Biological Safety and Biosecurity Manual

Transcription

1 Biological Safety and Biosecurity Manual Rev 01,

2 FOREWORD 2

3 FOREWORD... 2 Section 1. Introduction... 6 Section 2. Scope... 6 Section 3. Roles and Responsibilities... 6 Institutional Official... 6 Institutional Biosafety Committee... 6 The Biological Safety Officer... 6 Office of Environmental Health & Safety... 7 Health Services... 7 Principal Investigator... 7 Lab Personnel... 8 Ancillary Personnel and Visitors... 8 Section 4. Biological Safety... 8 Risk Groups... 8 Biological Safety Containment Levels... 8 Biosafety Level 1 (BSL-1)... 9 Biosafety Level 2 (BSL-2)... 9 Lab Specific Operating Procedure... 9 Biosafety Laboratory Requirements... 9 Risk Assessment Recombinant and Synthetic Nucleic Acids Section 5. Biosafety Facility Requirements General Biological Laboratory Facility Requirements Biosafety Level 2 Facility Requirements Section 6. Work Practice Requirements Standard Microbiological Practices for All Labs Work Practices for BSL-2 Laboratories BSL-2 Required Practices BSL-2 Additional Best Practices Section 7. Personal Protective Equipment Section 8. Waste

4 Section 9. Additional Lab Hazards Chemicals Ionizing Radiation Compressed or Liquid Gases Noise Nanotechnology Section 10. Disinfection and Decontamination Spills in the Open Laboratory Spills in a Biological Safety Cabinet Commissioning, Decommissioning or Relocating a Laboratory Section 11. Training Section 12. Medical Surveillance NIH Guidelines Requirements for Recombinant or Synthetic Nucleic Acid Materials Bloodborne Pathogens Section 13. Incidents Incident Reports Lab Acquired Infection Medical Evaluation Section 14. Import/Export Centers for Disease Control (CDC) Department of Agriculture (USDA) Exports Material Transport Agreements Section 15. Shipping Section 16. Biosecurity Infectious Agents Plants and Seeds Insects Rodents Section 17. References

5 Appendix 1: Biosafety Level-2 Lab Specific Operating Procedure Template (adapted with permission from the University of Vermont) Appendix 2: Risk Groups NIH Appendix B Appendix 3: Risk Assessment Guidelines for the Principal Investigator Appendix 4: Biosafety Level 1 and Biosafety Level 2 Requirements Appendix 5: Common Disinfectants and Guidelines Appendix 6: Biohazardous Waste Flowchart Appendix 7: Biological Incident Form Appendix 8: Select Agents and Toxins List Appendix 9: Examples of Infectious Agents That Pose a Higher Risk to Susceptible Individuals

6 Section 1. Introduction Biological safety and biosecurity are integral to the use of biological materials at the University of New Hampshire. Safety and security are achieved through the use of various containment principles, including controls, work practices and equipment which aim to protect the worker, community and environment from biological hazards. The Biological Safety and Biosecurity Manual serves as a resource document for working safely in Biosafety Level 1 (BSL-1) and Biosafety Level 2 (BSL-2) containment, as well as the security of all biological materials on campus. Section 2. Scope This manual covers use of biohazardous materials in research, teaching, diagnostics and commercial applications on the UNH Durham (UNHD), Manchester (UNHM) and Jackson Estuarine Lab (JEL) campuses. Together, all three campuses are considered UNH. This document covers safe practices for BSL-1 and BSL-2 laboratories. Individual labs are encouraged to use the information in this document to prepare a lab specific biosafety operating procedure that must meet or exceed requirements covered in this manual. Print and/or electronic copies of the biosafety manual or operating procedure must be available and accessible to lab personnel at all times. This manual is not meant to cover OSHA s Bloodborne Pathogens Standard (29 CFR ), but complements the work practices described in the UNH Exposure Control Plan. This Biological Safety and Biosecurity Manual is a resource document. It is a living document subject to edits for new programs and ongoing improvements. Submit all suggestions and comments regarding this manual to the Biological Safety Officer. Changes and edits are reviewed and approved by the Institutional Biosafety Committee. Section 3. Roles and Responsibilities Institutional Official The Institutional Official (IO) serves as the ultimate responsible official for biological safety within the institution and must provide continuing support for institutional biological safety and biosecurity. The IO has established an Institutional Biosafety Committee and appointed a Biological Safety Officer (BSO) to ensure that all regulatory requirements for biological safety and security have been met. The Senior Vice Provost for Research serves in this role for UNH. Institutional Biosafety Committee The Institutional Biosafety Committee (IBC) assumes the responsibility for ensuring biological safety at UNH. The Committee must fulfill all aspects of its mission as documented in the IBC Charter and Procedures. The IBC reviews and approves all recombinant and synthetic nucleic acid work; as well as reviews all work in BSL-2 containment. It is the IBC s remit to ensure compliance with the National Institutes of Health Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines), as well as guidelines published by the Centers for Disease Control and Prevention (CDC) and the regulations published by the Occupational Safety and Health Administration (OSHA). The Biological Safety Officer The primary goal of the Biological Safety Officer (BSO) is to carry out the policies set forth by the IBC to ensure compliance with the NIH Guidelines, the CDC and OSHA. The BSO partners with Principal Investigators to provide a complete biosafety and biosecurity program for each biological laboratory. 6

7 This includes: developing and delivering comprehensive training programs, providing practical expertise for regulatory questions, providing written operating procedures for biosafety measures, and performing risk assessments for all work with biohazardous agents. The BSO is a biosafety technical resource for the entire campus community. Office of Environmental Health & Safety The Office of Environmental Health and Safety (OEHS) provides technical expertise for regulatory compliance and safe work practices. For Biological Safety, OEHS implements IBC approved programs, including the Institutional Biosafety Committee Charter and Procedures, the Bloodborne Pathogen Exposure Control Plan and the Biohazardous Waste Disposal Plan. Laboratories registered with the IBC are inspected by the Biological Safety officer on a 3 year cycle concurrent with IBC protocol renewal. The Laboratory Safety Officer may inspect these laboratories on a more frequent basis. A summary of OEHS program areas and the contacts for each area is located on the OEHS webpage. Health Services UNH Health Services acts as the occupational health service provider for any worker on the UNH campus working with biohazardous or potentially biohazardous materials. A member of Health Services acts as an ad hoc committee member on the Institutional Biosafety Committee when a protocol is reviewed for which medical surveillance or prophylaxis might be necessary to keep workers safe from potential exposures. If UNH Health Services does not have the expertise in house to provide recommendations for certain protocols or post exposure consultation, a specialist will be sought to fulfill the needs of the protocol. Principal Investigator On behalf of the institution, the Principal Investigator and/or Principal Instructor (PI) are responsible for full compliance with the NIH Guidelines, the latest version of the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories (BMBL), and this document, when using biohazardous materials including recombinant DNA. It is the PI s responsibility to Determine if their work requires IBC approval based on the IBC s procedural document and the NIH Guidelines and to notify the IBC of any regulated work before that work begins. Make available to all laboratory staff the protocols that describe the potential biohazards and the precautions to be taken. Instruct and train laboratory staff in the practices and techniques required to ensure safety, and the procedures for dealing with accidents. Implement required safety practices as assigned by the IBC and in compliance with regulatory agencies. This includes implementing Universal Precautions when appropriate. Inform the laboratory staff of the reasons and provisions for any precautionary medical practices advised or requested (e.g., vaccinations or serum collection). Routinely monitor lab work to ensure safe practices are being practiced. Ensure compliance to all regulatory requirements within their laboratory. Inform lab associates of any special hazards as they apply to the work in their laboratory. Ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. Personnel must receive annual updates or additional training when procedural or policy changes occur. Enforce the institutional policies that control access to the laboratory. 7

8 Investigate and report significant problems pertaining to the operation and implementation of containment practices and procedures in writing to the Biological Safety Officer. Correct work errors and conditions that may result in the release of infectious materials, or recombinant or synthetic nucleic acid molecule materials. Ensure the integrity of the physical containment and the biological containment of the materials used in the lab. This includes annual certification of the Biological Safety Cabinet. Lab Personnel Laboratory personnel must: Be informed of safety guidelines and procedures and understand regulations as they apply to their work. Participate in safety training as required and assigned by their PI and OEHS. Perform all lab work in a safe manner as described in this manual and by specific operating procedures established for their laboratory. Practice all reasonable precautions to protect themselves and fellow lab workers from potential exposures while working in the lab in instances where lab hazards are not fully known. Ancillary Personnel and Visitors Personnel who enter laboratories to perform non-routine work, or visitors who work in a lab for any reason, are considered ancillary personnel. Ancillary personnel can be emergency responders, facilities, maintenance, or housekeeping personnel, as well as visiting guests who perform hands-on work. Ancillary personnel must have biosafety awareness training, at a minimum, when entering BSL-2 laboratory spaces. Guided tours or visitors who are escorted do not require training; however personal protective rules appropriate for the lab would apply at all times. Section 4. Biological Safety Risk Groups Biohazardous materials are classified according to risk levels, and through a risk assessment are placed into appropriate containment levels. Biological materials that some may not consider biohazardous may be regulated, so careful consideration must be given to all work with biological materials. There are four risk levels, called Risk Groups (RGs), and pathogens are classified according to their relative pathogenicity for healthy adults. The following are the NIH definitions of the four Risk Group levels: Risk Group 1 (RG1) agents are not associated with disease in healthy adult humans. Risk Group 2 (RG2) agents are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available. Risk Group 3 (RG3) agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available. Risk Group 4 (RG4) agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available. NIH assigns Risk Group ratings to various pathogens in Appendix B of the NIH Guidelines. These can also be found in Appendix 2 of this manual. Biological Safety Containment Levels Containment laboratories for infectious agents and laboratory animals are described by biosafety levels (BSL). Biosafety levels are designated in ascending order, by degree of protection provided to 8

9 personnel, the environment, and the community. Standard microbiological practices are common to all BSL laboratories. Special microbiological practices enhance worker safety, environmental protection, and address the risk of handling agents requiring increasing levels of containment. UNH operates biosafety level 1 (BSL-1) and biosafety level 2 (BSL-2) labs only; therefore only guidance for these types of laboratories are described. Biosafety Level 1 (BSL-1) Biosafety Level 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-1 laboratories are not necessarily separated from the general traffic patterns in a building. Work is typically conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is not required, but may be used as determined by appropriate risk assessment. Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist with training in microbiology or a related science. Biosafety Level 2 (BSL-2) Biosafety Level 2 builds upon BSL-1. BSL-2 is suitable for work involving agents that pose moderate hazards to personnel and the environment. It differs from BSL-1 in that: 1) laboratory personnel have specific training in handling pathogenic agents and are supervised by scientists competent in handling infectious agents and associated procedures; 2) access to the laboratory is restricted when work is being conducted; and 3) all procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets (BSCs) or other physical containment equipment. Lab Specific Operating Procedure It is strongly recommended that BSL-2 labs with special hazards adopt a lab specific biosafety operating procedure that is tailored to the specific hazards and procedures associated with the lab. A template for this is given in Appendix 1. Required BSL-2 procedures must be reflected in the lab specific operating procedure and additional practices may be implemented. A lab specific operating procedure should accompany an Institutional Biosafety Committee protocol submission. A copy of the lab specific operating procedure should be readily accessible in the laboratory in paper and/or electronic form. Biosafety Laboratory Requirements A summary of requirements for biosafety levels 1 and 2 is given below. The requirements are for activities involving biohazardous materials and laboratory animals. More specific information for Standard Microbiological Practices, Safety Equipment, Facilities and Special Practices are given in Sections 5 (Biosafety Facility Requirements) and 6 (Work Practice Requirements). Table 1: Summary of Biosafety Levels for Microbiological Agents and Laboratory Animal Work Biosafety Level Microbiology Practices Safety Equipment (primary barriers) Facilities (secondary barriers) BSL-1 Standard microbiological practices None required. PPE: lab coats and gloves; eye and face protection as needed Laboratory bench and sink required BSL-2 BSL-1 practice plus: Limited access Biohazard warning signs Sharps precautions Primary barriers: Biological safety cabinets or other physical containment devices used for all manipulations of agents that cause BSL-1 plus: Autoclave available 9

10 Animal Biosafety Level 1* (ABSL-1) Animal Biosafety Level 2* (ABSL-2) Biosafety operating procedure defining any special practices, waste decontamination or medical surveillance policies Standard animal care and management practices, including appropriate medical surveillance programs ABSL-1 practice plus: Limited access Biohazard warning signs Sharps precautions Biosafety operating procedure Decontamination of all infectious wastes and animal cages prior to washing splashes or aerosols of infectious materials PPE: lab coats, gloves, face and eye protection, as appropriate As required for normal care of each species PPE: lab coats and gloves, eye, face protection, as needed ABSL-1 equipment plus primary barriers: Containment equipment appropriate for animal species PPE: lab coats, gloves and face, eye and respiratory protection, as appropriate *For activities in which experimentally or naturally infected vertebrate animals are used. Standard animal facility: No recirculation of exhaust air Directional airflow recommended Hand washing sink is available ABSL-1 plus: Autoclave available Hand washing sink available Mechanical cage washer recommended Negative airflow into animal and procedure rooms recommended Risk Assessment In order to determine if a biological material is a biohazardous material and what level containment the biohazardous material requires for safe work and storage, a laboratory biological risk assessment must be completed. When determining the level of risk a biological material poses, evaluation of the Risk Group assigned to the agent is a good starting point. A list of organisms assigned to Risk Groups by the National Institutes of Health is located in Appendix 2. After determining Risk Group assignment, the following factors should be evaluated: Virulence; Pathogenicity; Infectious dose; Environmental stability; Route of spread; Communicability; Quantity handled; Availability of prophylaxis or treatments Gene product effects (if rdna) ; and Identification of susceptible populations. Attenuated strains of infectious agents or those proven to have lost known virulence factors may qualify to be handled at a lower containment level than that identified for a parent strain and therefore should be addressed in the risk assessment. Alternatively, some strains may prove to be more virulent than parent types, especially if genetically modified, so this must be taken into consideration during the risk assessment as well. An initial risk assessment is performed by the PI while planning experiments. Guidance for how to perform a risk assessment is given in Appendix 3. A final risk assessment is performed by the Biological 10

11 Safety Officer as a part of the IBC registration process. Assessments for work that fall under the purview of the IBC must be completed prior to commencing that work. A risk assessment of laboratory work can be requested at any time by the Principal Investigator, laboratory worker, or any member of the campus community by contacting the Biological Safety Officer. The Biological Safety Officer, in conjunction with the Investigator or Instructor, will assess biological materials in use, planned work procedures, personnel qualifications, facility set up and equipment used during lab processes. Once a risk assessment has been completed a biosafety level appropriate for the work will be determined and proposed to the IBC. A laboratory specific biosafety operating procedure is strongly encouraged based on the risk assessment. A Principal Investigator may use this Biological Safety and Biosecurity Manual as a resource and starting point to identify equipment and practices that make sense for their laboratory. All lab specific biosafety operating procedures must be approved by the Institutional Biosafety Committee as a part of the protocol approval. If a lab specific operating procedure is not adopted based on a risk assessment, the requirements listed in this manual must be followed. If insufficient data is available to identify risk, the default will always be a more conservative approach to working safely with biological materials. Recombinant and Synthetic Nucleic Acids Recombinant DNA (rdna) and synthetic nucleic acids are regulated by the NIH. UNH s Institutional Biosafety Committee is registered with NIH and presides over work with these agents. The IBC s procedures are detailed in the Institutional Biosafety Committee Charter and Procedures document, located on the IBC webpage. A registration must be filed with the IBC prior to beginning any work with rdna or synthetic nucleic acids. Section 5. Biosafety Facility Requirements General Biological Laboratory Facility Requirements All biological laboratories at UNH must meet a few standard requirements, no matter the biosafety level. These requirements include: Laboratories have doors for access control. Laboratory windows that open to the exterior should be fitted with screens. Each laboratory must have a sink for hand washing. The lab is designed so that it can be easily cleaned. Bench tops are impervious to water, chemicals, and moderate heat. Lab furniture is sturdy and easily cleanable. Cloth chairs are not allowed. Spaces between furniture are accessible for cleaning. An autoclave for decontamination of biological waste is available, or biological waste is collected for disposal by incineration or other approved method. Eyewash stations are readily available. 11

12 An insect and rodent control program is in place. A sign incorporating the universal biohazard symbol is posted at the entrance to the laboratory when infectious agents are present. The name and phone number of the Principal Investigator and at least one other emergency contact must be posted. The sign should include the name of the agents in use and any special precautions for entry. Biosafety Level 2 Facility Requirements In addition to the General Biological Laboratory Facility Requirements, BSL-2 lab facility requirements include: Laboratory doors should be self-closing and must have locks. Biosafety cabinets should be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. Locations away from doors, windows that can be opened, heavily traveled laboratory areas and other possible airflow disruptions are appropriate for cabinet placement. Biosafety cabinets must be certified annually and operated according to manufacturer s recommendations. Section 6. Work Practice Requirements Standard Microbiological Practices for All Labs Aseptic techniques and good microbiological practices must be practiced in all biological laboratories. Below are requirements for good microbiological practices that are standard and required in both BSL-1 and BSL-2 labs: Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption is not be permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose. Mouth pipetting is prohibited; mechanical pipetting devices must be used. Safe practices must be observed when using pipettes and pipetting aids. Safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware is required. Sharps should be used only when absolutely necessary because needles can be the cause of accidental injection or aerosol production. Whenever practical, a Principal Investigator or Instructor must adopt engineering and work practice controls that reduce the risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. Careful management of needles and other sharps, which includes storage in a capped manner, or in a puncture resistant container. Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal. Used disposable needles and syringes must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard walled container for transport to a processing area for decontamination, preferably by autoclaving. Broken glassware must not be handled directly. Instead, removal using a brush and dustpan, tongs or forceps is required. Plasticware should be substituted for glassware whenever possible. 12

13 Whenever possible, blunt needles or cannulas should be used and always with a LuerLock syringe. A needle/syringe unit should never be used in place of a standard pipette. Never use a syringe unit to expel a stream of biohazardous fluid forcefully into an open container to mix. A disposable needle/syringe unit is always preferred and should be disposed as a unit. Two handed recapping is never allowed and one handed or mechanical recapping should only be done when absolutely necessary. Self-sheathing needles and other safety engineered sharps are available from many manufacturers and should be used. Stocks of infectious agents in Risk Group 1 or Risk Group 2 categories that are human pathogens, or are environmentally or agriculturally significant pathogens, must be entered into the campus biological inventory database in UNHCEMS as soon as they are received. OEHS should be contacted for access to the biological inventory and for container barcodes. Cultures, stocks, recombinant DNA materials, and other potentially infectious materials must be decontaminated before disposal using an effective method. At UNH this could be via validated autoclave, chemical disinfection, or packaging for removal for incineration. If chemical disinfection is used, an EPA registered disinfectant is required. All biological liquids must be transported between labs in closeable, leak proof containers. Work surfaces must be decontaminated after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant. An EPA registered disinfectant is required. Materials removed from the facility for decontamination or destruction must be packed in accordance with applicable local, state, and federal regulations. See the UNH Biohazardous Waste Disposal Plan for proper packaging of biohazardous waste. Hand washing is required after working with potentially hazardous materials and before leaving the laboratory. Work Practices for BSL-2 Laboratories Practices listed below are standardly accepted work practices for a BSL-2 laboratory. BSL-2 lab practices build upon those listed above for all biosafety labs. Equipment and practices used in BSL-2 labs should be based on a risk assessment. BSL-2 labs are encouraged to adopt a lab specific biosafety operating procedure outlining the equipment and work practices approved for their facility. See Appendix 1 for a BSL-2 Lab Specific Operating Procedure template. BSL-2 Required Practices All Standard Microbiological Practices Principal Investigators/Instructors must ensure that laboratory personnel demonstrate proficiency in standard and lab specific practices before working with risk group 2 agents. Properly maintained biosafety cabinets or other physical containment devices and appropriate personal protective equipment must be used whenever: Procedures with a potential for creating infectious aerosols or splashes are conducted. High concentrations or large volumes of infectious agents are used. Liquid disinfectant traps and in-line HEPA filtration must be used to protect all vacuum lines. Hands must be kept away from the eyes, nose, face and hair when working with infectious agents. This will prohibit the potential for self-inoculation. 13

14 Disinfection of work surfaces must be performed daily, and immediately after any spill events. Routine housekeeping must be utilized to keep the laboratory orderly and without physical hazards. Clutter must be removed from the labs or kept at an absolute minimum. Floors should be kept clean and mopped on a regular schedule. The universal biohazard symbol must be used to alert to the actual or potential presence of biohazard materials in or on equipment, containers, rooms, or materials. Fluorescent orange or red is always used with a contrasting color such as black or white. The symbol must be prominently displayed on all refrigerators, freezers, incubators, microwaves and other equipment in the biological laboratory used to store or handle biohazardous materials. Transport containers must be labeled with the biohazard symbol for all materials coming out of a BSL-2 facility. Waste materials decontaminated outside of the immediate laboratory must be placed in a durable, leak proof container and secured for transport. BSL-2 Additional Best Practices Practices in this section are recognized as best practices and should be implemented based upon a risk assessment. Any additional best practices used should be detailed in the BSL-2 Lab Specific Operating Procedure, as appropriate. Personal electronics must be addressed in a risk assessment when used in the laboratory. Inanimate items, or fomites, can spread contamination via transfer from one individual to another. Electronics should be wiped down with an appropriate disinfectant prior to leaving the laboratory. If working with biohazardous fluids, pipetting may be required to be confined to a safety cabinet or hood. This requirement is based on the agent in use and the risk assessment. Biohazardous material should not be mixed by suction and expulsion through a pipette. Biohazardous material should be pipetted using good microbiological practices, such as pipetting down the side of a tube, not using considerable force to expel liquids out of a pipette, etc. A bench cover or diaper may be used on the work surface when biohazardous materials will be pipetted to prevent contamination. If used, these surface liners should be disposed at the end of every day. Discharge from pipettes should be as close as possible to the fluid or agar. Allow fluid to run down the wall of the tube or bottle and not be dropped from the top. Based on a risk assessment, contaminated pipettes may need to be placed in a puncture resistant sharps container, or be immersed in disinfectant prior to disposal. Pipettes used in a BSC should be discarded in a receptacle within the BSC. Water baths should be cleaned and disinfected regularly with an appropriate disinfectant. Aerosols should be minimized. Common lab practices that can create aerosols include the following: Blowing the last drop of a liquid culture or chemical from a pipette; Removing the cover from a blender or grinder shortly after completion of the blending or grinding operation; 14

15 Removing the cap from a bottle of a liquid culture or suspension immediately after vigorous shaking; Improper stoppering of volatile substances; Grinding tissue with mortar and pestle or glass tissue grinder; Decanting the supernatant fluid after centrifugation; Re-suspending packed cells by shaking or mixing; Inserting a hot wire loop in a culture; Withdrawing a culture sample from a sure/seal (vaccine-type) bottle; Opening a freeze dried preparation; Shaking and blending cultures and infected tissues in high speed mixers; Disrupting tissue cultures to release virus by shaking with glass beads; Streaking an inoculum on a rough agar surface Sonic disruption of cells Evacuating the atmosphere from a vacuum steam sterilizer prior to sterilization of contaminated material Opening a culture plate, tube or ampoule. Cultures of infectious agents should be opened and manipulated in the biological safety cabinet, based on a risk assessment. Vigorous shaking will create aerosols so it is best to gently swirl cultures to mix with minimal aerosol generation. When any culture is re-suspended, wait a few moments before opening the container to reduce the aerosol potential. The insertion of a hot wire loop or needle into a liquid, agar slant or agar plate will cause spatter and release of an aerosol. To minimize, cool the loop in air or by touching it to the inside of the container or agar surface prior to contact with the culture or colony. The use of a micro incinerator is the preferred method of sterilizing a loop, rather than using an open flame because the open flame causes aerosols. Disposable inoculating loops are also available from multiple commercial lab vendors. Petri dish cultures can accumulate condensate on the lid of the petri dish. This condensate can contain viable microorganisms and forms a film between the rim and lid of the inverted plate. Aerosols are dispersed when this film is broken by opening the plate. If plates are obviously wet, they should be opened in the biological safety cabinet. Opening microfuge tubes, screw top tubes, or culture tubes produce aerosol releases. Wrapping the cap in gauze, or disinfectant soaked gauze (for infectious agents) prevents the possibility of aerosol release. These tubes can also be opened in the biological safety cabinet. Centrifugation presents two serious hazards - mechanical failure and dispersion of aerosols. Balancing the centrifuge properly and using sealed rotors and safety cups can help prevent both types of centrifuge hazards. Centrifuge procedures are determined by a risk assessment. High speed centrifuges present additional hazards because of the higher stresses and forces on the system. High-speed rotor heads are prone to metal fatigue and rotors should be accompanied by a log book documenting the number of hours run at top or de-rated speeds. Failure to do this can result in dangerous and expensive disintegration if the rotor is run too long. If a rotor is treated with a disinfectant following a run or a spill, it should be rinsed with clean water and dried as soon as decontamination is complete. 15

16 Observe safety precautions whenever using a blender, mixer, sonicator, or cell disruptor. If possible, and based on a risk assessment, these operations with infectious materials should be done in a biosafety cabinet. Equipment should be capped. If an item cannot be capped, for example, a blender, then a paper towel soaked in disinfectant should be placed over the top to block aerosols. Stainless steel or polypropylene blender bowls are to be used with infectious material because of potential breakage. Glass bowls that can shatter can cause sharps injuries and should not be used with infectious agents. Allow aerosols to settle for at least 1 minute prior to opening the blender in the biological safety cabinet. Freezers should be defrosted periodically. When defrosted, freezers that contain infectious agents or chemicals should be checked for broken tubes or ampoules and disinfected. Requirements for BSL-1 and BSL-2 laboratories are given in checklist for in Appendix 4. Section 7. Personal Protective Equipment General requirements for laboratory personal protective equipment in BSL-1 or BSL-2 labs are as follows. Specific requirements are based upon a risk assessment and should be detailed in the lab specific biosafety operating procedure. Protective laboratory coats must be worn when working with hazardous materials to prevent contamination of personal clothing or skin. Laboratory coats are removed before leaving for non-laboratory areas (e.g., cafeteria, library, and administrative offices). Protective eyewear is required when conducting procedures that have the potential to create splashes of microorganisms or other hazardous materials when the microorganisms must be handled outside the BSC or other containment device. Many microbiologists and virologists may use chemicals that can cause blindness if splattered into the eye, such as concentrated quaternary disinfectants. Safety eyewear must always be used when working with hazardous chemicals. Infection can occur through the conjunctiva of the eyes if a pathogenic microorganism is splattered into the eye; therefore, safety glasses should be used whenever cultures are manipulated on the lab bench. Persons who wear contact lenses in laboratories should also wear eye protection. Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Gloves are changed when contaminated, glove integrity is compromised, or when otherwise necessary. Gloves are removed and hands washed with soap and running water when work with hazardous materials has been completed and before leaving the laboratory. Gloves must not be worn outside the laboratory. Never wash or reuse disposable gloves. Contaminated gloves are disposed with other contaminated laboratory waste. Double gloving is prudent if you must work with biohazardous materials with a cut, abrasion or open wound. 16

17 Laboratory clothing, such as lab coats, must not be taken home for laundering. Lab coats should be autoclaved if potentially contaminated with infectious agents and laundered on site at UNH or sent to a commercial laundering facility. Eye and face protection should be decontaminated before reuse, or disposed with other contaminated laboratory waste. Evaluate respiratory protection in rooms containing infected animals, when work is done outside of a BSC, or as determined by a risk assessment. The UNH Respiratory Protection Program should be consulted for requirements of respirator use. Section 8. Waste Biohazardous waste generated at UNH must be treated by either chemical disinfection or physical inactivation in a validated autoclave as soon as possible after generation. Personnel are required to follow the specific guidelines in the UNH Biohazardous Waste Procedures when handling biohazardous waste. Solid Waste The primary method to disinfect biohazardous solids at UNH is using a validated autoclave. Solid waste includes plastics, gloves, agar, or other solid or semi-solid materials used in the lab. Sharps and glass should not be placed in autoclave bags for disposal. Only autoclaves validated for biohazardous waste can be used for waste. Biohazardous waste must be contained in clear autoclave bags that have no distinguishing marks. When collecting biohazardous waste in a BSL-2 lab, the waste container must have a lid and remain closed at all times except when materials are being added. The waste container must have the biohazard symbol affixed to the side and top of the container. If the bag of waste is removed from the container and will not be autoclaved immediately, it must be tagged with a biohazard symbol, or placed in a secondary container with a biohazard symbol while awaiting waste treatment. Once the bag has been autoclaved, it is placed in a black trash bag and placed in the dumpster. Any biohazard tags that were applied while awaiting disinfection in the autoclave must be removed prior to disposal in the dumpster. Each waste autoclave cycle and its parameters must be recorded on the biological waste log sheet hanging next to the validated autoclave as described in the UNH Biohazardous Waste Disposal Plan. Alternatively, if a validated waste autoclave is not available in the area, a biohazard waste box (biobox, also known as red bag waste) must be used to dispose of biohazardous waste solids. Red autoclave bags can only be used with bioboxes for biohazardous waste disposal. Bioboxes are incinerated and leave no trace of red bags in landfills. Boxes and red biohazard bag liners can be obtained from the Office of Environmental Health and Safety. Chemical and radioactive materials must never be placed in any biohazardous waste stream. Liquid Waste Liquid biological waste that does not contain chemical or radiological components must be autoclaved or disinfected with bleach, or other EPA registered disinfectant appropriate for the agent, prior to sink disposal. For autoclaving, the liquid cycle parameters must be recorded on the biological waste log sheet as described in the UNH Biohazardous Waste Disposal Plan. 17

18 For chemical disinfection, the most common disinfectant for liquids is bleach. Add bleach to liquid waste at a 1:10 ratio for a final concentration of 10% (5,250 ppm sodium hypochlorite). Allow 30 minutes of contact time, mixing occasionally to ensure liquid waste and bleach have universally combined. Dispose into a laboratory sink. Run the water following the disposal to dissipate the bleach smell. If an alternative disinfectant is chosen, it must be an EPA registered disinfectant, appropriate for the agent being treated, and the manufacturer s guidelines followed for contact time. Not all disinfectants approved for surfaces are appropriate for liquid waste, so ensure that the disinfectant used is suitable for liquid waste. A list of common chemical disinfectants is given in Appendix 5. Sharps Needles, syringes, capillary tubes, microscope slides, cover slips, glass cuvettes, scalpels, razor blades and any broken glassware that is contaminated with biological material are considered sharps. Sharps contaminated with biohazardous materials must go into a red, rigid container labeled with the universal biohazard symbol. Biological sharps containers should be sealed when ¾ full and placed in a biohazard box for disposal. Contact OEHS for pickup. Sharps that are not biohazardous can go into a thick-walled, sturdy plastic or metal container. The container should have a tight fitting, screw-type lid which can be used to seal the sharps when ¾ full. Label the container with the words "SHARPS" and "DO NOT RECYCLE." Seal the container with heavyduty tape and contact OEHS for pickup. A waste flow chart summarizing waste procedures is included in Attachment 3. Section 9. Additional Lab Hazards A biological laboratory often has other types of hazard present in addition to biohazardous materials. A typical biological or biochemistry laboratory will have a number of hazardous chemicals, including corrosives, toxics and potential carcinogenic agents, as well as a potential to have radioactivity. Below are resources that address the other hazards that may be present in the laboratory. Chemicals Chemicals are often used in biological laboratories. Corrosive and toxic agents are common for various functions such as precipitation assays, tags or stains, preservation of specimen, disinfection, etc. See the UNH Chemical Hygiene Plan for instructions on safe handling and inventory of all chemical substances. Ionizing Radiation Some laboratories use radioisotopes for tagging and labeling proteins, DNA, RNA, etc. Typical isotopes used in the lab are tritium ( 3 H), Carbon 14 ( 14 C), Phosphorus 32 or Phosphorus 33 ( 32 P, 33 P), Sulfur 35 ( 35 S) or Iodine 125 or Iodine 129 ( 125 I, 129 I). See the UNH Radiation Safety Manual for detail on handling radioisotopes in a biosafety laboratory. 18

19 Compressed or Liquid Gases Compressed gases and liquid cryogens are often used in biological laboratories. Working with these materials often pose some risks and additional Personal Protective Equipment and specialized handling practices may be required for working with them. Refer to the UNH Chemical Hygiene Plan for details about handling compressed gases and liquid cryogens. Noise Ultrasonic cell disruptors (sonicators) are used in biological labs for a number of applications, including: cell lysis; cell extract preparation; disruption of various types of cells, including yeast, bacteria, plants and mammalian cells; protein extraction; preparation and isolation of enzymes; production of antigens; DNA extraction and/ or targeted fragmentation; and liposome preparation. Sonicators work by using high frequency sound to disrupt the cell by cavitation. Two hazards associated with sonication are: hearing loss due to high frequency sound; and aerosol production. Elimination of these two hazards is critical to safety. Hearing protection must always be available in a lab using ultrasonic cell disruptors. Ear muffs must be provided near the sonicator and a sign indicating Hearing Protection Required is necessary. If possible, a soundproof cabinet should be used. Do not sonicate in a room with people who are not wearing hearing protection. Close the door to the room while sonication is on-going. Inhalable aerosols can be avoided by capping samples, or using a sonicator enclosure. If sonicating a Risk Group 2 organism, the sonicator must be placed in a certified biological safety cabinet while being used. Nanotechnology The use of nanotechnology in biological laboratories is becoming more common. Nanomaterials are nanometer sized particles often referred to as nanoparticles and can be used in a variety of ways in biology, from drug delivery to labeling proteins and tissue engineering. The small size of the nanoparticles creates a potential health hazard due to the possibility of inhalation, absorption, or ingestion; therefore, engineering controls and personal protective equipment are quite important when working with these materials. The UNH Nanomaterials Safety Program must always be followed when using nanotechnology in conjunction with biological materials in the laboratory. Section 10. Disinfection and Decontamination Laboratory equipment should be routinely decontaminated. Decontamination must be completed after spills, splashes, or other potential contamination of laboratory equipment or lab areas. Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material. Equipment must always be decontaminated prior to repair, maintenance, or removal from the laboratory. Heat Sterilization There are hazards with handling hot solids and liquids that have been autoclaved for sterilization. Steam autoclaves use steam under pressure and scalding burns can result when opening the autoclave too 19

20 quickly, or handling items before allowing them to cool or without proper heat resistant PPE. Waste handling is addressed in the UNH Biohazardous Waste Disposal Plan. It is important that any item(s) placed in an autoclave for sterilization be in loads of manageable size and not too heavy. Fluids and agar sterilized with steam under pressure become superheated which can cause violent boiling of the contents that can boil over the containers and splash or scald as items are removed from the autoclave. Allowing sufficient cooling time reduces this risk. Additionally, the use of thermal gloves reduces the risk of burns on the hands when removing items from the autoclave. Spills in the Open Laboratory If biohazardous material is spilled in the laboratory, it is important to avoid inhaling any airborne material. If infectious material is spilled that has a known inhalation exposure route, it is beneficial for lab occupants to hold their breath and exit the lab immediately. If possible to do so safely, warn others to evacuate the immediate area. If clothing is known or suspected to be contaminated, remove the clothing with care, folding the contaminated area inward. Discard the clothing into a red biohazard bag, or place the clothing directly in an autoclave. Wash all potentially contaminated skin areas with soap and clean water. Safety showers are available in all lab buildings and should be utilized if necessary. If a safety shower is used, effluent must be treated as a biological spill. Do not re-enter the lab for 30 minutes, thus allowing aerosols to settle. Spill kits are generally located under every lab sink and a biological spill kit should include absorbent pads or paper towels, bleach, gloves, a disposable lab coat, safety goggles or a face shield and a dustpan/broom or forceps. PPE must be worn when entering the laboratory to clean the spill area. Step 1: Place absorbents over the spill. Be careful not to step in any area that may have droplets or settled aerosols from the spill. Step 2: Pour EPA registered disinfectant appropriate for the agent around the spill and into the spill. Avoid splashing. Step 3: Allow contact time as recommended by the disinfectant manufacturer. Step 4: Use paper towels to wipe up the disinfectant and spill, working toward the center of the spill. Discard paper towels into a red bag with the biohazard symbol. These red bags will be placed in a biohazard box for disposal. The paper towels cannot be autoclaved because chemical disinfectant has been used. Step 5: Disinfect the surface again once all spill materials have been removed from the area. Step 6: Remove PPE carefully, avoiding contamination with clean clothing and wash hands, arms and face thoroughly. Spills in a Biological Safety Cabinet A spill that occurs within an operating BSC presents low risk with the cabinet running, so if a spill occurs, keep the cabinet running throughout the spill cleanup. PPE including a lab coat, gloves and safety glasses must be worn during spill cleanup. Step 1: Immediately place absorbents over the spill. 20

21 Step 2: Pour EPA registered disinfectant appropriate for the agent around the spill and into the spill. Avoid splashing. If the spill has gotten into the grill, pour disinfectant solution into the grill to ensure that the drain pans and catch basins below the work surface are coated with disinfectant. Step 3: Allow contact time recommended by the manufacturer for the disinfectant used. Step 4: Use paper towels to wipe up the disinfectant and spill, working toward the center of the spill. If disinfectant was used in the grill, lift the front exhaust grill and tray and wipe all surfaces. Wipe the catch basin and drain the disinfectant into a container. Step 5: Wipe walls, work surfaces, and equipment with disinfectant. Step 6: Many disinfectants are corrosive to metal surfaces. Follow all disinfection steps with a water or 70% alcohol wipe down to remove disinfectant residue from stainless steel surfaces. Step 7: Discard paper towels into a red bag with the biohazard symbol. These bags will be placed in a biohazard box for disposal. Step 8: Remove PPE carefully, avoiding contamination with clothing and wash hands thoroughly. If the entire cabinet, including HEPA filter, has become contaminated, contact OEHS to schedule decontamination prior to using the cabinet. Commissioning, Decommissioning or Relocating a Laboratory When commissioning or relocating a laboratory, it is important to confirm that all physical aspects of the laboratory are appropriate for the biological safety level. OEHS should review all lab spaces prior to occupancy to ensure requirements are met. For decommissioning or moving, biological laboratories must: Disposition all biological materials either through disposal as biohazardous waste, moving biologicals to a new lab room or storage area, or transferring materials to another Principal Investigator. If moving or transferring, material locations must be updated in the biological inventory in UNHCEMS. Disinfect all equipment with an EPA registered disinfectant appropriate for the materials used or stored within them. If a biohazard label is present on equipment, the label must be removed or defaced following disinfection and prior to disposition to another lab or disposal. Based on a risk assessment, biological safety cabinets may need to be decontaminated with a gaseous disinfecting agent. Contact OEHS for evaluation prior to moving the cabinet. After all materials and equipment have been removed from the lab, all surfaces such as floors, lab benches and cabinet fronts must be wiped down with an EPA registered disinfectant that is appropriate for the materials used in the lab. Section 11. Training It is the responsibility of the Principal Investigator and/or Instructor to ensure all personnel working with biohazardous materials under their guidance are trained appropriately for the work they will be doing. The PI is encouraged to contact OEHS and speak with the Biological Safety Officer regarding the type of 21

22 training offered through the Office of Environmental Health and Safety. OEHS offers multiple biosafety training modules on-line through UNHCEMS, as well as live training given by the Biosafety Officer when requested. At a minimum, workers in a BSL-1 or BSL-2 lab must complete general biosafety training for the level of containment lab they are working in. Refresher training will be requested by OEHS periodically. Examples of training required for each biosafety level: Laboratory Biosafety Level Materials Used Training Required BSL-1 Risk Group 1 agents Biosafety Level 1 BSL-2 Risk Group 2 agents Biosafety Level 2 BSL-2 Human derived materials Biosafety Level 2 and Bloodborne Pathogens BSL-2 None (ancillary personnel or Biosafety awareness visitors) BSL-1 or BSL-2 Materials to be autoclaved Autoclaving Biohazardous Waste Contact the Biosafety Officer for guidance in determining which types of training are needed for a specific laboratory. Section 12. Medical Surveillance Medical surveillance is a pro-active approach to preventing workplace injuries. In the biological safety program, medical surveillance is an important tool used to monitor individuals for occupational disease acquired while on the job. The UNH medical surveillance program aims to: Provide active immunization where indicated by a health professional; Provide early detection of laboratory-acquired infections; and Exclude highly susceptible individuals (e.g. pregnant women or immunocompromised individuals) from highly hazardous/infectious laboratory work. This determination can be made during a risk assessment or medical consult with the occupational health professional. The Principal Investigator, IBC and BSO have combined roles in identifying personnel who should be included in the UNH medical surveillance program. The risk assessment process for IBC protocol registration is the time to address any items which might call for medical surveillance. UNH Health Services provides the professional services associated with medical surveillance, including identification of risk based on health history, prescription of prophylaxis or treatments, and follow up care when necessary. NIH Guidelines Requirements for Recombinant or Synthetic Nucleic Acid Materials The NIH Guidelines for Recombinant DNA Research state that it is the responsibility of the Institution to "determine, in connection with each project, the necessity for medical surveillance of recombinant DNA research personnel before, during, and after their involvement in this research". The Guidelines further require the principal investigator or instructor to investigate and report in writing to the IBC any serious or extended illnesses of a worker or any accident that results in inoculation of recombinant DNA materials through cutaneous penetration, 22

23 ingestion of recombinant DNA materials, probable inhalation of recombinant DNA materials following gross aerosolization, or any incident causing serious exposure to personnel or danger of environmental contamination. These activities constitute the minimum requirements for a medical surveillance program for recombinant DNA research. The Guidelines also recommend that the medical surveillance program provide for collection and maintenance of serum samples and for the immunization of all workers who may work with known pathogens for which an effective vaccine is available. The extent of any medical surveillance program will vary greatly, depending upon the risk assessment and taking into account the nature and size of the research project and the available medical facilities. Bloodborne Pathogens The Hepatitis B vaccine is recommended for all UNH personnel who handle human derived materials such as blood, blood products (including cells, cell lines and cell strains), unfixed tissue, and bodily fluids. OSHA s Bloodborne Pathogens Standard requires the employer to offer this vaccine, free of charge, to those employees covered by the UNH Exposure Control Plan. Additional information about the vaccine can be found in the Plan, in the OSHA regulation (29 CFR ) and on the CDC website. Section 13. Incidents Due to the infectious nature of biohazardous materials, incidents that may result in exposure must be evaluated and treated immediately. This is also true of incidents involving recombinant or synthetic nucleic acid molecules. All such incidents must be reported to the laboratory supervisor and OEHS as soon as possible after the incident. Medical evaluation and treatment should be provided as soon as practical and records of the evaluation maintained. Incident Reports An incident report is filed for all known or potential exposures, injuries and near misses. Prompt reporting is crucial to proper medical follow up and prophylaxis. The report should be filed by the individual and/or their supervisor within 24 hours of the incident. The report is forwarded to Human Resources for review. Following human resource s review, OEHS will investigate the cause of the incident and put in place corrective actions to help prevent reoccurrence of the same or similar incident. The investigation will include review of techniques, procedures and types of equipment that are used and may have been involved in the incident. Lab Acquired Infection Exposure to an infectious agent, or an agent carrying recombinant or synthetic nucleic acid molecules, may not always be evident to the worker right away. This is especially true for inhalation of aerosols. Because of this, it is important to investigate serious, unusual or extended illnesses of a lab worker doing research with any of these agents. Additionally, if multiple lab workers come down with the same type of illness all at once or over a number of months, it is prudent to track and investigate any patterns of illness from the lab. If investigation finds that exposure to an infectious agent or rdna has occurred there may be reporting requirements to NIH or the Department of Public Health. 23

24 Medical Evaluation There are medical conditions that may put a person at an increased risk of Lab Acquired Infection. Lab personnel that are immunocompromised or immunosuppressed due to medical treatment or illness may want to seek medical evaluation prior to working in a lab with infectious agents. Additionally, there are a number of infectious agents that are considered teratogenic, so pregnant workers may also want a medical evaluation prior to working in a lab where these agents are present. Section 14. Import/Export A variety of national and international regulations exist which require permits for the import, export and interstate movement of biologicals materials. Centers for Disease Control and Prevention (CDC) The Centers for Disease Control and Prevention s Import Permit Program (IPP) regulates the importation of infectious biological agents, infectious substances, and vectors of human disease into the United States. Items that most often require a permit from CDC are: Naturally occurring or bioengineered infectious biological agents capable of causing disease in a human; Any material that is known or reasonable expected to contain an infectious biological agent; and Vectors, including animals/animal products that are known to transfer or are capable of transferring an infectious biological agent to a human. Department of Agriculture (USDA) The United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) requires permits for the import of animal and plant materials due to the potential risk of exotic disease introduction into the U.S. or transmission of diseases across state lines. Examples of items requiring permits are: Infectious agents of livestock and biological materials containing animal material; Tissue culture materials and suspensions of cell culture grown viruses or other etiologic agents containing growth stimulants of bovine or other livestock origins; Plant pathogens, weeds and seeds Organism and Soil Permits Plants and Plant Products Permits Exports The export of various biological materials or agents of human, plant, and animal diseases may require a license from the Department of Commerce for export from the United States to areas abroad. Contact the Export Control Office for more information and guidance on export requirements. Material Transport Agreements Material Transfer Agreements (MTAs) address the exchange of research materials between individuals at separate organizations and address ownership, intellectual property, publications, and liability related to the research materials. UNHInnovation initiates an MTA, which is a binding written contract between parties that governs the use of exchanged material, when needed. Contact the Export Control Office about MTAs for guidance on this process. 24

25 Section 15. Biological Safety and Biosecurity Manual Shipping The Department of Transportation (DOT) regulates the shipment of infectious substances in commercial transportation to, from and within the United States. International Air Transport Association (IATA) regulates all dangerous good shipped by air. Categories of agents regulated by these agencies are in Division 6.2: UN 2814, Biological Substance, Category A UN 3373 Biological Substance, Category B The UNH Laboratory Safety Officer will train all personnel who are required to make a decision about shipping categories and preparing packages for shipment. Training must be completed prior to shipping any biological packages. Reference the UNH Shipment of Biologicals Material Manual for information about proper shipping. Section 16. Biosecurity Laboratory biosecurity measures are those instituted to prevent loss, theft, intentional misuse or unintentional release of biohazardous material from the laboratory. Biosecurity takes into account external and internal threats and employs a few basic principles: Creating and maintaining an accountability system for the storage and use locations of all infectious agents on campus; Controlling access so that external sources do not have contact with inventory; Understanding who has internal access, and for what purposes; and Ensuring materials are secure at all times when not in current use. Infectious Agents The accountability system set up at UNH is based on a biological inventory of human infectious agents or environmentally or agriculturally significant pathogens. This inventory is kept in UNHCEMS and will be entered by anyone who owns biological inventory. Access to inventory is granted by the inventory owner and it is the owner s responsibility to determine who in their laboratory should have access to the physical inventory. The Principal Investigator should assess the professional and ethical suitability of personnel who have regular access to biohazardous materials prior to authorizing access to those individuals. All pathogenic agents considered Risk Group 2 or higher must be secured at all times when not in use. A locked laboratory or a lock on the refrigerator or freezer where stocks are stored is considered appropriate security. Only personnel whose work includes handling these pathogens should be allowed access to the key or code for the secured storage areas. Inventory information that is tracked, at a minimum, includes: 1. pathogen or toxin name; 2. risk group identification; 3. material owner; 4. physical location of storage; and 5. physical location of use 25

26 If possible, an estimate of the amount of material should be tracked; this is especially important for biological toxins that can be tracked in gram or milligram quantities. For agents that are more difficult to track with concrete amounts, CFU, PFU or other quantity can be tracked for stock materials based on the type of agent it is. In the event of a security breach or known loss or theft of materials, notification to the owner of the materials, as well as to EHS must be made immediately. A Biological Incident form must be completed and an investigation by the Biological Safety Officer will be completed. The Biological Incident form is located in Appendix 7. Plants and Seeds Plant materials and seeds that are genetically modified with recombinant DNA techniques, or those that are invasive, non-native, or agriculturally significant must be accounted for at all times and not be released to the environment. Inventory of these materials may be tracked in UNHCEMS or in a similartype database or spreadsheet by the Principal Investigator. Insects Insects used in research or for instruction must also be continuously accounted for. Insects can be vectors of human or animal disease and those brought in for laboratory purposes must remain in the laboratory at all times. If loss, theft or accidental release (escape) of a laboratory insect occurs, immediate notification to the Biosafety Officer is required. Rodents As with insects, rodents can be vectors of human or animal diseases and it is critical to keep laboratory animals from mixing with wild populations. The Animal Resource Office maintains a set of Standard Operating Procedures which outline their biosecurity policy and integrated pest management plan. Section 17. References This manual was written from the below list of published guidance documents. Much of the wording was take verbatim in order to maintain the integrity of guidance from these agencies. 1. Biosafety in Microbiological and Biomedical Laboratories, Fifth edition, U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Institutes of Health, HHS Publication No. (CDC) , December World Health Organization Laboratory Biosafety Manual, Third Edition, Geneva, 2004, ISBN Canadian Biosafety Standards and Guidelines, First Edition, Public Health Agency of Canada, ISBN NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines), U. S. Department of Health and Human Services, National Institutes of Health, November Laboratory Biorisk Management CWA 16393:2012, European Committee for Standardization, CEN Workshop Agreement, Brussels 6. Laboratory Safety Monograph, Office of Research Safety, national Cancer Institute and the Special Committee of Safety and Health Experts, January 2, 1979, U.S. Department of Health, Education and Welfare, Public Health Service, National Institutes of Health 26

27 27

28 Appendix 1: Biosafety Level-2 Lab Specific Operating Procedure Template (adapted with permission from the University of Vermont) Investigator Name: Building and Lab Number: IBC Protocol Name & Number: Current Revision Date: Materials in use in the lab: Infectious Agent and Strain: Viral Vector: Biotoxin: Description of Risks to Laboratory Personnel: How and where was the biohazardous material obtained? Describe strain and any bioengineered or natural circumstances that make it more or less hazardous (examples: is it attenuated, more virulent, clinical sample, lab strain). Describe potential routes of exposure specific to the agent and how it is being used in the lab. Explain how risk of exposure from these routes will be reduced or eliminated in the lab through the use of engineering controls, personal protective equipment or administrative controls. Describe specific populations of susceptibles (examples: pregnant women or immunocompromised individuals) who may be at higher risk of becoming infected if exposed. A. Work Requirements 1. Pre-work requirements: [Add information for educating technicians, such as EHS on-line training, review of this operating procedure and how hazards will be communicated through the use of in-lab education, SOPs, signs, labels, etc.] All personnel working in laboratories listed on the IBC protocol must have completed the following Environmental Health and Safety Trainings: 28

29 On-line Trainings: Laboratory and Chemical Safety Biosafety Level 2 Bloodborne Pathogens Sharps Safety Autoclaving Biohazardous Waste Biohazardous Waste Management Biological Safety and Biosecurity Manual 2. Medical Surveillance (pre-exposure evaluations and testing) Laboratory personnel must be provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory. [Describe specific medical surveillance needed before working with infectious agent(s)in this lab: serology/antibody tests (and frequency), vaccinations and other laboratory tests needed. Include how this will be managed and documented.] 3. Safety practices and procedures for working with an infectious agent at BSL-2 Containment: [Address each section below] B. Standard Microbiological Practices [Add any lab specific general safe practices your lab may employ.] Do not work on this project until you have received and understood the required training outlined in section 1. The laboratory supervisor must address and enforce safety and security policies that control access to the lab Do not work with biohazardous materials outside of room(s) XXX Keep the door to room(s) ## closed while the work is ongoing Access to the laboratory is limited or restricted while biohazardous work is being conducted Persons wash their hands after they handle hazardous materials, after removing gloves and before leaving the laboratory. Eating, drinking, smoking, handling contact lenses, applying cosmetics and storing food for human consumption are not permitted in the work areas. Food must be stored outside the work area in designated storage areas. Mouth pipetting is prohibited Policies for the safe handling of sharps are instituted (see section F) All procedures are performed carefully to minimize the creation of splashes or aerosols. Work surfaces are decontaminated once a day and after any spill of viable material. All biohazardous waste is managed appropriately (see section J) A biohazard sign is posted at the entrance to the laboratory and on equipment or areas where materials are stored or in use. C. Procedures 29

30 [Describe specific laboratory procedures that may pose a risk of exposure to laboratory personnel (i.e., should reference all potential routes of exposure listed in the first section of this operating procedure)] D. Personal Protective Equipment [Describe what PPE will be used to reduce the risk of exposure (e.g., latex gloves, nitrile gloves, N95 respirator, disposable lab coat). Include when it needs to be worn, how to store reusable PPE (example: cotton lab coats), and frequency of cleaning (if reusable) or disposal] E. Decontamination and Disinfection [List and describe laboratory decontaminants and disinfectants that are used (if you are unsure as to what the appropriate disinfectant is, contact the Biosafety Officer for assistance). List all applications and the disinfecting procedure with regards to instrument or surface (example: lab bench), disinfectant used (example: 10% bleach solution), frequency of disinfectant preparation (example: freshly prepared bleach solution), allowed contact time before wiping (15 minutes is standard, but can vary based on organism), and frequency of decontamination (every day before and after work has been conducted)] F. Biological Safety Cabinets Class II biosafety cabinets (BSC) prevent the escape of particulates into the workers environment and prevent contaminants from the surrounding environment from entering the cabinet. All researchers listed on the IBC protocol should know and practice the following biosafety cabinet procedures: Type and location of BSC The BSC type is ## (example: Labconco, Class II A). The BSC is located in ## (example: left side of room 133). Preparing to work in the BSC Turn the UV light off and turn on the visible light Switch the cabinet blower on. Let the BSC run for 15 minutes prior to beginning work. If the cabinet malfunctions, do not start the experiment, and report to a supervisor and or the Biosafety Officer. Disinfect surfaces with prescribed disinfectant noted in section D. Place only the items you will need inside the cabinet Ensure that the front and rear grilles are not blocked by materials Working in the BSC Cover the work surface with absorbent paper Segregate the work area into clean and dirty (contaminated) sections. Keep contaminated material toward the rear of the cabinet. Place waste in a container lined with a biohazard waste bag inside the BSC. Work at least 6-8 inches inside the cabinet work surface. 30

31 Discard contaminated liquids in a 500 ml bottle containing 50 ml undiluted bleach (should be described in the section D). Keep containers closed when not in use to minimize spills. Always remove first pair of gloves before removing hands from the BSC. Vacuum Trap Trap consists of two flasks, the primary flask containing XXX (example: 10% bleach solution) and the secondary, overflow flask also containing XXX. A HEPA filter is located in line between the second (overflow) flask and the vacuum pump or house vacuum line. The trap system is set in a secondary container (tray) to contain potential spills or overflow. Preparing to shut down the BSC Spray (with a proper disinfectant described in section D) and remove all waste and materials before preparing for cleanup. Surface decontaminate all material and equipment with 70% ethanol or appropriate disinfectant before removing them from the cabinet. Wipe the cabinet surfaces with 70% ethanol after decontaminating with the appropriate disinfectant. Leave the blower on for at least 5 minutes to purge the cabinet. Prior to leaving the lab at the end of the procedure, switch off the visible light and turn on the UV lamp (if used). G. Safe Sharps Procedures If at all possible, do not use glass, needles and razor blades for procedures with biohazardous materials. Only needle locking syringes or disposable syringe needle units are used for injection or aspiration of infectious or biohazardous material. Used needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand prior to disposal. Use syringes which re-sheathe the needle, needleless systems, and other sharps safety devices whenever possible. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plasticware should be substituted for glassware whenever possible. Promptly dispose of all sharps in appropriate (labeled) sharps containers. Never fill sharps containers more than ¾ full Close and seal the top (with tape) of the full sharps container prior to placing it in a biohazard waste box for final disposal. H. Storage Keep isolated stocks of ## in a secondary leak-proof container (with cover) in the -80 C freezer in room ## Ensure that there is a biohazard label on the freezer and that the freezer is secure (i.e., either there is a lock on the freezer or the room where the freezer is housed is locked) at all times. Only people trained are authorized to have access. All stocks must be tracked in the UNHCEMS biological inventory system. I. Transportation 31

32 ## is transported in a primary container (e.g., vial), within a plastic (snap or screw top) secondary container for transport within the laboratory. ## is transported in a primary container (e.g., vial), within a plastic (snap or screw top) secondary container, within a tertiary, cooler for transport outside of the laboratory where the material is regularly stored and manipulated. J. Instrumentation If instruments other than a biosafety cabinet are used with the biohazardous material, describe the name of the instrument, where it is located (room # and location in the room), that it is labeled with a biohazard symbol, and how to decontaminate it. Specific information is listed below for centrifuge use. Incubator Sonicator Cell sorter or cytometer Centrifuge Perform all centrifugations in closed containers with sealed (o-ring) cups. Load and unload containers in a biosafety cabinet. Preparing for centrifugation Bring rotor to BSC Fill tubes and insert in holders, and screw caps Disinfect rotor with appropriate disinfectant before taking out of the BSC After centrifugation is completed Bring rotor to BSC Remove tubes Decontaminate the rotor with 70% ethanol Return rotor to centrifuge K. Biohazardous Waste Management Biohazard waste containers (for autoclave) Must be covered when not in use Must contain at least one clear autoclave bag with no distinctive markings When full, bag is sealed with tape, brought to the autoclave and sterilized. If the bag cannot be sterilized immediately, affix a biohazard label on the bag while it awaits processing. Labels are available in the autoclave rooms. Biohazard waste containers (for incineration) Must be covered when not in use Must contain two red biohazard bag liners Must not be overfilled or weigh over 55lbs Must not contain free liquid If collecting waste as part of a freezer clean out, contact the Biosafety Officer for assistance When full, the bag is sealed with tape, placed in the biobox and the box sealed with shipping tape. Contact EHS for pickup. 32

33 Pipettes, tips, tubes, etc. Decontaminate with appropriate disinfectant (listed in section D) for at least 15 minutes; or via autoclave. Decontaminate all non-sharp material (tubes, flasks, etc.) with disinfectant for at least 15 minutes; or via autoclave. Liquid medium and solutions Decontaminate via chemical disinfection or autoclave. For chemical disinfections, add bleach solution to a final concentration of 10% disinfectant. Leave for approximately 20 minutes Pour disinfected liquids down the drain. If using any disinfectant other than bleach, which may not be appropriate to pour down the drain, contact EHS for safe disposal options. For autoclave, use liquid cycle for 60 minutes. Contaminated PPE All disposable, contaminated PPE is disposed in a biohazard incineration box. L. Protocols for laboratory spills and emergencies [Describe protocol for cleaning up spills, maintaining staff safety, decontamination and disposal.] Small/Manageable spill procedures Protect yourself Wear PPE (e.g., lab coat, gloves, safety glasses or face shield) Contain the spill Notify others in lab and cordon off the spill area (keep people away) Disinfect the spilled material Cover the spill with paper towels or spill pads Pour (do not spray) freshly made bleach solution or other appropriate disinfectant onto the paper towels (working from the outside inward Allow to sit for 20 minutes Clean up Sweep up materials and place into a red biohazard bag Do not pick up sharps with your hands, use tongs, a dust pan or another available method Dispose of sharps in a sharps container Dispose of PPE in biowaste container If the spill is large or unmanageable, exit the lab, post a spill clean-up in progress sign at all entrances and call EHS at for assistance. M. Exposure Response Protocol Wash exposed area with water (eyes) or soap and water (subcutaneous) If in need of immediate medical treatment (serious cut), go to nearest Emergency Room Report exposure to laboratory supervisor or principle investigator (if readily available) During regular business hours (8AM 5PM, Mon.-Fri), go to UNH Health Services for follow up, evaluation and baseline serology (if serology is recommended for the agent you were exposed to) Report exposure to the Biosafety Officer, Complete a Biological Incident Form, located in the UNH Biosafety and Biosecurity Manual 33

34 I have read and understand the information within this BSL-2 lab operating procedure for IBC protocol ###-##-#. If I have questions about procedures I will ask my Principal Investigator immediately, or contact the Biosafety Officer at Student/Employee Name Student/Employee Signature Date 34

35 Appendix 2: Risk Groups ===================================================================================== The National Institutes of Health (NIH), along with other regulatory bodies around the world, have classified infectious agents according to Risk Group. The information below for Risk Group 1 and Risk Group 2 agents can be found in the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules Appendix B. The American Biological Safety Association has created a database compiling international regulatory information for risk group classification of infectious agents. Both of these sources should be consulted when doing a risk assessment. ===================================================================================== NIH Appendix B Risk Group 1 (RG1): Agents that are not associated with disease in healthy adult humans Examples of RG1 agents include asporogenic Bacillus subtilis or Bacillus licheniformis; adeno- associated virus (AAV all serotypes); and recombinant or synthetic AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus. A strain of Escherichia coli is a RG1 agent if it (1) does not possess a complete lipopolysaccharide (i.e., lacks the O antigen); and (2) does not carry any active virulence factor (e.g., toxins) or colonization factors and does not carry any genes encoding these factors. Those agents not listed in Risk Groups 2, 3 and 4 are not automatically or implicitly classified in RG1; a risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed. Risk Group 2 (RG2): Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available Risk Group 2 (RG2) - Bacterial Agents Acinetobacter baumannii (formerly Acinetobacter calcoaceticus) Actinobacillus Actinomyces pyogenes (formerly Corynebacterium pyogenes) Aeromonas hydrophila Amycolata autotrophica Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum) Arizona hinshawii - all serotypes Bacillus anthracis Bartonella henselae, B. quintana, B. vinsonii Bordetella including B. pertussis Borrelia recurrentis, B. burgdorferi Burkholderia (formerly Pseudomonas species) except those listed in Appendix B-III-A (RG3)) 35

36 Campylobacter coli, C. fetus, C. jejuni Chlamydia psittaci, C. trachomatis, C. pneumoniae Clostridium botulinum, C. chauvoei, C. haemolyticum, C. histolyticum, C. novyi, C. septicum, C. tetani Coxiella burnetii specifically the Phase II, Nine Mile strain, plaque purified, clone 4 Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale Dermatophilus congolensis Edwardsiella tarda Erysipelothrix rhusiopathiae Escherichia coli - all enteropathogenic, enterotoxigenic, enteroinvasive and strains bearing K1 antigen, including E. coli O157:H7 *Francisella tularensis specifically *F. tularensis subspecies novicida [aka F. novicida], strain Utah 112; *F. tularensis subspecies holarctica LVS; *F. tularensis biovar tularensis strain ATCC 6223 (aka strain B38) *For research involving high concentrations, BL3 practices should be considered (see Appendix G-II-C- 2. Special Practices (BL3)). Haemophilus ducreyi, H. influenzae Helicobacter pylori Klebsiella - all species except K. oxytoca (RG1) Legionella including L. pneumophila Leptospira interrogans - all serotypes Listeria Moraxella Mycobacterium (except those listed in Appendix B-III-A (RG3)) including M. avium complex, M. asiaticum, M. bovis BCG vaccine strain, M. chelonae, M. fortuitum, M. kansasii, M. leprae, M. malmoense, M. marinum, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi Mycoplasma, except M. mycoides and M. agalactiae which are restricted animal pathogens Neisseria gonorrhoeae, N. meningitidis Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. transvalensis Pseudomonas aeruginosa Rhodococcus equi Salmonella including S. arizonae, S. choleraesuis, S. enteritidis, S. gallinarum-pullorum, S. meleagridis, S. paratyphi, A, B, C, S. typhi, S. typhimurium Shigella including S. boydii, S. dysenteriae, type 1, S. flexneri, S. sonnei Sphaerophorus necrophorus Staphylococcus aureus Streptobacillus moniliformis Streptococcus including S. pneumoniae, S. pyogenes Treponema pallidum, T. carateum Vibrio cholerae, V. parahaemolyticus, V. vulnificus Yersinia enterocolitica Yersinia pestis specifically pgm( ) strains (lacking the 102 kb pigmentation locus) and lcr( ) strains (lacking the LCR plasmid) Risk Group 2 (RG2) - Fungal Agents Blastomyces dermatitidis Cladosporium bantianum, C. (Xylohypha) trichoides 36

37 Cryptococcus neoformans Dactylaria galopava (Ochroconis gallopavum) Epidermophyton Exophiala (Wangiella) dermatitidis Fonsecaea pedrosoi Microsporum Paracoccidioides braziliensis Penicillium marneffei Sporothrix schenckii Trichophyton Risk Group 2 (RG2) - Parasitic Agents Biological Safety and Biosecurity Manual Ancylostoma human hookworms including A. duodenale, A. ceylanicum Ascaris including Ascaris lumbricoides suum Babesia including B. divergens, B. microti Brugia filaria worms including B. malayi, B. timori Coccidia Cryptosporidium including C. parvum Cysticercus cellulosae (hydatid cyst, larva of T. solium) Echinococcus including E. granulosis, E. multilocularis, E. vogeli Entamoeba histolytica Enterobius Fasciola including F. gigantica, F. hepatica Giardia including G. lamblia Heterophyes Hymenolepis including H. diminuta, H. nana Isospora Leishmania including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana, L. peruviana, L. tropica Loa loa filaria worms Microsporidium Naegleria fowleri Necator human hookworms including N. americanus Onchocerca filaria worms including, O. volvulus Plasmodium including simian species, P. cynomolgi, P. falciparum, P. malariae, P. ovale, P. vivax Sarcocystis including S. sui hominis Schistosoma including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi Strongyloides including S. stercoralis Taenia solium Toxocara including T. canis Toxoplasma including T. gondii Trichinella spiralis Trypanosoma including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cruzi Wuchereria bancrofti filaria worms Risk Group 2 (RG2) - Viruses 37

38 Adenoviruses, human - all types Alphaviruses (Togaviruses) - Group A Arboviruses --Chikungunya vaccine strain 181/25 --Eastern equine encephalomyelitis virus --Venezuelan equine encephalomyelitis vaccine strains TC-83 and V Western equine encephalomyelitis virus Arenaviruses --Junin virus candid #1 vaccine strain --Lymphocytic choriomeningitis virus (non-neurotropic strains) --Tacaribe virus complex --Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV) Bunyaviruses --Bunyamwera virus --Rift Valley fever virus vaccine strain MP-12 --Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV) Caliciviruses Coronaviruses Flaviviruses - Group B Arboviruses --Dengue virus serotypes 1, 2, 3, and 4 --Japanese encephalitis virus strain SA Yellow fever virus vaccine strain 17D --Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV) Hepatitis A, B, C, D, and E viruses Herpesviruses - except Herpesvirus simiae (Monkey B virus) (see Appendix B-IV-D, Risk Group 4 (RG4) - Viral Agents) --Cytomegalovirus --Epstein Barr virus --Herpes simplex types 1 and 2 --Herpes zoster --Human herpesvirus types 6 and 7 Orthomyxoviruses --Influenza viruses types A, B, and C (except those listed in Appendix B-III-D, Risk Group 3 (RG3) - Viruses and Prions) --Tick-borne orthomyxoviruses Papilloma viruses --All human papilloma viruses Paramyxoviruses --Newcastle disease virus --Measles virus --Mumps virus --Parainfluenza viruses types 1, 2, 3, and 4 --Respiratory syncytial virus 38

39 Parvoviruses --Human parvovirus (B19) Picornaviruses --Coxsackie viruses types A and B --Echoviruses - all types --Polioviruses - all types, wild and attenuated --Rhinoviruses - all types Poxviruses - all types except Monkeypox virus (see Appendix B-III-D, Risk Group 3 (RG3) - Viruses and Prions) and restricted poxviruses including Alastrim, Smallpox, and Whitepox (see Section V-L, Footnotes and References of Sections I through IV) Reoviruses - all types including Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus) Rhabdoviruses --Rabies virus - all strains --Vesicular stomatitis virus non exotic strains: VSV-Indiana 1 serotype strains (e.g. Glasgow, Mudd- Summers, Orsay, San Juan) and VSV-New Jersey serotype strains (e.g. Ogden, Hazelhurst) Rubivirus (Togaviruses) --Rubella virus Risk Group 3 (RG3): Agents that are 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 3 is not currently allowed at University of New Hampshire. Risk Group 4 (RG4): Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk) Risk Group 4 is not allowed at University of New Hampshire. 39

40 Appendix 3: Risk Assessment Guidelines for the Principal Investigator A risk assessment takes into account various factors related to the agent in use, scale, anticipated handling practices and worker competence and health status. Use the list below to begin compiling your assessment of risk for your proposed biological work. AGENT FACTORS: Name Source Pathogenicity Virulence Infectious Dose Environmental stability Route of spread Communicability Availability of vaccine or treatment Recommended disinfectant Reports of Lab Acquired Illnesses PROTOCOL FACTORS: Scale Operations (e.g., centrifugation, aerosols, clinical diagnostic) Tissue culture Small animals Large animals Non-Human Primate RDNA GENE MODIFICATION FACTORS: Increased toxicity Broader host range Increased environmental stability Stability of genetic insert Physiological activity Allergenicity WORKER RELATED FACTORS: Experience with specific agent Expertise in specific protocols Training Health status 40

41 Appendix 4: Biosafety Level 1 and Biosafety Level 2 Requirements Biosafety containment level designation for the lab: Biosafety Level 1 (BSL-1): 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. Work is typically conducted on open bench tops using standard microbiological practices. Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist with training in microbiology or a related science. Biosafety Level 2 (BSL-2): for work involving agents that pose moderate hazards to personnel and the environment. Laboratory personnel have specific training in handling pathogenic agents and are supervised by scientists competent in handling infectious agents and associated procedures. Access to the laboratory is restricted when work is being conducted. All procedures in which infectious aerosols or splashes may be created are conducted in BSCs or other physical containment equipment. A. STANDARD MICROBIOLOGICAL PRACTICES Lab access limited/restricted when experiments or work with cultures/specimens are in progress. 2. Lab personnel wash hands after handling viable materials, removing gloves, or leaving lab. 3. No eating, drinking, smoking, handling contact lenses, applying cosmetics, or storing human food in lab. 4. Mechanical pipetting devices are used and no mouth pipetting is allowed Contact lens users wear safety glasses, goggles or face shields. 5. Sharps handling policies/practices in place. 6. Needles are not bent, sheared, broken, recapped, removed from syringes, or otherwise manipulated prior to disposal. 7. Sharps containers are labeled, conveniently located, and puncture resistant. 8. Non-disposable sharps containers are hard-walled and leak proof. 9. Broken glassware is only handled by mechanical means. 10. Plastic is substituted for glassware whenever possible. 11. Procedures minimize splashes/aerosols. 12. Work surfaces are decontaminated at least daily at completion of work and after any spill or splash. 13. Cultures/stocks/infectious material/wastes are decontaminated using an effective method (physical, chemical) before disposal. 14. Materials removed from the facility to be decontaminated are placed in durable, leak proof containers, are secured for transport, and are packed according to state, local and federal regulation. 15. A sign with the biohazard symbol is posted when infectious agents are present (sign lists emergency contacts & applicable warnings). 16. Insect/rodent control program in effect. 17. Lab supervisor has ensured personnel received appropriate training. B. SPECIAL PRACTICES Personnel entering the lab are advised of potential hazards and meet entry/exit requirements. 2. Medical surveillance and immunizations are provided as applicable for agents handled or present in the laboratory. 3. A lab specific biosafety operating procedure has been prepared and adopted as policy. The operating procedure is available and accessible. 4. Personnel at risk of acquiring infections or for whom infections may have serious consequences are denied access to lab. 5. Chemical disinfectants are appropriate for agents being used. 6. The lab supervisor ensures personnel are proficient in standard/special microbiological practices before working with BSL-2 agents. 7. Cultures, tissues, specimens, or infectious wastes are kept in covered, leak-proof containers during collection, handling, processing, storage, transport or shipment. 8. Lab equipment is routinely decontaminated and always after spills and splashes. 9. Spills involving infectious materials are contained, decontaminated and cleaned up by staff properly trained and equipped to work with these materials. 41

42 10. Equipment is decontaminated prior to repair, maintenance and removal from the laboratory. 11. Incidents that may result in exposure are reported, evaluated and treated as appropriate. 12. Animals not associated with work being performed are not permitted in the laboratory. 13. Plants not associated with work being performed are not permitted in the laboratory. 14. Procedures with infectious materials that may generate aerosols are conducted in a physical containment device (such as a BSC). C. SAFETY EQUIPMENT PPE (lab coats, gloves, safety eyewear) is appropriate for the work being done and potential hazards. 2. Lab coats designated for lab use are worn while working with hazardous materials and are removed prior to exiting for non-lab areas. 3. Soiled/used lab clothing is autoclaved before laundering. 4. Eye and face protection are used for anticipated splashes/sprays of hazardous materials when used outside of a containment device. 5. Contact lens wearers wear eye protection when handling hazardous materials. 6. Gloves must be worn to protect hands from exposure to hazardous materials. 7. Gloves are changed if contaminated, integrity is compromised, or when otherwise necessary. 8. Glove type is appropriate for the materials being handled. 9. Gloves are not worn outside of the laboratory. 10. Hands are washed after removal of gloves. 11. Disposable gloves are not washed or reused. 12. BSCs and other physical containment devices are properly maintained and certified for use. 13. BSCs are used for procedures with a potential to create infectious aerosols or splashes. 14. Centrifuge safety cups or sealed rotor heads are used with high concentrations or large volumes of infectious agents. 15. Eye, face and respiratory protection are used in rooms containing infected animals as determined by a risk assessment. D. LABORATORY FACILITIES Lab has doors for access control. 2. Lab doors are self-closing and have locks. 3. Lab has a sink for hand washing. 4. Lab is designed to be easily cleaned (no rugs or carpeting). 5. Lab furniture is capable of supporting anticipated loads and uses. 6. Spaces between benches, cabinets and equipment are accessible for cleaning.. 7. Bench tops are impervious to water and resistant to heat, solvents, corrosives and other chemicals. 8. Chairs are covered with non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant. 9. If lab has windows that open to the exterior, the windows are fitted with fly screens. 10. BSCs are installed so that fluctuations in air supply and exhaust do not interfere with proper operations. 11. Vacuum lines are protected with liquid disinfectant traps. 12. An eyewash station is readily available. 13. There is an inward flow of air to the lab without recirculation to spaces outside the laboratory (recommended, not required). 14. BSCs that recirculate air back into the laboratory are certified annually, or are exhausted via a thimble connection. 15. A method for decontamination of all lab wastes is available within the facility (autoclave, chemical disinfection, or other validated method). 42

43 Appendix 5: Common Disinfectants and Guidelines Practical Requirements Inactivation Efficacy Potential Disinfectants Category Quat. Ammon. Cpds Brand Effective Dilution Shelf Life C (dilute/open) Sink Disposal bactericidal virucidal fungicidal sporicidal tuberculocidal Application Skin disinfectant hard surfaces stainless steel liquid for disposal Characteristics % (1,000-50,000 ppm) Quatricide TB N/A (2100 ppm) Parvovircidal Quatricide PV 1:64 (750 ppm) Parvovircidal Quatricide 1:64 (780 ppm) d(dil/cl) Cetylcide II 1:64 (740 ppm) d(dil/op) Lysol I.C. spray N/A (1000 ppm) 2 years Lysol I.C. Quat 1:256 (660 ppm) 2 years Madacide-FD N/A (3080 ppm) Roccal 1:256 (920 ppm) Chlorine Cpds 50,000 ppm A + Clorox Germicidal bleach Clorox Germicidal bleach Leaves Residue Inactivated by organic matter 1:10 (5,000 ppm) 1 mo corrodes metal 1:5 (10,000 ppm) 1 mo corrodes metal Iodophor ppm A + Wescodyne 1:100 (125 ppm) years poor residual activity Betadine years poor residual activity Iodine years poor residual activity Phenolic Cpds % (10,000-50,000ppm) Sporicidin N/A (15,600 ppm) 6 mos Alcohols flammable 70 85% (700,000- Ethyl & Isopropyl 850,000 ppm) Envirocide N/A (202,000 ppm) 2 years Biodegradab le Opticide3 N/A (210,000 ppm) years lysol brand spray - houseold N/A (790,000 ppm) 2 years Aldehydes % (2,000-80,000 ppm) Cetylcide G 1:16 (34,000 ppm) Peroxide Cmpds Metricide Plus 30 N/A (34,000 ppm) (10h) Cidex OPA N/A (5,500 ppm) 2yr (14d) Cidex Plus 28 N/A (< 50,000 ppm) (28d) (32h) Hydrogen peroxide (L) 3-6% (30,000-60,000 ppm) (10h) (32h) (18h) Chlorhexidine Nolvasan 1:50 (400 ppm) 1 yr non-toxic Gas Ethylene Oxide 8 23 g/ft N A Paraformaldehyde 0.3 g/ft3 > N A Vapor-plasma Hydrogen Peroxide Sterrad Nx (90% H2O2) 58.0% to 59.5% C = closed and stored away from heat & light References: (reference The information on this disinfectant table was compiled from the individual product sheets and the references listed below. Block S. Disinfection, Sterilization, and Preservation, 4th Ed. Philadelphia: Lea and Febiger, 1991 McDonnell G. Antisepsis, Disinfection, and Sterilization: Types, Action and Resistance. Washington, DC: ASM Press, 2007 Biosafety in Microbiological and Biomedical Laboratories, Centers for Disease Control and Prevention/National Institutes of Health, 5th Ed. U.S. Department of Health and Human Services. Washington, DC: 2009 U.S. Department of Health, Education, and Welfare. NIH Laboratory Safety Monograph: A Supplement to the NIH Guidelines for Recombinant DNA Research. National Institutes of Health, Other 43

44 Appendix 6: Biohazardous Waste Flowchart 44