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8. principles of biosafety, biocontainment & program management (nbb160)
 

8. principles of biosafety, biocontainment & program management (nbb160)

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    8. principles of biosafety, biocontainment & program management (nbb160) 8. principles of biosafety, biocontainment & program management (nbb160) Presentation Transcript

    • Biocontainment LaboratoryBiosafety & Biocontainment Training
    • Principles of Biosafety,Biocontainment& Program Management
    • Learning Outcomes
      On successful completion of the course, the
      participant will be able to:
      Discuss the components of the BMBL 5th edition
      Discuss characteristics of BSL- 1, BSL-2, BSL-3, and BSL-4 laboratories
      Discuss and demonstrate standard laboratory practices as applied in BSL- 1, BSL-2, BSL-3, and BSL-4 laboratories
    • What Do You Think?
      Is the laboratory a hazardous work environment?
      2. Do you work with hazardous materials, i.e. biological, chemical?
      Then, why do people choose to work without gloves, safety glasses, buttoned lab coats etc. in the laboratory?
    • What’s Wrong With This Picture?
    • Historical Perspective
    • Historical Perspective
      First Recorded Laboratory-associated Infections
      Disease/agent Transmission Year
      Typhoid fever (Salmonella typhi) unknown 1885
      Tetanus (Clostridium tetani) syringe 1893
      Cholera (Vibriocholerae) pipette 1894
      Brucellosis (Brucellamelitensis) syringe 1897
      Diphtheria (Corynebacteriumdiphtheriae) pipette 1898
      Glanders (Burkholderiamallei) syringe 1898
    • Historical Perspective
      Fort Detrick, MD
      • 1943 - Establishment of offensive biological weapons program
      • 1943 - Developed Class III BSC prototype
      • 1950 - Installed first Class I BSC
      • 1954 - First Biological Safety Conference
      • Dr Arnold Wedum - director of safety from 1944 to 1969
    • Historical Perspective
      Development of BMBL
      1969 - BW program shut down (Nixon)
      1974 - CDC Classification of Etiological Agents on the Basis of Hazard
      1974 - NIH Standards for Research Involving Oncogenic Viruses
      1976 - Recombinant DNA Guidelines
      1984 - CDC/NIH Biosafety in Microbiological and Biomedical Laboratories
    • Agent Laboratory-associated Infections
      Brucella sp. 507
      Coxiella burnetii 456
      Mycobacterium tuberculosis 417
      Hepatitis viruses 380
      Salmonella sp. 324
      Francisella tularensis 225
      Hantavirus 169
      Venezuelan equine
      encephalomyelitis virus 150
      (Influenza A2 virus) (15)
      Laboratory Infections: Most Frequent (1930-1999)
    • Laboratory Accidents
      Splashes, spills - 27%
      Needlesticks - 25%
      Cuts - 16%
      Animal bites, scratches - 14%
      Mouth pipetting - 13%
      Unknown/other - 5%
      Pike, 1976
    • Laboratory Hazards & Risk
      Hazards
      • Animals
      • Biologicals
      • Chemicals
      • Radiation
      • Procedures
      • Equipment
      • Facility
      • People
      Risk
      • Absorption
      • Ingestion
      • Inhalation
      • Injection
      Hazard & risk must be communicated to ALL staff!
    • Risk Assessment
      Contamination?
      Containment?
      The “risk assessment” is the backbone
      of safe practice!
    • Laboratory Safety Programs
      Biosafety Program
      • Knowledgeable supervisor
      • Knowledgeable personnel
      • Aware of potential hazards
      • Proficient in practices & techniques
      • Lab specific safety manual
      • Policies
      • Exposure control plans/programs
      • Emergency response
      • Safety committee(s)
    • Laboratory Safety Programs
      Training Program
      • Regulatory
      • Federal
      • State
      • Local
      • Institutional
      • Site specific
      • Work specific
      • Relevant
      Get training for what you ACTUALLY
      do not what others THINK you do!
    • Regulations
      • Occupational Safety and Health Act
      • BloodbornePathogens Standard
      • Possession, Use and Transfer of Select Agents and Toxins
      • US Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training
      • Transportation of Dangerous Goods Acts and Regulations
      • Hazardous Waste Management Standard
      • Occupational Exposure to Hazardous Laboratory Chemicals Standard
      • Radiation Protection Standard
    • CDC/NIH Guidelines
      BMBL
    • CDC/NIH Guidelines
      Biohazard
      An agent of biological origin that has the capacity to produce deleterious effects on humans, i.e. microorganisms, toxins and allergens derived from those organisms; and allergens and toxins derived from higher plants and animals.
    • CDC/NIH Guidelines
      Biohazard Classification: Biosafety Levels
      BSL1 Agents not known to cause disease
      BSL2 Agents associated with human disease
      BSL3 Indigenous/exotic agents associated
      with human disease and with potential
      for aerosol transmission
      BSL4 Dangerous/exotic agents of life
      threatening nature
    • CDC/NIH Guidelines
      Biosafety
      The application of combinations of laboratory practice and procedure, laboratory facilities, and safety equipment when working with potentially infectious microorganisms.
    • CDC/NIH Guidelines
      Laboratory Classification: Biosafety Levels
      Combinations of laboratory practice and procedure, laboratory facilities, and safety equipment to provide containment
      Low Containment
      Biosafety Level 1
      Biosafety Level 2
      Biosafety Level 3
      Biosafety Level 4
      High Containment
    • CDC/NIH Guidelines
      Containment
      • Safe methods for managing infectious materials to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents
    • CDC/NIH Guidelines
      Primary Containment
      • Protection of personnel and immediate laboratory environment from exposure to infectious agents
      • Accomplished by:
      • Good microbiological technique
      • Appropriate safety equipment
    • CDC/NIH Guidelines
      Secondary Containment
      • Protection of external environment from exposure to infectious materials
      • Accomplished by:
      • Facility design
      • Operational practices
    • CDC/NIH Guidelines
      Biosafety Components
      Practice and procedure
      • Standard
      • Special
      Safety equipment
      • Standard
      • Special
      Facilities
      • Design & Construction
      • Function
    • Biosafety Level 1 (BSL-1)
      Work with microorganisms not known to consistently cause disease in healthy adult humans
      • Basic level of containment:
      • Standard microbiological practices
      • No special primary or secondary barriers recommended (other than a sink for hand washing)
      • No special practices
    • Biosafety Level 1 (BSL-1)
      Design
      • Access controls
      • Sinks
      • Impervious bench tops
      • Easily cleaned surfaces
      • Adequate illumination
    • Biosafety Level 1 (BSL-1)
    • Biosafety Level 2 (BSL-2)
      • Work with moderate-risk microorganisms
      and agents (i.e. Hepatitis B virus, HIV,
      Salmonella, etc.)
      • Basic level of containment:
      • Standard microbiological practices
      • Secondary barriers (i.e. hand washing sinks, segregated waste, BSCs available
      • Special practices (i.e. immunization program, access controls)
    • Biosafety Level 2 (BSL-2)
      Design
      • Lockable doors
      • Sinks with hands-free operation
      • Impervious bench tops resistant to
      moderate heat & organic solvents
      • Easily cleaned surfaces
      • Eyewash available
      • BSCs installed properly; away from doors
    • Biosafety Level 2 (BSL-2)
    • Biosafety Level 3 (BSL-3)
      • Work with indigenous or exotic agents with
      potential for respiratory transmission; may
      cause serious or lethal infection (i.e.
      Mycobacterium tuberculosis, St. Louis
      encephalitis virus, and Coxiella burnetii)
      • Basic level of containment:
      • Standard microbiological practices
      • Secondary barriers (i.e. BSCs required, directional air flow, anti-room entry)
      • Special practices (i.e. restricted access, solid front lab coat, autoclave required)
    • Biosafety Level 3 (BSL-3)
      Design
      • Restricted access; lockable doors –
      emergency “open”
      • Sealed windows
      • Hands-free sink near door
      • Surfaces resistant to chemicals
      • Monolithic floor; sealed room penetrations
      • Ducted exhaust
      • Decontamination method available
    • Biosafety Level 3 (BSL-3)
    • Biosafety Level 4 (BSL-4)
      • Work with dangerous and exotic agents that
      pose a high individual risk of life-threatening
      disease; may be transmitted via aerosols; no
      available vaccine or therapy (i.e. Ebola, Marburg)
      • Basic level of containment:
      • Standard microbiological practices
      • Secondary barriers (i.e. BSCs, personal protective suits or Class III BSCs, directional air flow)
      • Special practices (i.e. isolated/restricted access facility, sealed rooms, treated waste)
    • Biosafety Level 4 (BSL-4)
      Types
      • Class III biosafety cabinets (cabinet line)
      • Personal protective suits (suit lab)
      Design
      • Isolated facility; separate building
      • Daily inspections of containment parameters
      • Restricted access
      • Changing rooms & showers
      • Double door autoclaves
      • All room penetrations sealed
    • Biosafety Level 4 (BSL-4)
      Design
      • Dedicated non-re-circulating ventilation
      • HEPA filtered supply and double HEPA
      filtered exhaust air
      • Occupants wear one-piece personal
      protection suits
      • Redundant systems
    • Biosafety Level 4 (BSL-4)
      Suit Lab
    • Biosafety Level 4 (BSL-4)
      Cabinet Line
    • Biosafety Level 4 (BSL-4)
    • Biosafety Level 4 (BSL-4)
      CDC Maximum Containment Laboratory
    • Animal Facilities
      ABSL 1-4
      Standard practices
      Special practices
      Safety equipment
      Facilities
    • Standard Lab Practices
      Standard Practices?
      Those practices which you should perform whenever you enter/exit the lab/animal area and during your work while in the lab/animal area.
    • Standard Lab Practices
      • Limit access
      • Wash hands frequently
      • No eating, drinking, etc.
      in lab
      • No mouth pipetting
      • Handle sharps carefully
      • Contain hazardous
      materials & aerosols
    • Standard Lab Practices
      • Decontaminate work areas
      • Decontaminate waste
      materials
      • Post hazard warning signs
      • Personnel must be trained
      • Wear PPE; leave in lab
      • Develop/follow SOPs
    • Exercise #1
      Margurita was moving a rhesus monkey to another room when she was splashed in the eyes by a fluid coming from inside the cage. She wiped it off and told her supervisor. He told her to continue work as it seemed to be a minor incident. Two weeks later her eye became inflamed and she developed a fever with severe headache. She reported to the ER, was admitted, treated with acyclovir and released. Ten days later she was readmitted with weakness in her legs which progressed to overall paralysis. She was placed on a respirator, lapsed into a coma, and died. Testing revealed Herpes B virus infection.
      Margurita was moving a rhesus monkey to another room when she was splashed in the eyes by a fluid coming from inside the cage. She wiped it off and told her supervisor. He told her to continue work as it seemed to be a minor incident. Two weeks later her eye became inflamed and she developed a fever with severe headache. She reported to the ER, was admitted, treated with acyclovir and released. Ten days later she was readmitted with weakness in her legs which progressed to overall paralysis. She was placed on a respirator, lapsed into a coma, and died. Testing revealed Herpes B virus infection.
    • Personal Protective Equipment
    • Personal Protective Equipment
    • What’s Wrong?
    • Exercise #2
      Jason was working alone on Sunday with Neisseriameningitidis, concentrating it with a bench-top centrifuge prior to aliquoting it into cryovials. Several days later, he developed a high fever and chills, reported to the ER, and was admitted. He lapsed into a coma and came within two hours of dying from an acute case of meningitis.
      Jason was working alone on Sunday with Neisseriameningitidis, concentrating it with a bench-top centrifuge prior to aliquotingit into cryovials. Several days later, he developed a high fever and chills, reported to the ER, and was admitted. He lapsed into a coma and came within two hours of dying from an acute case of meningitis.
    • Laboratory Infections
      1965 Survey
      • Fewer than 20% of all reported infections
      were associated with a known accident
      • Exposure to infectious aerosols considered
      to be plausible but unconfirmed source for
      more than 80% of reported cases
      All lab operations, including repairs, should be
      performed to reduce or eliminate aerosol production!
    • Aerosols
      Aerosol Generating Lab Operations
      • Pipetting
      • Mixing
      • Pouring
      • Vortexing
      • Centrifuging
      • Loading syringes
      • Flaming loops
      • Lasers, cell sorters
    • Aerosols
      Experiment: Take a culture with known concentration (1010/ml culture) back to your lab and perform various lab operations as carefully as you can for 10 min. Then, sample area around where the operation was performed and culture for contamination. (Dimmick, et. al. 1973)
      Results Organisms Recovered
      Blender, open 106
      Sonicator with bubbling 106
      Pipetting, vigorous 106
      Dropping culture 3 X 105
      Splash on a centrifuge rotor 105
      Blender, opened after 1 minute 2 X 104
      Pipetting, carefully 104
    • Class I
      Class III
      Class II
      Class II
      Type A1
      Class II
      Type A2
      Class II
      Type B2
      Class II
      Type B1
      Aerosol/fume Containment Devices
      Biological
      Safety
      Cabinet
      Fume Hood
    • Biological Safety Cabinets
    • Biological Safety Cabinets
      Class I
      Single pass air
      Worker protection; no product protection
      Exhausts to outside (w/wo HEPA filter)
      Class II
      Re-circulated air
      Worker/product/environmental protection
      Laminar flow
      Exhausts inside/outside (w/wo HEPA filter)
      Suitable for work with BSL2-4 agents
      Class III
      Totally enclosed; air-tight
      Exhausted outside thru HEPA filters
      Suitable for work with BSL3/4 agents
    • Intake
      100 ft/min
      Biological Safety Cabinets
      Exhaust
    • Biological Safety Cabinets
      HEPA (High Efficiency Particulate Air) Filter
    • Biological Safety Cabinets
      HEPA Filter - Particle Capture Efficiency
      Anthrax (1u)
      99.97
      Smallpox
      Tularemia (.3u)
      Collection Efficiency (%)
      0.01
      0.1
      1.0
      10.0
      0.3
      Particle Diameter (u)
    • Biological Safety Cabinets
      Inward Airflow = ~100 ft/min
    • LAMINAR FLOW
      DOOR OPENING
      PERSON WALKING
      HVAC VENT
      ROOM AC
      FPM 100 200 300 400 500 600 700 800
      MPH 1 2 3 4 5 6 7 8
      Biological Safety Cabinets
      Comparative Velocities
      Peters and Peters 1990
    • Biological Safety Cabinets
      Airflow, Hand Movements & Walk-by
    • Biological Safety Cabinets
      Basic Operation
      • Turn BSC on
      • Check magnehelic gauge
      • Load BSC with work materials
      • Allow BSC to run for 4-5 min
      • Perform work
      • Remove materials from BSC
      • Disinfect inside of BSC
    • Biological Safety Cabinets
      What’s Wrong?
    • Biological Safety Cabinets
    • Biological Safety Cabinets
    • Biological Safety Cabinets
    • Biological Safety Cabinets
      What’s Wrong?
    • Biological Waste
    • Biological Waste
    • Biological Emergencies
      Surface Contamination
      • Alert others
      • Remove glass shards
      • Cover spill; apply appropriate disinfectant, i.e. Clorox
      • Allow adequate contact time (20”)
      • Properly dispose of materials
      • Notify supervisor
    • Biological Emergencies
      Personal Contamination
      Alert others
      Remove contaminated clothing
      Flush affected area (15-20”)
      Apply first aid if needed
      Report to Clinic/ER
      Notify supervisor
    • What’s Wrong?
    • What’s Wrong?
    • What’s Wrong?
    • What’s Wrong?
    • What’s Wrong?
    • What’s Wrong?
    • What’s Wrong?
    • Laboratory Contamination
    • Standard Lab Practices
      • Limit access
      • Wash hands frequently
      • No eating, drinking, etc. in lab
      • No mouth pipetting
      • Handle sharps carefully
      • Contain hazardous materials & aerosols
    • Standard Lab Practices
      • Decontaminate work areas
      • Decontaminate waste materials
      • Post hazard warning signs
      • Personnel must be trained
      • Wear PPE; leave in lab
      • Develop/follow SOPs
    • THINK
      before
      you act!
      Safety Is As Safety Does!
    • Biocontainment LaboratoryBiosafety & Biocontainment Training