclave Tape
Use on every item placed in the autoclave.
Indicates if temp. has been reached, but not duration




                                         untreated



                                         autoclaved


                                                         42
Chemical Integrators                 Biological Indicators

   Place a Thermalog-S                 Perform test at least monthly.
    integrator in center of each        Bacillus stearothermophilus (See
    autoclave load.                      UW Biosafety Manual Section IV pg 47
                                         for procedure)
   Attach a string to integrator
    for retrieval at end of cycle.
                                                                            43
               Temperature monitoring
   Operators must ensure minimum temperature of 250°F /
    121°C for at least ½ hour. ( > time/temp. may be required)
   Autoclave thermometer calibration at least annually



                    Structural Testing
   Must be inspected by a qualified pressure vessel inspector at
    least every two years

                                                                    44
Record Keeping for
Biohazardous Waste
 Maintain and post a user log

Date                     Type of container(s)
Time                     Closure on container(s)
Temperature              Pattern of loading
Pressure                 Water content
Type of waste            Maximum load quantity




        Complete the EH&S
       Quality Control Checklist
 http://www.ehs.washington.edu/forms/rbs/qcchecklist.doc




                                                           45
                       Visual Inspections
           Before loading and unloading the autoclave:
   Inspect biohazardous bags and
    containers for protruding sharps
    or lab glass
   Items with protruding sharps or
    lab glass must be placed in a
    secondary, leak-proof, container.




                                                         46
              Solid Biological Waste
 can be stored up to 14 days prior to decontamination



                                   NO




         NO



       Designate a space
avoid contaminating lab supplies                  YES


                                                        47
       Safe Practices
   Chemical Decontamination
   Pipetting
   Transportation
   Spills
   Exposures




                               48
Surfaces and Liquid Biohazardous Waste
Use an appropriate chemical decontaminant (see handout)


Points to Consider    Human Source Material – BBP

Concentration             Must be EPA registered against
                           Hepatitis B and HIV, or TB
Contact time
Stability             Example: Chlorine Bleach
Toxicity              1:10 spills
Corrosiveness         1:100 routine surface decontamination
Flammability
                       70% Ethanol alone does not meet BBP
                                   regulations
                                                              49
    Safe Pipetting

   Use pipette filters to prevent contamination of equipment
   Do not forcefully pipette up and down or blow out final volume
   Pipette down the side of wells, dishes, flasks
   Collect pipettes or micropipette tips in the BSC for disposal as
    contaminated biohazardous “lab glass” waste
   Recommended discard containers:




         Pipet keepers                         Bench top keepers
                                                                       50
     Transporting samples within the building
       Use a durable / leakproof secondary container


1)                                            2)




            Ziplock Bags


      Label with biohazard sign with
      contact information on container                      Coolers



     Decontaminate the outer container prior to leaving the lab
                                                                      51
 Shipping Biological Samples outside of the University
             Highly regulated (IATA, 49 CFR, State Regulations)
                     Specific shipping containers required
          Additional packaging, absorbent materials, labels, paperwork




Shippers must attend EH&S Class: Shipping and Transporting hazardous Materials
                                                                         52
        Transporting biohazardous waste
   Materials must be decontaminated prior to disposal
   Materials must be placed in a durable, leakproof container for
       transport from the lab.




                                                                     53
          Biological Spills
http://www.ehs.washington.edu/rbsbiosafe/spillbiokit.shtm

          Have a plan for your specific lab

                    Include:
             Chemical decontaminants
               Absorbent material
                Additional PPE
                Tongs / Forceps
              Biohazard waste bags




                                                            54
               Incidents / Exposures

Lifethreatening – Dial 911
Wash area immediately and thoroughly (15 minutes)
Contact Supervisor / Manager
Seek Care:
       UW Employee Health Clinic 685 1026 - UWMC ER
       Harborview Employee Health 731 3081 – Harborview ER
Report using the On-line Accident Reporting System (OARS)
        http://www.ehs.washington.edu/ohsoars/index.shtm#accidents




                                                                     55
          Animal Biosafety Level

BSL - 1   >>>>>>>   ABSL- 1
BSL - 2   >>>>>>>   ABSL- 2
BSL - 3   >>>>>>>   ABSL- 3
BSL- 4    >>>>>>>   ABSL- 4 (None at UW)




                                           56
              Additional Risks


 Animal sharps (teeth, fangs, claws, beaks, etc.)
 Size and weight of the animal

 Zoonotic diseases( e.g. Herpes B)

 Will an administered infectious agent
  potentially be shed from the animal?



                                                     57
              RPHA: Appendix A
            Hazardous material information for
                 animal husbandry staff

 Projectcontact information
 Hazardous material identification

 Additional PPE/ procedures required

 Exposure procedures




                                                 58
Medical/Occupational Surveillance

      Vaccination recommendations
      respirator usage/fit testing

      Animal Allergies

      Animal use medical Screening
       form: AUMS



                                      59
             Biosafety Lab Inspections
              Supports biological approval by the IBC


By who:             University Biosafety Officer

When:               New projects, renewal, significant changes

How do I Prepare:   Request a BSL-1 or BSL-2 checklist

What to expect:     Assess current facility and practices
                    Discuss research needs, compliance options
                    Provide additional training if necessary

Follow-up           E-mail summary to PI / lab manager
                    2nd inspection may be required
Common Deficiencies From Inspections
                (Increasing risk of exposure)

  Centrifuge safety cups not in use
  Lab coats not in use or designated for lab

  Updated Biosafety Manual needed (2003)

  Improper biohazardous waste disposal process

  Fabric upholstered chairs present in the lab

  Food present in the lab

  No Biohazard door sign




                                                  61
    How To Minimize Your Exposure Risk
    Current lab specific risk assessment
    Use your safety equipment & PPE (know benefits & limitations)
    Stay safety conscious
        Avoid drift of practices (Most convenient is usually not the safest)
    Plan ahead /stay well organized
    Address additional issues that may increase susceptibility
        Immuno-compromised, skin rashes, cuts, pregnancy
    Contact an EH&S Biosafety Officer or Occupational Health Nurse
       with questions 206 221 7770


                                                                               62

				
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