Biological Safety It s in Your Hands

1. Biological Safety…It’s in Your Hands

2. Biological Safety Training Module 1: Basics of Biosafety Module 2: Risk Assessment Module 3: Working with Infectious Agents Module 4: The Biosafety Levels Module 5: The Biosafety Audit

3. Working with Infectious Agents in the Lab What does your lab work with? Click the Button to Learn More Lentivirus/Retrovirus Adenovirus Human Cell Lines Attenuated E. coli Other Agents

4. Working with Lentivirus in the Lab Biosafety Level 2+ Perform experiments in a BL2 Lab Utilize BL3 procedures and precautions Determine the risks Which procedures will create risks to lab personnel? How can the procedures and precautions utilized in the lab reduce the risks?

5. Risks of Working with Lentivirus Blood-Borne Pathogens Update Training Annually http://ehs.uky.edu/classes/bloodborne/bptrain.html Usually spread through direct exposure to infected body fluids and tissue culture fluids Possibility of aerosol transmission if exposed to large quantity

6. Lab acquired infections have resulted from splashing of infected materials, skin exposure, and puncture wounds Extreme care should be taken to avoid spills and splashes Assume virus present on any surface that was in contact with infected materials Risks of Working with Lentivirus

7. Drying in environment reduces concentration of infectious virus on surfaces within several hours Steam sterilization and chemical decontamination are highly effective Susceptible to many disinfectants How Can You Reduce the Risk?

8. How Can You Reduce the Risk? Blood-Borne Pathogen Prevent contact of infected materials with gloves and gowns Protect mucous membranes with respirator, face shield, face mask, and/or goggles Prevent aerosol formation

9. Work with 3rd generation lentiviral vectors Safer than wild-type vectors Do not contain the full complement of genes of the HIV virus Only gag, pol, and rev genes are expressed in these systems making them much safer to use in the laboratory How Can You Reduce the Risk?

10. BSL2+ containment Personal Protective Equipment Use of BSC and Sealed Centrifuge Buckets or Sealed Rotors Wash Hands Decontaminate Work Surfaces How Can You Reduce the Risk?

11. Risk of Adenovirus in the Lab Pathogen of mucous membranes Even replication-defective can cause corneal and conjunctival damage Usually spread through droplet, aerosol, hand to mouth routes Can be complemented in vivo, becoming replication competent Quite stable Can be infective even in ether or chloroform

12. Working with Adenovirus in the Lab Biosafety Level 2 is required for Adenoviral work in the lab Keep infected animals segregated and contained as they can shed virus Test for replication competent recombination rate Use effective disinfectant 5% phenol 0.5% Sodium hypochlorite

13. Work with adenoviral vector systems that have deletions Safer than wild-type vectors Do not contain the full complement of genes of the wild-type virus E1, E2, or E3 cassettes may be removed Newly engineered vector systems are “gutless” containing few viral genes How Can You Reduce the Risk?

14. Reduce the Risk! Protect mucous membranes with respirator, face shield or goggles Prevent aerosol formation Personal protective equipment Gowns and gloves Wash hands

15. OSHA defines human cell lines as Other Potentially Infectious Materials (OPIMs) Many cell lines are infected with known or unknown viruses Laboratory work with human cell lines is considered Biosafety Level 2 and will usually be completed in a Biological Safety Cabinet Laboratories working with human cells must have an Exposure Control Plan in place and offer all employees a Hepatitis B Vaccination Working with Human Cell Lines

16. Working with Human Cell Lines General Guidelines: No Sandals, No Shorts Lab Coats and Gloves Decontaminate Waste Wash Hands Know Your Cell Line Tested for Infectious Agents? Primary or Immortalized? Tumor-Derived?

17. Working with Attenuated E. Coli Examples: K12, DH5a, Top10 Microbes that don’t cause disease in healthy humans BSL-1 Bench Work Decontaminate liquid waste with bleach prior to disposal Wipe down work surfaces on a daily basis with disinfectant No Shorts or Sandals Labcoat and Gloves

18. Other Infectious Agents in the Lab Determine the hazards associated with your lab’s specific agents What biosafety level should be used for this agent? What personal protective equipment should be utilized? What route of infection is most common with this agent? Contact the Department of Biological Safety for help determining specific risks for your agent

19. For more information on infectious agents: American Type Culture Collection (ATCC) http://www.atcc.org/ Health Canada MSDS for Infectious Agents http://www.phac-aspc.gc.ca/msds-ftss/index.html Biosafety in Microbiological and Biomedical Laboratories http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

20. Disinfectants Most Effective on Clean Surfaces Clean large amounts of biological material prior to disinfection Large amounts of protein quickly exhaust the disinfectant and prevent complete disinfection

21. Disinfectants Effective Disinfectants 0.5% Sodium Hypochlorite EPA Approved Chemical Disinfectants for Hospitals Iodophors Quaternary Ammonium Compounds Ineffective against spores Know What is Right for Your Lab

22. Disinfectants Less Effective Disinfectants Ethanol, Isopropanol 70% is more effective than 95-100% Combination of long contact time and short dry time makes ethanol difficult to use for effective disinfection of hard surfaces Effective for soaking instruments Never use ethanol as a hand wash, due to severe drying of skin Use ethanol in a BSC to remove residue of chlorine bleach

23. Factors Affecting Disinfectant Effectiveness Organic load present on surface to be contaminated or in liquid to be decontaminated Presence of spores versus vegetative bacteria Growth phase of organisms to be removed Freshly-prepared disinfectant pH of disinfectant Diluents utilized to prepare disinfectant

24. Can you recognize safe practices?What’s wrong with this picture?

25. Using a Biological Safety Cabinet Minimize storage of boxes and supplies in and around the BSC Never store items atop the BSC Wash hands before and after working in the BSC Wear a clean lab gown and gloves while working in the cabinet

26. Using a Biological Safety Cabinet Effectiveness of a BSC is a result of directional airflow that must not be disrupted Avoid rapid movement of arms in and out of the BSC that may disrupt airflow Close lab doors to prevent drafts Minimize traffic around BSC Walk slowly past BSC if necessary

27. Disinfect the work surface of the BSC before and after work Disinfect all items which go into and come out of the BSC Do not store any items in the BSC Allow the BSC to operate before work begins and after work ceases Using a Biological Safety Cabinet

28. Using a Biological Safety Cabinet Do not block air intake or exhaust Clean up all spills in the BSC immediately After spills and on a routine basis clean and disinfect the tray under the work surface Get More Training! http://ehs.uky.edu/classes/bsc/welcome.html

29. What’s wrong with this picture?

30. Want More Training on BSCs? The Department of Biological Safety has an on-line training program for biological safety cabinets. http://ehs.uky.edu/classes/bsc/welcome.html

31. Ultraviolet Light in the Lab Do not work in BSC when UV light is on Surfaces must be directly bathed in UV light to be effectively disinfected Do not rely on UV light as primary means of disinfection Many organisms have repair mechanisms for UV damage and cannot be effectively inactivated

32. UV light is not more effective the longer it is on 30 minutes is sufficient BSC sash should be fully closed and the blower turned off if UV light is on Do not work within 6-10 feet of cabinet when UV light is on if sash is not fully closed UV light will reflect off cabinet surfaces to outside of cabinet UV lights must be maintained for proper function Periodically wipe dust from bulb Measure wavelength on an annual basis to check that emission is in effective range Ultraviolet Light in the Lab

33. Let others in the lab know when you have UV devices in use, including handheld UV lights and transilluminators. Ultraviolet Light in the Lab

34. Use a face shield with UV light equipment to prevent damage to skin and eyes Unprotected exposure to UV light can cause serious damage to eyes and skin Eye Damage, Ultraviolet Keratosis Skin Damage, Erythema (Sunburn) Ultraviolet Light in the Lab

35. Ultraviolet Light in the Lab

36. Need More Information? For more information on the topics covered in this training check out these websites: CDC Primary Containment for Biohazards http://www.cdc.gov/od/ohs/biosfty/bsc/bsc.htm UK Department of Biological Safety http://ehs.uky.edu/biosafety/ Biosafety in Microbiological and Biomedical Laboratories http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm NIH Guidelines for Recombinant DNA http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html