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Biosafety Basics Training
- 1. MILWAUKEE SCHOOL OF ENGINEERING CONCEPTS OF BIOSAFETY, BIOCONTAINMENT & RISK ASSESSMENT MARIAN DOWNING, RBP, CBSP, SM(NRCM)
- 2. Objectives of this course • By the end of this course, you will: – Know why a risk assessment is necessary for all research with biological materials – Know the basic considerations for a risk assessment – Understand standard microbiological practices for the lab and use of the biosafety cabinet – Have a basic understanding of how to dispose of waste, clean up a spill, and what to do in an accidental exposure situation
- 4. What is Biosafety? • Fundamental objective: – Containment of potentially harmful biological agents • Purpose of containment: – Reduce or eliminate exposure of lab workers, other persons, and outside environment
- 5. Proliferation of Containment • BMBL Outlines: – Standard Microbiological Practices – Special Microbiological Practices – Safety Equipment – Facilities http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
- 6. What is a biohazard? – An agent of biological origin that has the capacity to produce deleterious 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 with biohazardous materials Mycobacterium tuberculosis
- 7. Risk Groups of Viruses, Bacteria, Fungi and Parasites Risk Group Agents 1 Not associated with disease in healthy adult humans Associated with human disease which is rarely serious 2 and for which preventive or therapeutic interventions are often available Associated with serious or lethal human disease for which 3 preventive or therapeutic interventions may be available (high individual risk but low community risk) Likely to cause serious or lethal human disease for which 4 preventive or therapeutic interventions are not usually available (high individual risk and high community risk)
- 8. Biosafety Levels Risk Group Biosafety Level Agents 1 Basic – BSL1 Not generally pathogenic 2 Basic – BSL2 Community pathogens 3 High Containment – BSL3 Agents spread by aerosol route 4 Maximum containment – Extreme pathogens, no antimicrobials BSL4 or other drugs
- 9. Biosafety Levels (BSLs) • Combinations of lab practices and techniques, safety equipment, and laboratory facilities • Specifically appropriate for: – Operations performed – Known or suspected routes of infection – Lab function or activity • NOTE: The Risk Group of an organism may not correspond with the Biosafety Level – This is determined by the Risk Assessment
- 10. How to Determine a Biosafety Level? • Conditions under which the agent ordinarily can be handled in the 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 absolute minimum the worker’s exposure to agents, their risk of an LAI (lab associated infection), and potential impact on the environment. • 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, West Nile) • Some infections more severe in adults (mumps, chicken pox, 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 - BSC’s, personnel protective gear, containment equipment Secondary - Room, systems Tertiary - Containment around systems • Access Control and Separation • Redundancy and Reliability • Decontamination
- 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, pcr testing (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 BSL2 Agents Not known to cause Associated with human disease, but not disease in life-threatening Healthy adults Practices Standard Microbiological BSL1 plus: Practices •Limited access •Biohazard signs •Sharps precautions •Biosafety manual with waste handling and medical surveillance Safety No special equipment Biosafety cabinets or other physical Equipment required containment for splashes or aerosols PPE: lab coats, gloves, eye/face protection Facilities Sink required BSL1 plus: •Autoclave 20
- 21. Biosafety Levels 1 & 2 BSL-1 BSL-2
- 23. Hierarchy Of Controls Most 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 containers BD 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 work areas • Do not store food in the laboratory • Minimize splashing, spraying, and generation of droplets • Do not reach into trash/sharps containers • 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 with disinfectant (daily)
- 27. Hand Decontamination • Hand washing is the single most important procedure for preventing infections http://www.cdc.gov/handhygiene/training.html#posters 27
- 28. Routine Hand Hygiene • Soap and water – Most of benefit is from removal of transient organisms on surface epidermal layers – Also removes soil, such as dirt, blood, feces – Soap and water, if available, are better than hand sanitizers in most cases
- 29. BD FACSAria-II Prevention 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 rubber diaphragm on vial or sampling port CDC/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 containment of 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 vessel forces 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 laundry bag – 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 with gloved hands (e.g., door knobs, phones) • Consider chemical compatibility (solvents, etc.) • Don’t 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 latex and nitrile) • Wash hands after removing • DO NOT wear outside the lab!
- 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 in horizontally 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 completely sash is fixed • May have solvent/chemical • Designed for seated work • Positive pressure – air storage underneath • Negative pressure blowing into face or breathing zone Pictures 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 HEPA filter • A2 may be used with nonvolatile toxic chemicals or radionuclides – minute levels of volatile toxic chemicals and radionuclides • If exhausted outside • A2 has airflow of 100 lfm Ethanol 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 horizontal Courtesy of MSOE
- 43. BSC Practices • Thoroughly clean BSC before/after use • Do not cover the front grills or block rear vent • Decontaminate all materials in and out • Discard containers inside BSC • Adjust chair height to look down through sash • Wait 15-20 minutes for HEPA filters to electrostatically charge before starting work
- 44. BSC Work Practices • Slow arm movements, move in and out in a straight 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 filter Collection vessel Overflow vessel with disinfectant
- 46. What’s Wrong With this Picture?
- 47. BSC Practices • Load only those materials needed inside cabinet – No withdrawing hands for discard or supplies • Wait 5 minutes after loading BSC before working • Do not cross hands while working or sway side to side • No Bunsen burners or alcohol burners in the BSC Corning products – Use disposable loops and needles – Bacti-cinerator or micro-burner
- 48. What Do You Notice About this Picture? CDC photo: James Gathany
- 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 manufacturer’s label instructions • Disinfect bench tops at least daily • Decontaminate equipment sent for repair 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 of safer devices annually
- 54. Sharps Precautions • Do not overfill sharps container – Seal before waste reaches indicator line on container • Keep upright • Have adjacent to work area • NOT for chemical disposal • DO NOT: – Recap needles before placing in sharps container – 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 OR chemically disinfected – ALL rDNA materials that have not been chemically disinfected – Contaminated gloves, spill cleanup materials, etc. • NOTE: ALL RECOMBINANT DNA CONTAMINATED MATERIALS MUST BE TREATED AS THOUGH THEY WERE INFECTIOUS!
- 56. Disposal/Handling of Contaminated Items Pipettes Pipette tips Solids Bulk Sharps Liquids Pipettes Pipette Biohazard Disinfectant Disposal Container Sharps Trays Box Container 10% bleach 15 minutes Autoclave Glassware Biohazard container Waste 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 taken home for cleaning – The lab technician will coordinate the 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 shipping regulations, 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 spills of biological material • Absorb liquid, preclean with detergent (to remove organic materials), 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 fingers Courtesy of MSOE
- 62. Emergency Procedures Accidental Exposure in the Lab • Remove PPE and provide immediate care to the exposure site: • Wash wounds and skin with soap and water for 15 minutes • 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 the Emergency Clinic
- 63. Thank You! Be sure to complete the associated quiz for this training module.
Editor's Notes
- Hepatitis Banc C – RG 2
- Decon into cabinet with alcohol or disinfectant (remove environmental contaminants)Decon out with disinfectant, especially if using infectious organisms. This includes vortex mixers, pipets, tip boxes, discard bin, plastic discard bag, etc.
- Moldy collection flaskIf there was any disinfectant in the flask, it is not sufficient or fresh (see previous slide)No in line filterNo secondary container (if outside BSC) to prevent breaking and leakageFlask should be netted if glass, or plastic
- When laboratorians request a BSC decon because of contamination problems, 99% of the time it is actually due a dirty cabinet, poor technique or incubator problem. It is very difficult to dislodge particles (viruses, bacteria, etc.) once they have lodged in the HEPA filter (Van der Waals forces) and the chances that the HEPA filter is the problem are minimal.
- She is actually working too close to the front of the cabinet!Risking infection from her materialsRisking contamination of her materials**Julie would like us to add another thing that is wrong: She generally doesn’t accept a face shield without safety glasses/goggles underneath
- Julie’s note: Students should notify the laboratory supervisor or lab technician only. As they will contact EHS (Julie). Students shouldn’t have to contact two sets of people.