1. Working with Biological Materials in the O’Connell Laboratory Grace O’Connell, PhD [Date Training Is Given]

2. Basic Requirements Research involving biological agents is regulated by the National Institutes of Health (NIH), Centers for Disease Control and Prevention, the US Department of Agriculture and the State of California. UC Berkeley requires a BUA approved by CLEB prior to the initiation of the experiment. CLEB works with campus Biosafety Officer Bob Hashimoto, who can provide additional guidance (bobhash@berkeley.edu or 643-6562). bobhash@berkeley.edu

3. Purpose of This Training UC Berkeley and CLEB require that the PI provide lab-specific biosafety training on the hazards of the specific experimental procedures used in their laboratory to members of their research group. One way to document this training is to record it in the minutes of a group meeting, listing all participants.

4. Contents of This Training CLEB Administrative Requirements CLEB Administrative Requirements Role of the Investigator/Lab Staff Role of the Investigator/Lab Staff Description of the Experiment-BUA Description of the Experiment-BUA –The Agents Used –The Template Risk Assessment/Biosafety Level Risk Assessment/Biosafety Level –Good Work Practices –Personal Protective Equipment –Engineering Controls Waste Disposal/Decontamination Waste Disposal/Decontamination Emergency Procedures Emergency Procedures

5. CLEB Administrative Requirements CLEB requires the following from every PI approved to use biological agents: CLEB requires the following from every PI approved to use biological agents: –Train all research group members on lab- specific hazards and procedures –Report incidents involving biological agents and clinical specimens immediately to EH&S –Submit a BUA amendment for any change in personnel or Scope of Work

6. Description of the Experiment-BUA The Scope of Work-from the O’Connell BUA narrative The scope of the research includes mechanical testing of animal and human samples and cell culture. The scope of the research includes mechanical testing of animal and human samples and cell culture. Spine sections will be acquired either from a local abattoir or through a donor bank (e.g. NDRI for human samples). Spine sections will be acquired either from a local abattoir or through a donor bank (e.g. NDRI for human samples). Care should be taken when using equipment for sample preparation and testing. Equipment should be considered as biohazard contaminated (performed in 2166 Etcheverry). Care should be taken when using equipment for sample preparation and testing. Equipment should be considered as biohazard contaminated (performed in 2166 Etcheverry).

7. Description of the Experiment-BUA The Agents Used The O’Connell lab uses the following: Human specimens Human specimens Animal specimens (bovine) Animal specimens (bovine) Human Cells Human Cells

8. Description of the Experiment-BUA The Risks That May Affect Containment Level These are some of the common experimental risks associated with our research: The primary route of exposure of our biological materials is through the bloodborne route. Procedures involving sharps, especially when handling human and animal subjects is of primary concern. The primary route of exposure of our biological materials is through the bloodborne route. Procedures involving sharps, especially when handling human and animal subjects is of primary concern. All employees and students are encouraged to receive vaccination against hepatitis B. All employees and students are encouraged to receive vaccination against hepatitis B. All used sharps should be placed in rigid hard plastic sharps containers that will be brought to the red bag consolidation area for disposal as medical waste. All used sharps should be placed in rigid hard plastic sharps containers that will be brought to the red bag consolidation area for disposal as medical waste. Reusable metal sharps shall be autoclaved at 121 degrees C for 30 minutes and 20 PSI. Reusable metal sharps shall be autoclaved at 121 degrees C for 30 minutes and 20 PSI.

9. Description of the Experiment-BUA The Risks That May Affect Containment Level All human clinical specimens shall be handled with standard Universal Precautions. Universal Precautions is an approach to infection control. According to the concept of Universal Precautions, all human blood and certain human body fluids are treated as if known to be infectious for HIV, HBV, and other bloodborne pathogens.Universal Precautions is an approach to infection control. According to the concept of Universal Precautions, all human blood and certain human body fluids are treated as if known to be infectious for HIV, HBV, and other bloodborne pathogens. Our lab will perform analytical and tissue culture studies with human and animal specimens. You should apply 10% bleach and 70% ethanol as disinfectants to clean up following an experiment.

10. Risk Assessment/Biosafety Level Summary of Good Work Practices Wash Hands Frequently Wash Hands Frequently Use Personal Protective Equipment and Engineering Controls Use Personal Protective Equipment and Engineering Controls Minimize Use of Sharps and if used, handle carefully Minimize Use of Sharps and if used, handle carefully Do Not Eat, Drink, or Smoke in the lab Do Not Eat, Drink, or Smoke in the lab Disinfect Work Areas Disinfect Work Areas

11. Risk Assessment/Biosafety Level Good Work Practices Centrifugation: During cell preparation, samples will need to be centrifuged in a sealed vial (the centrifuge will contain a cone and will be self contained). Centrifugation: During cell preparation, samples will need to be centrifuged in a sealed vial (the centrifuge will contain a cone and will be self contained). –Transport samples in sealed tubes placed in a tube rack. Care will be taken to avoid spillage.

12. Risk Assessment/Biosafety Level Good Work Practices Sample preparation: Spine samples will need to be dissected for biomechanical studies. Sample preparation: Spine samples will need to be dissected for biomechanical studies. –Proper safety attire shall be worn, including gloves, lab coat, safety goggles, and appropriate clothing Never work in the laboratory alone. Never work in the laboratory alone.

13. Risk Assessment/Biosafety Level Good Work Practices Room 5102 is additional wet lab space to perform assays. Room 5102 is additional wet lab space to perform assays. Sample Transportation: When transporting samples between rooms, make sure solutions are sealed and placed in a hard sided container. Sample Transportation: When transporting samples between rooms, make sure solutions are sealed and placed in a hard sided container. Samples should be fixed with 4% paraformaldehyde or previously frozen at -20 o C prior to transportation. Fixing and freezing are effective methods make the tissues no longer viable. Samples should be fixed with 4% paraformaldehyde or previously frozen at -20 o C prior to transportation. Fixing and freezing are effective methods make the tissues no longer viable. All solutions will be labeled and biohazard solutions will be labeled with a biohazard sticker. All solutions will be labeled and biohazard solutions will be labeled with a biohazard sticker.

14. Risk Assessment/Biosafety Level Good Work Practices Autoclave: Tools that will need to be used sterilely will be autoclaved. These items include medical tools, glass bottles, and pipette tips. Autoclave: Tools that will need to be used sterilely will be autoclaved. These items include medical tools, glass bottles, and pipette tips. Note: Biohazard waste should be disposed of in red biohazard bags and disposed of through EH&S. Biohazard waste should not be autoclaved. Note: Biohazard waste should be disposed of in red biohazard bags and disposed of through EH&S. Biohazard waste should not be autoclaved. Caution: Autoclaves can be a physical hazard due to the heat and steam so no one is permitted to use it unless they have been trained on the safety procedures and how to operate the settings. Caution: Autoclaves can be a physical hazard due to the heat and steam so no one is permitted to use it unless they have been trained on the safety procedures and how to operate the settings.

15. Risk Assessment/Biosafety Level Personal Protective Equipment The following personal protective equipment must be worn when manipulating tissue or using the biosafety cabinet: The following personal protective equipment must be worn when manipulating tissue or using the biosafety cabinet: –Gloves (latex or nitrile) –Water resistant lab coat –Goggles/safety glasses

16. Risk Assessment/Biosafety Level Engineering Controls The following engineering controls must be used whenever handling tissue in the laboratory: The following engineering controls must be used whenever handling tissue in the laboratory: –Biosafety cabinet –Sink for Hand Washing –Mechanical pipettes –Sealed vials –Sharps containers/Broken Glass box –Eyewash/deluge shower (use only in an emergency)

17. Waste Disposal/Decontamination Waste Disposal: Tissue culture plastic used for cell culture will be bleached then disposed of in a biohazard bag. Red bag waste shall be picked up by Environmental Health and Safety. Waste Disposal: Tissue culture plastic used for cell culture will be bleached then disposed of in a biohazard bag. Red bag waste shall be picked up by Environmental Health and Safety. Paraformaldahyde and any mixed biological and chemical waste solutions should be disposed of through EH&S as chemical waste. Paraformaldahyde and any mixed biological and chemical waste solutions should be disposed of through EH&S as chemical waste. Spill procedures (biological tissues/blood/waste): Clean spills by soaking it in 10% bleach for 20 minutes before cleaning up and disposing as biohazardous waste. Spill procedures (biological tissues/blood/waste): Clean spills by soaking it in 10% bleach for 20 minutes before cleaning up and disposing as biohazardous waste. –Notify Dr. O’Connell and EH&S of any spills within 8 hours.

18. Emergency Procedures In the event of a laboratory accident: In the event of a laboratory accident: –Tend to injured person, wash the wound with water. –Notify Dr. O’Connell or the lab supervisor IMMEDIATELY so that CLEB and the Biosafety Officer can be notified within 8 working hours. –Initiate clean up procedures. –Notify EHS, if the PI is not available, 642-3073 or 642-3333 (After Hours) –Seek medical attention at Tang or the Alta Bates ER at 2450 Ashby Avenue after hours.

19. Conclusion This training is designed to inform you of the regulatory requirements regarding use of biological materials and our lab’s implementation of the campus safety requirements. All experiments with human clinical specimens by CLEB and any changes require an approved amendment to the BUA. Contact the Biosafety Officer for assistance, if needed (Bob Hashimoto).