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  1. 1. Biosafety in Microbiological and Biomedical Laboratories S. Wongratanacheewin, Ph.D. Melioidosis Research Center, Faculty of Medicine, KKU. E-mail: sura_wng@kku.ac.th
  2. 2. September 11, 2001
  3. 3. ไทยรัฐ 19 กันยายน 2546 ไทยรัฐ 20 กันยายน 2546
  4. 4. Biosafety  Personal protection  Work place practices  Administrative  Engineering All play an equal role in protecting workers from occupational exposures in laboratories
  5. 5. Biosafety  Personal protection  PPE, respirotors, gloves, booties, eye and ear protections  Work place practices  Entry and exit practices, decontamination, emergency procedure, handle sharp.
  6. 6. Biosafety  Administrative  Training  Risk management  Immunization  Security, supervision  Engineering  System, Hepa filter, Accessible
  7. 7. CDC Bioterrorism Agents  Category A  Category B  Category C
  8. 8. Category A Diseases/Agents  can be easily disseminated or transmitted from person to person;  result in high mortality rates and have the potential for major public health impact;  might cause public panic and social disruption; and  require special action for public health preparedness.
  9. 9. Category B Diseases/Agents  Second highest priority agents include those that  are moderately easy to disseminate;  result in moderate morbidity rates and low mortality rates; and  require specific enhancements of CDC's diagnostic capacity and enhanced disease surveillance.
  10. 10. Category C Diseases/Agents  Third highest priority agents include emerging pathogens that could be engineered for mass dissemination in the future because of  availability;  ease of production and dissemination; and  potential for high morbidity and mortality rates and major health impact.
  11. 11. Containment  Primary containment, the protection of personnel and the immediate laboratory environment from exposure to infectious agents, is provided by both good microbiological technique and the use of appropriate safety equipment.  Secondary containment, the protection of the environment external to the laboratory from exposure to infectious materials,
  12. 12. Laboratory Practice and Technique  Develop or adopt a biosafety or operations manual that identifies the hazards that will or may be encountered, and that specifies practices and procedures designed to minimize or eliminate exposures to these hazards.
  13. 13. Principles of Biosafety  Laboratory personnel  Safety practices, and techniques must be supplemented  Appropriate facility design and engineering features  Safety equipment, and management practices.
  14. 14. Safety Equipment (Primary Barriers)  Biological safety cabinets (BSCs)  Enclosed containers,  Engineering controls designed to remove or minimize exposures to hazardous biological materials.
  15. 15. The biological safety cabinet (BSC)  Device used to provide containment of infectious splashes or aerosols.  Three types of biological safety cabinets (Class I, II, III) used in microbiological laboratories.  Open-fronted Class I and Class II biological safety cabinets are primary barriers which offer significant levels of protection to laboratory personnel.  Safety centrifuge cup, an enclosed container designed to prevent aerosols.
  16. 16. Personal Protection  gloves, coats, gowns,  shoe covers, boots,  respirators,  face shields,  safety glasses, or goggles.
  17. 17. Facility Design and Construction (Secondary Barriers)  Depend on the risk of transmission of specific agents.  Separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and handwashing facilities.  If an infectious aerosol is present, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment.
  18. 18. Biological Safety  Biological Hazards  Divided into 4 biosafety levels  Levels define the lab requirements, protective equipment, and work practices  4 animal biosafety levels also
  19. 19. Biosafety Level 1  Definition  Work with agents not known to cause disease in healthy adults  Minimal potential hazard to personnel and environment
  20. 20. Biosafety Level 1  Requirements  Wash hands before leaving lab  No eating, drinking, etc. in lab  Follow safe sharps procedures  Decontaminate waste and work surfaces
  21. 21. Biosafety Level 1  Examples  Escherichia coli K12  Klebsiella oxytoca  Lactobacillus  Saccharomyces
  22. 22. Biosafety Level 2  Definition  Work with agents of moderate potential hazard to personnel and the environment. Agents associated with human disease which is rarely serious and for which preventative or therapeutic interventions are often available.
  23. 23. Biosafety Level 2  Requirements  All requirements for BSL1 plus:  Training to handle organisms  Personal protective equipment (PPE)- lab coats, eyewear, and gloves  Minimize aerosol generation  Biohazard sign posted on door
  24. 24. Biosafety Level 2  Examples  Pathogenic E. coli  Salmonella sp.  Streptococcus sp.  Hepatitis B and C Viruses
  25. 25. Biosafety Level 3  Definition  Agents associated with serious or lethal disease for which preventative or therapeutic intervention may be available. High individual risk, low community risk. Often risk is by inhalation route.
  26. 26. Biosafety Level 3  Requirements  All requirements for BSL2 plus:  Limited lab access  2 doors in series to access lab  Able to wash entire lab  Special exhaust ventilation- not recirculated
  27. 27. Biosafety Level 3  Requirements  All requirements for BSL2 plus:  High level of training  Personnel receive vaccinations if available  Work in biosafety cabinets
  28. 28. Biosafety Level 3  Examples  HIV  Mycobacterium tuberculosis
  29. 29. BSL 3 cabinet
  30. 30. Class I High Efficiency Particulate Air filter Removes over 99.95% of particles 0.3 mm in diameter or larger HEPA Filters
  31. 31. Class II A Class II B1
  32. 32. Connection to building exhaust system required. Class II B2
  33. 33. Connection to building exhaust system required. Class II B3
  34. 34. Biosafety Level 4  Definition  Agents likely to cause serious or lethal disease for which preventative or therapeutic intervention is usually NOT available. Dangerous or exotic agents.
  35. 35. Biosafety Level 4  Requirements  All requirements for BSL3 plus:  Class III biosafety cabinet or positive pressure suits  Shower/change rooms - clothing autoclaved before laundering  Air locks
  36. 36. Biosafety Level 4  Examples  No Bacteria or Fungi  Ebola Virus  Monkey B Virus  Marburg Virus
  37. 37. Safe Work Practices  Wash hands after work; before leaving lab; when removing gloves  No eating, drinking, applying cosmetics, handling contact lenses in lab  Maintain labs in clean, orderly fashion  Limit access to lab when work with organisms is in progress  Use good microbiological techniques  No mouth pipetting  When possible use plastic instead of glass
  38. 38. Personal Protective Equipment  Contaminated personal protective equipment (PPE) should remain in the work area- do not wear in any "clean" areas  PPE that is contaminated must be discarded as infectious waste or disinfected prior to routine laundering
  39. 39. Use of Equipment  Minimize airflow disturbances (moving in/out of cabinet, people walking by, opening doors, blocking grilles with equipment)  Wipe down surfaces with alcohol before and after work. Let run 10 minutes to clean it  Minimize use of flames in cabinet  Try not to use chemicals in cabinets- only use in Class I or II hoods that are ducted- contact DOHS  Do not store excess equipment in cabinet
  40. 40. Centrifuge Safety  Aerosol generation from broken tubes, opening tubes, decanting supernatant, resuspending materials  Balance load  Use sealed tubes, safety buckets or rotors when possible  When possible fill & open centrifuge tubes or buckets in BSC
  41. 41. Centrifuge Accidents  If a tube breaks-  Turn off centrifuge  Leave the lab  Call Officers  If you find a tube broke when you open the centrifuge-  Close the lid  Leave the lab  Call Officers
  42. 42. Shipping/ Transporting  Who regulate transportation of biologicals????  Includes infectious materials, diagnostic specimens, dry ice  Program being developed- to include mandatory training
  43. 43. Disinfection  All equipment, environmental, and working surfaces must be cleaned and decontaminated after work with infectious materials  Broken glass or sharps shall be cleaned up using mechanical means- NOT by hand  Different levels of cleaning- decontamination, sanitization, disinfection, sterilization
  44. 44. Decontamination  Use appropriate disinfectant for agents in use  Follow instructions, paying attention to dilution, shelf life, and contact time to assure effective kill  Care must be used to ensure mixing of incompatible materials does not occur
  45. 45. Infectious Waste  What is infectious waste  Procedures for disposal  Solid waste  Liquid waste  Sharps  Autoclaving waste  Containers and pick-ups  Mixed waste
  46. 46. Emergencies  Safety equipment  in labs  Showers  Eye washes
  47. 47. BSL2 Spills  Small spills-  Wipe up with disinfectant-soaked paper towel then clean surface with suitable disinfectant  Large BSL2 Spill in BSC  Keep cabinet running during and 10 minutes after cleanup  Don PPE  Cover spill with disinfectant- let set- wipe up  Clean catch basin under work surface if spill ran there  Remove PPE when done and wash  Leave the lab, close door, post lab off-limits  Decon people if necessary  After 30 minutes, put on PPE and assemble supplies
  48. 48. (building name & room number, list subrooms, if any) Hazards Within: List hazards such as Flammables, Acids, Bases, General Chemicals, high pressure compressed gases, Biohazards, Radioactive materials, etc Primary Contact: (name & office phone) Second Contact: (name & office phone) Building Monitor/Safety: (name & office phone) Department Head: (name & office phone) Fire/Police/Ambulance: 911 Envir. Health & Safety (or RSO, if needed): 646-3327
  49. 49. Acknowledgements  T2 (Thailand Tropical Diseases Research Program).  TRF (Thailand Research Fund).  NSTDA  ONSITE, Emory University  Faculty of Science, MU  The Virology Association of Thailand  Faculty of Medicine, KKU.

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