Biosafety Manual

2408 Wanda Daley Drive Ames, Iowa 50011-3602 (515) 294-5359 | Copyright © Reviewed 2022 Biosafety Manual

Biosafety Manual 2

Biosafety Manual 3 Directory of Service and Emergency Providers Services Environmental Health and Safety 2408 Wanda Daley Drive | (515) 294-5359 Iowa State University Occupational Medicine Department G11 Technical and Administrative Services Facility (TASF), 2408 Pammel Drive | (515) 294-2056 McFarland Clinic PC, Occupational Medicine 1018 Duff Avenue | (515) 239-4496 Thielen Student Health Center 2647 Union Drive | (515) 294-5801 Emergency Emergency - Ambulance, Fire, Police 911 Department of Public Safety / Iowa State University Police Armory, 2519 Osborn Drive | (515) 294-4428 Mary Greeley Medical Center 1111 Duff Avenue | (515) 239-2011

Biosafety Manual 4 Table of Contents Directory of Service and Emergency Providers 3 A. Introduction 7 Definition of Biohazardous Materials 7 Purpose 7 Responsibilities 8 Iowa State University (ISU) 8 Environmental Health and Safety (EH&S) 8 Supervisors 8 Personnel 8 Institutional Biosafety Committee (IBC) 8 B. Recombinant or SyntheticNucleicAcidMolecules, Human,Animal, andPlant Pathogens, Biological Toxins: Institutional Biosafety Committee (IBC) 10 C. Medical Survelliance 12 Workplace Exposure Assessment 12 Exposure Monitoring 12 Vaccinations and Testing 13 Exposure to Biohazardous Materials 13 Work-Related Injuries, Illnesses and Exposures 14 Reporting 14 D. Biosafety Practices and Procedures 16 Work Practices (First Line Of Defense) 16 Laboratory Biosafety Level Criteria 16 Biosafety Level 1 (BSL–1) Minimum Criteria 18 Biosafety Level 2 (BSL–2) Minimum Criteria 19 Biosafety Level 3 (BSL–3) Minimum Criteria 20 Biosafety Level 4 (BSL–4) 21 Laboratory Decommissioning 22 Training and Education 22 Signs and Labeling 23

Biosafety Manual 5 Security 23 Personal Protective Equipment (PPE) 24 Laboratory Practice and Technique 26 Pipetting 27 Centrifugation 28 Using Needles, Syringes, and Other Sharps 28 Blending, Grinding, Sonicating, Lyophilizing, and Freezing 29 Open Flames 29 Flow Cytometry 30 Evaluating Laboratory Safety 30 Animal Handling 31 Animals on Campus 31 Animals in the Field 32 Arthropod Research 32 Arthropod Containment Levels 33 Cell and Tissue Culture 34 Safety Equipment (Primary Containment) 35 Biosafety Cabinets (BSC) 35 What is a Biosafety Cabinet? 35 When Must I Use a BSC? 36 Open Flames in a BSC 36 Decontamination and Ultraviolet Lights in a BSC 36 Annual Certification Testing 37 Moving or Repairs 37 Purchasing and Installing a New BSC 37 Facility Design (Secondary Containment) 38 E. Disposal and Disinfection of Biohazardous Materials 39 University Policies 39 Supplies 39 What If I Do Not Have Waste Handling Facilities? 39

Biosafety Manual 6 Autoclaves 39 Elements Required for Effective Autoclave Use 39 Autoclave Safety 41 Pressure Vessel Monitoring 41 Chemical Disinfectants 42 Choosing a Chemical Disinfectant 42 Types of Chemical Disinfectants 43 Inactivation, Verification, and Biocontainment of Infectious Agents and Toxins 46 Prion Inactivation and Biocontainment Procedures 46 USDA Recommendations for Inactivation of Prions Affecting Livestock 46 BMBL Recommendations for Inactivation of Prions Affecting Humans 46 Precautions in Using NaOH or Sodium Hypochlorite Solutions in Autoclaves 47 Biocontainment and Working Procedures 47 F. Biohazard Spill Clean-up 48 Biohazard Spill Kit 48 Biohazard Spill Response Procedure 49 G. Transporting and Shipping Biohazardous Materials 51 On-Campus Transport of Biohazardous Materials 51 Off-Campus Transport of Biohazardous Materials by Commercial Carriers 51 Permit Requirements 52 Animals, Plants, Introduction of Genetically Modified Organisms 52 Human Pathogens or Biological Toxins 52 CDC Importation Permits for Etiologic Agents 52 Select Agents and Toxins 53 Packaging and Paperwork Requirements 54 Off-Campus Transport of Biohazardous Materials by Non-Commercial Routes 55 H. Biological Material Inventory and Biohazardous Materials Security 57 Bioinventory 57 Biosecurity 57 Non-discrimination Statement 58

7 Biosafety Manual A. Introduction Definition of Biohazardous Materials Biohazardous materials are those materials of biological origin that could potentially cause harm to humans, domestic, or wild animals, or plants. Examples include recombinant or synthetic nucleic acid molecules, transgenic animals or plants, human, animal, or plant pathogens, biological toxins (such as tetanus toxin), human blood, and certain human body fluids, and human or primate cell cultures. Purpose The purpose of the Iowa State University (ISU) Biosafety Program is to assist in protecting faculty, staff, and students; minimize exposure to biohazardous materials, prevent the release of biohazardous materials that may harm humans, animals, plants or the environment, and protect the integrity of experimental materials. To better fulfill these goals, biosafety staff members serve on the Institutional Biosafety Committee (IBC) and the Institutional Animal Care and Use Committee (IACUC), manage the Bloodborne Pathogen Exposure Control Plan, and conduct exposure assessments for the Occupational Medicine (Occ Med) program. Environmental Health and Safety (EH&S) biosafety staff also: • coordinate the certification of biosafety cabinets, • advise faculty, staff, and students who work with biohazardous materials about applicable regulatory guidelines, • assist researchers in determining appropriate practices and facilities for biocontainment and proper biohazardous waste disposal methods, • oversee proper disposal of biohazardous waste, • provide assistance with obtaining regulatory permits and shipping biohazardous materials, and • oversee the Select Agents and Toxins program. The Biosafety Manual outlines appropriate practices, university policies, and regulatory requirements for working safely with biohazardous materials. For a comprehensive overview of the core requirements that must be followed in all laboratories at ISU, please see the Laboratory Safety Manual.

8 Biosafety Manual Responsibilities Iowa State University (ISU) The president of Iowa State University is ultimately responsible for all environmental, health and safety issues. This responsibility is exercised through the normal lines of authority within the university by delegating the charge for ensuring safe work practices and adherence to established policies and guidelines to the senior vice president and provost, deans, directors, department chairs, principal investigators, supervisors, and, ultimately, each employee. Environmental Health and Safety (EH&S) EH&S is responsible for the development and oversight of proper management practices for all biohazardous materials at Iowa State University, including developing and implementing policies for ISU. EH&S is also responsible for ensuring that affected departments are aware of the university policies and regulatory guidelines regarding the proper use of biohazardous materials. Supervisors Principal Investigators (PIs), instructors, and supervisors are primarily responsible for ensuring that the policies and guidelines established in this manual are strictly followed by all personnel under their jurisdiction, including collaborating researchers. Personnel Individuals who work with biohazardous materials have a responsibility to follow the guidelines presented in this manual and to consult with their supervisors regarding the safe handling and proper disposal of specific biohazardous materials used in their work area. Individuals who are pregnant, immunocompromised, or have other health conditions are advised to consult the safety data sheets (SDS) for all hazardous chemicals, radioactive materials, and pathogenic organisms in their workplace environment in order to determine if any risks exist. They should also consult with their supervisor, Occupational Medicine, or their physician of choice concerning potential risks and how to manage those risks.

9 Biosafety Manual Institutional Biosafety Committee (IBC) The IBC is appointed by the Office of the Vice President of Research and serves as the review committee in all matters involving recombinant or synthetic nucleic acid molecules studies, as required by the National Institutes of Health’s (NIH) Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines). The IBC is responsible for reviewing the biological safety and public health programs at ISU, including: • any use of human, animal, or plant pathogens or biological toxins, • administration of experimental biological products (vaccines, sera, etc.) to animals, and • field releases of plant pests or genetically engineered organisms. The IBC also makes policy recommendations to the Office of the Vice President for Research to ensure compliance with federal, state and local regulations and guidelines. The IBC has the authority to require operational changes to ensure compliance with required conditions.

10 Biosafety Manual B. Recombinant or Synthetic Nucleic Acid Molecules, Human, Animal, and Plant Pathogens, Biological Toxins: Institutional Biosafety Committee (IBC) The Institutional Biosafety Committee (IBC) must approve any teaching or research project that involves: • Recombinant or synthetic nucleic acid molecules, including transgenic animals or plants. • Human, animal, or plant pathogens (such as bacteria, viruses, fungi, prions, or parasites). • Toxins of biological origin (such as tetanus toxin or aflatoxin). • Administration of experimental biological products to animals. • Field releases of plant pests or genetically modified organisms. The IBC is administered by the Office of Research Ethics (ORE). The IBC was established under the NIH Guidelines, and its authority is derived from federal regulations and from the Iowa State University Office of the Senior Vice President and Provost. The IBC is appointed by the Vice President for Research, as one of the standing committees of the university. The committee serves as campus authority in all matters involving recombinant or synthetic nucleic acid molecules studies as required by the Federal Register, May 7, 1986, vol.51, #88, pages 16958-16985, and subsequent guidelines which supersede earlier versions. The committee also reviews projects involving other hazardous biological materials. Compliance with the NIH Guidelines is important to promote the safe conduct of research involving recombinant or synthetic nucleic acid molecules. Compliance with the NIH Guidelines is mandatory as a condition of receiving NIH funding. Institutions that fail to comply risk suspension, limitation, or termination of financial assistance for noncompliant NIH projects and risk NIH funding for other recombinant or synthetic nucleic acid molecules research at the Institution. It is also possible the institution would have to obtain prior NIH approval for any recombinant or synthetic nucleic acid molecules projects. The IBC is composed of several experts including bacteriologists, entomologists, plant pathologists, diagnostic laboratory virologists, the university biosafety officer, laboratory technicians, zoonotic disease experts, public health experts, and two non-institutional members.

11 Biosafety Manual Additional Resources NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) • Federal requirements for all recombinant or synthetic nucleic acid molecules • Guidelines for Institutional Biosafety Committees NIH Guidelines Training

12 Biosafety Manual C. Medical Survelliance The Occupational Medicine Program is designed to minimize personnel health risks from workplace hazards. Hazards may include chemicals such as formaldehyde or benzene; physical hazards such as excessive noise or lasers; human pathogens, tissues, and cell lines; animal handling; and radioactive materials or devices. The program includes workplace exposure assessments, exposure monitoring and medical surveillance. All Iowa State University (ISU) personnel, including part-time and student workers, are encouraged to participate in the Occupational Medicine Program, which is provided at no charge. Refer to the Occupational Medicine Guidelines for more information. Workplace exposure to human blood, tissues, cell lines, and other potentially infectious materials (OPIM), as defined by the OSHA Bloodborne Pathogen Standard (29 CFR1910.1030), requires medical surveillance and annual Bloodborne Pathogen Exposure Control Training. Iowa State University’s written bloodborne pathogen exposure control plan is the Bloodborne Pathogens Manual. Workplace Exposure Assessment Participation in the Occupational Medicine Program requires completion of a Hazard Inventory form. The online form must be completed by new employees who are exposed to hazards as part of their assigned job duties and/or current employees who have changes to their hazards or personnel information. Environmental Health and Safety (EH&S) will use this information to determine the need for enrollment in the ISU Occupational Medicine Program. Individuals and supervisors will receive an email after EH&S has completed the evaluation of the hazards, and can login to see the results. If it is determined that the individual’s workplace hazards require medical monitoring or training, the individual will receive a notice from the ISU Occupational Medicine Program with further instructions. Exposure Monitoring As part of the workplace exposure assessment, exposure monitoring may be performed by EH&S to quantify the level of exposure experienced by employees at ISU. Monitoring results are used to determine if medical surveillance of an employee will be required and whether control measures should be implemented to ensure a safe work environment. Each department and laboratory supervisor is responsible for ensuring that any recommended control measures are implemented. EH&S may perform additional monitoring to determine the effectiveness of control measures. EH&S is available to conduct occupational exposure monitoring

13 Biosafety Manual whenever a possible exposure or potential health hazard is suspected in the work environment. Vaccinations and Testing Personnel who work with human pathogens must be given the option of being vaccinated, provided a vaccine is available, and informed of the risks associated with the vaccine. • Personnel working with human blood, tissues, cell lines, or OPIM must be offered the hepatitis B vaccination. • High-risk personnel, such as health care workers, must also be offered a titer test two months after the final hepatitis B vaccine dose. • Personnel whose job duties potentially expose them to tuberculosis must be offered routine testing to monitor exposure. Vaccinations and tuberculosis testing will be administered by the Occupational Medicine office and billed to the appropriate PI or department. Affected personnel choosing to receive a vaccination will need to schedule an appointment with Occupational Medicine, (515) 2942056. They should bring a completed Intramural Purchase Order form with them to their appointment. Affected personnel choosing not to receive a vaccination must complete the Decline to Vaccinate portion of the Consent or Decline of Vaccination Form. The department supervisor must ensure that the completed and signed decline form is placed in the individual’s department personnel file and a copy sent to the Occupational Medicine doctor’s office. Information about specific vaccines and exposure tests commonly given to ISU personnel can be found on the Centers for Disease Control and Prevention (CDC) website. The following are vaccines and procedures offered by Occupational Medicine: • hepatitis B • influenza, inactivated vaccine • rabies • tetanus/diphtheria • tuberculosis testing Exposure to Biohazardous Materials Before working with human pathogens, blood, tissues, cell lines, or OPIM, all applicable safety information, such as the SDS for a specific pathogen, must be reviewed and documented. Human

14 Biosafety Manual pathogen SDSs are available at the Public Health Agency of Canada. Familiarity with exposure routes, symptoms, and treatment methods will provide better preparation in the event of exposure to the human pathogens, blood, tissues, cell lines, or OPIM. If exposure to human pathogens, blood, tissue, cell lines, or OPIM occurs or is suspected to have occurred while at work, appropriate medical treatment must be sought immediately. Work-Related Injuries, Illnesses and Exposures Iowa State University employees exposed or injured while at work or in the course of employment must seek medical attention at the McFarland Clinic PC, Occupational Medicine Department, 1215 Duff Ave, Ames, IA; (515) 239-4496. Supervisors should call the McFarland Clinic Occupational Medicine Department during regular work hours to schedule an appointment for the employee. Any relevant safety information such as an SDS should accompany the employee to the appointment. Reporting All work related injuries, illnesses, or exposures must be reported to the employee’s supervisor, even when medical attention is not required or is refused by the employee: An Incident Report (IR) must be completed by the injured employee and/or the supervisor through the ISU Incident Portal within 24 hours of the incident. Upon submission of the report, the supervisor will receive an email requesting information relating to the Accident Investigation as part of the incident reporting process. The supervisor is asked to reply directly to the email with answers to the questions asked within 24 hours of receiving the email. Questions regarding the form may be forwarded to University Human Resources at (515) 294-3753. Contact EH&S at (515) 294-5359 for guidance and assistance, especially when a serious injury or major loss occurs. Student Accidents and Injuries Students not employed by Iowa State University who are exposed or injured in the classroom or laboratory should seek medical attention at the Thielen Student Health Center, 2647 Union Drive, (515) 294- 5801. All accidents and injuries sustained by ISU students while in academic classes or events sponsored by the university must be reported to Risk Management by the student and a university representative using the ISU Incident Portal. Refer to the Accidents and Injuries web page for more information.

15 Biosafety Manual Additional Resources OSHA Bloodborne Pathogen Standard (29 CFR1910.1030) Bloodborne Pathogens Manual CDC Public Health Agency Canada, Laboratory Biosafety and Biosecurity

16 Biosafety Manual D. Biosafety Practices and Procedures Work Practices (First Line Of Defense) Safe work practices are the most critical part of preventing exposure when working with biohazardous materials. The best laboratory and safety equipment available cannot provide protection unless personnel use good work practices and have adequate training. Additional safe work practices may be critical at preventing exposure to non biohazardous materials. Safe work practices that apply to all laboratories at ISU are covered in the Laboratory Safety Manual. Laboratory Biosafety Level Criteria The four biosafety levels (BSL) provide guidelines to ensure appropriate protection for laboratory users and the environment based on biological risk. Biological risk is related to the infectious agent used, the pathogenicity of the agent, and the mode of transmission. A wide variety of requirements for both physical containment and procedural details comes with increasing levels of protection. The Biosafety in Microbiological and Biomedical Laboratories (BMBL), published by CDC and NIH, lists proper practices, procedures, and facilities for each biosafety level. • BSL-1 is suitable for work involving well-characterized agents not known to consistently cause disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel and the environment. • BSL-2 builds upon BSL-1. BSL-2 is suitable for work involving agents associated with human disease that pose moderate hazards to personnel and the environment. • BSL-3 is applicable to clinical, diagnostic, teaching, research, or production facilities where work is performed with indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposure. • BSL- 4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease that is frequently fatal, for which there are no vaccines or treatments, or a related agent with unknown risk of transmission.

Biosafety Manual 17 BIOSAFETY LEVELS - THE SHORT VERSION (adapted from BMBL with Iowa State University policies incorporated) BSL Agents Practices Safety Equipment (Primary Barriers) Facilities (Secondary Barriers) 1 Well-characterized agents not known to consistently cause disease in immunocompetent adult humans and present minimal potential hazard to laboratory personnel and the environment. Standard microbiological practices Refer to Iowa State University policy on minimum personal protective equipment (PPE) for labs: lab coats, gloves, eye and/or face protection Open bench top Hand-washing sink required 2 Associated with human disease Hazard: percutaneous injury, ingestion, mucous membrane exposure BSL-1 practice plus: Biosafety Cabinets (BSCs) Leak-proof container for sample transport Primary barriers: Class I or II Biosafety Cabinets or other physical containment devices used for all manipulations of agents that may cause splashes or aerosols of infectious materials PPE: lab coat, gloves, eye and/or face protection BSL-1 plus: Autoclave available 3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences BSL-2 practice plus: Controlled access Decontamination of all wastes Decontamination of lab clothing before laundering Baseline serum Primary barriers: Class I or II BSCs or other physical containment devices used for all open manipulations of agents PPE: protective lab clothing, gloves, respiratory, eye and/ or face protection BSL-2 plus: Physical separation from access corridors Self-closing, double door access Exhaust air not recirculated Directional airflow into laboratory 4 Materials requiring BSL-4 facilities and practices are not used at Iowa State University. Materials requiring BSL-4 facilities and practices are not used at Iowa State University. Materials requiring BSL4 facilities and practices are not used at Iowa State University. Materials requiring BSL4 facilities and practices are not used at Iowa State University. Reference: BMBL, current edition and NIH Guidelines Animal biosafety levels describe similar levels for containment facilities and practices necessary when vertebrate animals are infected with human pathogens (ABSL-2, ABSL-3, ABSL-4). Plant biosafety levels describe similar levels for greenhouse containment facilities and practices necessary for recombinant plants and plants infected with plant pathogens or plant pests (BL1-P, BL2-P, BL3-P, BL4-P). Most laboratories on campus qualify as BSL-1 or BSL-2. Four ABSL-3/BSL-3 facilities serve the ISU research community.

18 Biosafety Manual The following lists summarize the minimum criteria for laboratories operating at Biosafety Levels 1-3. These criteria are detailed in the current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL), a joint publication of the CDC and NIH. Biosafety Level 1 (BSL–1) Minimum Criteria • An eyewash station is readily available in the laboratory. • An effective integrated pest management program is implemented. • Long hair is restrained so that it cannot contact hands, specimens, containers, or equipment. • Personnel are trained in analytical methods, standard operating procedures (SOPs), spill response, potential hazards, and applicable safety training. • Access to the laboratory is limited or restricted at the discretion of the laboratory director when work with cultures or specimens is in progress. • Laboratory walls, floors, and ceilings are designed to be easily cleaned. • Benchtops are impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals. • Chairs used in laboratory work are covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant. • Laboratory windows, that open to the exterior, are fittedwith screens. • Lumination is adequate for all activities and avoids reflections and glare that could impede vision. • Laboratory is outfitted with a hand-washing sink with soap and towels. • Appropriate PPE is used in the laboratory (for example lab coats, safety glasses/goggles, closed-toe shoes, and gloves). • Where research animals are present in the laboratory the risk assessment considers appropriate eye, face, and respiratory protection, as well as potential animal allergens. • Laboratory personnel are enrolled in the respiratory protection program when warranted by risk assessment. • Only animals or plants associated with current studies are present in the laboratory. • Personnel wash their hands after handling biohazardous agents or animals, after removing gloves, or before leaving the room. • Eating, drinking, smoking, applying cosmetics, handling contact

19 Biosafety Manual lenses, and storing food for human consumption are prohibited in the laboratory. • Mouth pipetting is prohibited. Mechanical devices are used for pipetting. • Policy is in place for safe handling of sharps. • First aid kit is accessible and suitably stocked. • Procedures are in place to minimize splashes and the creation of aerosols. • Work surfaces are decontaminated with an appropriate disinfectant at least once a day and after a spill of viable material. • Laboratory equipment is decontaminated routinely and after contamination; and before repair, maintenance, or removal from the laboratory. • Cultures, stocks, and regulated waste are decontaminated by an effective method before disposal. • Proper biological waste labeling is in place for off-site decontamination. • Appropriate biohazard containers are used for containment of biohazardous waste. • Laboratory has a rodent and pest control program in place. • A biohazard sign indicating the required biosafety level, required PPE, exit procedures, required immunizations, and the PI’s name and phone number is posted at the laboratory entrance during work with human pathogens. Biosafety Level 2 (BSL–2) Minimum Criteria • BSL-1 minimum criteria are followed. • An autoclave is available for decontaminating laboratory wastes. • All personnel, and particularly those of reproductive age and/or thosehavingconditions thatmaypredispose themto increased risk for infection (e.g., organ transplant, medical immunosuppressive agents), are provided information regarding immune competence and susceptibility to infectious agents. Individuals having such conditions are encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance. • Personnel have been trained in the specific hazards associated with pathogenic agents and Recombinant or Synthetic Nucleic Acid Molecules in accordance with SOPs and protocols. Training will be documented and available for inspection. • At risk laboratory personnel have received appropriate

20 Biosafety Manual immunizations if available. • A biosafety manual and SOPs are written to incorporate specific biosafety precautions appropriate to the laboratory. • Procedures for handling a spill or accident, including required followup and documentation, are in place and available to lab personnel. • Exhaust HEPAfilters or equipment containing HEPAfilters, including biosafety cabinets and cage racks, must be certified annually. • Certified biosafety cabinets, located away from high-traffic areas and drafts, are used for procedures with a potential for creating infectious aerosols or splashes and for handling large volumes of pathogenic materials. • Centrifuges having sealed rotor heads or centrifuge safety cups are used. • Personnel wear safety glasses, goggles, or a face shield during operations posing potential for splashes or aerosols. • Protective clothing (such as lab coats) is kept inside the laboratory. • Appropriate gloves are worn when handling hazardous materials. • Laboratory personnel are enrolled in the respiratory protection programwhenwarrantedby riskassessment.The riskassessment considers whether respiratory protection is needed for the work with hazardous materials. If needed, relevant staff are enrolled in a properly constituted respiratory protection program. • Doors are self-closing and lockable. • Illumination is adequate for all activities and avoids reflections and glare that could impede vision. • Vacuum lines are protected with liquid disinfectant traps. • Potentially infectious materials must be placed in a durable, leakproof container during collection, handling, storage, and transport. Biosafety Level 3 (BSL–3) Minimum Criteria • BSL-2 minimum criteria are followed. • Access to the laboratory is through two consecutive self-closing doors. • Laboratory doors are lockable in accordance with institutional policies. • Every consideration has been given to the physical construction of the BSL-3 laboratory (for example sealed penetrations, smooth walls, sealed joints, and cove bases). • Depending on the biohazardousmaterials used or special handling conditions, HEPA (High Efficiency Particulate Air) filtration may be

21 Biosafety Manual required for air exiting the room to the outdoors. • Directional airflow, flowing from clean areas to contaminated areas, is provided. • All experimental manipulations are done inside a certified biosafety cabinet (primary containment). • Every consideration is given to alternative forms of needles or glassware to prevent sharps injuries. • Equipment exposed to BSL-3 agents is decontaminated before any repair, service or disposal. • Protective clothing consists of solid-front gowns, scrub suits, or coveralls. • Respiratory and face protection is used when in rooms containing infected animals. • A hands-free sink with soap and paper towels is available for use near the exit door. • If present, all windows are closed and sealed. • All cultures, stocks, biological waste, gloves, gowns, and other contaminated articles are decontaminated in the laboratory. • Vacuum lines are protected with liquid disinfectant traps and HEPA filters. • All procedures for the facility designandoperationaredocumented. • The BSL-3 facility design, operational parameters, and procedures are verified and documented prior to operation. • The BSL-3 facility is tested annually or after significant modification to ensure operational parameters are met. Verification criteria are modified as necessary by operational experience. • Exhaust HEPA filters and equipment containing HEPA filters must be certified annually. Biosafety Level 4 (BSL–4) Use of materials requiring BSL-4 facilities and practices must be conducted within a certified BSL4 facility. Additional Resources U.S. Department of Agriculture (USDA) Regulations for Animals and Animal Products (9 CFR 001-199) • Import/transport permits are issued by theAnimal and Plant Health Inspection Service (APHIS) Veterinary Services (VS) branch • Quick reference and applications for import and interstate

22 Biosafety Manual transport permits are available at the USDA Import-Export Directory for USDA-APHIS Veterinary Services USDA Agricultural Bioterrorism Protection Act of 2002: Possession, Use and Transfer of Biological Agents and Toxins (9 CFR 121) • Registration program for possession and transfer of pathogens or biological toxins defined as USDA VS Select Agents Biosafety in Microbiological and Biomedical Laboratories (BMBL) • Guidelines for human pathogen use published by the Centers for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH). current edition U.S. Public Health Service (USPHS) Foreign Quarantine (42 CFR 71) and Etiologic Agents, Hosts, and Vectors (Part 71.54) Regulations • CDC Importation Permits for Etiologic Agents CDC Possession, Use and Transfer of Select Agents and Toxins (42 CFR 72-73, 42 CFR 1003) • Registration program for possession and transfer of pathogens or biological toxins defined as Department of Health and Human Services (DHHS) Select Agents Pathogens SDSs from Public Health Agency of Canada • Quick safety references for pathogenic microorganisms in an SDS format from Health Canada’s Laboratory Centre for Disease Control. Laboratory Decommissioning When a laboratory is shut down, decommissioned, transferred to another researcher, or re-purposed, established procedures must be strictly followed. The Laboratory Checkout Form may be used for this purpose. If assistance is required, please call EH&S, (515) 294-5359. Training and Education Anyone planning to use biohazardous materials must be adequately trained before beginning work. Annual laboratory-specific training is required to be conducted and documented by the supervisor to ensure continued safety. Information communicated in the laboratoryspecific training must include: • A discussion of the Iowa State University Biosafety Manual and how it applies to activities conducted in specific work areas. • An explanation of the health hazards and signs and symptoms of exposure to biohazardous materials used in specific work areas. • Adescription of actions personnel can take to protect themselves from exposure, such as special work practices, use of safety equipment, vaccinations, emergency procedures, etc.

23 Biosafety Manual EH&S offers a variety of biosafety and other safety-related on-line and classroom training courses. Visit Learn @ISU for more information or to register for classes. Signs and Labeling Anyone entering areas where biohazardous materials are used must be aware of the potential hazards. Specific door signs for this purpose are provided by EH&S through the door signage app (https://ask.ehs. ; call (515) 294-5359 if you require assistance. A biohazard symbol with a red background indicates “human biohazards,” and must be posted at the entrance of rooms where microorganisms or biological toxins known to cause disease in humans are used. This includes microorganisms classified as Biosafety Level 2 (BSL2) or greater and human blood, tissues, cell lines, or OPIM. Red or orange biohazard labels must be placed on containers and storage units (refrigerators, freezers, incubators, waste containers, etc.) used for microorganisms or biological toxins causing disease in humans, or human blood, tissues, cell lines, or OPIM. Contaminated equipment and biohazardous waste must be labeled in the same manner. A biohazard symbol with a yellow background indicates “animal biohazards,” and must be posted at the entrance of rooms where strict animal pathogens are used. A biohazard symbol with a dark green background indicates “plant biohazards,” and must be posted at the entrance of rooms where strict plant pathogens or pests are used, or where certain Genetically Modified (GM) organisms or microorganisms are grown or processed. Where multiple biohazards are present, human hazards generally take precedence over animal and plant hazards when choosing which sign to use. To create the correct signs, go to Door Signage. The emergency contact information and minimum requirements for working in each space, shall be displayed at the laboratory entrance. The door signage must be tailored to the specific hazards in the laboratory environment. The door signage program requires a current chemical inventory. If you have questions about required signage, please contact EH&S at (515) 294-5359. To maintain compliance with OSHA’s Bloodborne Pathogen Standard, the entrance to any laboratory or area where Human Immunodeficiency Virus or Hepatitis B Virus are used or stored must have a door sign which is predominantly fluorescent orange or orange-red with lettering and symbols in a contrasting color. These signs must include the universal biohazard symbol and the word “biohazard.” These signs must include the name of the potentially infectious material, any special instructions for entering or exiting the area, and the name and telephone number of the area supervisor. These door signs may be requested from EH&S.

24 Biosafety Manual Security Some level of security is warranted for all laboratories, based on the existing risks and regulatory requirements. Each laboratory should conduct a risk assessment to determine appropriate security measures. Some examples of security measures include locked buildings, locked laboratories, locked storage units, limiting distribution of brass keys, proximity cards or key codes, and personnel background checks. For detailed information on biohazardous materials security requirements, refer to the Biosecurity section of this manual. Personal Protective Equipment (PPE) Appropriate PPE is chosen by considering the potential routes of exposure that need to be protected to prevent exposure and infection. It is essential that PPE be removed before leaving the room where biohazardous materials are used. PPE must never be taken home. PPE is removed in a manner that minimizes personal contamination. It should be disposed of or decontaminated in the work area where it is used. Please refer to the Laboratory Safety Manual for more information regarding PPE. Lab Coats and Uniforms Lab coats, scrub suits, gowns, and closed-toe shoes prevent biohazardous materials from reaching skin and, more importantly, any cuts, dermatitis, etc. that may be present. They prevent biohazardous materials from contaminating street clothing. They also prevent the normal flora present on the skin from contaminating laboratory cultures. • At minimum, a long-sleeved lab coat worn over clothing and closed-toe shoes must be worn in any laboratory. Long sleeves minimize contamination of skin and street clothes and reduce shedding of microorganisms from the skin. Closed-toe shoes protect the feet from spills and injuries from dropped sharps. • Lab coats must remain in the laboratory when personnel leave the laboratory. This keeps any contamination on the lab coat in the laboratory instead of spreading it to other work areas or homes. • PPE that is sent for commercial laundering, such as lab coats, must be properly contained and labeled. A proper label must have the name of the biological agent of potential exposure, type of decontamination used, and the date when it was last used. • Elastic-cuffed lab coats help prevent spills that can be caused by catching a loose cuff on laboratory equipment.

25 Biosafety Manual When working with biohazardous materials inside a biosafety cabinet, elastic cuffs or double gloving (second pair over cuff) prevent contaminated air from being blown up the lab coat sleeve onto clothing. Gloves Gloves prevent exposure of the skin and any cuts, dermatitis, etc. that may be present, to biohazardous materials. • Both latex and nitrile disposable gloves will prevent exposure tomicroorganisms. However, nitrile glovesmust bewornwhen handling chemicals, since latex provides little to no protection from chemical exposure. EH&S laboratory personnel, can provide assistance with choosing appropriate gloves. • For best protection, the cuffs of the gloves should overlap the lower sleeves of the lab coat. • Consider the need for bite and/or scratch resistant gloves. • Disposable gloves must not be reused. They are designed for disposal after one use or if exposed to a chemical (they offer limited chemical protection). Dispose of used gloved with other contaminated laboratory waste. Utility gloves, such as rubber dish washing gloves, may be disinfected for re-use if they do not show signs of wear or degradation. • Change gloves when contaminated, when glove integrity is compromised, or when otherwise necessary. • For information concerning the chemical resistance of the different types of gloves, access the Ansell Chemical Resistance Guide. • EH&S can provide assistance with finding an alternative for personnel with allergic reactions to gloves (most common with latex) and/or the powder they contain. Eye and Face Protection Eye and face protection prevent splashes into the eyes, nose and mouth (mucous membrane exposure), and onto the skin. • Goggles or safety glasses must be worn when working with laboratory hazards. • Prescription safety eyewear is available through approved providers. • Face shields should be used for full face protection. • N-95 masks provide splash protection for the mouth and nose.

26 Biosafety Manual Respirators Respirators prevent the inhalation of aerosolized microorganisms (inhalation exposure) when safety equipment designed to contain infectious aerosols, such as a biosafety cabinet, is not available. Respirators also reduce the inhalation of animal allergens when primary containment of animals is not possible or practical. • EH&S can assist in determining if a respirator is needed and which type, call (515) 294-5359. • Personnel who are required to use dust masks or other types of respirators for personal protection must participate in annual respirator training and fit testing. Medical approval to wear respiratory protection is required before training and fit testing can occur. More information is available on the Respirator web page. • The PI or laboratory supervisor is responsible for conducting hazard assessments, training, and coordinating the use of PPE. Completion of a hazard assessment in association with a standard operating procedure allows individual laboratory PPE requirements to be determined and justified by PIs or laboratory supervisors. Document PPE selection on a standard operating procedure developed for the experiment or laboratory operation. Laboratory Practice and Technique Personnel can be infected with organisms they come in contact with in the workplace. In order for infection to occur, there must be an adequate number of organisms to cause disease (infectious dose) and a route of entry into the body. Knowing how infectious organisms are transmitted and the infectious doses can help in evaluating risk and avoiding infection. Information about the organism(s) must be gathered prior to starting work with them. Safety information about pathogens can be obtained through Pathogen Safety Data Sheets and the BMBL. Infectious agents are transmitted through one or more routes of exposure: • Sharps (parenteral) injuries (needlesticks, cuts with contaminated broken glass, etc). • Inhalation of aerosols (microscopic solid or liquid particles (5 micrometers or less) dispersed or suspended in air). • Ingestion (oral-fecal routes of contamination are a common source of infection; hand-washing is imperative). • Mucous membrane exposure (including the eyes, inside of the mouth and nose, and the genitals).

27 Biosafety Manual Using work practices that block routes of exposure can prevent workplace infection. Good microbiological techniques must always be used in the laboratory: • Wearing appropriate PPE blocks potential routes of exposure. • Eating, drinking, smoking, chewing tobacco, applying cosmetics, or storing food in laboratories is strictly prohibited. Potentially contaminated hands must be kept away from the mouth, eyes, and non-intact skin. • Hands must be washed frequently, even after wearing gloves, and scrubbed vigorously with soap and water for a full 30 seconds (as long as it takes to sing “Happy Birthday” or the “Iowa State Fight Song”). The physical removal of organisms from the skin is just as important as using a disinfectant. • Work surfaces and equipment must be decontaminated after using biohazardous materials. The following are suggestions for common laboratory procedures to follow when conducting work with biohazardous materials. Each will help prevent biohazardous materials from entering the body through common exposure routes. Pipetting Pipetting can cause the creation of aerosols and splashing. Micropipettors may also create aerosols. • Mouth pipetting is prohibited. Mechanical pipetting aids must be used. • All biohazardous materials must be pipetted in a biosafety cabinet if possible. • Cotton-plugged pipettes should be used. Cotton-plugged micropipette tips are also available. • Biohazardous materials must never be forcibly discharged from pipettes. “To deliver” (TD) pipettes must be used instead of pipettes requiring blowout. • To avoid splashing, biohazardous material should be dispensed from a pipette or micropipettor by allowing it to run down the receiving container wall. • After use, pipettes should be placed horizontally in a pan filled with enough liquid disinfectant to completely cover them. Allow adequate disinfection time before disposal of pipettes. • Plastic micropipette tips and pipettes are sharp and should be disposed of in a puncture-resistant container after decontamination.

28 Biosafety Manual • When working in a biosafety cabinet, all waste and/or disinfecting containers must be kept inside the cabinet while they are being used. • Use proper PPE. Centrifugation Improper use of a centrifuge can cause the release of aerosols. • Leaks can be prevented by not overfilling centrifuge tubes. The outsides of the tubes should be wiped with disinfectant after they are filled and sealed. • Sealed tubes, O-ring sealed rotors, or O-ring sealed safety buckets must be used. To avoid spills from broken tubes, the tubes, lids, O-rings, buckets, and rotors should be inspected for damage before each use. • Ensure that rotors are balanced before centrifugation. • Rotors and centrifuge tubes must be opened inside a biosafety cabinet. If a biosafety cabinet is not available, a minimum of 10 minutes settling time must be allowed before opening. • Use proper PPE. • Centrifuges and accessories must be cleaned and disinfected regularly or when contaminated. Using Needles, Syringes, and Other Sharps The greatest risks when using sharps are accidental injections, lacerations, and the creation of aerosols. • Needles and syringes may only be used when there is no reasonable alternative. Safety needles and syringes must be used in these instances. • Substitute plasticware for glassware when possible. • Sharps must be kept away from fingers as much as possible. Sharps must never be bent, sheared, or recapped. Needles should never be removed from syringes after use. If a contaminated needle must be recapped or removed from its syringe, a mechanical device, such as a forceps, must be used. • Air bubbles should be minimized when filling syringes. • A pad moistened with disinfectant must be placed over the tip of a needle when expelling air. Work must be performed in a biosafety cabinet whenever possible. • An appropriate sharps container must be kept close to the

29 Biosafety Manual work area to avoid walking around with contaminated sharps. Care must be taken not to overfill sharps containers. They are considered full when they are 2/3 filled. The Sharps and BiohazardousWasteProcedure details proper disposal methods. • Non-disposable sharps are placed in a hard-walled container for transport and decontamination. • Do no handle broken glassware directly. Remove using a brush and dustpan, tongs, or forceps. • Use proper PPE. Blending, Grinding, Sonicating, Lyophilizing, and Freezing The greatest risk when using any of these devices is the creation of aerosols. • Blenders, grinders, sonicators, lyophilizers, etc. must be operated in a biosafety cabinet whenever possible. Shields or covers must be used whenever possible to minimize aerosols and splatters. • Safety blenders should be used. Safety blenders are designed to prevent leakage from the bottom of the blender jar and to withstand sterilization by autoclaving. They also provide a cooling jacket to avoid biological inactivation. • Avoiding glass blender jars prevents breakage. If a glass jar must be used, it must be covered with a polypropylene jar to contain the glass in case of breakage. • A towel moistened with disinfectant must be placed over the top of the blender while operating. This practice can be adapted to grinders and sonicators as well. • Aerosols must be allowed to settle for five minutes before opening the blender jar (or grinder or sonicator container). • Lyophilizer vacuum-pump exhaust must be filtered through HEPA filters or vented into a biosafety cabinet. • Polypropylene tubes should be used in place of glass ampoules for storing biohazardous material in liquid nitrogen. Ampoules can explode, causing eye injuries and exposure to the biohazardous material. • Use proper PPE. Open Flames When sterilizing inoculating loops in an open flame, aerosols that may contain viable microorganisms can be created. Open flames are also an obvious fire hazard.

30 Biosafety Manual • A shielded electric incinerator or hot bead sterilizer should be used instead of an open flame. • Disposable plastic loops and needles are also excellent alternatives. • Open flames should not be used in biosafety cabinets because they disrupt the laminar airflow and may be a fire hazard. Flow Cytometry Flow cytometers operate under pressure, generating aerosols. When flow cytometry is used to study known or potentially biohazardous materials, such as unfixed human or primate cells or known pathogens, operators may be at risk of exposure to aerosolized materials. When possible, all biological samples should be fixed (for example, with formalin) before being run through the flow cytometer. When performing flow cytometry on known or potentially biohazardous materials cannot be avoided, the following guidelines must be followed to prevent personal exposure. • Flow cytometry must be conducted in a laboratory that meets BSL-2 criteria at minimum. • Flow cytometry must be conducted in either a certified chemical fume hood, certified biosafety cabinet, or other approved negative-exhaust ventilation system. • Personnel must wear proper PPE, including gloves, a lab coat, and eye protection. • The catch basin should have an appropriate disinfectant added when the unit is in use. • The flow cytometer and lab bench must be cleaned and disinfected after each use. Refer also to Wiley Cytometry Guidelines for additional references regarding flow cytometry biosafety. Note the article “Biosafety Guidelines for Sorting of Unfixed Cells.” Evaluating Laboratory Safety The Laboratory Safety Survey includes criteria for work with infectious agents (from the current edition of BMBL) and for work with recombinant or synthetic nucleic acid molecules (from the NIH guidelines) Additional criteria for general laboratory safe work practices that apply to all laboratories at ISU are covered in the Laboratory Safety Manual. A Laboratory Safety Survey should be completed by laboratory personnel annually to help ensure that good laboratory safety practices are being used.