Biosafety Protocol STEVENS INSTITUTE OF TECHNOLOGY. Prepared and Approved by: Institutional Biosafety Committee

Transcription

1 Biosafety Protocol STEVENS INSTITUTE OF TECHNOLOGY Prepared and Approved by: Institutional Biosafety Committee Rev: 02/2010

2 This document was written by Dr. Ann Aguanno for the express purpose of compiling federal, state, local and standard biohazard compliance protocols to be applied and utilized solely by Stevens Institute of Technology. This protocol is the property of Stevens Institute of Technology and cannot be copied without the permission of Stevens Institute. This document is considered Administrative Literature, according to the Stevens Institute of Technology Copyright Policy. Context of this protocol is derived from multiple federal, state and commercial sources. Appropriate citations and acknowledgements of those sources are indicated within this document. Rev: 02/2010 II

3 This document describes the Biosafety Protocol for Stevens Institute of Technology. This plan conforms to regulations set forth by the Code of Federal Regulations, Title 29 (Labor), Chapter XVIII (Office of Safety and Health Administration), Part 1910, Subpart I, Sections.132 (29 CFR ) Personal Protective Equipment, Subpart Z, Sections.1030 (29 CFR ) Bloodborne Pathogens and.1450 (29 CFR ) Occupational Exposures in the Laboratory, Title 32 (National Defense), Parts 626 and 627, (Biological Defense Safety Program; Administrative and Work Practices Controls and Technical Safety Requirements, respectively)(32 CFR 626 and 32 CFR 627), Title 40 (Protection and Environment), Part 725 (40 CFR 725) Reporting Requirements and Review process for Microorganisms and Title 49 (Transportation), Chapter I, Part 173, Sections and the New Jersey Community Right to Know Act, the Federal Emergency Planning and Community Right to Know Act of 1986 (EPCRA ) and the New Jersey Administrative Code Title 7, Chapter 26 (G), 7:26A. In order to comply with these standards, Stevens Institute has established this Biosafety Plan to ensure that employees and students are informed about and protected from any health hazards that are associated with biohazradous materials they may be exposed to in the laboratory or other teaching or research environments. This plan is made readily available to the entire Stevens community in hard copy and internally on the World Wide Web ( the Web ). A review and evaluation of this Biosafety Plan is conducted annually and is updated as necessary. Questions regarding this plan should be directed to: Tsan-Liang Su, Research Associate Professor, Committee Chair, CES, x5697, t1su@stevens.edu (name, title, campus address, phone and ) The individuals responsible for implementation of this plan are members of the Institutional Biosafety Committee (IBC), a division of the Stevens Safety Committee. Members of the IBC are: (name, title, campus address, phone and ) Phil Leopold, Dept. Director and Professor, CCBBME, x8957, phil.leopold@stevens.edu Frank Cannavale, Technical Assistant, CCBBME, x8291, frank.cannavale@stevens.edu Xioajun Yu, Assistant Professor, CCBBME, x5256, xioajun.yu@stevens.edu Joseph Glavy, Assistant Professor, CCBBME, x8193, joseph.glavy@stevens.edu Barbara DeHaven, IO, Executive Director, Office of Sponsored Research, x8762, bdehaven@stevens.edu Rev: 02/2010 III

4 Table of Contents Page # Contact Information Foreword Overview What are biological hazards and biosafety What activities are addressed in this Protocol? Who must adhere to these guidelines? What are the responsibilities of the Stevens community? The Institution s responsibilities The Institutional Biosafety Committee s responsibilities The Biological Safety Officer s responsibilities The Principal Investigator s responsibilities The lab personnel, TA s & student s responsibilities List of Abbreviations i ii iii iv v vi vii ix x xii xiii Section I Introduction to Biosafety (A) Biosafety Containment 1 (B) Biosafety Levels 2 -BL-1, BL-2, BL-3 -Practical Description of BL-1, BL-2, BL-3 Biosafety Levels 3 -Table 1; Biosafety Levels (B1) Biosafety Levels for Animal Work5 (B2) Biosafety Levels for Recombinant DNA 5 (C) Biohazard Definitions 6 (D) Categories of Biohazards (E) Recombinant DNA 7 (F) Human Gene Therapy and Transgenic Animals (G) Human and Animal Pathogens 8 (G1) Introduction to Microorganisms/Infectious Agents -Viruses -Bacteria 9 -Fungi -Prions 10 -Parasites -Bloodborne Pathogens V

5 Page # (G2) Classification of Pathogenic Agents into Risk Groups 11 -RG1, RG2, RG3, RG4 (H) Other Potentially Hazardous Biological Materials 12 (H1) Human Blood, Blood Products, Body Fluids, Tissues (H2) Animal Use (H3) Tissue Culture/Cell Lines (H4) Tuberculosis 13 (H5) Wild Rodents (I) Experiments Covered by this Protocol 14 Section II Control Measures (A) Laboratory Practices and Techniques 15 (A1) General Laboratory Safety Practices 16 -General Rules -Personal Hygiene 17 -General Housekeeping 18 -Safety Practices with Glassware 19 -Centrifuge Safety 20 (A2) Biohazard Laboratory Safety Practices 21 -Understanding Routes of Exposure -Means of Exposure 22 -Table 2; Concentration and Particle Size of Aerosols General Control Measures to Prevent Exposure 24 -Other Recommended Laboratory Practices (A3) Transport of Biological Materials 26 (A4) Decontamination Procedures 27 -Forms of Decontamination -Physical and Chemical Means of Decontamination 28 -Heat -Autoclave Use -Liquid Disinfection 29 -General Considerations -General Types -Table 3a; Properties and Applications of Disinfectants 31 -Table 3b; Properties and Applications of Disinfectants 32 -Table 3c; Properties and Applications of Disinfectants 33 -Vapors and Gases 34 -Radiation V

6 Page # Engineering Controls (B) Safety Equipment 35 (B1) General Safety Equipment (B2) Personal Protective Equipment 37 (B3) Biohazard Specific Safety Equipment 39 -Biohazard Warning Sign and Posting -Figure 1; Biohazard Symbol 39 -Biological Safety Cabinet 40 -Safe and Effective Use of Biological Safety Cabinet 41 (C) Laboratory Facilities 42 Administrative Controls (D) Registration of Biohazardous Activities with the IBC 43 (E) Medical Surveillance 44 (F)Personnel Communication and Training 45 (G) Shipping of Biohazards 46 (H) Laboratory Inspection and Maintenance (I) Record Keeping 47 Section III Biohazard Waste (A) Categories of Biohazard Waste 48 (B) General Labeling, Packaging, Storage and Disposal 49 (B1) Labeling (B2) Packaging (B3) Storage (B4) Disposal 50 -Autoclave Procedure (C) Waste Specific Procedures 51 (C1) BL-1 and BL-2 (C2) BL-3 (C3) Pathological Waste (D) New Jersey State Guidelines 52

7 Page # Section IV Emergency Response IN THE EVENT OF AN EMERGENCY 53 (A)How to Respond to a Biohazard Accident 54 (A1) Biohazard Spill-Kit (A2) Biological Organism Spill -Spills on the Body -Spills Inside the Laboratory 55 -Spills Inside a BSC 56 -Spills Inside a Centrifuge -Spills Outside the Laboratory (A3) Blood Spills (B)How to Respond to a Chemical Accident 57 (C)How to Respond to a Radiation Accident (D)Reporting an Accident 58 Appendix A Reference Information Reference Agencies Reference Documents Resources on the Internet Appendix B BL1-4 for Biohazard Agents (excerpt from NIH Guidelines) Appendix C BL1-4 for Animals Appendix D rdna Biosafety Procedures Appendix D2 Exemptions to the NIH Guidelines for certain rdna activities Appendix E Risk Groups; Risk Group and Biosafety Level definitions Appendix E1 Table E1 Bacterial Risk Groups Appendix E2, Table E2 Viral Risk Groups Appendix E3 Table E3 Fungal Risk Groups Appendix E4, Table E4 Parasitic Risk Groups Appendix E5 Restricted Agents by the CDC Appendix F Biological Safety Cabinet Information Table F1, Cabinet Selection Table F2 Comparison of BSCs Figure F1A Horizontal Clean Bench Figure F1B Vertical Clean Bench Figure F2 Class I BSC Figure F3A Class II, Type A, BSC

8 Figure F3B Class II, Type I BSC Figure F3C Class II, Type B2 BSC Figure F4 Class III BSC Figure F5 Typical Layout for working in BSC Figure F6 Protecting the House Vacuum Appendix G Disposal of Biohazards Appendix H Forms Form 1; Laboratory Inspection Report Form 2; Annual Protocol for Use of Biohazardous Material Form 2a; Quarterly update on Biohazard Use Form 3; Registration Document for rdna Research From 3a; Registration Document for rdna Research (NIH) Form 4; Declaration of Pregnancy Form 5; Medical Consultation Form 6; Biohazard Accident Report Form 7; Statement of Training Form 8; Animal Use Control Form Rev: 02/2010 VIII

9 Contact Information All Emergencies On Campus Phone x5105 Or, dial (May also use red phones located in corridors on campus or follow instructions posted near conventional phones) Biological Safety Officer Frank Cannavale Institutional Biosafety Committee Chair: Tsan-Liang Su x8291 x5697 Risk & Safety Oversight Task Force Committee Chair: Keith Sheppard, Professor, Material Science & Engineering Tony Blazini, Associate Director, Residence Life Frank Cannavale, Technical Assistant, Dept. of Chem. & Chem Biology Jane Cannavale, Safety Officer James Collins, Deputy Chief of Campus Police Norman Forster, Director of Maintenance, Physical Plant Timothy Griffin, Chief, Campus Security Mark Samolewicz, Vice President, Human Resources Chuck Shaw, Director of Risk Management, Office of Finance Tsan-Liang Su, Research Associate Professor, Center for Environ. Sys. Safety and Environmental Officer Jane Cannavale Biohazard Waste and Chemical Waste Disposal Information Frank Cannavale, Technical Assistant Stevens Health Center Custodial Services Norman Forster, Director of Maintenance, Physical Plant Radiation Safety Officer Tsan-Liang Su, Research Associate Professor, Center for Environ. Sys. Campus Police Risk Management, Office of Finance Chuck Shaw x5260 x5130 x8291 x5276 x5106 x5278 x5107 x5122 x8568 x5697 x5276 x8291 x5678 x5278 x5697 x5105 x8568

10 Foreword This manual (Biosafety Protocol) has been developed as part of Stevens Institute of Technology s Biosafety program. The manual sets forth a Biosafety Protocol, which provides a complete program of administration controls, medical surveillance, vaccination and containment strategies for reducing risk posed by biohazardous agents. This program has been established so that the Stevens campus presents an environment that is suitable for high quality research and teaching while maintaining a safe work place. This is accomplished by protecting personnel and students from exposure to infectious agents, preventing environmental contamination, and complying with applicable federal, state and local requirements. The Biosafety program ensures this by providing Institute-wide safety guidelines, polices, and procedures for the use and manipulation of biohazards. Although implementation of these procedures is the responsibility of the Principal Investigator (PI) or course Instructor, its success depends largely on the combined efforts of laboratory supervisors, personnel and students. Therefore, planning for implementation of biological safety must be part of every laboratory activity in which biohazardous materials are used. It is important to remember that although this manual provides assistance in the evaluation, containment and control of biohazards, it is imperative that any individuals involved receive the proper supervision and training. THE MOST IMPORTANT ELEMENT IN MAINTAINING A SAFE WORK ENVIRONMENT IS STRICT ADHERENCE TO GOOD MICROBIOLOGICAL AND LABORATORY PRACTICES AND TECHNIQUES. ALL INDIVIDUALS MUST BE AWARE OF POTENTIAL RISKS AND MUST BE TRAINED AND PROFICIENT IN THE PRACTICES AND TECHNIQUESS REQUIRED FOR HANDLING BIOHAZRDOUS MATERIAL. IT IS THE RESPONSIBILITY OF THE PRINCIPAL INVESTIGATOR OR COURSE INSTRUCTOR TO PROVIDE AND/OR ARRANGE FOR APPROPRIATE TRAINING OF ALL PERSONNELL. NOTE: as NIH guidelines are continually updated and amended, the reader is directed to the NIH website, and the Code of Federal Regulations website, for any updates made to the NIH guidelines, upon which this Protocol is based. Rev: 02/2010 ii

11 OVERVIEW The following section presents a brief overview of the information presented in this Biosafety Protocol (the Protocol), the reference documents cited within it, the NIH Guidelines and Biosafety in Microbiological and Biomedical Laboratories. It is intended as a summary only; the reader is urged to obtain full, detailed information pertaining to each subject from within the text of this Protocol from the pertinent reference documents cited or from the NIH Guidelines. Use this information as a checklist for compliance rules, regulations, responsibilities and procedures. Where relevant, topics are accompanied by a reference to the sections within this Protocol. What are Biological hazards and Biosafety? A Biohazard is an agent of biological origin that has the capacity (or is perceived) to produce deleterious affects on humans; e.g. recombinant DNA molecules, their hosts and sources, bloodborne pathogens, etiological agents, genetically engineered organisms, cell cultures and tissues. Biosafety is a program of administrative controls, medical surveillance and containment strategies for reducing risks of exposure to biohazards. (for details, see Section I, Introduction to Biosafety). Rev: 02/2010 iii

12 What activities are addressed in this Protocol? This Protocol details guidelines which must be adhered to if any of the following activities are conducted (see also Section I, I): transfer of drug resistance trait into microbes which may compromise the use of the drug to control disease agents in humans, veterinary medicine or agriculture formation of recombinant DNA molecules which biosynthesize lethal toxin molecules (Section I,E ) transfer of recombinant DNA or RNA into human subjects (Section I,E) use of Risk Group 2, 3 or 4 agents or restricted agents (Section I,G2 & Appendix E) cloning of DNA from Risk Group 2, 3 or 4 agents into nonpathogenic prokaryotic or lower eukaryotic organisms agents (Section I,G2 & Appendix E) use of infectious DNA or RNA viruses in tissue culture systems (Section I,H) experiments involving whole animals or plants whose genome has been altered through the introduction of recombinant DNA molecules agents (Section I,H) experiments involving more than 10 liters of culture of any kind (NIH Guidelines Appendix K) research that is conducted at or is sponsored by an institution that receives any support for recombinant DNA research from the NIH research directly supported by NIH funds v

13 Who must adhere to these guidelines? All individuals who are involved in experiments which include any of the above activities or come in contact with any aspect of these activities or facilities, instrumentation, containment or storage systems and waste products generated from these activities. Individuals include Principal Investigators, laboratory supervisors Instructors of educational laboratories Teaching assistants, other laboratory aides and laboratory staff Graduate and undergraduate students working in research laboratories or attending educational laboratory courses Custodial and maintenance staff performing work-related activities in research or educational facilities

14 What are the responsibilities of the members of the Stevens Community regarding Biological Safety? The Institution (Stevens) must ensure that any recombinant DNA or other types of biohazard research funded by the NIH is in compliance with the NIH Guidelines as summarized in this Protocol. The Institution s responsibilities include; Establish and implement policies that provide safe conduct of biohazard research and ensure compliance with these guidelines Establish an Institutional Biosafety Committee (IBC) to carry out policies Appoint a Biological Safety Officer (a member of the IBC) if the institution conducts rdna research at Biosafety Level (BL) 3 or higher or engages in large scale (greater than 10 liters) research (see Appendices B & C and NIH Guidelines Appendix K) Appoint an individual with expertise in plants or animal containment to the IBC if rdna research involving plants or animals is conducted (Section I,H & Appendix C and NIH Guidelines Appendix P &Q) Ensure that the IBC has adequate expertise and training if activities involving transfer of rdna into humans are conducted (NIH Guidelines Appendix M) Ensure appropriate training of IBC, PI, laboratory and maintenance staff regarding biological safety and implementation of these guidelines Establish and maintain a health surveillance program for personnel involved in large scale research and activities involving organisms of Biosafety Level 3 or 4 (see Laboratory Safety Monograph for components of such a program; available from IBC or the ORDA, NIH) Report any significant problems, violations or significant research-related accidents within 30 days to the NIH, unless report filed by PI or IBC

15 Rev: 02/2010 vii

16 (What are the responsibilities of the members of the Stevens Community regarding Biological Safety? con t) Institutional Biosafety Committee (IBC) is similarly responsible for ensuring that all biohazardous activities conducted at or sponsored by the institution is in compliance with these guidelines. Compliance is ensured by the review, approval and oversight of all projects involving these activities. Who s on the IBC? A minimum of five members; two members, not affiliated with Stevens, who represent the interests of the surrounding community with respect to health and protection of environment; one member who represents the Stevens laboratory technical staff. The members should have collective experience and expertise in rdna technology, biological and general safety, and physical containment techniques and procedures. No member can review or approve their own work. In addition, a consultant with knowledge of institutional commitments and policy, applicable laws, standards of professional practice, community attitudes and the environment should be available. IBC must have a rotating Chair, a Biological Safety Officer, and plant, animal and human gene therapy experts, if applicable. The IBC responsibilities include; Establish Biosafety policies and the Biosafety Protocol to insure safe conduct of work involving biohazardous materials and revise and update the Biosafety Protocol as necessary File annual report with the NIH/ORDA including a roster of IBC members, along with biographical sketches Conduct open regular meetings and make minutes available Review and approve all proposed projects/course material involving biohazardous materials for compliance with this Protocol. Review to include assessment of containment levels, facilities, procedures, practices, training and expertise and should ensure compliance with surveillance requirements, data and adverse event reporting (Section II & Appendix F) Rev: 02/2010 viii

17 (IBC Responsibilities con t) Review and approve all purchases or transfer of materials (gifts, donations collaboration reagents, etc) associated with biohazard activities and notify PI/Instructor of results (Section II & Appendix F) Conduct periodic (yearly, until biohazard activities increase at Stevens) reviews of ongoing activities to ensure compliance (Appendix H, Form 1) Adopt emergency plans for accidental spills and personnel containment from biohazard activities (Section IV) Report significant violation and biohazard activity-related accidents to the NIH Provide technical consultation to PI and instructors Establish and monitor procedures for the disposal, storage and transport of biohazardous materials and update when necessary (Section III) Provide basic biosafety training sessions for the Stevens community and consultation when requested Establish guidelines for the use, handling and disposal of animals (Section I, Appendix C & NIH Guideline Appendix Q) Keep records of committee activities, applicable laws and regulations, proposals, maintenance and training records, and accident reports. x

18 (What are the responsibilities of the members of the Stevens Community regarding Biological Safety? con t) Biological Safety Officer (BSO) is required if the institution engages in large-scale research activities involving viable organisms containing rdna of level BL-3 or BL-4. The BSO responsibilities include; Periodic (yearly, until Stevens biohazard activities increase) inspections of laboratories and facilities to ensure compliance (Appendix H, Form 1) Report to the IBC any significant problems, violations and research-related accidents Determining emergency plans for handling accidental spills, personnel contamination and investigation of laboratory accidents involving biohazards Provide advice on laboratory/facility security Provide technical advice to PI and IBC (See Laboratory Safety Monograph, available from the IBC or the ORDA and NIH, for more details regarding BSO)

19 (What are the responsibilities of the members of the Stevens Community regarding Biological Safety? con t) Principal Investigator, Course Instructor or Facility Supervisor has the primary responsibility for complying with these guidelines. The Principal Investigator s responsibilities include; Be adequately trained in good microbiological techniques and Stevens Biosafety training Determine the types of biohazardous agents that will be encountered in the laboratory or research facility and assess the biological characteristics of agents used and/or created (see Section I and Appendix H, Form 1) Plan and implement the appropriate biosafety protocol to insure the safe use of these biohazards, including the required level of physical and biological containment (Sections I & I, DI and Appendix H, Forms 2, 3) Before performing research or educational activities, submit for review and approval a description of the appropriate biohazards protocols that will be employed in the research or teaching facility (Section II,D and Appendix H, Forms 2, 2a & 3) Submit a yearly description, with quarterly updates, to the IBC of all work involving biohazardous materials, including appropriate biosafety protocols utilized (Section II,D and Forms 2, 2a & 3) Maintain communication with the IBC and notify it of any changes in research activities that would alter their approved Biohazard protocol (Appendix H, Forms 2, 2a, 3) Comply with protocols specific to their research activities, the Stevens Biosafety Protocol, the NIH Guidelines and any applicable federal and local laws

20 (PI responsibilities, con t) Assure that personnel working with biohazardous materials are appropriately trained so they are aware of the hazards and are proficient in the practices and techniques required for safe handling (Appendix H, Form 7) Inform staff/student/ta of any advisable precautionary medical practices and relate when it may be inadvisable for pregnant or immune compromised individuals to work with biohazardous materials. Decline access when deemed necessary (Appendix H, Form 4) Ensure the integrity of physical and biological containment systems (Section II) Assure required hazard warning signs are posted and biohazard containers are labeled to educate all visitors, including custodial and maintenance staff Assure that biohazardous waste is treated and disposed of properly (Section III) Comply with shipping requirements for biohazard molecules (Section II, F) Immediately report any biohazard accident to the BSO and IBC (Appendix H, Forms 5 & 6) Assure that all biohazardous materials are properly disposed of and all relevant biohazardous information is fully disclosed to IBC before closing or leaving a laboratory facility (Appendix H, Form 8) Rev: 02/2010 xii

21 (What are the responsibilities of the members of the Stevens Community regarding Biological Safety? con t) Laboratory Personnel, Teaching Assistants and Students, although under the supervision of PI, instructor or lab supervisor, also bear specific responsibilities including; Receive biosafety training from either the Stevens IBC or laboratory or course supervisor Comply with all written and oral rules pertaining to the work being conducted Report any biohazardous accidents immediately to supervisor Use PPE as directed by protocol or supervisor, or insure appropriate PPE use (Section II,B) Rev: 02/2010 xiii

22 LIST OF ABBREVIATIONS NIH Guidelines- Guidelines for Research Involving Recombinant DNA Molecules The Protocol- Stevens Biosafety protocol BL- Biosafety Level BSC- Biological Safety Cabinets BSE- Bovine Spongiform Encephalopathy CDC- Centers for Disease Control HPS- Hantavirus Pulmonary Syndrome HEPA- High Efficiency Particulate Air IBC- Institute Biosafety Committee NIH- National Institutes of Health ORDA- Office of Recombinant DNA activities PI-Principal Investigator PPE- Personal Protective Equipment rdna- Recombinant DNA RG- Risk Group RMW- Regulated Medical Waste TA- Teaching Assistant TCE- Transmissible Spongiform Encephalopathy USDHHS- U.S. department of Health and Human Services UV- Ultraviolet Rev: 02/2010 xiii

23 SECTION I Introduction to Biosafety Individuals who work in scientific laboratories or similar type research facilities are exposed to many kinds of hazards. In fact, this can be said of most workplaces; in some the hazards are well-recognized (fire, for example) and the appropriate precautions are obvious. However, some hazards are not so readily apparent and may call for precautions not ordinarily encountered elsewhere. The purpose of this manual is to address the biohazardous agents that may be encountered in the occupational and educational settings present at Stevens; namely research and teaching laboratories and research facilities. The intent is to provide basic information on biohazards and the control of biohazard exposures that can be applied within these settings. This manual does not address radiation or chemical hazards. For information regarding protocols designed to handle these materials the reader is directed to the Radiation Safety Officer or the Stevens Chemical Hygiene Plan, respectively. (A) Biosafety Containment Biological safety or biosafety, is the application of knowledge, techniques and equipment to prevent personal, laboratory and environmental exposure to potentially infectious agents, or biohazards. Biosafety protocols utilize containment or barrier strategies to accomplish this. The three elements of containment include laboratory practice and technique, safety equipment (primary barrier), and facility design (secondary barrier). Primary Barriers (safety equipment) provide protection of personnel and the immediate laboratory environment through good microbiological technique (laboratory practice) and the use of appropriate safety equipment. Secondary Barriers (facilities) provide protection of the environment external to the laboratory through a combination of facility design and operational controls.

24 (B) Biosafety Levels The level of containment or barrier required protecting the individual and/or the environment from exposure to the biohazardous agent is defined by a rating termed the Biosafety Level (BL), of which four currently exist. Biosafety Level 1 (BL-1) is the least restrictive while Biosafety Level 4 (BL-4) requires a special containment facility, which is currently not available at Stevens. Most research and teaching involves Biosafety Levels 1-3; therefore this manual will only focus on these three levels. Fo0r information pertaining to BL4, the reader should consult the IBC, Biosafety in Microbiological and Biomedical Laboratories and Biosafety Reference Manual (see References Cited) The following is a summary of the Biosafety Levels 1-through 3. Biosafety Level 1 (BL-1): includes agents not known to cause disease in healthy adults. It requires standard microbiological practices with no safety equipment (primary barrier) and only an open bench-top sink (secondary barrier). BL-1 practices, safety equipment, and facilities are appropriate for undergraduate teaching laboratories and other work using defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans (e.g. Bacillus subtilis, Naegleria gruberi). NOTE: Many agents not normally associated with disease in humans are opportunistic pathogens and may cause infection in the young, the aged and the immune compromised individual. In addition, vaccine strains that have undergone multiple in vivo passages should not be considered a virulent. Biosafety Level 2 (BL-2): agents associated with human disease through inoculation, ingestion or mucous membrane exposure hazards. It requires BL-1 practices plus limited access, biohazard warning signs, Sharps precautions (see Section IIIA) and a biosafety manual. Primary barriers include Biological Safety Cabinets (BSC; see Section IIB) or other physical containment devices and Personal Protective Equipment (PPE; see Section II, B2) as needed, and an autoclave as a secondary barrier. BL-2 practices, safety equipment and facilities are applicable to clinical, diagnostic, teaching and research facilities where work involves a broad spectrum of indigenous, moderate-risk agents present in the community and associated with human disease of varying severity (e.g. Hepatitis B, Salmonella spp. And Toxoplasma spp.). With good microbiological techniques, these agents can be used safely on the bench-top if the aerosol potential is kept low; however procedures with high aerosol potential must be conducted in primary containment equipment. This level of biosafety practice is consistent with the concept of Universal Precautions, which requires the treatment of all materials as if they are infectious.

25 Biosafety Level 3 (BL-3): indigenous or exotic agents with potential for aerosol infection and associated with serious or lethal disease in humans. It requires BL-2 practice plus controlled access, decontamination of all waste and lab clothing, the use of primary barriers, such as BSC and PPEs (Section IIB) for all agent manipulations, and physical separation from access corridors and non-recirculated or negative airflow into laboratory. BL-3 practices, safety equipment and facilities are applicable to clinical, diagnostic, teaching, research and production facilities which work with agents associated with serious or lethal disease which pose autoinoculation or ingestion exposure risks. Examples include Mycobacterium tuberculosis and Coxiella burnetti. Practical Description of BL-1, BL-2 and BL-3 Biosafety Practices To aid the reader in determining what safety precautions should be employed in their laboratory situation, a simplified summary of the appropriate guidelines follows, including specific recommendations for PPE and other barriers. These guidelines apply to biomedical and microbiological research and teaching laboratories, including laboratories working with recombinant DNA. BL-1 and BL-2 Laboratories Standard microbiological practices in these laboratories requires the wearing of laboratory coats, gowns or uniforms to prevent contamination or the soiling of street clothes, and the removal of these before leaving the laboratory area. These practices prohibit eating, drinking, smoking, applying cosmetics and storing food in the work area. Most research, teaching and diagnostic laboratories operate under BL-1 and BL-2 safety precautions. BL-3 Laboratories These laboratories rely heavily on primary and secondary barriers and engineering controls. The wearing of laboratory coats or gowns is required with a decontamination step prior to laundering necessary. In addition, entry must be through a controlled access with no individuals under the age of 16 permitted. No plants or animals not included in the research are allowed. These Biosafety Level requirements are described in greater detail in Table 1. For in-depth descriptions of Biosafety Levels as put forth by the USDHHS, CDC and NIH, see Appendix B for an excerpt from HHS Publication No , Biosafety in Microbiological and Biomedical Laboratories.

26 Insert Table 1 Here

27 (B1) Biosafety Levels for Animal Work Biosafety levels are also defined for activities with experimental animals. As Stevens does not support animal experimentation, these practices will not be described here. For more information see Appendix C, Table C1, and the CDC publication Biosafety in Microbiological and Biomedical Laboratories. (B2) Biosafety Levels for Recombinant DNA Work Containment for large-scale recombinant DNA experiments or production (greater than 10 Liters) is described in the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines). Four levels of containment are described in Appendix K of that publication. See Reference list, Appendix A for Internet Address of this publication. A hard copy of this publication is also available. Contact the Biosafety Officer or the IBC for its location.

28 (C) Biohazard Definition For the purpose of this manual, a biohazard is an agent of biological origin that has the capacity to produce deleterious effects on humans (Heinsohn, 1995). In general, a biohazard presents a risk or potential risk to the health of both humans and animals, either through direct infection or indirectly through damage to the environment. These agents include both a variety of microorganisms and multicellular organisms and the toxins and allergens derived from them (see next section). It is important to note that the presence of any of these organisms or their biologically derived substances in the workplace does not necessarily represent a hazard. The hazard potential depends on complex relationships among the agents, hosts and environment, which must all be considered when assessing and dealing with risk potential. (D) Categories of Biohazards The following is a list of biohazard agents or potentially infectious material: Human, animal and plant pathogens including bacteria, fungi, viruses, parasites and prions (see below for more detail). All human blood, blood products, tissues and certain body fluids Cultured cells (all human and certain animal) and potentially infectious material that these cells may contain Allergens Toxins (bacterial, fungal, plant, etc) Clinical specimens Infected animals and animal tissues Certain recombinant products, including recombinant DNA

29 (E) Recombinant DNA (rdna): Definition of rdna molecules: as defined by the NIH Guidelines, recombinant DNA molecules (rdna) are molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cells or molecules that result from the replication of rdna molecules. Experiments involving the generation of rdna may require registration and approval by the IBC, in addition to the NIH. The NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) is the definitive reference for rdna research in the U.S For an overview of laboratory practices appropriate for handling rdna and the associated Biosafety Level ratings, see Appendix D1. If the experimental protocol is not covered by the NIH Guidelines, contact the Biosafety Officer or the IBC for further information. For specific questions about particular host-vector system not covered in the NIH Guidelines, call the Office of Recombinant DNA Activities, NIH or visit the Federal Register web site (Appendix A). There are some rdna molecules that are exempt from the NIH Guidelines and are not required to be registered with the ORDA (see Appendix D2) ; however registration with the IBC is still required (see the following paragraph). As a condition of funding for recombinant DNA research, Stevens Institute must ensure that research conducted at or sponsored by Stevens must comply with the NIH Guidelines. However, it is the standard practice at Stevens to comply with the most current Guidelines, irrespective of funding source. Therefore, all experiments involving recombinant DNA must be registered to the IBC by the PI or Course Instructor by submitting Form 3, Registration Document for Recombinant DNA Research (see Appendix H). This form provides information regarding Host strain Source of DNA/RNA Recombinant vector Transgene introduction The use of large-scale fermentation of recombinant organisms Release of these organisms into the environment Containment conditions Use of transgenic plants or animals (F) Human Gene Therapy and Transgenic Animals

30 All protocols involving human gene therapy or the creation of transgenic animals or plants must be approved locally by the IBC prior to submission to outside agencies. Consult the NIH Guidelines (Appendices L, M, P, Q) for further details. (G) Human and Animal Pathogens (G1) Introduction to Microorganisms/Infectious Agents A detailed understanding of the dynamics of microorganisms within various settings is required to effectively realize and control their potential hazard. Microorganisms or microbes include the viruses, bacteria, some fungi and the recently identified prions. Although there is a large array of these organisms, only a small number of them are pathogenic to humans. Each organism requires a specific set of parameters for efficient growth, metabolism, development and reproduction. It is important to note that in the past 50 years there has been a change in the agents responsible for laboratory associated infections with a shift from bacterial associated illness to viral illness. Viruses A submicroscopic, subcellular agent consisting of a nucleic acid core (either DNA or RNA) surrounded by a protective protein coat sometimes surrounded by a lipoprotein membrane. Since viruses are incapable of generating energy or conducting biosynthetic mechanisms without a host organism, they are considered a host-dependent living organism. They are obligate intracellular parasites. When presented with the appropriate host organism, the virus invades the host cell and commandeers it to perform all functions necessary for viral replication. Size: microns (comparable to large protein macromolecule) Classification: based on shape, protein coat composition, presence or absence of a lipoprotein membrane, type of nucleic acid and host specificity Examples: Bacteriophage (or phage) are viruses that parasitize bacteria. Retroviruses are animal viruses that have an RNA molecule as their primary nucleic acid.

31 Bacteria (Prokaryotes) A cellular, simple organism which lacks a nucleus and membrane bound organelles. They have an outer cell wall composed of peptidoglycan, sometimes surrounded by a slime sheath which provides protection from host defenses. They reproduce asexually by fission. The bacterial chromosome is comprised of a single circular strand of DNA. In addition, they may incorporate DNA molecules from another bacteria through the process of conjugation or by the uptake of free DNA released by dead bacteria in their environment. Their nutritional modes vary greatly and the can live under extreme conditions. When faced with unfavorable conditions, bacteria can form an endospore; a dehydrated bacterial cell encased in heavy protective spore coats which allows it to survive under extremely harsh conditions. Frequently described in terms of colony-forming units (CFU) where colonies result from the growth of an individual bacteria cell. Size: microns by 2.5 micron Classification: based on shape, or composition of the cell wall as determined by ability for bacteria to uptake an aniline dye (Gram staining); either Gram-positive (GPB) or Gram-negative (GNB). Also classified by their nutritional characteristics. Fungi Mostly multicellular eukaryotes, with varied structures that share a common mode of nutrition. May reproduce sexually through spore formation (a nonmotile, reproductive cell that can grow directly into a new organism) which provides considerable resistance to various environmental conditions and will develop under favorable conditions. May also reproduce asexually through budding. A number of subtypes include: Yeast (single-celled) Size: microns diameter ovals Filamentous fungi Size: micron diameter fibers Classification: based on reproductive mode and the type of spore producing structure.

32 Prions A protein particle that lack any type of nucleic acid. They are believed to reproduce by structurally converting other proteins normally found in cells. Examples of characterized prions include the agents which cause the animal disease Scrapie and the Bovine Spongiform Encephalopathy (BSE) which causes mad cow disease. Parasites An organism that lives off of and obtains nutrition from another organism called a host. This is a broad category of organism that includes the viruses (e.g. HIV), bacteria (e.g. streptococcus), protists and fungi. Bloodborne Pathogens An additional means of classification of some biohazard agents is the term bloodborne pathogens. These agents include the hepatitis viruses, human immunodefiency virus (HIV), malaria, and syphilis. These are commonly transmitted through body fluids, either human or other animal. It is important to remember that hazardous biological agents described above have the ability to replicate, which is in sharp contrast to hazardous chemical agents. Therefore there is no such thing as a safe dose of biohazardous agentsthere is NO safe level.

33 (G2) Classification of Pathogenic Agents into Risk Groups Risk assessment is ultimately a subjective process. There are several systems for classifying human and animal pathogens according to the hazard they present to the individual and the community. In general, however, the pathogenicity of the organism, its mode of transmission and host range, and effective preventive measures and/or treatment are criteria considered when establishing classification groups. The most current classification is found in the NIH Guidelines for Research Involving Recombinant DNA Molecules. In this classification scheme, biohazard agents are placed into four Risk Groups (RG), where Risk Group 1 (RG-1) contains agents of low or no hazard and Risk Group 4 (RG-4) contains highly infectious agents. Risk Group 1 (RG-1) contains agents that are not associated with disease in healthy adult humans (e.g. Escherichia coli K-12, Saccharomyces cerevisiae). Risk Group 2 (RG-2) contains agents associated with human disease which are rarely serious and for which preventive or therapeutic interventions are available. Risk Group 3 (RG-3) contains agents associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk). Risk Group 4 (RG-4) contains agents likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual and community risk) A comprehensive list of Risk Group agents and their Biosafety Level definitions can be found in Appendix E, Tables E1 through E4. It is important to realize that none of these lists are inclusive. Any unlisted agent needs to be subjected to a risk assessment based on the known and potential properties of the agent and its relationship to agents that are listed. In addition, a listing of organisms and toxins which are restricted by the CDC due to their infectious nature are also provided in Appendix E. Use of any of these organisms must be registered with the IBC and the CDC. Determining the RG of a biological agent is a part of the biosafety risk assessment and is important for assigning the correct Biosafety Level for containment. In general, RG-2 agents are handled at BL-2 and RG-3 are handled at BL-3. However, large quantities may alter containment conditions. For more information see the NIH Guidelines for Research Involving Recombinant DNA Molecules, Biosafety in Microbiological and Biomedical Laboratories or contact the Biosafety Officer or the IBC.

34 (H) Other Potentially Hazardous Biological Materials (H1) Human Blood, Blood Products, Body Fluids and Tissues Biosafety Level 2 practices and procedures (or Universal Precautions), must be followed when handling these materials because of the infectious agents they may contain. If a highly infectious agent might be encountered in the human blood or body products, (e.g. Tuberculosis, Ebola Virus), utilize BL-3 precautions (H2) Animal Use The use of animals in research requires compliance with the Animal Welfare Act and any state and local regulations. As Stevens does not support animal experimentation, the biohazards associated with this type of work will not be discussed here. (H3) Tissue Culture/Cell Lines When cell cultures are known to contain a biohazard agent, the cell line can be classified as the same level as that recommended for the agent. Cell lines of human origin should be handled at Biosafety Level 2. Non-primate or normal primate origin cell lines, which do not harbor primate viruses, nor are contaminated with bacteria, mycoplasma, or fungi, may be handled at Biosafety Level 1. If cells lines are from tumor or lymohoid tissue, or are transformed by an oncogenic virus they should be handled at Biosafety Level 2. Note: Recent product recalls for bovine serum have raised awareness of the potential Bovine Spongiform Encephalopathy (BSE) or Transmissible Spongiform Encephalopathy (TCE) contamination of those sera. For more information on the testing or purity of bovine serum, contact the supplier.

35 (H4) Tuberculosis Work with Mycobacterium tuberculosis or M. bovis cultures must performed at BL-3 and requires the approval of the IBC. For more information about working safely with Mycobacterium sp. in the laboratory, see the CDC publication Guidelines for Preventing the Transmission of Tuberculosis in Health-Care Facilities. (H5) Wild Rodents If course subject matter or research activities require individuals to work/study outdoors where wild rodents may be encountered, precautions against contracting hantavirus pulmonary syndrome (HPS) should be taken. Generally, the likelihood of exposure through inhalation is low. All outdoor excursions and exposure risks should be planned with the IBC or the BSO well in advance of the activity.

36 (I) Experiments Covered by this Protocol The guidelines described in this Protocol apply to the following activities: (I1) (I2) (I3) (I4) (I5) (I6) (I7) Experiments involving the transfer of a drug resistance trait to micro-organisms that are not known to acquire the trait naturally if such acquisition would compromise the use of the drug to control disease agents in humans, veterinary medicine or agriculture. Formation of rdna molecules containing genes for the biosynthesis of toxin molecules lethal for vertebrates at a lethal dose (LD) LD 50 of less than 100 nanograms per kilogram body weight Experiments involving the transfer of rdna or rrna into human subjects Experiments using Risk Group 2, 3 or 4 agents or restricted agents as host or vector systems Experiments in which DNA from Risk Group 2, 3 or 4 agents or restricted agents is cloned into nonpathogenic prokaryotic or lower eukaryotic organisms. Experiments involving whole animals or whole plants whose genome has been altered by the stable introduction of rdna (transgenic) or who are used to test rdnamodified organisms. Experiments involving more than 10 liters of culture (see Appendix K of the NIH Guidelines) (see the NIH Guidelines, Section III, for more details regarding the above activities All of these activities must be reported to and approved by the IBC using Form 3, Registration for Recombinant DNA Research and Form 2, Annual Protocol for Use of Biohazardous Materials. In addition, activities (I1) and (I2) require that Form 3 also be submitted to the NIH for approval (see Section II, D)

37 Section II Control Measures (For more details regarding containment practices see Biosafety in Microbiological and Biomedical Laboratories) The term biosafety describes a complete program of administrative controls, medical surveillance and containment strategies for reducing the risk of potential exposure to infectious agents or other biologically derived materials. Effective application of a biosafety protocol involves four elements: Laboratory Practice and Technique Safety Equipment Facility Design Engineering Controls Administrative Controls (A) Laboratory Practice and Technique The most important element of containment is adherence to good laboratory practices. Individuals working with biohazards must be proficient in the practices and techniques required for the safe handling of those materials. When standard laboratory practices are not sufficient to control the hazards, additional measures may be required. It is the responsibility of the PI or the course Instructor or supervisor to select additional practices (see Section II, A2) and ensure the correct application of these practices. Regardless of the specific biohazard agent(s) encountered, however, there are standard laboratory safety guidelines which must always be followed to insure the safety of all those working in the facility.

38 (A1) General Laboratory Safety Practices The following is a list of some general safety considerations and is presented solely as a reminder and guideline for working in a laboratory/research setting. Details pertaining to some of these safety rules and guideline may be found elsewhere (e.g. Stevens Chemical Hygiene Plan). For purposes of this Protocol, the laboratory or research facility is the area where any biohazard exposure may occur. This includes the location of activities/experiments, storage of biohazards, transfer locales and waste storage. General Rules No running, jumping or horseplay in laboratory/research areas No one shall work alone at any time when performing a task that is considered exceptionally hazardous by a supervisor or a safety officer Spills shall be cleaned immediately (see Section III). Water spills are included, since they pose a slip potential and potential flooding and equipment damage (particularly to the floor below). Exercise caution with step stools Make certain the laboratory is left clean after work is performed. All animals (including pets) should be excluded from the Institute.