1. FDS Biosafety Training Shulin Chen Lab Developed and posted 11/14/08 http://www.bio-safety.wsu.edu/biosafety/

2. Outline Introduction Biosafety Levels  Practices  Facilities Biosafety Cabinet Case Study Resources Regulations

3. Biosafety Introduction Practicing Science Safely Means That Before Any Activity...  YOU KNOW the risks  YOU KNOW the worst things that could happen  YOU KNOW what to do if they should happen  YOU KNOW AND USE the prudent practices, protective facilities, and protective equipment needed to mitigate the risks

4. Biosafety Levels – Know the Risks BSL 1: Material not known to consistently cause disease in healthy adults. BSL 2: Associated with human disease. Hazard is from percutaneous injury, ingestion, or mucous membrane exposure. Some agents with environmental or agricultural impact. BSL 3: Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences. BSL 4: Dangerous/exotic agents which pose a high risk of life- threatening disease, aerosol-transmitted lab infections or related agents with unknown risk of transmission

5. Appropriate Biosafety Level Agent Risk Group + Risk Assessment = Appropriate Biosafety Level Agent Risk Group Resource: http://www.absa.org/riskgroups/index.html Risk Group Examples: RG-1 E. coli (standard host vector systems) Most plant pathogens e.g., Aschochyta spp. Adenovirus type 1-4, most human cell lines RG-2 Klebsiella, Listeria, E. coli O157, Human Adenovirus, Candida spp. Giardia lamblia, Some human cell lines HEK-293 HeLa (characterized agents) RG-3 Mycobacterium tuberculosis, M. bovis, Coccidioides immitis, HIV, R. richettsii

6. A Complete Risk Assessment includes Agent Characterization (Risk Group RG) Personnel Factors (experience) Work Activity Factors Environmental Factors Equipment Factors Risk Consequences Probability Profile

7. Risk Assessment Risk of Activity – same agent can have different containment levels:  Procedures that produce aerosols have higher risk  Procedures using needles or other sharps have higher risk  Handling blood, serum or tissue samples may have lower risk  Purified cultures or cell concentrates may have higher risk  Large volumes (>10 L) have higher risk

8. Protection Should Match the Risk: What is the Expected Route of Infection?

9. AGENT / LAI* *LAI= Laboratory Acquired Infections PRIMARY HAZARDS Klebsiella spp. LAI’s have been reported Direct contact of mucous membranes with contaminated objects, inhalation of infectious aerosols; accidental parenteral inoculation; ingestion Staphylococcus epidermidis Direct contact of mucous membranes, accidental parenteral inoculation.

10. AGENTPathogenicity/Epidemiology Klebsiella spp.Pathogenicity: Frequent cause of nosocomial urinary and pulmonary infections; wound infections, secondary infection in lunch of patients with chronic pulonary disease, enteric pathogenicity, (enterotoxin). Epidemiology: Worldwide distribution. 2.3 of all infection due to Klebsiella spp. Are hospital- acquired: causes 3% of all acute bacterial pneumonia; common source of nosocomial outbreaks. Staphylococcus epidermidisPathogenicity: Generally considered to be part of normal Human flora but has been documented as a pathogen in numberous cases of bacteremia, surgial wounds, urinary tract and ophthalmologic infections especially in immunocompromised hosts. Epidemiology: World wide distribution. Associated with nosocomial infections.

11. Know What to Do What Constitutes a Biological Exposure Risk? What to do When There has Been a Potential Exposure? How to Clean up a Biological Spill? When Should you Seek Assistance With a Spill Clean up?

12. What Constitutes a Biological Exposure Risk? Breach in Primary or Secondary Containment What is Primary Containment? What is Secondary Containment? What to do when there is a breach? Tell your supervisor and the Biosafety Manager for WSU. Fill out an incident report, and all other paper work as required. What to do when there is a near miss? Fill out an incident report.

13. Primary Containment Lab practices – standard lab practice, limited access, biohazard warning sign, sharps/needle precautions, SOPs, decontamination, waste. Safety equipment – biosafety cabinets (BSC), sharps containers, sealed rotors. Personal protective equipment (PPE) – lab coat, gloves, goggles, respirators.

14. Establishing Precautions to Minimize Risk Primary containment

15. Practices/Equipment PPE Risk Assessment Immunization Surveillance

16. Secondary Containment - Facilities Facility provides containment through traditional construction BSL-1 & BSL-2  Best practices HVAC provides negative pressure in BSL-2 labs. Depending on risk assessment this may be required. Facility provides containment through special design features BSL-3  Anterooms  Double doors  Alarmed redundant HVAC systems

17. Possible Biological Release Scenarios Is This a Release? 1. Open a centrifuged tube at the open bench that contains Klebsiella spp.? 2. Using a wire transfer loop and a bunsen burner pick a Klebsiella colony and streak for isolation work in the BSC. Is This a Release? 3. Transfer liquid cultures with a transfer pipette to produce BSL-2 organism serial dilutions. Work performed at the open bench.

18. Answers to Release Scenarios 1. Open a centrifuge tube of Klebsiella at open bench Centrifugation causes the pressure inside the centrifuge tube to be altered from the ambient pressure. When opening the lid aerosols are formed. Performing this action at the open bench exposes you and your coworkers to potentially infectious aerosols 2. Bunsen burner in the BSC Bunsen burners cause air turbulence in the Biosafety Cabinet which may lead to escape of bio-aerosols and subsequent exposure to infectious aerosols to the cabinet worker and others in the laboratory.

19. Answers to Release Scenarios 3. Infectious liquid transfer at bench Any manipulation of infectious liquids potentially creates aerosols. To perform manipulations of infectious liquids safely this work should be done in the Biosafety Cabinet Summary In all three of these case studies there has been a potential release of infectious aerosols. NOTE: The following slide shows a picture of aerosol generation when expelling a liquid from a pipette.

21. Spill Clean up of Biological Agents Surface Contamination:  Alert co-workers  Define/isolate contaminated area  Put on appropriate PPE (personal protective equip.) to include gloves, lab coat and face shield (if appropriate)  Remove/glass/glass shards with forceps or scoop  Apply absorbent towels to spill – Do NOT apply disinfectant directly to the spill as this may aerosolize the agent  Apply disinfectant to towel surface  Allow adequate contact time (generally 20 minutes)  Remove towels, mop up; clean with alcohol or soap/water or other agent as appropriate.  Dispose of materials in biohazardous waste  Notify lab instructor

22. Spill in a BSC If the spill of an infectious agent was enough to create puddles or liquid in the drain pan then the following procedure should be followed:  a.Leave the cabinet running and close the view screen for about 5 minutes. This will allow aerosols to settle before starting cleanup.  b.The drain pan should be flooded with appropriate disinfectant. Leave the disinfectant in the pan for required contact time, longer if the spill involved a high organic load and 10% bleach is used. The disinfectant then needs to be drained out and the surfaces thoroughly cleaned with water to prevent corrosion.

23. Spill Clean up of Biological Agents Personal Exposure: Clean exposed surface with soap/water, (1 minute), eyewash (eyes) 15-20 min., or rinse mouth 3x’s with water Apply first aid and treat as an emergency Notify lab instructor – fill out incident report and other forms as requested If appropriate report to medical clinic for treatment/counseling

24. Major Spills This is a spill of a potentially biohazardous material that will take more than 30 minutes to clean up (not including the agent deactivation period of 10-60 minutes depending on the biological agent and disinfectant in use) Call 911 for assistance

25. General Guidelines for Surface Decontamination AGENTDISINFECTANTINACTIVATION TIME Recombinant DNA10% Bleach20 minutes Bacterial Spores>6% Hydrogen Peroxide30 minutes Vegetative Bacteria10% Bleach20 minutes Viruses & Viroids10% Bleach20 minutes Fungi10% Bleach20 minutes Feline Parvovirus20% Bleach30 minutes Free Living Cryptosporidium 70% Ethanol10 minutes Most Parasites10% bleach30 minutes Prions50% bleach60 minutes

26. Practical Disinfectants for use in Recombinant DNA Research NE=Not Effective, b=variable results dependent on virus Reference: NIH Guidelines for working with Recombinant DNA Lab Safety Monograph (Appendix D updated) Liquid Disinfectants Practical RequirementsInactivatesImportant Characteristics Use/DilutionContact time minutes Vegetative Bacteria Lipo- viruses Nonlipid Viruses Bacterial Spores Effective Shelf life > 1week Effective for Surface Decontam ination Liquids For Discard Category Lipo virusBroad Spectrum Quaternary Ammonia Compounds 0.1-2.0%10NE++++ Phenolic Compounds 1.0-5.0%10NE++b++ Chlorine Compounds 500ppm(a)1030++++++ Iodophor25- 1600ppm(a) 1030++++++ Alcohol, Ethyl70-85%10NE++b++ Alcohol, isopropyl 70-85%10NE++b++ Formaldehyde0.2-8.0%1030++++++ Glutaraldehyde 2.0%1030++++++

27. Know and Use prudent practices, protective facilities, and protective equipment needed to mitigate risks

28. Recommended Biosafety Containment & Practices – Infectious Agents BSLAgentsPractices Primary Containment Safety Equipment Primary Barriers Facilities (Secondary Barriers) 1 Not known to consistently cause disease in healthy adults Wash hands after handling viable materials No smoking, eating, handling contact lenses, applying cosmetics No mouth pipetting Minimize aerosols& splashes Decontaminate work surfaces daily Do NOT wear gloves out of lab Remove gloves before touching door handles phones etc. None required Doors Sink required Work surfaces easily cleaned & impervious to water Sturdy furniture

29. Recommended Biosafety Containment & Practices – Infectious Agents BSLAgentsPractices (Primary Containment) Safety Equipment (Primary Barriers) Facilities (Secondary Barriers) 2Associated with human disease, hazard = percutan- eous injury, inges- tion, mucous mem- brane exposure BSL-1 practice plus: Limited access  Lab entry and exit policies. Biohazard warning signs at entrance of lab “Sharps” precautions Decontaminate stock cultures prior to disposal BSM - SOP Report Spills & Accidents Leak-proof containers for transport PPE required (as approp.) Documented training Primary barriers = BSC I or II or other physical barriers, e.g. splash shields, covered centrifuges etc. -For all open manipulations of agents that cause splashes or aerosols HEPA filters required on vacuum lines Personal Protective Equipments PPE’s: laboratory coats; gloves; face protection as needed BSL-1 plus: Autoclave available Locking Doors BSC (as needed) L Eyewash readily available Negative pressure in laboratory (ideal)

30. Biosafety Cabinet What is a Biosafety Cabinet (BSC)? Understand Equipment Limitations Safe Use of a BSC  Why & When to Use a BSC SOP for BSC BSC Maintenance / Certification  Decontamination

31. What is a Biosafety Cabinet? A BSC is a piece of equipment that is only as good as the users understanding of how to appropriately use and maintain it There are different Classes of BSC’s  Class I – user protection but no product protection  Class II – product, user and environment protection Provides a ~100ft/min protective air barrier  Class III- Air tight - use with Risk Group 3/4 agents Fume hoods and laminar flow clean work benches are NOT Biosafety Cabinets.

32. What is a Biosafety Cabinet

33. Outline What is a Biosafety Cabinet (BSC)? Understand Equipment Limitations Safe Use of a BSC  Why & When to Use a BSC SOP for BSC BSC Maintenance / Certification  Decontamination

34. Understand Equipment Limitations Fume Hoods Not for use with infectious materials or environmentally dangerous organisms Exhaust air not HEPA filtered; not easily decontaminated Laminar Clean Air  Horizontal Air blows through HEPA at rear of work surface across work surface and into face of user NOT for use with lab animals, potentially infectious materials, drug formulations.  Vertical Air blows through HEPA on top of work surface downward Air may flow under a sash and into the room. Some models don’t even have sashes Turbulence can distribute aerosols into user’s breathing zone Not for use with potentially infectious materials  Reverse-flow Pull air from front of cabinet through pre-filter and HEPA at rear Used to reduce user’s exposure to animal urine, dander etc. (with PPE) Not for work with biohazards (no containment)

35. Understand Equipment Limitations BSC class I  Inward airflow protects worker  Exhaust to outside w/wo HEPA filter  No product protection  Not for use with tissue culture BSC class II  “ Sterile” work surface  Protects worker, product and environment  For use with RG 2-3 agents  Restrictions for flammables and chemicals depending on type of class II BSC

36. Understand Equipment Limitations Class II BSC  Type A2 – 30% exhausted to the room Not recommended for hard ducting or chemical usage  All Type B cabinets – air flow from the rear grill is discharged into the exhaust system so activities that may generate chemical vapors should be conducted towards the rear of the BSC  Type B3 – 30% exhausted to outside Minute chemical usage allowed  Type B1 – 70% exhausted to outside Minute amount of volatile chemicals allowed  Type B2 – 100% exhausted to outside Small amount of volatile chemicals allowed Not a good choice for tissue culture work

37. Outline What is a Biosafety Cabinet (BSC)? Understand Equipment Limitations Safe Use of a BSC  Why & When to Use a BSC SOP for BSC BSC Maintenance / Certification  Decontamination Resources

38. Safe Use of a Biosafety Cabinet  Must be isolated from other work areas, lab entry, high traffic areas and away from air ducts  Must be operated properly to minimize risk (more info later in SOP section) Sash must be kept at manufacturers recommended height while in use with biological agents.

39. Safe Use of a Biosafety Cabinet  Flames are not a constant temperature and therefore can cause air turbulence inside a BSC Compromise protective air barrier integrity  Are not spark/fire proof. Gas Bunsen burners should not be used in BSC’s 70% recirculated (Class II type A2) Uncombusted gas (yellow flame) captured in plenums. UV light – microscopic holes in gas tubing. Net result high explosion hazard

40. Bunsen Burner - BSC Fire

41. Safe Use of a Biosafety Cabinet  UV lights Cause skin cancer and cataracts. Therefore eyes and skin should not be exposed. Not reliable as primary decontamination  Line of site  Poor penetration  UV light turns on at 80 micro watts /square cm  UV light only kills at 160 micro watts/square cm Secondary decontamination  15 minutes generally sufficient  More than 15 minutes eats up plastics and glue in HEPA filter and in long term can destroy BSC integrity and cause cabinet leak test to fail

42. Safe Use of a Biosafety Cabinet Do’s  Keep supplies to a minimum and ~6” from sides  Discard all infectious materials inside the BSC (disinfectant)  Understand how the BSC works  Turn the cabinet on for manufacturer recommended time before using it  Inform your supervisor if you are immuno-compromised  Wear a mask if you are coughing or sneezing Don’ts  Rely on the UV light to decontaminate  Put anything on the front grill  Move quickly  Use sweeping motions  Keep supplies close to the sides and back  Move the sash below or above the recommended standard  Use a gas flame in the hood  Remove material before disinfecting. When working with potentially infectious material

43. FDS Safe Use of a Biosafety Cabinet 1. What happens if the front sash is up too high?

44. Safe Use of a Biosafety Cabinet 1. Question: Front sash up too high Answer: Down flow of clean (HEPA filtered) air will provide more pressure than inflow of air through the front sash and safety is compromised i.e. agent contamination of the user and environment is likely.

45. FDS 2. What Happens If The Front Sash Is Down Too Low?

46. Safe Use of a Biosafety Cabinet 2. Question: Front sash down too low. Answer: In flow of air through front sash is stronger than down flow of air. Net result is compromised product integrity, i.e. outside air flows into the cabinet providing a source of potential contamination.

47. FDS 3. What Happens If Objects Are Placed On The Front Grill Of The BSC?

48. Safe Use of a Biosafety Cabinet 3. Question: Effect of objects placed on the front grill. Answer: The front grill is one of the dirtiest areas of the entire lab so placing items on this grill is not a good idea. Additionally this interferes with air balance and causes too much down flow of air which creates safety issues, i.e. release of agent outside of the cabinet.

49. FDS 4. What Happens When Your Arm Moves Over The Work Inside The BSC?

50. Safe Use of a Biosafety Cabinet 4. Question: Arm moves over work area. Answer: Down flow air moves contaminants on your arm/lab coat down onto the work surface compromising product sterility. Note: If you are right handed the waste container should be on the right side of the BSC.

51. FDS 5. What Happens When People Walk Behind The BSC?

52. Safe Use of a Biosafety Cabinet 5. Question: Person walks behind the Biosafety Cabinet. Answer: Protective air current is ~100 feet / minute = a little over one mile an hour. People walk at 2-3 miles per hour and this can disrupt the protective air current of the biosafety cabinet.

53. Outline What is a Biosafety Cabinet (BSC)? Understand Equipment Limitations Safe Use of a BSC  When to Use a BSC SOP for BSC BSC Maintenance / Certification  Decontamination Resources

54. When to Use a BSC Product Protection  Tissue Culture User /Environmental Protection  Aerosolizing activities with Risk Group 2 agents.

55. Aerosol Producing Activities Sonication, blending, mixing, vortexing Almost any liquid manipulation Centrifuging Pouring Pipetting Opening containers at non-ambient pressures, (e.g. fermenters, freezer vial) Loading syringes and injections

56. Aerosol Producing Activities (cont.) Intranasal inoculation of animals Changing animal bedding Harvesting tissue, eggs Tissue grinder/homogenizers Lasers Cell sorters Necropsy

57. Aerosol Control Procedures Biosafety Cabinet (BSC) properly maintained, used and certified No blow-out of pipettes Sidewall contact when delivering liquids Use of aerosol-minimizing tips Secondary enclosure for sonicators, etc. Capped centrifuge tubes, sealed cups, carriers and rotors

58. Outline What is a Biosafety Cabinet (BSC)? Understand Equipment Limitations Safe Use of a BSC  Why Use a BSC SOP for BSC BSC Maintenance / Certification  Decontamination Resources

59. Why Use a BSC? – User Protection Account for 50% of LAI’s (Lab Acquired Infections) 1. Inhalation exposure to infectious aerosols Account for 20% of LAI’s 1. Parenteral Inoculation (to include animal bites and scratches) 2. Spills, splashes, skin and mucous membrane exposure 3. Ingestion

60. Outline What is a Biosafety Cabinet (BSC)? Understand Equipment Limitations Safe Use of a BSC  Why & When to Use a BSC SOP for BSC BSC Maintenance / Certification  Decontamination Resources

61. BSC Standard Operating Procedure Start up Procedure Pre-plan to minimize supplies and movements in and out of the cabinet Turn on for manufacturer’s recommended time prior to use Ensure that back grill paper catch is clear Start up Procedure Verify air flow Verify drain valve is closed Disinfect cabinet surfaces Place all materials inside the cabinet (disinfect as appropriate) Verify view screen height is appropriate

62. BSC Standard Operating Procedure Work in the Cabinet Wash hands and arms Do NOT cover the front or back grill with anything Use slow movements Avoid door and people movement beside cabinet Avoid open flames Work in the Cabinet Place pipettes exposed to infectious materials in disinfectant inside the cabinet before removing Safe work area is 6 inches from sides of cabinet Enclose all potentially contaminated material & disinfect surfaces before removing from BSC Disinfect work surfaces

63. Standard Operation Procedure - BSC Develop from Manufacturer’s guidelines Baker SOP available in BSL-2 Biosafety Manual Template (supplemental resource D)  http://www.bio-safety.wsu.edu/forms.asp http://www.bio-safety.wsu.edu/forms.asp

64. Outline What is a Biosafety Cabinet (BSC)? Understand Equipment Limitations Safe Use of a BSC  Why & When to Use a BSC SOP for BSC BSC Certification Resources

65. BSC Certification Certification Must be certified every year / after installation / and after the BSC is moved NSF certifier to provide certification to NSF standards requires  2 HP compressor  *Pump designed for aerosol challenge Certification Check air-flows  100-110 ft/min in-flow (A2)  75 ft/min in-flow (A1)  65-75 ft/min down-flow Check HEPA filter  Particle generator* – 18-20 lbs pressure to produce 0.3 micron particle size (99.9% efficiency test)

66. Outline What is a Biosafety Cabinet (BSC)? Understand Equipment Limitations Safe Use of a BSC  Why & When to Use a BSC SOP for BSC BSC Maintenance Resources

67. BSC Maintenance Maintenance Keep the cabinet clean If cabinet is off keep front sash down to minimize dust Clean up spills that get under the cabinet immediately http://www.bio- safety.wsu.edu/biosafety/ http://www.bio- safety.wsu.edu/biosafety/ Disinfect before & after use Bleach pits stainless steel Monitor minihelic gauge Maintenance Don’t store materials in the hood Verify that the front grill/back grill and paper catch are unobstructed Once a month disinfect the bottom of the hood and then drain >10-12 year old BSC’s - stop stocking parts

68. Outline What is a Biosafety Cabinet (BSC)? Understand Equipment Limitations Safe Use of a BSC  Why & When to Use a BSC SOP for BSC Decontamination Resources

69. Decontamination Microorganisms can be grouped in terms of resistance to disinfectants. Highly Susceptible Susceptible Resistant Highly Resistant Extremely Resistant

70. Microbial Chemical Resistance Highly Susceptible Mycoplasmas Susceptible Gram +&- bacteria Fungal Spores Resistant Non-Enveloped Viruses Mycobacteria Highly Resistant Bacterial Endospores Protozoal Oocytes Extremely Resistant Prions

71. Effectiveness of Chemical Disinfectants Effectiveness is influenced by:  Composition of chemical disinfectants  Concentration of microorganisms and chemical disinfectants  Contact time with the Disinfectant  Presence of organic matter  Presence of interfering substances  Temperature at which they are used

72. Summary of Disinfectant Activities Disinfectant Disinfection Level Bacteria Lipophil. Viruses Hydro- Philic Viruses M. tuberculosis Fungi Comments Quaternary Ammonium (0.5-1.5%) low++--+/Ineffective against bacterial spores. May be ineffective against Pseudomonas and other gram negative bacteria; recommendation limited to environmental sanitation Alcohols (ethyl and isopropyl) 60-85% Intermed.++-+/+ Ethyl or isopropyl alcohol at 70-80% concentration is a good general purpose disinfectant; not effective against bacterial spores., high concentrations of organic matter diminish effectiveness; flammable Phenolics (0.4%-5%) Intermed.+++/-++ Not sporicidal; phenol penetrates latex gloves; eye/skin irritant; remains active upon contact with organic soil; may leave residue Chlorine (100- 1,000 ppm) Intermed.++++/+Not generally sporicidal; inactivated by organic matter; fresh solutions of hypochlorite (chlorox) should be prepared weekly; corrosive; irritating to eyes and skin Iodophors (30- 1,000 ppm iodine) Intermed. ++++/ Recommended for general use. Wescodyne diluted 1 to 10 is a popular disinfectant for washing hands. Inactivated by organic matter Glutaraldehyde (2-5%) high+++++ Used to sterilize surgical instruments that can not be autoclaved; strong odor; use with adequate ventilation. Not for use on environmental surfaces. Because it is a sensitizer and causes asthma it is not recommended for laboratory use.

73. Case Study – Putting it all Together Work with M. bovis.  rDNA  M. bovis culture Agent Risk Group http://www.absa.org/riskgroups/bacteriasearch.php?genus=Mycobacterium http://www.absa.org/riskgroups/bacteriasearch.php?genus=Mycobacterium Risk Assessment Appropriate BSL Special Considerations

74. Practicing Science Safely Means That Before Any Activity...  YOU KNOW the risks MSDS  YOU KNOW the worst things that could happen Sick/Die  YOU KNOW AND USE the prudent practices, protective facilities, and protective equipment needed to mitigate the risks Risk Assessment  YOU KNOW what to do Follow Biosafety Manual

75. A Complete Risk Assessment includes Agent Characterization (Risk Group RG) Personnel Factors (experience) Work Activity Factors Environmental Factors Equipment Factors Risk Consequences Probability Profile

76. A- Agent Characterization Pathogenicity of material – disease incidence and severity Routes of Transmission – parenteral, airborne or ingestion Agent Stability – ease of decontamination Infectious Dose – LD50 Concentration – infectious material/volume & working volume Origin of material - Wild Type, exotic, primary cells Availability of effective prophylaxis – Hepatitis B vaccine Medical surveillance – effectiveness & availability of treatment

77. A- Agent Characterization Pathogenicity of material – 4 th most common LAI, MDR strain used increases the risk. Routes of Transmission – parenteral, airborne & ingestion Agent Stability – Resistant to many disinfectants Infectious Dose – 10 bacilli by inhalation Concentration – cultures grown on plates Origin of material - Wild Type Availability of effective prophylaxis – INH (Isoniazid) available, ineffective for most MDR strains Medical surveillance – treatment of MDR Mycobacterium problematic

78. B - Personnel Factors Biosafety training SOP training Health Assessment (immunosuppressed) Experience with the Agent (B, I, A)  Beginner, Intermediate, Advanced MSDS read Experience with the procedures (B,I,A) Use of PPE training Job hazard analysis undertaken Allergies (animal, environmental)

79. C- Work Activity Factors Aerosol generating potential Potential for self- inoculation (needle stick, lesion) Sample origin and concentration Volume of pathogen used Animal use (types, potential viral shedding, bites and scratches) Replication competency Recombinants Cell line characteristics Toxin production (y/n, MSDS)) Modification of pathogen (y/n, result / implication) Vector use (y/n, describe) Inventory Records Contingency plan (exposure, accidental releases / spills) Techniques – cryogenics, cytometry Disinfectant used as directed

80. D – Environmental Factors Level of containment available Degree of monitoring of containment factors Impact of external activities Biosecurity (access and inventory control) Availability and status of emergency support Housekeeping and Trades Personnel Access by public

81. E – Equipment Factors Equipment Maintenance (frequency, status) Manual Reservoirs empty &disinfected Standard Operating Procedures Location of use Ventilation Consideration

82. F - Rating of Risk Consequences Rating of Risk Consequences ClassRatingConsequences 4CatastrophicPeople: fatalities, evacuation outside site area Environment: irreversible, long-term damage outside site area Business: total loss: > $2 million Interruption: > 2 months image: severely damaged, > 1 week, national 3CriticalPeople: serious injuries, effects outside site area Environment: reversible, short-term damage outside site area Business: total loss: > $100,000 - $2 million Interruption: > 2 – 8 weeks image: damaged, > 1 week, regional 2MarginalPeople: minor injuries, annoyance outside site area Environment: only site area effected Business: total loss: > $5,000 - $100,000 Interruption: > 1 – 2 weeks Image: < 1 week, local 1NegligiblePeople: no effects Environment: only building effected Business: total loss: < $ 10,000 Interruption: > 1 week Image: no effects

83. F - Rating of Risk Probability Table 2 - Rating of Probability ClassRatingProbabilityDefinition AFrequentMore than once a yearLikely to occur repeatedly in life cycle system BModerateOnce per yearLikely to occur several times in life cycle system COccasionalOnce in 5 yearsLikely to occur sometime in life cycle system DRareOnce in 25 years (e.g. once in the life cycle of the system) Not likely to occur in system life cycle, but possible EUnlikelyOnce in 100 yearsWill occur once in a lifetime of a site FVery UnlikelyOnce in 1,000 yearsAlmost impossible to occur

84. G -Probability Profile Risk Profile/Probability Consequences Probability Negligible 1 Marginal 2 Critical 3 Catastrophic 4 Frequent A Moderate B Occasional C Rare D Unlikely E Very Unlikely F

85. Resources - People WSU – Biosafety Pullman and offsite campuses  Lorraine McConnell 509-335-4462 lorrmcc@wsu.edu lorrmcc@wsu.edu WSU- EH&S Pullman  Mike Kluzik509-335-9553mkluzik@wsu.edumkluzik@wsu.edu

86. Resources Biosafety in Microbiological and Biomedical Laboratories: http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc. htm http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc. htm American Biosafety Association: http://www.absa.org/XriskgroupsX/index.html http://www.absa.org/XriskgroupsX/index.html Canadian Biological MSDS’s: http://www.phac- aspc.gc.ca/msds-ftss/index.htmlhttp://www.phac- aspc.gc.ca/msds-ftss/index.html WSU Biosafety Web Page: http://www.bio- safety.wsu.edu/biosafetyhttp://www.bio- safety.wsu.edu/biosafety

87. Web Resources - BSC CDC / NIH Guidelines on Biosafety Cabinets:  http://www.ors.od.nih.gov/ds/pubs/bsc/contents.html http://www.ors.od.nih.gov/ds/pubs/bsc/contents.html AIHA – BSC Safety Information:  http://www2.umdnj.edu/eohssweb/aiha/accidents/BSC.ht m#Biosafety%20Cabinet http://www2.umdnj.edu/eohssweb/aiha/accidents/BSC.ht m#Biosafety%20Cabinet CDC / NIH 2 nd ed. “Primary Containment for Biohazards”:  file:///Z:/Biosafety%20Cabinets/CDC- Primary%20Containment%20for%20Biohazards%20(BS C's).htm file:///Z:/Biosafety%20Cabinets/CDC- Primary%20Containment%20for%20Biohazards%20(BS C's).htm Ohasis – Office of Health & Safety Primary Containment for Biohazards Section V.  file:///Z:/Biosafety%20Cabinets/Primary%20Containment %20for%20Biohazards.htm file:///Z:/Biosafety%20Cabinets/Primary%20Containment %20for%20Biohazards.htm

88. Web Resources - BSC CDC – Laboratory Biosafety Guidelines 3 rd edition:  http://209.85.173.104/search?q=cache:h373QUWmYfEJ: www.phac-aspc.gc.ca/publicat/lbg-ldmbl- 04/pdf/lbg_2004_e.pdf+CDC+2007+3rd+edition+of+Selec tion+Installation+and+Use+of+BSC&hl=en&ct=clnk&cd= 5&gl=us&client=firefox-a http://209.85.173.104/search?q=cache:h373QUWmYfEJ: www.phac-aspc.gc.ca/publicat/lbg-ldmbl- 04/pdf/lbg_2004_e.pdf+CDC+2007+3rd+edition+of+Selec tion+Installation+and+Use+of+BSC&hl=en&ct=clnk&cd= 5&gl=us&client=firefox-a ABSA Position Paper on the Use of Ultraviolet Lights in Biological Safety Cabinets:  http://209.85.173.104/search?q=cache:tbuGD9FOadAJ:w ww.ehs.umass.edu/ABSA%2520UV%2520light%2520pap er.pdf+position+paper+on+the+use+of+ultraviolet+lights +in+biological+safety+cabinets&hl=en&ct=clnk&cd=1&g l=us&client=firefox-a http://209.85.173.104/search?q=cache:tbuGD9FOadAJ:w ww.ehs.umass.edu/ABSA%2520UV%2520light%2520pap er.pdf+position+paper+on+the+use+of+ultraviolet+lights +in+biological+safety+cabinets&hl=en&ct=clnk&cd=1&g l=us&client=firefox-a

89. Web Resources - BSC BMBL Biosafety Cabinet Appendix A (4 th ed):  http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4aa.htm http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4aa.htm The Baker Company  http://www.bakerco.com/resources/intro.php http://www.bakerco.com/resources/intro.php NUAIRE How to select a BSC  http://www.nuaire.com/products/biological_safety_cabin ets/select_a_bio_cabinet.htm http://www.nuaire.com/products/biological_safety_cabin ets/select_a_bio_cabinet.htm NSF Accreditation Standards  http://www.nsf.org/business/biosafety_accreditation/ind ex.asp?program=BiosafetyCabCert http://www.nsf.org/business/biosafety_accreditation/ind ex.asp?program=BiosafetyCabCert

90. Regulations OSHA Bloodborne Pathogen Standard: http://www.osha.gov/SLTC/bloodbornepathogens/ind ex.html http://www.osha.gov/SLTC/bloodbornepathogens/ind ex.html Centers for Disease Control and Prevention (CDC): http://www.cdc.gov/od/sap/ http://www.cdc.gov/od/sap/ NIH Recombinant DNA Guidelines: http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html CDC Interstate Shipment of Etiological Agents: http://www.cdc.gov/od/ohs/biosfty/shipregs.htm http://www.cdc.gov/od/ohs/biosfty/shipregs.htm CDC Etiologic Agent Import Permit Program: http://www.cdc.gov/od/eaipp/ http://www.cdc.gov/od/eaipp/ APHIS – USDA: http://www.aphis.usda.gov/vs/ncie/http://www.aphis.usda.gov/vs/ncie/

91. THANK YOU