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

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

  1. 1. Biocontainment LaboratoryBiosafety & Biocontainment Training<br />
  2. 2. Principles of Biosafety,Biocontainment& Program Management<br />
  3. 3. Learning Outcomes<br />On successful completion of the course, the<br />participant will be able to: <br />Discuss the components of the BMBL 5th edition<br />Discuss characteristics of BSL- 1, BSL-2, BSL-3, and BSL-4 laboratories<br />Discuss and demonstrate standard laboratory practices as applied in BSL- 1, BSL-2, BSL-3, and BSL-4 laboratories<br />
  4. 4. What Do You Think?<br />Is the laboratory a hazardous work environment?<br />2. Do you work with hazardous materials, i.e. biological, chemical?<br />Then, why do people choose to work without gloves, safety glasses, buttoned lab coats etc. in the laboratory? <br />
  5. 5. What’s Wrong With This Picture?<br />
  6. 6. Historical Perspective<br />
  7. 7. Historical Perspective<br />First Recorded Laboratory-associated Infections<br />Disease/agent Transmission Year<br />Typhoid fever (Salmonella typhi) unknown 1885<br />Tetanus (Clostridium tetani) syringe 1893<br />Cholera (Vibriocholerae) pipette 1894<br />Brucellosis (Brucellamelitensis) syringe 1897<br />Diphtheria (Corynebacteriumdiphtheriae) pipette 1898<br />Glanders (Burkholderiamallei) syringe 1898<br />
  8. 8. Historical Perspective<br />Fort Detrick, MD<br /><ul><li>1943 - Establishment of offensive biological weapons program
  9. 9. 1943 - Developed Class III BSC prototype
  10. 10. 1950 - Installed first Class I BSC
  11. 11. 1954 - First Biological Safety Conference
  12. 12. Dr Arnold Wedum - director of safety from 1944 to 1969</li></li></ul><li>Historical Perspective<br />Development of BMBL<br />1969 - BW program shut down (Nixon)<br />1974 - CDC Classification of Etiological Agents on the Basis of Hazard<br />1974 - NIH Standards for Research Involving Oncogenic Viruses<br />1976 - Recombinant DNA Guidelines<br />1984 - CDC/NIH Biosafety in Microbiological and Biomedical Laboratories<br />
  13. 13. Agent Laboratory-associated Infections<br />Brucella sp. 507<br />Coxiella burnetii 456<br />Mycobacterium tuberculosis 417<br />Hepatitis viruses 380<br />Salmonella sp. 324<br />Francisella tularensis 225<br />Hantavirus 169<br />Venezuelan equine <br /> encephalomyelitis virus 150<br />(Influenza A2 virus) (15)<br />Laboratory Infections: Most Frequent (1930-1999)<br />
  14. 14. Laboratory Accidents<br /> Splashes, spills - 27%<br /> Needlesticks - 25%<br /> Cuts - 16%<br /> Animal bites, scratches - 14%<br /> Mouth pipetting - 13%<br /> Unknown/other - 5%<br />Pike, 1976<br />
  15. 15. Laboratory Hazards & Risk<br />Hazards<br /><ul><li>Animals
  16. 16. Biologicals
  17. 17. Chemicals
  18. 18. Radiation
  19. 19. Procedures
  20. 20. Equipment
  21. 21. Facility
  22. 22. People</li></ul>Risk<br /><ul><li> Absorption
  23. 23. Ingestion
  24. 24. Inhalation
  25. 25. Injection</li></ul>Hazard & risk must be communicated to ALL staff! <br />
  26. 26. Risk Assessment<br />Contamination?<br />Containment?<br />The “risk assessment” is the backbone <br />of safe practice! <br />
  27. 27. Laboratory Safety Programs<br />Biosafety Program<br /><ul><li>Knowledgeable supervisor
  28. 28. Knowledgeable personnel
  29. 29. Aware of potential hazards
  30. 30. Proficient in practices & techniques
  31. 31. Lab specific safety manual
  32. 32. Policies
  33. 33. Exposure control plans/programs
  34. 34. Emergency response
  35. 35. Safety committee(s)</li></li></ul><li>Laboratory Safety Programs<br />Training Program<br /><ul><li>Regulatory
  36. 36. Federal
  37. 37. State
  38. 38. Local
  39. 39. Institutional
  40. 40. Site specific
  41. 41. Work specific
  42. 42. Relevant</li></ul>Get training for what you ACTUALLY <br />do not what others THINK you do! <br />
  43. 43. Regulations<br /><ul><li>Occupational Safety and Health Act
  44. 44. BloodbornePathogens Standard
  45. 45. Possession, Use and Transfer of Select Agents and Toxins
  46. 46. US Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training
  47. 47. Transportation of Dangerous Goods Acts and Regulations
  48. 48. Hazardous Waste Management Standard
  49. 49. Occupational Exposure to Hazardous Laboratory Chemicals Standard
  50. 50. Radiation Protection Standard</li></li></ul><li>CDC/NIH Guidelines<br />BMBL<br />
  51. 51. CDC/NIH Guidelines<br />Biohazard<br />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.<br />
  52. 52. CDC/NIH Guidelines<br />Biohazard Classification: Biosafety Levels<br />BSL1 Agents not known to cause disease<br />BSL2 Agents associated with human disease<br />BSL3 Indigenous/exotic agents associated<br /> with human disease and with potential<br /> for aerosol transmission<br />BSL4 Dangerous/exotic agents of life<br /> threatening nature<br />
  53. 53. CDC/NIH Guidelines<br />Biosafety<br />The application of combinations of laboratory practice and procedure, laboratory facilities, and safety equipment when working with potentially infectious microorganisms.<br />
  54. 54. CDC/NIH Guidelines<br />Laboratory Classification: Biosafety Levels<br />Combinations of laboratory practice and procedure, laboratory facilities, and safety equipment to provide containment <br />Low Containment<br />Biosafety Level 1<br />Biosafety Level 2 <br />Biosafety Level 3<br />Biosafety Level 4 <br />High Containment<br />
  55. 55. CDC/NIH Guidelines<br />Containment<br /><ul><li>Safe methods for managing infectious materials to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents</li></li></ul><li>CDC/NIH Guidelines<br />Primary Containment<br /><ul><li>Protection of personnel and immediate laboratory environment from exposure to infectious agents
  56. 56. Accomplished by:
  57. 57. Good microbiological technique
  58. 58. Appropriate safety equipment</li></li></ul><li>CDC/NIH Guidelines<br />Secondary Containment<br /><ul><li>Protection of external environment from exposure to infectious materials
  59. 59. Accomplished by:
  60. 60. Facility design
  61. 61. Operational practices</li></li></ul><li>CDC/NIH Guidelines<br />Biosafety Components<br /> Practice and procedure<br /><ul><li>Standard
  62. 62. Special</li></ul> Safety equipment<br /><ul><li>Standard
  63. 63. Special</li></ul> Facilities<br /><ul><li>Design & Construction
  64. 64. Function</li></li></ul><li>Biosafety Level 1 (BSL-1)<br />Work with microorganisms not known to consistently cause disease in healthy adult humans<br /><ul><li>Basic level of containment:
  65. 65. Standard microbiological practices
  66. 66. No special primary or secondary barriers recommended (other than a sink for hand washing)
  67. 67. No special practices</li></li></ul><li>Biosafety Level 1 (BSL-1)<br />Design<br /><ul><li> Access controls
  68. 68. Sinks
  69. 69. Impervious bench tops
  70. 70. Easily cleaned surfaces
  71. 71. Adequate illumination</li></li></ul><li>Biosafety Level 1 (BSL-1)<br />
  72. 72. Biosafety Level 2 (BSL-2)<br /><ul><li> Work with moderate-risk microorganisms</li></ul> and agents (i.e. Hepatitis B virus, HIV,<br /> Salmonella, etc.) <br /><ul><li>Basic level of containment:
  73. 73. Standard microbiological practices
  74. 74. Secondary barriers (i.e. hand washing sinks, segregated waste, BSCs available
  75. 75. Special practices (i.e. immunization program, access controls)</li></li></ul><li>Biosafety Level 2 (BSL-2)<br />Design<br /><ul><li> Lockable doors
  76. 76. Sinks with hands-free operation
  77. 77. Impervious bench tops resistant to</li></ul> moderate heat & organic solvents<br /><ul><li> Easily cleaned surfaces
  78. 78. Eyewash available
  79. 79. BSCs installed properly; away from doors</li></li></ul><li>Biosafety Level 2 (BSL-2)<br />
  80. 80. Biosafety Level 3 (BSL-3)<br /><ul><li> Work with indigenous or exotic agents with</li></ul> potential for respiratory transmission; may<br /> cause serious or lethal infection (i.e.<br />Mycobacterium tuberculosis, St. Louis<br /> encephalitis virus, and Coxiella burnetii) <br /><ul><li>Basic level of containment:
  81. 81. Standard microbiological practices
  82. 82. Secondary barriers (i.e. BSCs required, directional air flow, anti-room entry)
  83. 83. Special practices (i.e. restricted access, solid front lab coat, autoclave required)</li></li></ul><li>Biosafety Level 3 (BSL-3)<br />Design<br /><ul><li>Restricted access; lockable doors –</li></ul> emergency “open”<br /><ul><li> Sealed windows
  84. 84. Hands-free sink near door
  85. 85. Surfaces resistant to chemicals
  86. 86. Monolithic floor; sealed room penetrations
  87. 87. Ducted exhaust
  88. 88. Decontamination method available</li></li></ul><li>Biosafety Level 3 (BSL-3)<br />
  89. 89. Biosafety Level 4 (BSL-4)<br /><ul><li> Work with dangerous and exotic agents that </li></ul> pose a high individual risk of life-threatening<br /> disease; may be transmitted via aerosols; no<br /> available vaccine or therapy (i.e. Ebola, Marburg)<br /><ul><li>Basic level of containment:
  90. 90. Standard microbiological practices
  91. 91. Secondary barriers (i.e. BSCs, personal protective suits or Class III BSCs, directional air flow)
  92. 92. Special practices (i.e. isolated/restricted access facility, sealed rooms, treated waste)</li></li></ul><li>Biosafety Level 4 (BSL-4)<br />Types<br /><ul><li> Class III biosafety cabinets (cabinet line)
  93. 93. Personal protective suits (suit lab)</li></ul>Design<br /><ul><li> Isolated facility; separate building
  94. 94. Daily inspections of containment parameters
  95. 95. Restricted access
  96. 96. Changing rooms & showers
  97. 97. Double door autoclaves
  98. 98. All room penetrations sealed</li></li></ul><li>Biosafety Level 4 (BSL-4)<br />Design<br /><ul><li> Dedicated non-re-circulating ventilation
  99. 99. HEPA filtered supply and double HEPA</li></ul> filtered exhaust air<br /><ul><li> Occupants wear one-piece personal</li></ul> protection suits<br /><ul><li> Redundant systems</li></li></ul><li>Biosafety Level 4 (BSL-4)<br />Suit Lab<br />
  100. 100. Biosafety Level 4 (BSL-4)<br />Cabinet Line<br />
  101. 101. Biosafety Level 4 (BSL-4)<br />
  102. 102. Biosafety Level 4 (BSL-4)<br />CDC Maximum Containment Laboratory<br />
  103. 103. Animal Facilities<br />ABSL 1-4 <br />Standard practices<br />Special practices<br />Safety equipment<br />Facilities<br />
  104. 104. Standard Lab Practices<br />Standard Practices?<br />Those practices which you should perform whenever you enter/exit the lab/animal area and during your work while in the lab/animal area.<br />
  105. 105. Standard Lab Practices<br /><ul><li>Limit access
  106. 106. Wash hands frequently
  107. 107. No eating, drinking, etc. </li></ul> in lab<br /><ul><li>No mouth pipetting
  108. 108. Handle sharps carefully
  109. 109. Contain hazardous</li></ul> materials & aerosols<br />
  110. 110. Standard Lab Practices<br /><ul><li>Decontaminate work areas
  111. 111. Decontaminate waste </li></ul> materials<br /><ul><li>Post hazard warning signs
  112. 112. Personnel must be trained
  113. 113. Wear PPE; leave in lab
  114. 114. Develop/follow SOPs</li></li></ul><li>Exercise #1<br />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.<br />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.<br />
  115. 115. Personal Protective Equipment<br />
  116. 116. Personal Protective Equipment<br />
  117. 117. What’s Wrong?<br />
  118. 118. Exercise #2<br />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.<br />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.<br />
  119. 119. Laboratory Infections<br />1965 Survey<br /><ul><li> Fewer than 20% of all reported infections</li></ul> were associated with a known accident<br /><ul><li> Exposure to infectious aerosols considered</li></ul> to be plausible but unconfirmed source for<br /> more than 80% of reported cases<br />All lab operations, including repairs, should be<br />performed to reduce or eliminate aerosol production!<br />
  120. 120. Aerosols<br />Aerosol Generating Lab Operations<br /><ul><li>Pipetting
  121. 121. Mixing
  122. 122. Pouring
  123. 123. Vortexing
  124. 124. Centrifuging
  125. 125. Loading syringes
  126. 126. Flaming loops
  127. 127. Lasers, cell sorters</li></li></ul><li>Aerosols<br />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)<br />Results Organisms Recovered<br />Blender, open 106<br />Sonicator with bubbling 106<br />Pipetting, vigorous 106<br />Dropping culture 3 X 105<br />Splash on a centrifuge rotor 105<br />Blender, opened after 1 minute 2 X 104<br />Pipetting, carefully 104<br />
  128. 128. Class I<br />Class III<br />Class II<br />Class II<br />Type A1<br />Class II<br />Type A2<br />Class II<br />Type B2<br />Class II<br />Type B1<br />Aerosol/fume Containment Devices<br />Biological<br />Safety<br />Cabinet<br />Fume Hood<br />
  129. 129. Biological Safety Cabinets<br />
  130. 130. Biological Safety Cabinets<br />Class I<br />Single pass air<br />Worker protection; no product protection<br />Exhausts to outside (w/wo HEPA filter)<br />Class II<br />Re-circulated air<br />Worker/product/environmental protection<br />Laminar flow<br />Exhausts inside/outside (w/wo HEPA filter)<br />Suitable for work with BSL2-4 agents<br />Class III<br />Totally enclosed; air-tight<br />Exhausted outside thru HEPA filters<br />Suitable for work with BSL3/4 agents<br />
  131. 131. Intake<br />100 ft/min<br />Biological Safety Cabinets<br />Exhaust<br />
  132. 132. Biological Safety Cabinets<br />HEPA (High Efficiency Particulate Air) Filter<br />
  133. 133. Biological Safety Cabinets<br />HEPA Filter - Particle Capture Efficiency <br />Anthrax (1u)<br />99.97<br />Smallpox<br />Tularemia (.3u)<br />Collection Efficiency (%)<br />0.01<br />0.1<br />1.0<br />10.0<br />0.3<br />Particle Diameter (u)<br />
  134. 134. Biological Safety Cabinets<br />Inward Airflow = ~100 ft/min<br />
  135. 135. LAMINAR FLOW<br />DOOR OPENING<br />PERSON WALKING<br />HVAC VENT<br />ROOM AC<br />FPM 100 200 300 400 500 600 700 800 <br />MPH 1 2 3 4 5 6 7 8 <br />Biological Safety Cabinets<br />Comparative Velocities<br />Peters and Peters 1990<br />
  136. 136. Biological Safety Cabinets<br />Airflow, Hand Movements & Walk-by<br />
  137. 137. Biological Safety Cabinets<br />Basic Operation<br /><ul><li>Turn BSC on
  138. 138. Check magnehelic gauge
  139. 139. Load BSC with work materials
  140. 140. Allow BSC to run for 4-5 min
  141. 141. Perform work
  142. 142. Remove materials from BSC
  143. 143. Disinfect inside of BSC</li></li></ul><li>Biological Safety Cabinets<br />What’s Wrong?<br />
  144. 144. Biological Safety Cabinets<br />
  145. 145. Biological Safety Cabinets<br />
  146. 146. Biological Safety Cabinets<br />
  147. 147. Biological Safety Cabinets<br />What’s Wrong?<br />
  148. 148. Biological Waste<br />
  149. 149. Biological Waste<br />
  150. 150. Biological Emergencies<br />Surface Contamination<br /><ul><li>Alert others
  151. 151. Remove glass shards
  152. 152. Cover spill; apply appropriate disinfectant, i.e. Clorox
  153. 153. Allow adequate contact time (20”)
  154. 154. Properly dispose of materials
  155. 155. Notify supervisor</li></li></ul><li>Biological Emergencies<br />Personal Contamination<br />Alert others<br />Remove contaminated clothing<br />Flush affected area (15-20”)<br />Apply first aid if needed<br />Report to Clinic/ER<br />Notify supervisor<br />
  156. 156. What’s Wrong?<br />
  157. 157. What’s Wrong?<br />
  158. 158. What’s Wrong?<br />
  159. 159. What’s Wrong?<br />
  160. 160. What’s Wrong?<br />
  161. 161. What’s Wrong?<br />
  162. 162. What’s Wrong?<br />
  163. 163. Laboratory Contamination<br />
  164. 164. Standard Lab Practices<br /><ul><li>Limit access
  165. 165. Wash hands frequently
  166. 166. No eating, drinking, etc. in lab
  167. 167. No mouth pipetting
  168. 168. Handle sharps carefully
  169. 169. Contain hazardous materials & aerosols</li></li></ul><li>Standard Lab Practices<br /><ul><li>Decontaminate work areas
  170. 170. Decontaminate waste materials
  171. 171. Post hazard warning signs
  172. 172. Personnel must be trained
  173. 173. Wear PPE; leave in lab
  174. 174. Develop/follow SOPs</li></li></ul><li>THINK <br />before <br />you act!<br />Safety Is As Safety Does!<br />
  175. 175. Biocontainment LaboratoryBiosafety & Biocontainment Training<br />

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