1.MILWAUKEE SCHOOL OF ENGINEERINGCONCEPTS OF BIOSAFETY, BIOCONTAINMENT & RISKASSESSMENTMARIAN DOWNING, RBP, CBSP, SM(NRCM)

2. Objectives of this course By the end of this course, you will: Know why a risk assessment isnecessary for all research withbiological materials Know the basic considerations fora risk assessment Understand standardmicrobiological practices for thelab and use of the biosafetycabinet Have a basic understanding ofhow to dispose of waste, clean upa spill, and what to do in anaccidental exposure situation 3. BIOSAFETY BASICS 4. What is Biosafety? Fundamental objective: Containment of potentially harmful biological agents Purpose of containment: Reduce or eliminate exposure of lab workers, otherpersons, and outside environment 5. Proliferation of Containment BMBL Outlines: Standard Microbiological Practices Special Microbiological Practices Safety Equipment Facilitieshttp://www.cdc.gov/biosafety/publications/bmbl5/index.htm 6. What is a biohazard? An agent of biological origin that has the capacity to producedeleterious effects in humans HBV, HCV, HIV, West Nile Virus (WNV), Malaria, Toxoplasma gondii, Mycobacterium tuberculosis, Staph aureus Infectious clinical specimens Infected animals Toxins and allergens derived from living organisms Materials or equipment that have come in contact withbiohazardous materialsMycobacterium tuberculosis 7. Risk Groups of Viruses, Bacteria, Fungi and ParasitesRisk GroupAgents1Not associated with disease in healthy adult humans Associated with human disease which is rarely serious2and for which preventive or therapeutic interventions are often available Associated with serious or lethal human disease for which3preventive or therapeutic interventions may be available (high individual risk but low community risk) Likely to cause serious or lethal human disease for which4preventive or therapeutic interventions are not usually available (high individual risk and high community risk) 8. Biosafety LevelsRisk Group Biosafety Level Agents1Basic BSL1Not generally pathogenic2Basic BSL2Community pathogens3High Containment BSL3 Agents spread by aerosol route4Maximum containment Extreme pathogens, no antimicrobials BSL4or other drugs 9. Biosafety Levels (BSLs) Combinations of lab practices and techniques, safetyequipment, and laboratory facilities Specifically appropriate for: Operations performed Known or suspected routes of infection Lab function or activity NOTE: The Risk Group of an organismmay not correspond with theBiosafety Level This is determined by the RiskAssessment 10. How to Determine a Biosafety Level? Conditions under which the agent ordinarily can be handled inthe laboratory Based on risk assessment Established by responsible scientists Resources include: Published data Biological safety officer (BSO) Institutional Biosafety Committee (IBC) 11. Risk Assessment Consideration of: Route of infection Infectious dose Manipulations to be done Volumes handled Virulence and pathogenicity Antibiotic resistance patterns Vaccine and treatment availability Other factors 12. Assessing risk Helps to assign the biosafety level that reduces to an absoluteminimum the workers exposure to agents, their risk of an LAI(lab associated infection), and potential impact on theenvironment. Will also assist in QA/QC and product protection. 13. Chain Of Infection Model What is the primary risk of working with agents? infecting self and others release of agent to the environment What factors have to be in place to cause an infection? presence of an infectious agent infectious dose of agent virulence of agent route of infection susceptible host All 5 factors must be present for an infection to occur Only 1 factor needs to be eliminated to avoid infection! 14. Routes of Exposure Parenteral (needlestick, scratch) Exposure to non-intact skin Inhalation Droplet Ingestion Mucous membranes (trans dermal) Absorption (e.g., toxins) Animal bites and scratches 15. Individual Risk Factors Susceptibility to Infection Age Some infections more severe in children (RSV, WestNile) Some infections more severe in adults (mumps, chickenpox, measles) Immune function usually decreases > middle age Sex TB = greater infectivity in women Listeria monocytogenes greater risk for women Mumps infection in men (testicle inflammation)CDC/James Gathany 16. Susceptibility to Infection Genetics Inherited immune problems Healthy Adult Factors affecting immune status: CDC/James Gathany Chemotherapy, smoking, immune suppressive drugs, Lupus, HIV, etc. Vaccination Status Reduces chance of infection Reduces severity of infection (chickenpox) Never reduce risk 100% Reproductive Risks Risks to fetus: Toxoplasma, Listeria, CMV, Rubella, HIV Mother may be asymptomatic, but fetal effects are severe 17. Biocontainment Concepts Containment Barriers Primary - BSCs, personnel protective gear, containment equipment Secondary - Room, systems Tertiary - Containment around systemsAccess Control and SeparationRedundancy and ReliabilityDecontamination 18. Biosafety Level 1 Basic lab for working with non-infectious agents Not known to cause disease in healthy adults May still have hazardous chemicals, radiation, magnets Work with established animal tissue culture cell lines, pcrtesting (polymerase chain reaction), E. coli, yeast, etc. Low individual risk, low community risk May still be able to cause an infection Immune suppressed individuals 19. Biosafety Level 2 Hospital clinical lab or standard R&D Lab Work with infectious agents Non-aerosol spread (HIV, HCV, Shigella) Moderate individual risk, low community risk Usually treatments or vaccines available Eyewash station Waste decontamination available BSCs installed as needed Inward air flow Recirculated to lab areas only Bacillus anthracis Photo: CDC 20. Biosafety Levels 1 & 2 BSL1 BSL2Agents Not known to cause Associated with human disease, but not disease in life-threatening Healthy adultsPracticesStandard Microbiological BSL1 plus: PracticesLimited accessBiohazard signsSharps precautionsBiosafety manual with waste handling andmedical surveillanceSafety No special equipment Biosafety cabinets or other physicalEquipmentrequired containment for splashes or aerosolsPPE: lab coats, gloves, eye/face protectionFacilities Sink requiredBSL1 plus:Autoclave20 21. Biosafety Levels 1 & 2 BSL-1 BSL-2 22. METHODS OF CONTROL 23. Hierarchy Of ControlsMost effective Avoidance Engineering Controls Work Practices PPE Least effective 24. Engineering Controls Centrifuges with aerosol safety cups Substitution, replacing sharps with non-sharp objects biosafety cabinets Hand and eye washing facilities Safe needles/scalpels Sharps containersBD Disposable Scalpel 25. Safe Work Practices Fill line Do not bend or recap needles Place sharps in the appropriate container Do not eat, drink, smoke, apply cosmetics or handle contact lenses in the workareas Do not store food in the laboratory Minimize splashing, spraying, andgeneration of droplets Do not reach into trash/sharpscontainers Minimize glassware/sharps hazards 26. Laboratory Work Practices, continued Wash hands frequently Keep your hands away from your face May infections are spread by mucous membrane contact Do not mouth pipet Also mucous membrane contact Wipe down work area withdisinfectant (daily) 27. Hand Decontamination Hand washing is the single most important procedure forpreventing infectionshttp://www.cdc.gov/handhygiene/training.html#posters 27 28. Routine Hand HygieneSoap and water Most of benefit is fromremoval of transientorganisms on surfaceepidermal layers Also removes soil, such asdirt, blood, feces Soap and water, if available,are better than handsanitizers in most cases 29. BD FACSAria-IIPrevention of Aerosols Waring Examples of aerosol-generating equipment: Centrifuge Sonicator Vortex mixer Bead beater Homogenizer Tissue grinder Blender Fermenter Pipette Cell sorter Laser 30. Other Aerosol Generators Vigorous shaking Any fluid manipulation Pouring Opening lyophilized cultures Flaming loops/needles (Bunsen burner) Removing needle from rubberdiaphragm on vial or sampling portCDC/Greg Knobloch 31. Aerosols Lab equipment Produces aerosols more efficiently than natural methods (coughing, sneezing, etc.) Materials encountered Higher titered than in natural setting Modest aerosol-producing activity could be infectious Larger quantities being manipulated Agent that is not normally aerosol-transmissible may be transmitted under these circumstances Class I or II BSC for containmentof small equipment(blenders, homogenizers, etc.)Banthrax Versa Dome 32. Safe Pipeting Never blow out last drop in pipette Use pipette aids with filters Cotton plugged pipettes provide some protection Horizontal pipette collection tray w/ disinfectant Disinfectant in upright pipet collection vesselforces aerosols out top of pipette into the air Pipet over disinfectant-soaked pad Never mix by suction + expulsion Discharge liquid down side of container Deliver as close as possible to contents 33. Personal Protective Equipment (PPE) Safety glasses with side shields Wear when entering the work area Lab coats Leave in the work area If contaminated, decontaminate before placing in laundrybag Wear buttoned with sleeves rolled down Closed toe shoes in the laboratory no sandals 34. Gloves Check for pinholes before wearing Do not touch common items withgloved hands (e.g., door knobs, phones) Consider chemical compatibility (solvents, etc.) Dont reuse disposable gloves Waterproof needed for working with human-sourced materials Do not use powdered latex gloves NOTE: if double gloving, can combine different types (like latexand nitrile) Wash hands after removing DO NOT wear outside the lab! 35. BIOLOGICAL SAFETY CABINETS 36. Know Your Hoods!Chemical Fume Hood Biological Safety Cabinet Laminar Flow Clean Air Center Closes completely: either Fixed sash opening (8 in.) HEPA filter visible inhorizontally or vertically(alarmed) rear or top of unit Not meant for sitting Sash moves up but does Usually no sash or Negative pressure not close completelysash is fixed May have solvent/chemical Designed for seated work Positive pressure airstorage underneath Negative pressure blowing into face orbreathing zonePictures are courtesy of MSOE 37. Biological Safety Cabinets (BSCs) Role: To protect the user from the samples To protect the environment from the samples To protect the samples from external elements Efficacy Dependent on scrupulous work practices Read BSC instructions in Biosafety Manual 38. Biological Safety Cabinets (Continued) There are 3 classes of BSC Class I: not often used MSOE Certification Sticker Class IIA and B: most often found in laboratories Class III: not often used Certification: Annually When moved After repairs, filter changes 39. Class II BSC Most commonly found BSC in laboratories HEPA-filtered vertical laminar air flow User and sample protected HEPA-filtered exhausted air User and environment protected There are 2 types and 2 subtypes (A1-2-B1-2) 40. Class II A2 BSCs 70% recirculated air through HEPAfilter A2 may be used with nonvolatiletoxic chemicals or radionuclides minute levels of volatile toxic chemicals and radionuclides If exhausted outside A2 has airflow of 100 lfmEthanol fire in BSC (AIHA) 41. Class II A2 BSC Courtesy of MSOE CDC drawing The Class II, Type A2 BSC may be connected to the building exhaust system. 42. Clean Bench Not to be confused with Class I BSC Inflow air is HEPA filtered Exhaust air is not filtered Used for Microbiology clean preparation (making agar plates)and molecular work (PCR) Air flow can be vertical or horizontalCourtesy of MSOE 43. BSC PracticesThoroughly clean BSC before/after useDo not cover the front grills or block rear ventDecontaminate all materials in and outDiscard containers inside BSC Adjust chair height to look down through sash Wait 15-20 minutes for HEPA filtersto electrostatically charge beforestarting work 44. BSC Work Practices Slow arm movements, move in and out in astraight line, do not sweep. Work over absorbent toweling Clean spills immediately, wait for BSC to purge Open items held at an angle, cap in hand BMBL, 5th ed. 45. BSC Work Practices Work towards the rear of the cabinet Equipment in back 1/3 of cabinet cease other activities while operating equipment Do not rely on UV decontamination Bulb must be dusted and tested regularly Vacuum trap and filter setup:BMBL, 5th ed.In line HEPA filterCollection vessel Overflow vessel withdisinfectant 46. Whats Wrong With this Picture? 47. BSC Practices Load only those materials needed insidecabinet No withdrawing hands for discard orsupplies Wait 5 minutes after loading BSC beforeworking Do not cross hands while working or swayside to side No Bunsen burners or alcohol burners inthe BSCCorning products Use disposable loops and needles Bacti-cinerator or micro-burner 48. What Do You Notice About this Picture? CDC photo: James Gathany 49. OTHER LABORATORY ISSUES 50. Disinfection Solutions commonly used 2 % bleach Prepare fresh daily 70% ethanol For cleaning only not considered an appropriate disinfectant for work with human specimens Cavicide BACDOWN Detergent Disinfectant Dispatch Surfaces Bench top BSC Fume hoods Centrifuges Equipment Incubators Floors 51. Disinfectants Must follow manufacturers labelinstructions Disinfect bench tops at least daily Decontaminate equipment sent forrepair or disposal Use Decontamination Verification Tag 52. Sharps Any item capable of puncturing the skin needles scalpel blades razor blades glass Pasteur pipettes broken contaminated glass In Wisconsin syringes with needles attached Eliminate, or substitute safer engineered sharps! 53. Examples of Sharps Alternatives Hypodermic needles Use safe needles or safe syringes Pasteur pipet Use plastic transfer pipet Scissors Use blunt-tipped safety scissors Box Cutters Use self-retracting utility knife Document consideration ofsafer devices annually 54. Sharps Precautions Do not overfill sharps container Seal before waste reaches indicator lineon container Keep upright Have adjacent to work area NOT for chemical disposal DO NOT: Recap needles before placing in sharpscontainer Remove needles by hand 55. Waste Disposal Regular Trash Items that are not contaminated Paper Disposables that have been chemically decontaminated E.g. tissue culture flask, tubes, plastic vials, paper towels, gloves Autoclave Microbial cultures Tissue culture materials that have not been ORchemically disinfected ALL rDNA materials that have not beenchemically disinfected Contaminated gloves, spill cleanupmaterials, etc. NOTE: ALL RECOMBINANT DNACONTAMINATED MATERIALS MUST BETREATED AS THOUGH THEY WEREINFECTIOUS! 56. Disposal/Handling of Contaminated ItemsPipettesPipette tips Solids Bulk Sharps Liquids Pipettes PipetteBiohazard Disinfectant Disposal ContainerSharps TraysBox Container10% bleach15 minutesAutoclave GlasswareBiohazard containerWaste Sewer 57. Laundry Requirements Change lab coats regularly No sorting in the workplace If there are spills on the lab coat: Remove Spray with disinfectant Rinse with water Lab coats cannot be takenhome for cleaning The lab technician will coordinatethe laundering of lab coats 58. Labeling and Shipping Label vials, tubes, etc. Biohazard label Or, label box or carrier Transfer internally in a zip lock bag with absorbent inside. Need to ship? Only trained employees allowed to package materials for shipping Renewed every 2 years Know status of materials to be shipped Most MSOE materials will be exempt from shippingregulations, but must still be packaged appropriately 59. All Biosafety Levels Post policies and procedures for entry Provide appropriate warning signs 60. EMERGENCIES 61. Spills Clean up and report spills per Biosafety Manual Procedure Lab workers/students must be able and equipped to handle spillsof biological material Absorb liquid, preclean with detergent (to remove organicmaterials), then wipe with an approved disinfectant Use disinfectant appropriate for agent Use full strength disinfectant on large spills (dilution effect) NEVER pick up broken sharps with fingersCourtesy of MSOE 62. Emergency ProceduresAccidental Exposure in the Lab Remove PPE and provide immediatecare to the exposure site: Wash wounds and skin with soap and water for 15minutes Flush mucous membranes with water for 15 minutes Notify the Laboratory Supervisor or the Lab Technician Follow directions of Supervisor, or, if after hours, visit theEmergency Clinic 63. Thank You! Be sure to complete theassociated quiz for this training module.