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

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  • 1. Biocontainment LaboratoryBiosafety & Biocontainment Training
  • 2. Principles of Biosafety,Biocontainment& Program Management
  • 3. 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
  • 4. 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?
  • 5. What’s Wrong With This Picture?
  • 6. Historical Perspective
  • 7. 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
  • 8. Historical Perspective
    Fort Detrick, MD
    • 1943 - Establishment of offensive biological weapons program
    • 9. 1943 - Developed Class III BSC prototype
    • 10. 1950 - Installed first Class I BSC
    • 11. 1954 - First Biological Safety Conference
    • 12. 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
  • 13. 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)
  • 14. Laboratory Accidents
    Splashes, spills - 27%
    Needlesticks - 25%
    Cuts - 16%
    Animal bites, scratches - 14%
    Mouth pipetting - 13%
    Unknown/other - 5%
    Pike, 1976
  • 15. Laboratory Hazards & Risk
    Hazards
    Risk
    Hazard & risk must be communicated to ALL staff!
  • 26. Risk Assessment
    Contamination?
    Containment?
    The “risk assessment” is the backbone
    of safe practice!
  • 27. Laboratory Safety Programs
    Biosafety Program
    • Knowledgeable supervisor
    • 28. Knowledgeable personnel
    • 29. Aware of potential hazards
    • 30. Proficient in practices & techniques
    • 31. Lab specific safety manual
    • 32. Policies
    • 33. Exposure control plans/programs
    • 34. Emergency response
    • 35. Safety committee(s)
  • Laboratory Safety Programs
    Training Program
    Get training for what you ACTUALLY
    do not what others THINK you do!
  • 43. Regulations
    • Occupational Safety and Health Act
    • 44. BloodbornePathogens Standard
    • 45. Possession, Use and Transfer of Select Agents and Toxins
    • 46. US Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training
    • 47. Transportation of Dangerous Goods Acts and Regulations
    • 48. Hazardous Waste Management Standard
    • 49. Occupational Exposure to Hazardous Laboratory Chemicals Standard
    • 50. Radiation Protection Standard
  • CDC/NIH Guidelines
    BMBL
  • 51. 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.
  • 52. 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
  • 53. CDC/NIH Guidelines
    Biosafety
    The application of combinations of laboratory practice and procedure, laboratory facilities, and safety equipment when working with potentially infectious microorganisms.
  • 54. 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
  • 55. 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
    • 56. Accomplished by:
    • 57. Good microbiological technique
    • 58. Appropriate safety equipment
  • CDC/NIH Guidelines
    Secondary Containment
    • Protection of external environment from exposure to infectious materials
    • 59. Accomplished by:
    • 60. Facility design
    • 61. Operational practices
  • CDC/NIH Guidelines
    Biosafety Components
    Practice and procedure
    Safety equipment
    Facilities
    • Design & Construction
    • 64. Function
  • Biosafety Level 1 (BSL-1)
    Work with microorganisms not known to consistently cause disease in healthy adult humans
    • Basic level of containment:
    • 65. Standard microbiological practices
    • 66. No special primary or secondary barriers recommended (other than a sink for hand washing)
    • 67. No special practices
  • Biosafety Level 1 (BSL-1)
    Design
    • Access controls
    • 68. Sinks
    • 69. Impervious bench tops
    • 70. Easily cleaned surfaces
    • 71. Adequate illumination
  • Biosafety Level 1 (BSL-1)
  • 72. Biosafety Level 2 (BSL-2)
    • Work with moderate-risk microorganisms
    and agents (i.e. Hepatitis B virus, HIV,
    Salmonella, etc.)
    • Basic level of containment:
    • 73. Standard microbiological practices
    • 74. Secondary barriers (i.e. hand washing sinks, segregated waste, BSCs available
    • 75. Special practices (i.e. immunization program, access controls)
  • Biosafety Level 2 (BSL-2)
    Design
    • Lockable doors
    • 76. Sinks with hands-free operation
    • 77. Impervious bench tops resistant to
    moderate heat & organic solvents
    • Easily cleaned surfaces
    • 78. Eyewash available
    • 79. BSCs installed properly; away from doors
  • Biosafety Level 2 (BSL-2)
  • 80. 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:
    • 81. Standard microbiological practices
    • 82. Secondary barriers (i.e. BSCs required, directional air flow, anti-room entry)
    • 83. 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
    • 84. Hands-free sink near door
    • 85. Surfaces resistant to chemicals
    • 86. Monolithic floor; sealed room penetrations
    • 87. Ducted exhaust
    • 88. Decontamination method available
  • Biosafety Level 3 (BSL-3)
  • 89. 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:
    • 90. Standard microbiological practices
    • 91. Secondary barriers (i.e. BSCs, personal protective suits or Class III BSCs, directional air flow)
    • 92. Special practices (i.e. isolated/restricted access facility, sealed rooms, treated waste)
  • Biosafety Level 4 (BSL-4)
    Types
    • Class III biosafety cabinets (cabinet line)
    • 93. Personal protective suits (suit lab)
    Design
    • Isolated facility; separate building
    • 94. Daily inspections of containment parameters
    • 95. Restricted access
    • 96. Changing rooms & showers
    • 97. Double door autoclaves
    • 98. All room penetrations sealed
  • Biosafety Level 4 (BSL-4)
    Design
    • Dedicated non-re-circulating ventilation
    • 99. 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
  • 100. Biosafety Level 4 (BSL-4)
    Cabinet Line
  • 101. Biosafety Level 4 (BSL-4)
  • 102. Biosafety Level 4 (BSL-4)
    CDC Maximum Containment Laboratory
  • 103. Animal Facilities
    ABSL 1-4
    Standard practices
    Special practices
    Safety equipment
    Facilities
  • 104. 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.
  • 105. Standard Lab Practices
    • Limit access
    • 106. Wash hands frequently
    • 107. No eating, drinking, etc.
    in lab
    • No mouth pipetting
    • 108. Handle sharps carefully
    • 109. Contain hazardous
    materials & aerosols
  • 110. Standard Lab Practices
    • Decontaminate work areas
    • 111. Decontaminate waste
    materials
    • Post hazard warning signs
    • 112. Personnel must be trained
    • 113. Wear PPE; leave in lab
    • 114. 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.
  • 115. Personal Protective Equipment
  • 116. Personal Protective Equipment
  • 117. What’s Wrong?
  • 118. 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.
  • 119. 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!
  • 120. Aerosols
    Aerosol Generating Lab Operations
  • 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
  • 128. 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
  • 129. Biological Safety Cabinets
  • 130. 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
  • 131. Intake
    100 ft/min
    Biological Safety Cabinets
    Exhaust
  • 132. Biological Safety Cabinets
    HEPA (High Efficiency Particulate Air) Filter
  • 133. 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)
  • 134. Biological Safety Cabinets
    Inward Airflow = ~100 ft/min
  • 135. 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
  • 136. Biological Safety Cabinets
    Airflow, Hand Movements & Walk-by
  • 137. Biological Safety Cabinets
    Basic Operation
    • Turn BSC on
    • 138. Check magnehelic gauge
    • 139. Load BSC with work materials
    • 140. Allow BSC to run for 4-5 min
    • 141. Perform work
    • 142. Remove materials from BSC
    • 143. Disinfect inside of BSC
  • Biological Safety Cabinets
    What’s Wrong?
  • 144. Biological Safety Cabinets
  • 145. Biological Safety Cabinets
  • 146. Biological Safety Cabinets
  • 147. Biological Safety Cabinets
    What’s Wrong?
  • 148. Biological Waste
  • 149. Biological Waste
  • 150. Biological Emergencies
    Surface Contamination
    • Alert others
    • 151. Remove glass shards
    • 152. Cover spill; apply appropriate disinfectant, i.e. Clorox
    • 153. Allow adequate contact time (20”)
    • 154. Properly dispose of materials
    • 155. 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
  • 156. What’s Wrong?
  • 157. What’s Wrong?
  • 158. What’s Wrong?
  • 159. What’s Wrong?
  • 160. What’s Wrong?
  • 161. What’s Wrong?
  • 162. What’s Wrong?
  • 163. Laboratory Contamination
  • 164. Standard Lab Practices
    • Limit access
    • 165. Wash hands frequently
    • 166. No eating, drinking, etc. in lab
    • 167. No mouth pipetting
    • 168. Handle sharps carefully
    • 169. Contain hazardous materials & aerosols
  • Standard Lab Practices
    • Decontaminate work areas
    • 170. Decontaminate waste materials
    • 171. Post hazard warning signs
    • 172. Personnel must be trained
    • 173. Wear PPE; leave in lab
    • 174. Develop/follow SOPs
  • THINK
    before
    you act!
    Safety Is As Safety Does!
  • 175. Biocontainment LaboratoryBiosafety & Biocontainment Training