Biosafety Training Shulin Chen Lab

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

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?

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. Primary containment Establishing Precautions to Minimize Risk

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? 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. 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.

18. Answers to Release Scenarios 1. Open a centrifuge tube of Klebsiella at open bench 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. • 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

19. Answers to Release Scenarios 3. Infectious liquid transfer at bench 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. • Any manipulation of infectious liquids potentially creates aerosols. To perform manipulations of infectious liquids safely this work should be done in the Biosafety Cabinet

21. Spill Clean up of Biological AgentsSurface 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 AgentsPersonal 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

26. Practical Disinfectants for use in Recombinant DNA ResearchNE=Not Effective, b=variable results dependent on virusReference: NIH Guidelines for working with Recombinant DNA Lab Safety Monograph (Appendix D updated)

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

28. Recommended Biosafety Containment & Practices – Infectious Agents

29. Recommended Biosafety Containment & Practices – Infectious Agents

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. 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 Safe Use of a Biosafety Cabinet

43. Safe Use of a Biosafety Cabinet 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. 2. What Happens If The Front Sash Is Down Too Low?

46. Safe Use of a Biosafety Cabinet • 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. 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. 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.