1	Biosafety in Biological and Biomedical Laboratories Marshall University Feb 9 2007
2	Major Safety Concerns General Biosafety Recombinant DNA (rDNA) Management of infectious agents and biohazardous waste Bloodborne Pathogen Exposure Human Subject Protection (IRB, Dr. C. Winger) Radioisotope Use (Dr. W. McCumbee) Use of Animals in Research (Dr. Howard) Chemical Safety (Dr. K. Guyer, Brian Carrico)
3	General Principles of Biosafety Containment: to reduce or eliminate exposure of lab workers and others to hazardous biological agents Bacteria, viruses, fungi, parasites Recombinant DNA Potentially dangerous cell lines
4	General Principles of Biosafety Three elements of containment: Safety equipment (personal protective equip) Facility design Lab practice and techniques Extent of containment depends of level of risk and nature of agent.
5	Examples of Safety Equipment Biological Safety Cabinets (Laminar Flow Hood) Safety Centrifuge Cup Gloves, lab coats, gowns, face shield, goggles, shoe covers, respirators
6	Four Biosafety Levels (BSLs) Combinations of lab practices and techniques, safety equipment and lab facilities Appropriate for operations performed and routes of transmission of infectious agents/rDNA BSL1-4 represent conditions under which the agent can be safely handled
7	Biosafety Levels 1-2 BSL 1: appropriate for work with organisms known to not cause in healthy adult humans. E coli, Bacillus spp, Exempt categories of rDNA work BSL 2: indigenous moderate risk agents. Primary Hazard: Skin break, mucous membrane exposure or ingestion. Examples Hepatitis B, HIV, some Salmonellae Human derived blood and blood products Cell culture work and some rDNA
8	Biosafety Levels 3-4 BSL 3: work with indigenous or exotic agents with potential for respiratory transmission or lethal consequences. Examples: M. tuberculosis, St. Louis encephalitis virus Primary and Secondary barriers to protect personnel in contiguous areas BSL 4: lethal exotic agents esp. where there is no vaccine or therapy. Marburg Virus BSL 4 facility is separate facility or HVAC isolated zone
9	BL 1 practices: Standard Microbiological Practices Access to lab is limited or restricted when experiments are in progress Workers wash hands after handling viable organisms, after removing gloves and before leaving the lab Eating, drinking, smoking, handling contact lens, brushing teeth, storing food is absolutely forbidden. Inspectors routinely look through the regular trash.
10	BL1 practices, part 2 Mouth pipetting is prohibited- must use mechanical pipetting devices Safe handling of sharps (disposal) Procedures minimize splashing and aerosols Decontaminate work surfaces on a daily basis All cultures, stocks and other infectious materials are decontaminated by an approved method (usually autoclaving). Biohazard signs are posted at lab entrance. Insect and rodent control program in place.
11	Biosafety Level 2 Similar to BSL-1 and suitable for work with agents of moderate hazard Lab personnel get specific training in handling pathogens Access to the lab is limited when work is being conducted. Greater precautions taken with contaminated sharps Specific procedures for reducing aerosols or splashes
12	BSL 3 and 4 Levels of personal protection increase Access is restricted Lab design becomes more critical Animal Biosafety Levels: special sets of procedures
13	Which Biosafety Level should you be using in your lab? Discuss specific safety issues with your mentor and safety committee chairs Lab heads should prepare written safety protocols that are lab/experiment specific Read the entire Biosafety in Microbiological and Biomedical Laboratories Section VI or the NIH rDNA guidelines Contact Dr. Primerano at 696-7338
14	General Biosafety Advice Get a lab coat and other safety equipment immediately. Don’t wear open shoes!!! Keep hair short or tied back Glove allergies: nitrile may help
15	Transformed or cancer cell lines Any experiment involving transformed or cancer cell lines falls under Biosafety Level 2 (BL2) practices. This work should be done under a biological safety cabinet. Special precautions are taken to limit aerosols. Cells and media must be treated with 10% bleach before autoclaving. Decontaminate spills with bleach and wipe down hood after working with cells. Protective clothing (lab coat and gloves) should be worn when working with the cells. Restrict access to the lab. See “Safe Handling of Cell Lines” on the IBC web site
16	USEFUL WEB SITES Biosafety in Microbiological and Biomedical Laboratories (BMBL) 4th Edition (April 1999) http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm http://www.musom.marshall.edu/biosafety/ University of Kentucky Lab Safety http://www.uky.edu/Services/EHS/handbook/
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18	The NIH Guidelines
19	The NIH Guidelines Apply to… rDNA research that is funded by the NIH, OR performed at or sponsored by an institution that receives any NIH funding for rDNA research. Means that all rDNA research at MU must comply with guidelines Failure to comply can result in (i) suspension, limitation, or termination of financial assistance for the noncompliant NIH-funded research project and of NIH funds for other rDNA research at the institution, or (ii) a requirement for prior NIH approval of any or all rDNA projects at the institution. Rationale: For biosafety to be meaningful, it has to be observed by all investigators at an institution
20	NIH Guidelines “Guidelines” does not mean “optional” They are one of several conditions of NIH funding
21	rDNA (rDNA) as defined by the NIH Guidelines rDNA molecules are either Molecules constructed outside cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, OR Molecules that result from the replication of those described above
22	Organization of the NIH Guidelines Section I – Scope Section II – Safety Considerations Section III – Types of Experiments Covered (6) IIIA – IBC Approval, RAC Review, NIH Director Approval Mandatory IIIB – NIH/OBA and IBC Approval Mandatory IIIC – IBC and IRB Approval, RAC Review Mandatory IIID – IBC Approval Before Initiation IIIE – IBC Notification At Initiation IIIF – Exempt Experiments Section IV – Roles and Responsibilities
23	SECTION II. SAFETY CONSIDERATIONS Section II-A. Risk Assessment Section II-A-1. Risk Groups (Appendix A) Section II-A-2. Criteria for Risk Groups Section II-A-3. Comprehensive Risk Assessment Section II-B. Containment
24	Risk Group Classifications Agents are classified according to their relative pathogenicity for healthy adult humans (from least to most pathogenic) Risk Group 1 (RG1) agents are not associated with disease in healthy adult humans. Risk Group 2 (RG2) agents are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available. Risk Group 3 (RG3) agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available. Risk Group 4 (RG4) agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available.
25	ABSA Web site If don’t which risk group your agent falls into: Call or email Don Primerano OR Consult the ABSA website: http://www.absa.org/resriskgroup.html
26	6 categories of rDNA experiments based on whose approval is needed IBC approval, RAC review, and NIH Director approval before initiation (Section III-A), NIH/OBA and IBC approval before initiation (Section III-B), IBC and Institutional Review Board approvals and RAC review before research participant enrollment (Section III-C), IBC approval before initiation (Section III-D), IBC notification simultaneous with initiation (Section III-E), and Exempt from the NIH Guidelines (Section III-F).
27	Section III-F. Exempt Experiments (6 Exemptions) The following rDNA molecules are exempt from the NIH Guidelines and registration with the IBC is not required: Section III-F-1. Those that are not in organisms or viruses. (e.g. PCR and genotyping reactions) Section III-F-2. Those that consist entirely of DNA segments from a single nonchromosomal or viral DNA source. Section III-F-3. Those that consist entirely of DNA from a prokaryotic host including its indigenous plasmids or viruses when propagated only in that host (or a closely related strain of the same species), or when transferred to another host by well established physiological means.
28	Section III-F. Exempt Experiments (Category 6) The following rDNA molecules are exempt from the NIH Guidelines and registration with the IBC is not required: Section III-F-4. Those that consist entirely of DNA from an eukaryotic host including its chloroplasts, mitochondria, or plasmids (but excluding viruses) when propagated only in that host (or a closely related strain of the same species). Section III-F-5. Those that consist entirely of DNA segments from different species that exchange DNA by known physiological processes, though one or more of the segments may be a synthetic equivalent. List of exchangers given in Appendix A. Section III-F-6. Those that do not present a significant risk to health or the environment as determined by the NIH Director, with the advice of the RAC. List of exemptions in Appendix C, Exemptions under Section III-F-6.
29	IBC Website
30	Marshall University rDNA Application Form 1. Program Director: 2. Principal Investigator(s): 3. Grant Title: 4. rDNA Registration Title (leave blank): 5. Description of Project: (in layman's terms) 6. Does this project involve transfer of drug resistance genes to a microorganism that does not acquire the gene naturally? Yes ____ No____ If yes consult section III-A-1. 7. Does this project involve the cloning of toxin molecules with LD50 less than 100 ng per kg body weight? Yes ____ No____ If yes consult section III-B-1.
31	Marshall University rDNA Application Form 8. Does this project involve the deliberate transfer of DNA or DNA or RNA derived from rDNA into one or more human research participants? Yes ____ No____ If yes consult section III-C-1. 9. Does this project involve: (a) the use of Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as Host-Vector Systems (Yes ____ No____ If yes consult section III D-1), (b) experiments in Which DNA From Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents is Cloned into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector Systems (Yes ____ No____ If yes consult Section III-D-2), (c) experiments Involving the Use of Infectious DNA or RNA Viruses or Defective DNA or RNA Viruses in the Presence of Helper Virus in Tissue Culture Systems (Yes ____ No____ If yes consult section III-D-3), (d) introduction of rDNA into whole animals (Yes ____ No____ If yes consult section III-D-4), (e) introduction of rDNA into whole plants (Yes ____ No____ If yes consult section III-D-5), (f) the use of more than 10 liters of culture (Yes ____ No____ If yes consult section III-D-6).
32	Marshall University rDNA Application Form 10. If you answered Yes to any question in part 9, please provide the following information (i) source(s) of DNA, (ii) nature of the inserted DNA sequences, (iii) host(s) and vector(s) to be used. 11. Will an rDNA gene be expressed and if so what expression system will be used and which protein that will be produced? 12. Experiment Category, Agent Classification and Host Vector Class and Containment Level For your research, please list the appropriate experiment category, agent classification and containment level(s) for your research. You will need to read sections I, II and III and some appendices of the NIH Guidelines to complete this part. 12A. Experiment Category: ___Select from NIH Guidelines subsections IIIA - IIIF. These are the six categories 12B. Agent Classification: ___ Select from NIH Guidelines Appendices A, B and C Input a specific Appendix e.g. appendix A-II 12C. Containment Levels: ___ Select from NIH Guidelines Appendix G and I Input a specific BL1-4 + Appendix e.g. Appendix G-II-A
33	Marshall University Lab Personnel Form: Program Director: Principal Investigator(s): Grant Title: rDNA Registration Title (leave blank): Institution: Department: Laboratory room no.: PI Tel#: Emergency Tel. no.: Laboratory Staff: (Please list) ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________ ________________________________
34	Marshall University School of Medicine Lab Safety Form I. rDNA approval is only valid for the PI(s) listed on the registration application. This title is not transferable. Have you ever been approved for rDNA Research as Principal Investigator at Marshall University? Yes No rDNA approval is given by title only. If for grant purposes you wish to change the title of an rDNA application that has been approved previously you may do so if: A. Containment levels remain the same as previously approved. B. Probes are the same. C. Host-Vector-Donor System is unchanged. Under the above conditions if you wish to make a change of title(s). A. Give approval # of referred registration. B. List new title(s) _______________________________________ _______________________________________ _______________________________________ III. As directed under the NIH Guidelines Section IV_B_1_I, should you (PI) and your staff undergo medical surveillance? Yes No If yes, contact IBC Chairman's Office 304- 696-7338 IV. I have read the NIH rDNA Guidelines and I agree to adhere to its rules and regulations regarding safe laboratory practices. ___________________________ _______________________ _______________________ Principal Investigator's Name Signature Date
35	Levels of Responsibility NIH Institution (University) IBC Principal Investigator
36	IBC Review In a nutshell, what must IBCs review? rDNA research for conformity with the NIH Guidelines Potential risk to environment and public health What do IBCs assess in reviewing rDNA research? Containment levels per NIH Guidelines Adequacy of facilities, SOPs, PI and lab personnel training Institutional and investigator compliance; e.g., adverse event reports
37	P.I. Responsibilities Determine the category of rDNA research Submit rDNA applications if and when necessary Report problems or violations to the IBC Report issues with the NIH Guidelines Be trained in good microbiological techniques and provide training in lab specific protocols to all lab personnel Adhere to IBC approved plans for handling cell lines and spills Comply with all facets of the rDNA guidelines including shipping requirements
38	IBCs and Exempt Research Who determines whether research is exempt? A matter of institutional policy according the guidelines. At Marshall, the PI (lab head) makes the determination whether a protocol falls into the exempt category If you as PI decide that a given protocol is exempt you do not need to complete an rDNA application BUT you must notify the IBC Chair by email. Give a short description of the protocol and tell why it is exempt. NIH OBA and IBC Chair can help with determinations
39	rDNA Regulations and Biosafety Levels Select BSL1-4 based on the category and specific nature of your protocol. Example: work with RG2 organisms must be conducted at BL2. Consult rDNA guidelines in all cases. BL1+BL2 are appropriate for much rDNA work at Marshall (but not all). A minimum of BL1 should be used even for exempt protocols. Each investigator must be familiar with the guidelines.
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41	Infectious Medical Waste
42	INFECTIOUS WASTE MANAGEMENT
43	64CSR56 Infectious Medical Waste
44	64CSR56 Infectious Medical Waste
45	Infectious Medical Waste
46	INFECTIOUS WASTE MANAGEMENT
47	TYPES OF INFECTIOUS WASTE Cultures of microorganisms & biologicals Human blood and blood products Pathological wastes All Contaminated Sharps Contaminated animal carcasses, body parts, bedding and related wastes Materials (soil, water, or other debris) which result from the cleanup of a spill of any infectious medical waste. Waste contaminated by or mixed with infectious medical waste.
48	Cultures and stocks of microorganisms and biologicals
49	Human Blood & Blood Products All human blood (wet or dried) Products from human blood. Bloodborne pathogens
50	Sharps Any article that can puncture or cut Sterilize if sharps have been used in human patient care or treatment OR if used to deliver infectious agents Examples: needles, syringes, scalpel blades, razors, forceps
51	Pathological Waste Human pathological wastes – tissues, organs, body parts, containers of body fluids All pathological waste should be packaged by the investigator and picked up by Stericycle. Stericycle provides containers and liners. Containers and liners are located in Room 116 BBSC. Contact Julia Schreiber or Connie Berk if you need packing materials.
52	Animal Waste Contaminated animal carcasses, body parts, animal bedding known to have been exposed to infectious agents during research These carcasses should be taken to the animal facility where Julia or Connie will assist you in preparing for pickup by Stericycle..
53	INFECTIOUS WASTE MANAGEMENT
54	WV BUREAU OF PUBLIC HEALTH LICENSE FOR BBSC
55	INFECTIOUS WASTE MANAGEMENT
56	BBSC INFECTIOUS WASTE MANAGEMENT PLAN
57	BBSC INFECTIOUS WASTE MANAGEMENT PLAN
58	INFECTIOUS WASTE MANAGEMENT OVERVIEW
59	PACKAGING DISPOSABLE INFECTIOUS WASTE
60	SPECIAL PACKAGING RULES Glassware
61	DELIVERING DISPOSABLE WASTE TO ROOM 119A
62	DELIVERING REUSABLE CONTAMINATED ITEMS TO ROOM 119 BBSC
63	STERILIZATION OF INFECTIOUS WASTE
64	IN THE EVENT OF A SPILL
65	DON’T PUT NONINFECTIOUS ITEMS IN ORANGE BAGS bubble wrap paper towels, either from drying your hands after washing them or from wiping down the bench with a disinfectant scalpel blade wrappers needle wrappers Benchkote or any other bench protector gloves packaging materials such as cardboard, Styrofoam “peanuts”, etc. paper (copy paper, etc. may be recycled in any of the large blue wheeled BFI bins on all floors and in the copy room) pipettes food wrappers pop cans (can be recycled in the blue bins)
66	PATHOLOGICAL WASTE POLICY
67	PATHOLOGICAL WASTE POLICY- STERICYCLE, INC. PICK-UP Pathological waste materials are collected in lined bins provided by Stericycle, Inc. Bins are located in the Animal Facility walk-in refrigerator. Materials are collected by Stericycle, Inc. at scheduled times. We are charged ~0.60 cents per pound for disposal of pathological waste and other nonsterile materials.
68	Ethidium Bromide Waste
69	Uncontaminated Pipette Tips Do not toss in the regular trash Put in a black bag inside a box When full seal the box and place in the hallway
70	Issuance of Keys for the locked unautoclaved waste bin Numbered Keys will be issued (one per lab) in the next few weeks