Appendix A: Project Registration And Approval

Appendix B: Standard Operating Procedures

Appendix C: Recommended Practices and Procedures

Appendix D: Training

Appendix E: Treatment, Handling and Disposal Of Biological Materials

Appendix F: Classification of Human Etiologic Agents on the Basis of Hazard

Appendix G: CDC Select Agents and Toxins

Appendix H: Plant Biosafety Level Criteria

This manual, in conjunction with the latest edition of the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories, outlines the requirements for safely working with biohazardous materials at the University of Wisconsin - Milwaukee. The Institutional Biosafety Committee (IBC) is responsible for the regulation of biohazardous materials on the UWM campus. Registration with the IBC is required for all instructional, research, and outreach projects involving potentially pathogenic microorganisms, etiologic agents of RG2 or higher, infectious agents, oncogenic viruses, human tissue and bloodborne pathogens, and in-vitro construction or propagation of recombinant DNA molecules. The IBC may be contacted through the Department of University Safety and Assurances, Biological Safety Program, at 229-4275.

The concern with all pathogens is the safety of personnel working with the biohazardous agent and the prevention of release of that agent. These are the primary concerns of the IBC. Traditionally, microorganisms have been classified as to the biological containment they require. Four levels of containment have been defined and termed Biosafety Levels (BSLs 1 4), thus an organism requiring, for example, biosafety level 2 containment is termed a BL2 or a Class 2 agent or organism. The containment requirements of the biosafety levels are found in CDC/NIH Biosafety in Microbiological and Biomedical Laboratories which is an integral component of this manual. NIH has introduced the concept of “risk groups” in which agents are classified into one of four risk groups based on their relative pathogenicity for healthy adult humans. In almost all instances, the risk group correlates with the recommended containment level. Thus, a Risk Group 2 organism is handled using BL 2 containment. Under some circumstances, the containment level required may be raised or lowered as a result of a comprehensive risk assessment. UWM has adopted the concept of risk groups in this manual. A discussion of risk groups is found in Chapter 1.

An integral component of the UWM Biosafety program is a group of generally accepted Standard Operating Procedures (SOPs). These standard operating procedures are included in Appendix B. Anyone working with a biohazardous agent or biohazardous material at any facility of UWM is expected to follow these standard operating procedures.

Many investigators may work with vertebrate animals and/or radioisotopes in conjunction with their work with some biohazardous agents. All vertebrate animal work conducted at UWM must be approved by the Institutional Animal Care and Use Committee (IACUC) which can be contacted through the campus Animal Care Program at 229-6016. All work with radioisotopes conducted at UWM must be authorized through the campus Radiation Safety Program which can be contacted at 229-4275.

Inevitably there are special situations that the existing program is not designed to accommodate. In those situations, consult with the Institutional Biosafety Committee or Biological Safety Officer before initiation of the work to prevent misunderstandings after work begins, whether the work is research, teaching or outreach.

The Principal Investigator involved in teaching, research, and/or outreach activities involving biohazardous materials has the primary ethical and legal responsibility to ensure the safety of students, faculty, staff, visitors and the environment. For that reason PIs are urged to carefully read this manual and understand its contents. If there are specific questions concerning this manual or the UWM Biological Safety Program please feel free to contact us at 229-4275.

The most recent editions of NIH Guidelines for Research Involving Recombinant DNA Materials and the CDC/NIH Biosafety in the Microbiological and Biomedical Laboratories provide the technical requirements for working with biohazardous materials at UWM. The remainder of this manual is UWM policy regarding working with biohazardous materials within university facilities.

The consideration of biological safety issues is of campus wide concern. With the passage of recent legislation regarding certain biological materials, the need for standard biological safety policies and over-site procedures becomes even more critical. A clear, consistent understanding of the organisms involved is an important first step in program organization. Traditionally, microorganisms have been classified according to the biological containment they require. Four levels of containment have been defined and termed Biosafety Levels (BLs 1 4); thus an organism requiring, for example, biosafety level 2 containment is termed a BL2 or a Class 2 agent or organism. More recently, the National Institutes of Health (NIH) has introduced the concept of “risk groups” in which agents are classified into four Risk Groups (RGs) based on their relative pathogenicity for healthy adult humans. The following table lists the four groups and the basis for classification.

For consistency, in this manual all organisms will be termed as “risk group” organisms. In almost all instances, the risk group correlates with the recommended containment level. Thus, a risk group 2 organism is handled using BL2 containment. Under some circumstances, the containment level required may be raised or lowered as a result of a comprehensive risk assessment as detailed in Section II A 3 of the NIH Guidelines for Research Involving Recombinant DNA Molecules. If you have any concerns regarding the risk group or appropriate level of containment for an organism consult the NIH Guidelines or call the campus Biological Safety Program.

1. Institutional Biosafety Committee

   The Institutional Biosafety Committee (IBC) is charged by the University Chancellor to formulate policy and procedures related to the use of biohazardous agents, including: human, animal, and plant pathogens, other infectious agents, toxins, and recombinant DNA (rDNA). As mandated by the NIH, experiments involving human gene therapy, formation of transgenic animals or plants, and the generation and/or use of rDNA must be registered and approved by the IBC. UWM also requires IBC registration and approval for use of Risk Group 2 or higher biohazardous agents.
   
   

   1. Organization Structure And Responsibilities

      The Chancellor, upon the recommendation of the Vice Chancellor for Research and Dean of the Graduate School, will appoint members to the IBC and designate one member to serve as chairperson. In order to provide the quality of input needed for in depth consideration of research activities presenting real or potential hazards, the membership shall be composed of Faculty, Laboratory Staff, Community Members, and Continuing Members. The specific composition and membership terms of the IBC shall be as follows:
      
      

      1. Faculty

         Five (5) faculty members shall be appointed for rotating three year terms. Faculty shall be selected on the basis of experience and expertise in infectious disease research, experience and expertise in rDNA technology, and the capability to assess the safety of biological research and to identify any potential risk to public health or the environment. Research academic staff with PI status are considered faculty for the purpose of this policy.

      2. Community Members Not Otherwise Affiliated with the University

         Two outside members who represent the interest of the surrounding community with respect to health and protection of the environment (e.g. officials of state or local public health or environmental protection agencies, members of other local governmental bodies, or persons active in medical, occupational health, or environmental concerns in the community) shall be appointed. These will be three (3) year membership appointments.

      3. Laboratory Staff

         One member representing laboratory research staff such as a research associate/research assistant, medical technologist, or laboratory technician shall be appointed. This will be a rotating 3 year membership.

      4. Continuing Members

         The following will be continuing committee members:
         
         

         • Asst. Director, University Safety and Assurances
         • Biological Safety Officer
         • Campus Veterinarian
         • Campus Medical Officer

      The IBC has the responsibility of assessing risks and potential environmental impacts associated with investigations involving biological agents and making recommendations for safe conduct of such studies. It also functions on behalf of the institution to ensure that the experimental work is performed in compliance with current policies and guidelines promulgated by government granting and regulatory agencies. The Committee does not monitor activities which are appropriately the concern of other established programs, e.g., Radiation Safety Program or Animal Care Program; however, it will closely interact with these groups in a concerted effort to minimize health risks to University personnel, students, and the general public.

      Registrations approved by the IBC will be active for 3 years from the date of approval. Written notification that the registration will expire will be sent out at least 30 days prior to the expiration date. The PI will then be required to submit a new registration application for review and approval by the IBC.

      The Committee shall meet at least once in the Fall and Spring Semesters and/or as necessary to conduct business. The Department of University Safety and Assurances shall provide staff support for the Institutional Biosafety Committee (IBC).

2. Definition Of Biohazardous Materials

   For the purposes of this policy, a biohazardous material is any biological material capable of causing harm to humans, animals or plants, including both biohazardous agents and non replicating materials such as toxins. In addition, this term may also be used to refer to material that harbors a biohazardous agent. A biohazardous agent is a pathogen capable of replication and is a disease causing microorganism (bacteria, chlamydia, fungi, parasites, prions, rickettsiae, viruses, etc.) capable of causing diseases in humans, animals, or plants. Toxic, mutagenic, and teratogenic chemicals are not considered biohazards for the purpose of this policy.

   Research involving experimentation and manipulation of various types of potentially infectious systems can be divided into four general categories. They are those that expose the worker or the environment to: (1) blood, bodily fluids, and organs/tissues of human origin; (2) infectious agents and their potentially infectious products; (3) recombinant DNA molecules and their products; and (4) miscellaneous biohazardous materials.
   
   

   1. Human Blood and Other Potentially Infectious Materials

      Experimentation or manipulations of human blood or other materials of human origin, including, but not limited to excreta, secreta, blood and its components, unfixed tissue, and tissue fluids, all of which may or may not contain an infectious agent, may place the worker at risk of exposure to bloodborne pathogens. Examples which could result in exposure would be clinical sciences laboratories performing tests or analyses on human blood or other potentially infectious materials or research labs performing experiments and/or manipulations with human blood or unfixed tissues or organs.

   2. Infectious Agents and Materials

      Infectious agents, pathogens or substances are defined as those substances containing viable microorganisms or their toxins which are known or suspected to cause disease in animals, plants or humans. Pathogens are classified as bacteria, fungi, rickettsiae, viruses, parasites, oncogenic viruses, and prions. Any materials which come in contact with infectious agents or their byproducts must be handled and disposed of in the same manner appropriate for disposing of the infectious agents themselves.

   3. Recombinant DNA Molecules and Products

      Research involving experiments with recombinant DNA materials includes, but is not limited to: commonly used host vector systems such as E. coli; recombinant DNA experiments using whole animals or plants; recombinant DNA or RNA experiments involving infectious animal or plant viruses; the production of transgenic animals; and the deliberate transfer of DNA or RNA into human subjects (requires human studies approval and approval from federal agencies, including NIH). Regardless of the cloning method utilized, precautions must be taken to assure that the systems neither cause disease in the operator nor release recombinant molecules into the environment.

   4. Miscellaneous Biohazardous Materials

      These include materials not directly covered by the above definitions, such as allergens, cultured animal cells and their potentially infectious agents, tissues from experimental animals (including animal dander), plant viruses, bacteria and fungi, toxins (bacterial, plant, etc.), and those as yet unnamed elements or agents which may produce disease. In regard to allergens, it is not the intent of the IBC to require the registration of research projects involving only allergens (i.e., projects involving allergens not listed as biohazardous agents or materials). Allergens are almost ubiquitous in the environment. However, it is important that PIs consider the hazard associated with allergens because manipulations of these allergens or materials containing allergens may result in human exposures to allergens at concentrations orders of magnitude greater than would normally occur. Because severe allergenic reactions may be life threatening, the control of allergens in any teaching, research or outreach project must be an important consideration in experimental/procedural design.

3. Review And Approval Of Biohazardous Studies

   The IBC can effectively carry out its designated functions only if it has adequate prior knowledge of potentially hazardous research projects. Therefore, all instructional, research, and outreach projects involving potentially pathogenic microorganisms; etiologic agents of RG2, RG3 and RG4; infectious agents; oncogenic viruses; human tissue and bloodborne pathogens; and in vitro construction or propagation of recombinant DNA molecules must be registered with and approved in writing by the Committee.

   It is not the purpose of the Committee to pass judgment on scientific merits or even to consider "risk" versus "expected benefits" of potentially hazardous research projects involving an organism of RG1 or RG2 to be undertaken on the University’s premises. Rather, it is the concern of this Committee to know whether or not the safety precautions proposed for the experimental work are adequate for protection of personnel and the environment. In general, the review process will focus on (1) qualifications of the investigator, (2) agents to be employed, (3) risks presented by experimental procedures, (4) adequacy of containment equipment and facilities, (5) training level of persons directly associated with the work, and (6) other factors relevant to safe conduct of the study.

   RG 3 agents, which normally require BL3 containment, pose a special risk to workers and the environment. Therefore, a more stringent evaluation procedure will be instituted by the IBC for its oversight of this type of work. At the present time, UWM has no facilities that would allow the use of RG3 agents. Until such time that BL3 facilities exist and have been inspected and certified for the containment of RG 3 agents at UWM, no such materials can be purchased, mailed, stored, used or disposed of in any campus laboratory or facility. It is the responsibility of a PI’s home Department or School/College to provide BL3 facilities.

   UWM does not allow RG 4 organisms or biohazardous materials requiring BL4 containment or facilities on the campus or at any of its outlying units or off campus locations. There are a limited number of approved and certified BL4 facilities within the U.S., such as those at the Centers for Disease Control and Prevention in Atlanta, GA and the U.S. Army Medical Research Institute into Infectious Diseases (USAMRID) in Fort Detrick, MD.

   The Principal Investigator and the Committee must concur on all matters relating to containment requirements, safe practices, and handling and disposal procedures for biohazardous agents. In event of non-concurrence, the recommendations of the Committee shall prevail until such time as they are modified or rescinded by appellate decision of an administrative review which may include outside reviewers. Questions relating to recombinant DNA studies that are not covered by the NIH Guidelines will be referred to the NIH Office of Recombinant DNA Activities for resolution.

   Some laboratories maintain cultures and/or archival samples of biohazardous agents. In these instances, a “generic” registration for maintenance and storage of reference or other samples/cultures shall be submitted. An inventory of agents should be attached and updated whenever significant changes are made, either additions or deletions. Significant changes include additional species or additional strains of inventoried agents that demonstrate a need for more stringent containment.

4. Biological Safety Officer

   The Biological Safety Officer (BSO) is primarily responsible for implementation and the day-to-day management of the biological safety program. Major duties or activities of the BSO include the following:

   1. Monitor compliance with University safety practices and procedures regarding potentially infectious and biohazardous materials.
   2. Assist in the preparation and periodic updating of a biosafety manual which is in accordance with University policy and consistent with government regulatory guidelines.
   3. Provide consultation to investigators on matters relating to laboratory safety, appropriate handling and containment of biohazardous agents, decontamination, and disposal of biohazardous wastes.
   4. Aid investigators in the development of appropriate emergency measures for dealing with accidental spills and contamination.
   5. Conduct surveillance of laboratories in which biohazardous agents are employed to ensure compliance with the approved protocol, prescribed safety guidelines, prescribed safety training and rectification of any deficiencies.
   6. Monitor all intra campus transport to ensure compliance with the rigorous containment procedures described herein. Provide information for off campus shipment of biohazardous materials.
   7. Develop and conduct training programs for laboratory personnel using biohazardous agents, specifically to promote techniques for the safe handling and disposal of biohazardous materials.
   8. Serve as liaison between the University and outside regulatory agencies concerned with the use of biohazardous agents.

   The Biological Safety Officer, with concurrence from the Chair of the IBC, or with concurrence of three (3) members of the IBC if the Chair is unavailable, may stop any work with microbial agents or any hazardous research project that creates an unreasonable hazard to personnel or involves experiments prohibited by the institution. The entire Committee then will review the problem and will complete the review within a working week then forwarding written recommendation(s) to the Vice Chancellor for Research and Dean of the Graduate School and the Provost for final action.

   In the event of a mechanical malfunction, systems breakdown or shutdown of any nature, or preventive maintenance of primary containment equipment or components, the Biological Safety Officer must be notified immediately. In the case of an unplanned event and if Physical Plant mechanical staff is not already on the scene, the Biological Safety Officer will notify appropriate Physical Plant staff. Proper precautions must be taken immediately. All experiments must be halted and the biological agents secured (e.g., containers sealed or containers placed in freezer or refrigerator). The area must be cordoned off during the entire time of the shut down. No further activities will be allowed until the Biological Safety Officer certifies that the facility is safe to use.

5. Principal Investigators

   The PI is expected to know and understand all relevant safety guidelines described in this manual, to personally monitor day to day operation of the laboratory, and to take all necessary steps for protection of staff, students, and the general public against undesirable consequences of experimental work conducted within UWM facilities. Ultimate responsibility for the safe conduct of research involving biohazardous agents rests with the PI. The PI is aware of the complexities of the research and its associated hazard and must adequately inform all who are working in the research laboratory or otherwise involved in the research of the hazards involved. Because working with biohazardous agents may pose special risks, the PI must assure compliance with the biohazardous materials standard policies and procedures.

   Additional duties of the investigator are as follows:
   
   

   1. File a registration form with and obtain written approval of the IBC before performing work involving the use of oncogenic viruses, infectious agents, recombinant DNA or experiments involving the use of human blood, bloodborne pathogens or other potentially infectious materials such as unfixed human tissues, primary human cell lines, and certain bodily fluids. The document submitted for this purpose should describe: (1) the agents and experiments to be performed; (2) the level of containment judged necessary for the work; (3) the containment facilities available; and (4) the training level of relevant laboratory personnel.

   2. Thoroughly inform and train all persons (including research personnel such as postdoctoral fellows, graduate students, undergraduate students, technicians and laboratory researchers) directly involved in potentially hazardous experiments, e.g., infectious agents and harmful chemicals, of the potential health risk presented and the safety procedures necessary to minimize exposure. Training requirements are outlined later in this manual. Each laboratory must maintain a current inventory book of biological agents, maintain a current record of personnel training, post safety standards for the required biosafety level and maintain safety information for each human pathogen in inventory.
   3. Establish emergency procedures to be followed in the event of an overt spill or contamination with potentially hazardous biological material. These procedures should be posted in a prominent place in the laboratory. Moreover, it is recommended that a responsible member of the laboratory staff be designated to handle emergency situations whenever the PI is absent from the premises. Chapter 4 of this manual provides guidelines for spill clean up.
   4. Immediately report any unusual incident, such as spill, break in containment, or overt contamination to the Biosafety Program at 229-4275.
   5. Post working areas and facilities with biohazard warning signs. Standardized signs will be provided by University Safety. The PI should consult the Biosafety Program if assistance is required in placement of signs.
   6. All research personnel including postdoctoral fellows, graduate students and undergraduate students under the direction of the PI are expected to participate in the safety training and to use safety procedures established by the PI.
   7. Cooperate with the Biosafety Officer during inspection visits.
6. Laboratory Personnel

   1. Laboratory personnel including all staff, faculty and students, shall perform all tasks using established safety practices and shall comply with the safety guidelines for the work being performed.

   2. Laboratory personnel are responsible for keeping themselves informed of the risks in the laboratory and to ask for and participate in training that increases their knowledge and ability to deal safely with the risks.
   3. Laboratory personnel shall report all unsafe practices to the PI.
   4. Laboratory personnel shall report all accidents and injuries to the PI.
7. Biosafety In Microbiological And Biomedical Laboratories

   The latest edition of the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories(BMBL), is considered an integral component of the UWM Biological Safety Manual. All descriptions of containment and handling of specific agents contained in the BMBL have been adopted as UWM containment and handling requirements. Copies of these documents are available by contacting the Biological Safety Program at 229-4275 or on line at the link shown above.

1. Categories Of Biohazards Or Potentially Infectious Materials
   
   
   1. Human, animal and plant pathogens:
      
      

      • Bacteria, including those with drug resistance plasmids

      • Rickettsiae
      • Fungi
      • Viruses, including oncogenic viruses and viroids
      • Parasites
      • Prions
   2. All human and/or non-human primate blood, blood products, tissues and certain body fluids. Researchers contemplating use of human blood, blood products, tissues and body fluids should also contact the Institutional Review Board at 229-3173.
   3. Cultured cells (all human and certain animal species) and potentially infectious agents these cells may contain.
   4. Biological toxins (bacterial, fungal, botanical, etc.)
   5. Certain recombinant products
   6. Infected animals and animal tissues

   A list of biological agents classified according to risk may be found in Appendix F.

2. Recombinant DNA (rDNA)

   All projects involving rDNA must comply with the NIH Guidelines (described below). Contact the Biosafety Program at 229-4275 or the NIH web site for a current copy of the NIH guidelines.

   1. Generation of rDNA

      UWM requires that all experiments involving the generation and/or use of rDNA require IBC registration, EVEN IF IT IS EXEMPT from the NIH Guidelines. The National Institutes of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules is the definitive reference for rDNA research in the United States. There may be rDNA experiments which are not covered by the guidelines that require review and approval by outside agencies before initiation or funding. These experiments are most common in the environmental sciences. If you have specific questions about a particular host vector system not covered by the guidelines, please call the Office of Recombinant DNA Activities (ORDA), National Institutes of Health at (301) 496 9838 or FAX (301) 496 9839.

   2. Transgenic Plants

      Experiments to genetically engineer plants by rDNA methods require IBC registration. The NIH rDNA guidelines provide specific plant biosafety containment recommendations for experiments involving the creation and/or use of genetically engineered plants.

      There are four plant biosafety levels, designated Plant Biosafety Level 1 through 4 (BL1 P through BL4 P), for work with plant diseases in whole plants or transgenic plants. The levels are combinations of practices, safety equipment and facilities for experiments on plants infected with agents which produce or may produce disease in cultivated crops or transgenic plants. In general, the biosafety level recommended for working with an infectious agent in vivo and in vitro are comparable. At this time, UWM policy does not allow work requiring BL3 P or BL4 P containment.

      The use of any campus greenhouse facility for research with transgenic plants must be approved by the IBC and greenhouse management personnel. Plant Biosafety Levels are discussed in Appendix H.

3. Other Potentially Hazardous Biological Materials

   All animal protocols involving the use of rDNA; infectious or transmissible agents; human blood, body fluids or tissues; or toxins must be submitted to the IBC for registration and approval prior to final approval by the Institutional Animal Care and Use Committee (IACUC). The PI must notify the Animal Care Program coordinator in writing prior to initiation of experimentation at Animal Biosafety Level 2. IACUC guidelines are available from the Animal Care Program at 229-6016. Investigators who are uncertain how to categorize agents should contact the Biosafety Program at 229-4275.

The University of Wisconsin - Milwaukee adheres to the NIH Guidelines for Research Involving Recombinant DNA Molecules with regard to all uses of recombinant DNA at the University. Experiments involving recombinant DNA are divided into categories denoted as classes. Presently there are 6 classes (III-A, III-B, III-C, III-D, III-E and III-F). It is the responsibility of the PI to read the NIH Guidelines in order to determine which class is applicable to the work conducted. UWM requires that all recombinant DNA work done at the University be registered with the Institutional Biosafety Committee (IBC) EVEN IF IT IS EXEMPT from the NIH Guidelines.

It is the policy of the University that the PI is responsible for complying with the NIH Guidelines for Research involving Recombinant DNA Molecules, regardless of the source of funds supporting that research. It is not possible to summarize the whole of those guidelines, but there are three groups of experiments that probably encompass the majority of work being done on campus.

1. Exempt Experiments

   To register exempt rDNA research complete “Registration Cover Sheet”, noting the reason the research is exempt and a “Recombinant DNA Registration Form for Exempt Research” and send to the BSO. The IBC via subcommittee review will certify the project as exempt. All such research must be conducted using Biosafety Level 1 (BL-1) Practices. This group includes, but is not limited to, experiments that involve:

   • Work that is not in an organism, e.g., sequencing of DNA and polymerase chain reaction.

   • DNA propagated solely in the same species (this exemption can not be used if elements of the construct such as promoters, enhancers and marker traits are derived from another organism.
   • Gene transfer between species known to exchange DNA by known physiological means.
   • rDNA molecules containing less than ½ of any eukaryotic virus genome that are propagated and maintained in cells in tissue culture.
   • Host vector systems using E. Coli K-12, Saccharomyces cerevisiae, Saccharomyces uvarum, Bacilllus subtilis, Neuospora crassa, or Psuedomonas putida and their plasmids
   • Work that does not present a risk, as determined by the NIH Director. It is not sufficient for an investigator to declare that the research does not present a risk to humans, animals, or the environment. This decision is made through special correspondence with the NIH Director.
2. Experiments Requiring IBC Notice Simultaneous with Initiation

   Some recombinant DNA work requires IBC review and approval simultaneous with initiation (prior approval is not required). To register non-exempt rDNA research complete “Registration Cover Sheet”, and a “Recombinant DNA Registration Form for Non-Exempt Research” and send to the BSO. Examples include:

   • Recombinant DNA molecules containing no more than 2/3 of the genome of any eukaryotic virus (with some restrictions) propagated and maintained in cells in tissue culture. It must be demonstrated that the cells lack helper virus for the specific families of defective viruses being used.

   • Many, but not all, experiments involving whole plants.
3. Experiments Requiring Prior Approval

   The following experiments require approval from either the NIH, Recombinant DNA Advisory Committee (RAC), Food and Drug Administration (FDA) and/or the IBC. To register these projects complete “Registration Cover Sheet”, and a “Recombinant DNA Registration Form for Non-Exempt Research” and send to the BSO with the applicable supporting documentation.

   • Gene transfer experiments in humans

   • Genes for toxins lethal for vertebrates
   • Release of genetically engineered organisms to the environment
   • Those using human or animal pathogens (Biosafety level 2 or higher) as host-vector systems, including adenovirus vectors and murine retroviruses that infect human cells
   • Cloning DNA from human or animal pathogens (Biosafety level 2 or higher) into non-pathogen host vector system
   • Cultures involving more than 10 liters
   • Experiments involving whole plants¹ or animals, including transgenic organisms

   ¹ Some plant experiments do not require prior approval. Work with recombinant DNA in plants or any work with plant pathogens must also comply with USDA and EPA

1. Administrative Controls
   
   
   1. Biohazard Warning Signs and Posting

      Each laboratory must clearly display a sign that provides safety information to visitors and service personnel. Signs must contain designations for all laboratory hazards in use with the laboratory (carcinogens, acutely toxic agents, reproductive hazards, biohazards, radioactive materials, lasers, etc). Contact University Safety and Assurances for more information.
      
      

      1. All areas and laboratories which contain biohazardous agents must be posted with a biohazard sign. The sign must be red/orange in color with a biohazard symbol and lettering in black.

         
      2. All areas and laboratories which contain biohazardous or toxic agents must clearly display signs stating "EATING, DRINKING, SMOKING PROHIBITED IN THIS AREA."

      There are no Biosafety Level 3, Biosafety Level 4, Animal Biosafety Level 3, Animal Biosafety Level 4, Plant Biosafety Level 3 or Plant Biosafety Level 4 facilities on the UWM campus. UWM policy prohibits use of Risk Group 3 or Risk Group 4 organisms on campus or at any off campus locations.

   2. Infectious and Biological Waste Management

      All disposal of infectious waste, autoclave bags, pipettes, sharps and biological waste must be performed in accordance with Wisconsin Department of Natural Resources (WDNR) medical waste regulations. UWM has developed procedures for the proper disposal of biological waste materials. Specific information on the disposal of biological materials and biological waste management is found in Appendix E.
      
      

      1. Mixed Waste

         Mixed wastes are potentially infectious waste contaminated with other types of waste, e.g., radioisotopes or toxic/carcinogenic compounds. Because of the difficulty in disposal of wastes regulated by more than one set of requirements and regulatory agencies, it is critical that provision be made for proper management prior to the initiation of any research that might result in mixed waste. Mixed wastes may require special containers, labeling, storage, etc. Contact University Safety prior to initiation of any research that might result in potentially infectious waste with multiple hazards.

      2. Animals

         All methods of disposal for research animals and animal parts that are considered to be infectious waste must be approved by the campus Animal Care Program (229-4275).

      3. Microorganism Cultures

         All cultures of microorganisms should be inactivated, using appropriate procedures, before disposal. This includes cultures of Risk Group 1 organisms. This is considered good laboratory practice.

   3. Biohazard Spill Clean Up Procedures

      The following procedures are provided as a guideline to biohazardous spill cleanup. Additional information regarding an emergency plan is in Appendix F of CDC/NIH Biosafety in the Microbiological and Biomedical Laboratories.

      1. Inside the Biosafety Cabinet (BSC):
         
         

         • Wear lab coat, safety goggles and gloves during cleanup.

         • Allow biosafety cabinet to run during spill cleanup.
         • Apply disinfectant and allow a minimum of 20 minutes contact time.
         • Wipe up spillage with disposable disinfectant soaked cloth.
         • Wipe the walls, work surface and any equipment in the cabinet with a disinfectant soaked cloth.
         • Discard contaminated disposable materials in an appropriate biohazardous waste container(s) and autoclave before discarding.
         • Place contaminated reusable items in autoclave bags, autoclavable pans with lids or wrap in newspaper before autoclaving and cleanup.
         • Expose non autoclavable materials to disinfectant, 20 minute contact time, before removal from the BSC.
         • Remove protective clothing used during cleanup and place in an autoclave bag for autoclaving.
         • Run cabinet 10 minutes after cleanup before resuming work or turning cabinet off.
      2. In the Lab, Outside of Biosafety Cabinet (BSC):

         • Clear area of all personnel. Wait for aerosol to settle before entering spill area. The time required will depend on ventilation within the area but a general rule of thumb is to wait approximately 30 minutes.

         • Remove any contaminated clothing and place in autoclave bag to be autoclaved.
         • Don a disposable gown, safety goggles and gloves.
         • Initiate cleanup with disinfectant as follows:

           1. Soak paper towels in disinfectant and place over spill.

           2. Encircle the spill with additional disinfectant, being careful to minimize aerosolization while assuming adequate contact.
           3. Decontaminate all items within spill area.
           4. Allow 20 minutes contact time to ensure germicidal action of disinfectant.
           5. Wipe equipment with 1:10 bleach followed by water then 70% alcohol.
           6. Place disposable contaminated spill materials in appropriate biohazardous waste container(s) for autoclaving.
           7. Place contaminated reusable items in autoclave bags, autoclavable pans with lids or wrap in newspaper before autoclaving and cleanup.
      3. Inside Centrifuge

         If a centrifuge tube breaks while the centrifuge is running, turn off motor. Allow the machine to be at rest for 30 minutes before opening. Or, if breakage is discovered after the machine has stopped, re close the lid immediately and allow the unit to be at rest for 30 minutes.

         • Unplug centrifuge before initiating clean up.

         • Don strong, thick rubber gloves and other personal protective equipment (PPE) before proceeding with clean up.
         • Flood centrifuge bowl with a germicidal disinfectant. Place paper towels soaked in a disinfectant over the entire spill area. Allow 20 minute contact time.
         • Use mechanical means (such as forceps) to remove broken tubes and glass fragments. Place them in a sharps container for autoclaving and disposal as infectious waste.
         • Remove buckets, trunnions and rotor and place in disinfectant for 24 hours or autoclave. Place paper towels soaked in a disinfectant over the equipment if transport is necessary.
         • Unbroken, capped tubes may be placed in disinfectant and recovered after 20 minute contact time or autoclaved.
         • Use mechanical means to remove remaining disinfectant soaked materials from centrifuge bowl and discard as infectious waste.
         • Place paper towels soaked in a disinfectant in the centrifuge bowl and allow it to soak overnight, wipe down again with disinfectant, wash with water and dry. Discard disinfectant soaked materials as infectious waste.
         • Remove protective clothing used during cleanup and place in a biohazard bag for autoclaving. Wash hands whenever gloves are removed.
      4. Outside Lab, During Transport

         • Transport biohazardous material in an unbreakable well sealed primary container placed inside of a second unbreakable lidded container labeled with the biohazard symbol (cooler, plastic pan or pail).

         • Should a spill occur in a public area, do not attempt to clean it up without appropriate personal protective equipment.
         • As an interim measure, wear gloves and place paper towels, preferably soaked in disinfectant, directly on spilled materials to prevent spread of contamination. To assure adequate contact, surround the spill with disinfectant, if available, taking care to minimize aerosols.
         • Call University Safety at 229-6339 to assist in cleanup. Call 9-911 outside normal working hours and request that the police contact a member of the University Safety staff.
2. Recommended Practices And Procedures

   UWM has developed general practices and procedures for work in a laboratory containing biohazardous agents. These are included in Appendix C, “Recommended Practices and Procedures”.

ALL outgoing shipments of hazardous materials (biological, chemical, radioisotopes) must be coordinated through University Safety and Assurances. All shippers of hazardous materials must have documented, DOT mandated training. There are significant fines associated with improper packaging and shipping of hazardous materials. Please contact University Safety at 229-6339 for further information.

1. Packaging And Shipping Of Biological Agents

   Regulations on the transportation of biological agents are aimed at ensuring that the public and the workers in the transportation chain are protected from exposure to any agent that might be in the package. Protection is achieved through (a) the requirements for rigorous packaging that will withstand rough handling and contain all liquid material within the package without leakage to the outside, (b) appropriate labeling of the package with the biohazard symbol and other labels to alert the workers in the transportation chain to the hazardous contents of the package, (c) documentation of the hazardous contents of the package should such information be necessary in an emergency situation, and d) training of workers in the transportation chain to familiarize them with the hazardous contents so as to be able to respond to emergency situations.

   Current resources regarding the packaging and shipment of biological agents are found in Appendix C of the CDC/NIH Biosafety in the Microbiological and Biomedical Laboratories.

   http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4ac.htm

2. Transportation In Between Buildings Or Locations On Campus

   Infectious materials and recombinant DNA molecules need to be packaged in a sealed, leakproof primary container (e.g., glass tube), which is securely positioned in a secondary leakproof and closable container (e.g., cooler, ice chest) containing a clearly visible biohazard symbol on the outside. Please contact University Safety regarding the transport of infectious agents and recombinant DNA molecules over public highways utilizing a state of Wisconsin vehicle. The use of private cars for the transportation of such materials on or off campus is prohibited.

3. Importation/Exportation Of Biological And Infectious Agents

   Receiving and sending animals, animal-derived materials, infectious or biohazardous agents, biological toxins, and genetically modified organisms require the approval of federal agencies such as the Centers for Disease Control and Prevention (CDC), the US Department of Agriculture (USDA), or the US Fish and Wildlife Service (USFWS). Regulations that govern the transfer of biological materials help to minimize or eliminate the possible threats to public health and agriculture.

   1. Importation of Infectious Agents

      For agents infectious to humans, CDC permit applications are found online at: http://www.cdc.gov/od/ohs. These agents include any infectious agent known or suspected to cause disease in humans, unsterilized specimens of human or animal tissues (including blood and other fluids), or vectors including infectious animals, bats, insects, arthropods and snails.

   2. Importation of Plant/Agricultural Pests

      A USDA/APHIS permit is required to import or domestically transfer a plant pest, plant biological agent, or any material that might contain them. Some items that are included are bees, biological control organisms, butterflies and moths, genetically engineered plants and microorganisms, certain fruits and vegetables, noxious weeds, snails and slugs, soil and wood products. Consult the following web page for more information and permit applications: http://www.aphis.usda.gov/ppq/permits

   3. Export Guidelines for Infectious Agents of Humans, Animals, Plants and Related Materials

      The export of infectious agents and related materials is governed by the following federal regulation: 15 CFR Parts 730 - 799. An export license is required from the Department of Commerce, when exporting infectious agents of human, plant, and animal diseases, including genetic material, and products which might be used for culture of large amounts of agents. Consult the following web page for specific items and procedures: http://www.bis.doc.gov/index.htm

4. CDC “select Agents”

   The US Department of Health and Human Services (HHS) and the US Department of Agriculture (USDA) have developed a list of select biological agents and toxins that have the potential to pose a severe biosecurity threat to public health, animals, and agricultural crops. A list of those materials currently classified as a “select agent” is provided in Appendix G. As directed by the US Patriot Act, HHS and USDA have adopted strict regulations for the obtaining, possession, use, or transfer of any of these selected agents. Failure to comply with the established regulations can result in significant civil and criminal penalties. Therefore, any plans for obtaining such materials must be discussed with the University Biosafety Officer and approved by the Biosafety Committee well in advance of any planned use. Also anyone contemplating select agent use should understand that a decision to do so means accepting a significant level of personal responsibility for meeting all aspects of the requirements mandated by the federal rules.

   HHS regulations in 42 CFR Part 73 Possession, Use, and Transfer of Select Agents and Toxins and the companion USDA regulations in 9 CFR Part 121 require federal registration and inspection; restricted lab access; development of written and strictly followed safety and security plans; personnel background checks (including fingerprinting) and training; accurate records and/or reporting of agent use, transfer, loss, or destruction.

   Because this is a newly established regulatory program and at the time of this writing there are no select agents on campus, any plans for use of select agents could easily take several months to get the appropriate permits and approvals and establish the security and protocols necessary to comply with the regulations. The source of research funds to cover the costs of facility security improvements necessary for compliance would also have to be identified.

5. Forbidden Agents

   UWM policy prohibits the use or possession of Risk Group 3 or Risk Group 4 organisms on campus or at any off campus locations. The only exclusion to this policy is reference material necessary for identification purposes. Reference material must be labeled as such and stored in an inactive state, either frozen or lyophilized. All freezers and storage cabinets must be clearly labeled with signs that include the biohazard symbol, the name of the organism, and emergency information (e.g., PI phone number). A list of all such viable reference material must be provided to the IBC.

University Researchers are required to obtain Institutional Biosafety Committee (IBC) review and approval for work involving:

• Recombinant DNA (including gene therapy, clinical trials and the creation of transgenic animals) - All work must be registered with the IBC. The Biosafety Officer (BSO) will process for subcommittee review (exempt work) or place on the agenda for full committee review (non-exempt work). Submit: Recombinant DNA Cover Sheet and Registration Form (either exempt or non-exempt)
• Infectious Biological Agents (organisms in Risk Group 2 or above - see Appendix F of Biological Safety Policy manual.) This includes a broad spectrum of organisms able to cause human disease. Submit: Biological Agent Registration Form
• Biologically Derived Toxins - Examples include endotoxins, staphlococcus, entertoxins, saxitoxin, cholera toxin, botulinum toxin, etc. Submit: Biological Toxin Registration Form

  To initiate the review process, University researchers must submit an IBC registration form to the Biological Safety Officer. Obtain the appropriate form by contacting Sharron Daly at (414) 229-4275 or by consulting the University Safety and Assurances web site: http://www.uwm.edu/Dept/EHSRM/BIO/index.html Project protocols which include the following items must accompany the completed form:

  1. Identification of potential hazards (such as aerosol generation, use of sharps, excretion of animals, etc.)

  2. Staff training and investigator experience.
  3. Safety procedures that will be employed to minimize risks (such as protective clothing, use if Biological Safety Cabinets, sharps disposal, etc.)
  4. Accidental spill and/or exposure procedures specific to the project.

  Send the documents to the BSO, who will process the form for subcommittee review¹ or place it on the agenda for full review by the IBC. The IBC meets semiannually² to review projects. The principal investigator will be invited to attend the IBC meeting to summarize the project and answer any questions the IBC may have. The Committee may request modification of the procedures or request the BSO to inspect labs or other facilities before granting approval. A copy of the approved form will be sent to the principal investigator.

  ¹Subcommittee review will be completed for projects determined to be “exempt” from the NIH guidelines.

  ² The IBC will not hold a meeting during any semester in which there are no projects that require full review.

Personal Protective Equipment (PPE)

Personal protective equipment such as gloves, safety glasses and a laboratory coat must be worn whenever biological work is conducted in the laboratory.

Handwashing

Hands must be washed immediately or as soon as feasible after removing gloves or other personal protective clothing.

Use of Sharps

Minimize the use and exposure to sharps in the workplace. Never recap, bend, or shear needles. When possible, replace glassware with less damaging materials such as plastic. Keep sharps containers readily available in all locations where sharps waste may be generated.

Clean Areas

Eating, drinking, smoking, applying cosmetics or lip balm, and handling contact lenses in the laboratory are prohibited in work areas where there is a reasonable likelihood of occupational exposure.

Aerosol Generation

Any procedures that could potentially generate aerosols or other inhalation hazards must be performed in a manner that will minimize airborne pathogen transmission.

Proper Labeling

Place a color coded label incorporating the universal biohazard label on the work surface of any potentially contaminated equipment or work surface to warn others of biohazard contamination which may not be easily visible. This includes freezers, refrigerators, and incubators.

Autoclave Safety

Always wear heat resistant gloves, goggles or safety glasses, and a laboratory coat when opening an autoclave. Be sure to allow the superheated steam to exit before attempting to remove the contents.

Spills

Always clean spills from the periphery of the spill towards the center. All cleaning materials must be disposed of in an appropriate manner.

Mouth Pipetting

Mouth pipetting may lead to accidental ingestion of biological specimens and is strictly prohibited.

Decontamination Procedures

A 0.5% sodium hypochlorite (a freshly prepared 1:10 dilution of household bleach) must be used to decontaminate equipment and work surfaces. In locations where bleach would cause corrosion, an iodophor (e.g., Wescodyne) must be used to decontaminate.

Local Transport of Infectious Materials

All infectious materials transported to and from the laboratory must be enclosed in a primary container with sealed lid or top, which must then be enclosed in a secondary leak proof, non breakable container (e.g., Coleman cooler) appropriately labeled with the biohazard symbol. Any specimens transported to and from off campus satellite facilities must be handled by a trained laboratory worker.

Storage

All infectious materials to be stored must be clearly labeled with the universal biohazard symbol. The storage space (e.g., freezer, refrigerator) must also be similarly labeled.

Bloodborne Pathogens

All PI's using human or non human primate blood or blood products, unfixed tissue, body fluids or organ or cell cultures of human or non human primate origin, must follow the procedures outlined in the UWM Bloodborne Pathogen Plan.

Human Organ and Cell Culture

All PI's using human organ or cell cultures (primary cultures, cell strains, cell lines), must handle all such cultures under BL2 conditions and in accordance with the Bloodborne Pathogen Standard unless the IBC has specifically approved a lower standard of containment.

Waste Disposal

All biologically infectious waste must be autoclaved or otherwise treated before disposal.

Biohazardous spills

• For spills that staff are able to clean up safely, a person wearing protective equipment (gloves, goggles, long sleeved lab coat) must first disinfect the area before wiping up the spill with disposable paper towels and disposing of all spill materials properly. Broken glass should be handled only by remote means such as tongs or forceps.

• For spills that staff may not be able to clean up safely, the room must be evacuated and personnel must be prevented from entering the area. The lab director or supervisor and University Safety (229-6339) must be contacted immediately. After 5:00 p.m., dial 9-911. The UWM Police will then contact a member of the University Safety staff.

Injuries

All injuries and accidental autoinoculation, ingestion or inhalations of infectious agents must be reported immediately to the lab director or supervisor and University Safety (229-6339). Affected employees should be sent to their primary care physician for evaluation, possible treatment and/or possible referral. Dial 9-911 immediately for any medical emergency.

Shipments

All domestic and international shipments of biological agents, infectious substances and diagnostic specimens must follow University policy and all applicable Federal and International regulations. Proper permits/licenses must be obtained as required before importing or exporting biological material.

Other Emergencies

In case of natural disasters, fires, or power failure the following precautions must be taken:

• In power failures, immediately discontinue all work until power is restored. If a tissue culture hood is being used, then all open containers must be closed, gas turned off and hood sash closed.

• In case of fire, personnel must immediately follow standard emergency procedures (evacuate area, call 9-911). Temperatures sufficient to ignite materials will inactivate infectious agents used in the laboratory. However, emergency personnel should don personal protective equipment before entering the lab and follow disinfecting procedures described above for decontaminating any released infectious materials not involved in the fire.
• In natural disasters, personnel must immediately follow standard emergency procedures (take shelter, wait for further instructions). Upon return to facility, personal protective equipment (PPE) must be used when entering a lab to decontaminate any disaster related release of infectious material. Contain released material using spill procedures. Emergency personnel should don personal protective equipment before entering lab and/or areas housing infected animals.

1. Engineering Controls

   1. Biological Safety Cabinets (BSCs)

      Biological safety cabinets are designed to contain aerosols generated during work with infectious material through the use of laminar air flow and high efficiency particulate air (HEPA) filtration. Consult CDC/NIH Biosafety in the Microbiological and Biomedical Laboratories for a discussion of the types and uses of biological safety cabinets.

   2. Safety Equipment

      Safety equipment includes items for personal protection such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses or goggles. Personal protective equipment (PPE) is often used in combination with biological safety cabinets and other devices which contain the biohazardous agents, animals or materials. When it is impractical to work in biological safety cabinets, personal protective equipment may form the primary barrier between personnel and infectious include certain animal studies, animal necropsy, agent production activities and activities relating to maintenance, service or support of the laboratory facility.

      Other safety equipment such as safety centrifuge cups and safety blenders are enclosed containers designed to prevent aerosols from being released during centrifugation or homogenization of infectious material.

      Containment controls such as biological safety cabinets, safety centrifuge cups and blenders must be used transmitted through the aerosol route of exposure. A description of the effective use of biological safety cabinets and information on other safety equipment may be found in the “Recommended Work Practices” section of this appendix.

      For more information on proper use and selection of a biological safety cabinet or other safety equipment contact the Biosafety Program at 229-4275.

2. Recommended Work Practices
   
   
   1. Pipettes and Pipetting Aids

      Pipettes are used for volumetric measurements and transfer of fluids that may contain infectious, toxic, corrosive or radioactive agents. Laboratory associated infections have occurred from oral aspiration of infectious materials, mouth transfer via a contaminated finger and inhalation of aerosols. Exposures to aerosols may occur when liquid from a pipette is dropped onto the work surface, when cultures are mixed by pipetting or when the last drop of an inoculum is blown out. A pipette may become a hazardous piece of equipment if improperly used. The safe pipetting techniques outlined below are required to minimize the potential for exposure to hazardous materials.

      1. Never mouth pipette. Always use a pipetting aid.

      2. If working with biohazardous or toxic fluid, confine pipetting operations to a biosafety cabinet.
      3. Always use cotton plugged pipettes when pipetting biohazardous or toxic materials, even when safety pipetting aids are used.
      4. Do not prepare biohazardous materials by bubbling expiratory air through a liquid with a pipette.
      5. Do not forcibly expel biohazardous material out of a pipette.
      6. Never mix biohazardous or toxic material by suction and expulsion through a pipette.
      7. When pipetting, avoid accidental release of infectious droplets. Place a disinfectant soaked towel on the work surface and autoclave the towel after use.
      8. Use "to deliver" pipettes rather than those requiring "blowout."
      9. Do not discharge material from a pipette at a height. Whenever possible allow the discharge to run down the container wall.
      10. Place contaminated, reusable pipettes horizontally in a pan containing enough liquid disinfectant to completely cover them. Do not place pipettes vertically into a cylinder.
      11. Discard contaminated disposable pipettes in an appropriate sharps container. Contact the campus Environmental Affairs Program at 229-4999 for more information on the disposal of pipettes or sharps.
      12. Pans or sharps containers for contaminated pipettes should be placed inside the biosafety cabinet, if possible.
   2. Syringes and Needles

      Syringes and hypodermic needles are dangerous instruments. The use of needles and syringes should be restricted to procedures for which there is no alternative. Blunt cannulas should be used as alternatives to needles wherever possible (i.e., procedures such as oral or intranasal animal inoculations). Needles and syringes should never be used as a substitute for pipettes. When needles and syringes must be used, the following procedures are recommended:

      1. Use disposable needle locking syringe units whenever possible.

      2. When using syringes and needles with biohazardous or potentially infectious agents:

         • Work in a biosafety cabinet whenever possible.

         • Wear gloves.
         • Fill the syringe carefully to minimize air bubbles.
         • Expel air, liquid and bubbles from the syringe vertically into a cotton pledget moistened with disinfectant.
         • Do not use a syringe to mix infectious fluid forcefully.
         • Do not contaminate the needle hub when filling the syringe in order to avoid transfer of infectious material to fingers.
         • Wrap the needle and stopper in a cotton pledget moistened with disinfectant when removing a needle from a rubber stoppered bottle.
      3. Bending, recapping, clipping or removal of needles from syringes is prohibited. If it is essential that a contaminated needle be recapped or removed from a syringe, the use of a mechanical device or the one-handed scoop method must be used. The use of needle nipping devices is prohibited.
      4. Use a separate pan of disinfectant for reusable syringes and needles. Do not place them in pans containing pipettes or other glassware to eliminate sorting later.
      5. Used disposable needles and syringes must be placed in appropriate sharps disposal containers and discarded as medical waste. Contact the campus Environmental Affairs Program at 229-6339 for more information on the disposal of sharps.
   3. Safe and Effective Use of Biosafety Cabinets

      In general:

      • Make sure your biological safety cabinet is certified when it is installed or after it is moved, and annually thereafter. Check the magnahelic gauge regularly for an indication of a problem.

      • Understand how your cabinet works.
      • Do not disrupt the protective airflow pattern of the biological safety cabinet. Such things as rapidly moving your arms in and out of the cabinet, people walking rapidly behind you, and open lab doors may disrupt the airflow pattern and reduce the effectiveness of the biological safety cabinet.
      • Plan your work.
      • Minimize the storage of materials in and around the biological safety cabinet.
      • Always leave the biological safety cabinet running.

      Operational directions:

      • Before using, wipe work surface with 70% alcohol. Wipe off each item you need for your procedures and place in cabinet.

      • DO NOT place objects over the front air intake grille. DO NOT block the rear exhaust grille.
      • Segregate contaminated and clean items. Work from "clean to dirty."
      • Place a pan with disinfectant and/or a sharps container inside the biological safety cabinet for pipette discard. DO NOT use vertical pipette discard canisters on the floor outside cabinet.
      • It is not necessary to flame items. This creates turbulence in airflow and will compromise sterility; heat buildup may damage the filters.
      • Move arms slowly when removing or introducing new items into the biological safety cabinet.
      • If you use a piece of equipment that creates air turbulence in the biological safety cabinet (such as a centrifuge, blender) place equipment in the back 1/3 of the cabinet; stop other work while equipment is operating.
      • Protect the building vacuum system from biohazards by placing a HEPA cartridge filter or its equivalent between the vacuum trap and the source valve in the cabinet.
      • Clean up all spills in the cabinet immediately. Wait 10 minutes before resuming work.
      • When work is finished, remove all materials and wipe all interior surfaces with 70% alcohol.
      • Remove lab coat and wash hands thoroughly before leaving laboratory.
   4. Cryostats

      Frozen sections of unfixed tissue infected with an etiologic agent pose a risk because accidents can occur. Freezing tissue does not necessarily inactivate infectious agents. Freezing propellants under pressure should not be used for frozen sections as they may cause spattering of droplets of infectious materials. Gloves should be worn during preparation of frozen sections. When working with biohazardous material in a cryostat, the following is recommended:

      • Consider the contents of the cryostat to be contaminated and decontaminate it frequently with 70% ethanol.

      • Consider trimmings and sections of tissue that accumulate in the cryostat to be potentially infectious and remove them during decontamination.
      • Defrost and decontaminate the cryostat with a tuberculocidal hospital disinfectant once a week and immediately after tissue known to contain bloodborne pathogens, M. tuberculosis or other infectious agents is cut.
      • Handle microtome knives with extreme care, Stainless steel mesh gloves should be worn when changing knife blades.
      • Consider solutions for staining potentially infected frozen sections to be contaminated.
   5. Centrifuge Equipment

      Hazards associated with centrifuging include mechanical failure and the creation of aerosols. To minimize the risk of mechanical failure, centrifuges must be maintained and used according to the manufacturer’s instructions. Users should be properly trained and operating instructions that include safety precautions should be prominently posted on the unit.

      Aerosols are created by practices such as filling centrifuge tubes, removing plugs or caps from tubes after centrifugation, removing supernatant, and re-suspending sedimented pellets. The greatest aerosol hazard is created if a tube breaks during centrifugation. To minimize the generation of aerosols when centrifuging biohazardous material, the following procedures should be followed:

      • Use sealed tubes and safety buckets that seal with O rings. Before use, inspect tubes, O rings and buckets for cracks, chips, erosions, bits of broken glass, etc. Do not use aluminum foil to cap centrifuge tubes because it may detach or rupture during centrifugation.

      • Fill and open centrifuge tubes, rotors and accessories in a biological safety cabinet. Avoid overfilling of centrifuge tubes so that closures do not become wet. After tubes are filled and sealed, wipe them down with disinfectant.
      • Add disinfectant to the space between the tube and the bucket to disinfect material in the event of breakage during centrifugation.
      • Always balance buckets, tubes and rotors properly before centrifugation.
      • Do not decant or pour off supernatant. Use a vacuum system with appropriate in line reservoirs and filters.
      • Work in a biological safety cabinet when re-suspending sedimented material. Use a swirling rotary motion is necessary, wait a few minutes to permit the aerosol to settle before opening the tube.

      Small low speed centrifuges may be placed in a biological safety cabinet during use to reduce the aerosol escape. High speed centrifuges pose additional hazards. Precautions should be taken to filter the exhaust air from vacuum lines, to avoid metal fatigue resulting in disintegration of rotors and to use proper cleaning techniques and centrifuge components. Manufacturers’ recommendations must be meticulously followed to avoid metal fatigue, distortion and corrosion.

      Avoid the use of celluloid (cellulose nitrate) tubes with biohazardous materials. Celluloid centrifuge tubes are highly flammable and prone to shrinkage with age. They distort on boiling and can be highly explosive in an autoclave. If celluloid tubes must be used, an appropriate chemical disinfectant must be used to disinfect them.

   6. Personal Protective Equipment (PPE)

      Personal protective equipment is used to protect personnel from contact with hazardous materials and infectious agents. Appropriate clothing may also protect the experiment from contamination. Personal protective equipment must be provided without cost to personnel. The following personal protective equipment is recommended for regular use:

      1. Face Protection

         Goggles in combination with masks, or chin length face shields or other splatter guards are required whenever there is the possibility of splashes, sprays or splatters of infectious or other hazardous materials to the face.

      2. Laboratory clothing

         This category includes: laboratory coats, smocks, scrub suits, and gowns. Long sleeved garments should be used to minimize the contamination of skin or street clothes and to reduce shedding of microorganisms from the arms. In circumstances where it is anticipated that splashes may occur, the garment must be resistant to liquid penetration (in order to protect clothing from contamination). If the garment is not disposable, it must be capable of withstanding sterilization in the event it becomes contaminated. Additional criteria for selecting clothing are: comfort, appearance, closure types and location, anti-static properties and durability. Protective clothing must be removed and non laboratory areas. Disposables should be available for visitors, maintenance and service workers in the event it is required. All protective clothing should be either discarded in the laboratory or laundered by the facility. Personnel must not launder laboratory clothing at home.

      3. Gloves

         Gloves must be selected based on the hazards involved and the activity to be conducted. Gloves must be worn when working with biohazards, toxic substances and other physically hazardous agents. Temperature resistant gloves must be worn when handling hot material or dry ice. Delicate work requiring a high degree of precision dictates the use of thin walled gloves. Protection from contact with toxic or corrosive chemicals may also be required.

         When working with hazardous materials, the lower sleeve and the cuff of the laboratory garment should be overlapped by the glove. A long sleeved glove or disposable arm shield may be worn for further protection of the garment.

         In some instances “double gloving” may be appropriate. If a spill occurs, hands will be protected after the contaminated outer gloves are removed. Gloves must be disposed of when contaminated, removed when work with infectious material is completed and not worn outside the laboratory. Disposable gloves must not be washed or reused.

      4. Respirators

         In certain instances additional personal protective equipment may be required. Respirator selection is based on the hazard and the protection factor required. Personnel who require respiratory protection must contact University Safety for inclusion in the UWM Respiratory Protection Program. The program provides: a medical examination to ensure no health conditions exist that would be exacerbated by respirator usage; annual fit testing to ensure proper respirator size and type; and training to ensure proper respirator use and maintenance. Under no circumstances shall anyone wear a respirator unless he/she is a participant in the program.

         Contact University Safety for assistance in selection of other personal protective equipment.

   7. Blenders, Ultrasonic Disrupters, Grinders and Lyophilizers

      The use of any of these devices results in considerable aerosol production. Blenders, grinders and cell disruption equipment should be used in a biological safety cabinet when working with biohazardous materials.

      Safety blenders, although expensive, are designed to prevent leakage from the bottom of the blender jar, provide a cooling jacket to avoid biological inactivation and to withstand sterilization by autoclaving. If blender rotors are not leak-proof, they should be tested with sterile saline or dye solution prior to use with biohazardous material. The use of glass blender jars is not recommended because of the breakage potential. If they must be used, glass jars should be covered with a polypropylene jar to prevent spraying of glass and contents in the event the blender jar breaks. A towel moistened with disinfectant should be placed over the top of the blender during use. Before opening the blender jar allow the unit to rest for at least one minute to allow the aerosol to settle. The device should be decontaminated promptly after use.

      Depending on the lyophilizer design, aerosol production may occur when material is loaded or removed from the lyophilizer unit. If possible, sample material should be loaded in a biological safety cabinet. The vacuum pump exhaust should be filtered to remove any hazardous agents or, alternatively, the pump can be vented into a biological safety cabinet. After lyophilization is completed, all surfaces of the unit that have been exposed to the agent should be disinfected. If the lyophilizer is equipped with a removable chamber, it should be closed off and moved to a BSC for unloading and decontamination. Handling of cultures should be minimized and vapor traps should be used wherever possible.

      Opening an ampoule containing liquid or lyophilized culture material should be performed in a biological safety cabinet to control the aerosol produced. Gloves must be worn. To open, nick the neck of the ampoule with a file, wrap it in disinfectant soaked towel, hold the ampoule upright and snap it open at the nick. Reconstitute the contents of the ampoule by slowly adding liquid to avoid aerosolization of the dried material. Mix the contents without bubbling and withdraw it into a fresh container. Discard the towel and ampoule top and bottom as infectious waste.

      Ampoules used to store biohazardous material in liquid nitrogen have exploded causing eye injuries. The use of polypropylene tubes eliminates this hazard. These tubes are available dust free or pre-sterilized and are fitted with polyethylene caps with silicone washers. Heat sealable polypropylene tubes are also available.

   8. Loop Sterilizers and Bunsen Burners

      Sterilization of inoculation loops or needles in an open flame generates small particle aerosols which may contain viable microorganisms. The use of a shielded electric incinerator minimizes aerosol production during loop sterilization. Alternatively, disposable plastic loops and needles may be used for culture work where electric incinerators or gas flames are not available. The loops are semi-quantitative and can be used for counting bacteria.

      Continuous flame gas burners should not be used in biological safety cabinets. These burners can produce turbulence which disturbs the protective airflow patterns of the cabinet. Additionally, the heat produced by the continuous flame may damage the HEPA filter. If a gas burner must be used, one with a pilot light should be selected.

   9. Housekeeping

      Good housekeeping in laboratories is essential to reduce risks and protect the integrity of biological experiments. Routine housekeeping must be relied upon to provide work areas free of significant sources of contamination. Housekeeping procedures should be based on the highest degree of risk to which personnel and experimental integrity may be subjected.

      Laboratory personnel are responsible for cleaning laboratory benches, equipment and areas that require specialized technical knowledge. Additional laboratory housekeeping concerns include:

      • Keeping the laboratory neat and free of clutter; surfaces should be clean and free of infrequently used chemicals, glassware and equipment. Access to sinks, eyewashes, emergency showers and fire extinguishers must not be blocked.
      • Proper disposal of chemicals and waste old and unused chemicals should be disposed of promptly and properly. Contact the Environmental Affairs Program at 229-4999 for details.
      • Providing a workplace that is free of physical hazards aisles and corridors should be free of tripping hazards. Attention should be paid to electrical safety, especially as it relates to the use of extension cords, proper grounding of equipment, avoidance of overloaded electrical circuits and avoidance of the creation of electrical hazards in wet areas.
      • Removing unnecessary items on floors, under benches or in corners.
      • Properly securing all compressed gas cylinders.
      • Never using fume hoods for storage of chemicals or other materials.

      Practical custodial concerns include:

      • Dry sweeping and dusting which may lead to the formation of aerosols is not permitted.
      • The usual wet or dry industrial type vacuum cleaner is a potent aerosol generator and, unless equipped with high efficiency particulate air (HEPA) filter, must not be used in the biological research laboratory.
3. Decontamination

   Decontamination is a term used to describe a process or treatment that renders a medical device, instrument, or environmental surface safe to handle. A decontamination procedure can range from sterilization to simple cleaning with soap and water. Sterilization, disinfection and antisepsis are all forms of decontamination.

   Sterilization is the use of a physical or chemical procedure to destroy all microbial life, including highly resistant bacterial endospores.

   Disinfection eliminates virtually all pathogenic non sporeforming microorganisms but not necessarily all microbial forms on inanimate objects (work surfaces, equipment, etc.). Effectiveness is influenced by the kinds and numbers of organisms, the amount of organic matter, the object to be disinfected and chemical exposure time, temperature and concentration.

   Antisepsis is the application of a liquid antimicrobial chemical to skin or living tissue to inhibit or destroy microorganisms. It includes swabbing an injection site on a person or animal and hand washing with germicidal solutions. Although some chemicals may be utilized as either a disinfectant or an antiseptic, adequacy for one application does not guarantee adequacy for the other. Manufacturers’ recommendations for appropriate use of germicides should always be followed.

   1. General Procedures
      
      
      1. All infectious materials and all contaminated equipment or apparatus should be decontaminated before being washed, stored or discarded. Autoclaving is the preferred method. Each individual working with biohazardous material should be responsible for its proper handling.
      2. Biohazardous materials should not be placed in autoclave overnight in anticipation of autoclaving the next day.
      3. Autoclaves should not be operated unattended or by untrained personnel.
      4. Special precautions should be taken to prevent accidental removal of material from an autoclave before it has been sterilized or simultaneous opening of both doors on a double door autoclave.
      5. Dry hypochlorites, or any other strong oxidizing material, must not be autoclaved with organic materials such as paper, cloth or oil. REMEMBER: OXIDIZER + ORGANIC MATERIAL + HEAT = POSSIBLE EXPLOSION
   2. Methods

      There are four main categories of physical and chemical means of decontamination. They are heat, liquid disinfection, vapors and gases, and radiation. Each category is discussed briefly below.

      1. Heat
         
         
         • Wet heat is the most dependable method of sterilization. Autoclaving (saturated steam under pressure of approximately 15 psi to achieve a chamber temperature of at least 250° F for a prescribed time) is the most convenient method of rapidly achieving destruction of all forms of microbial life. In addition to proper temperature and time, prevention of entrapment of air is critical to achieving sterility. Material to be sterilized must come in contact with steam and heat. Chemical indicators, e.g. autoclave tape, must be used with each load placed in the autoclave. The use of autoclave tape alone is not an adequate monitor of efficacy. Autoclave sterility monitoring should be conducted on a regular basis using appropriate biological indicators (B. stearothermophilus spore strips) placed at locations throughout the autoclave. The spores, which can survive 250° F for 5 minutes but are killed at 250º F in 13 minutes, are more resistant to heat than most, thereby providing an adequate safety margin when validating decontamination procedures. Each type of container employed should be individually tested with these spores because efficacy varies with the load, fluid volume, etc.
         • Dry Heat is less efficient than wet heat and requires longer times and/or higher temperatures to achieve sterilization. It is suitable for the destruction of viable organisms on impermeable non organic surfaces such as glass, but it is not reliable in the presence of shallow layers of organic or inorganic materials which may act as insulation. Sterilization of glassware by dry heat can usually be accomplished at 160 - 170° C for periods of 2 to 4 hours. Dry heat sterilizers should be monitored on a regular basis using appropriate biological indicators [Bacillus subtilis (globigii) spore strips].
         • Incineration is another effective means of decontamination by heat. As a disposal method incineration has the advantage of reducing the volume of the material prior to its final disposal.
      2. Liquid disinfection

         The most practical use of liquid disinfectants is for surface decontamination and, when used in sufficient concentration, as a decontaminate for liquid wastes prior to final disposal in the sanitary sewer. If liquid disinfectants are used, they must have been shown to be effective against the organism(s) present.

         Liquid disinfectants are available under a wide variety of trade names. In general, these can be classified as halogens, acids, alkalis, heavy metal salts, quaternary ammonium compounds, phenolic compounds, aldehydes, ketones, alcohols and amines. The more active a compound is, the more likely it is to have undesirable characteristics such as corrosivity. No liquid disinfectant is equally useful or effective under all conditions and for all viable agents.

      3. Vapors and gases

         A variety of vapors and gases possess decontamination properties. Vapors and gases are primarily used to decontaminate biological safety cabinets and associated systems, bulky or stationary equipment not suited to liquid disinfectants, instruments or optics which might be damaged by other decontamination methods, and rooms, buildings and associated air handling systems. Agents included in this category are: glutaraldehyde and formaldehyde vapor, ethylene oxide gas, peracetic acid and hydrogen peroxide vapor. When used in closed systems and under controlled conditions of temperature and humidity, excellent disinfection can be obtained. Great care must be taken during use because of the hazardous nature of many of these compounds. Contact University Safety and Assurances for monitoring requirements if these compounds are to be used.

      4. Radiation

         Although ionizing radiation will destroy microorganisms, it is not a practical tool for laboratory use. Non-ionizing radiation in the form of ultraviolet radiation (UV) is used for inactivating viruses, bacteria and fungi. It will destroy airborne microorganisms and inactivate microorganisms on exposed surfaces or in the presence of products of unstable composition that cannot be treated by conventional means.

         UV lamps are not recommended for decontamination unless they are properly maintained. Because UV lamp intensity or destructive power decreases with time, it should be checked with a UV meter yearly. Frequent lamp cleaning (at least every few weeks) is necessary to prevent accumulation of dust and dirt which drastically reduces its effectiveness. If UV must be used, it should be used when areas are not occupied.

4. Infectious Waste Management

   All disposal of infectious waste, autoclave bags, pipettes, sharps and biological waste must be performed in accordance with Wisconsin Department of Natural Resources (WDNR) medical waste regulations. UWM has developed procedures for the proper disposal of biological waste materials. Please consult Appendix E for specific information of the treatment, handling and disposal of biological materials.

   1. Mixed Waste

      Mixed wastes are potentially infectious waste contaminated with other types of waste, e.g., radioisotopes or toxic/carcinogenic compounds. Because of the difficulty in disposal of wastes regulated by more than one set of requirements and regulatory agencies, it is critical that provision be made for proper management prior to the initiation of any research that might result in mixed waste. Mixed wastes may require special containers, labeling, storage, etc. Contact University Safety prior to initiation of any research that might result in potentially infectious waste with multiple hazards.

   2. Animals

      All methods of disposal for research animals and animal parts that are considered to be infectious waste must be approved by the campus Animal Care Program (229-6016).

   3. Microorganism Cultures

      All cultures of microorganisms should be inactivated, using appropriate procedures, before disposal. This includes cultures of Risk Group 1 organisms. This is considered good laboratory practice.

As discussed in Chapter 1, the Principal Investigator (PI) is responsible for the training of everyone working in his/her laboratory. Training is possibly the single most important action a PI can take to promote a safe and healthy laboratory.

1. Laboratory Specific Biosafety Training

   Laboratory specific biosafety training shall be provided by the Principal Investigator (PI) and will include generalized training for the biosafety level at which the laboratory operates and specialized training for specific hazards present in that laboratory. For workers in laboratories operating at Biosafety Level 1 (BL1), it is suggested that information included as Appendix D-1 be reviewed with each worker. The document should be signed by both the laboratory worker and the Principal Investigator. This training shall be reviewed annually. If the suggested training form is not used, an equivalent document shall be developed to document that training in all standard and special practices, safety equipment and facilities related to work at BL1 has been provided.

   For workers in laboratories operating at Biosafety Level 2 (BL2), it is suggested that information included as Appendix D-2 be reviewed with each worker. The document should be signed by both the laboratory worker and the Principal Investigator. Note that if CDC select agents are used in the laboratory their presence should be noted on training documentation. This training shall be reviewed annually. If the suggested training form is not used, an equivalent document shall be developed to document that training in all standard and special practices, safety equipment and facilities related to work at BL2 has been provided.

   The signed training document should be retained by the PI as record of appropriate laboratory worker training.

2. Bloodborne Pathogens Training

   Bloodborne pathogens training is necessary for any laboratory worker who works with human blood, human blood components, products made from human blood, human organs or human body fluids. Bloodborne Pathogens Training is provided by the Department of University Safety and Assurances. Please contact the campus Safety Training Officer at 229-2736 to schedule bloodborne pathogens training.

3. Teaching Laboratories

   Instructors in teaching laboratories should provide specific training for the hazards expected to be encountered in the laboratory procedures utilized. Because of the brevity of courses and the large number of students involved it is recommended that each instructor develop a streamlined training form emphasizing the particular elements required for their course. Both the instructor and student should sign the training form.

Biosafety Level 1 is suitable for work involving well characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is neither required nor generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science.

The following standard and special practices, safety equipment and facilities apply to agents assigned to Biosafety level 1:

1. Standard Microbiological Practices
   
   
   1. Access to the laboratory is limited or restricted at the discretion of the primary investigator when experiments or work with cultures and specimens are in progress.
   2. Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.
   3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.
   4. Mouth pipetting is prohibited; mechanical pipetting devices are used.
   5. Policies for the safe handling of sharps are instituted.
   6. All procedures are performed carefully to minimize the creation of splashes or aerosols.
   7. Work surfaces are decontaminated at least once a day and after any spill of viable materials.
   8. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable leak proof container and closed before removal from the laboratory.
   9. A biohazard sign must be posted at the entrance to the laboratory whenever infectious agents are present. The sign must include the name of the agent(s) in use and the name and home phone number of the primary investigator.
2. Special Practices: None
3. Safety Equipment (Primary Barriers)
   
   
   1. Special containment devices or equipment such as a biological safety cabinet are generally not required for manipulations of agents assigned to Biosafety Level 1.
   2. It is recommended that laboratory coats, gowns, or uniforms are worn to prevent contamination or soiling of street clothes.
   3. Gloves should be worn if the skin on the hands is broken or if a rash is present. Alternatives to powdered latex gloves should be available.
   4. Protective eyewear should be worn for conduct of procedures in which splashes of microorganisms or other hazardous materials is anticipated.
4. Laboratory Facilities (Secondary Barriers)
   
   
   1. Laboratories should have doors for access control.
   2. Each laboratory contains a sink for handwashing.
   3. The laboratory is designed so that it can be easily cleaned. Carpets and rugs in laboratories are not appropriate.
   4. Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surface and equipment.
   5. Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning.
   6. If the laboratory has windows that open to the exterior, they are fitted with fly screens.

Certification:

I have received and understand the biological safety training as outlined in this document.

Biosafety Level 2 is similar to Biosafety level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs from BSL 1 in that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists; (2) access to the laboratory is limited when work is being conducted; (3) extreme precautions are taken with contaminated sharp items; and (4) certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.

The following standard and special practices, safety equipment and facilities apply to agents assigned to Biosafety level 2:

1. Standard Microbiological Practices
   
   
   1. Access to the laboratory is limited or restricted at the discretion of the primary investigator when experiments are in progress.
   2. Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.
   3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.
   4. Mouth pipetting is prohibited; mechanical pipetting devices are used.
   5. Policies for the safe handling of sharps are instituted.
   6. All procedures are performed carefully to minimize the creation of splashes or aerosols.
   7. Work surfaces are decontaminated at least once a day and after any spill or splash of viable material with disinfectants that are effective against the agents of concern.
   8. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable leak proof container before removal from the laboratory
2. Special Practices
   
   
   1. Access to the laboratory is limited or restricted by the primary investigator when work with infectious agents is in progress. In general, persons who are at increased risk of acquiring infection, or for whom infection may have serious consequences, are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immuno-suppressed may be at increased risk of acquiring infections. The primary investigator has the final responsibility for assessing each circumstance and determining who may enter the laboratory or animal room.
   2. The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential hazards and meet specific entry requirements (e.g., immunization) may enter the laboratory.
   3. A biohazard sign must be posted on the entrance to the laboratory when etiologic agents are in use. Appropriate information to be posted includes the agent(s) in use, the Biosafety level, the required immunizations, the primary investigator’s name and telephone number, any personal protective equipment that must be worn in the laboratory, and any procedures required for exiting the laboratory.
   4. Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).
   5. When appropriate, considering the agent(s) handled, baseline serum samples for laboratory and other at risk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the facility.
   6. Biosafety procedures are incorporated into standard operating procedures or in a Biosafety manual adopted or prepared specifically for the laboratory by the laboratory director. Personnel are advised of special hazards and are required to read and follow instructions on practices and procedures.
   7. The primary investigator ensures that laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates or additional training as necessary for procedural or policy changes.
   8. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scapels.
      
      
      • Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plastic ware should be substituted for glassware whenever possible.
      • Only needle locking syringes or disposable syringe needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they 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.
      • Syringes, which re sheathe the needle, needleless systems, and other safety devices, are used when appropriate.
      • Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass are decontaminated before disposal, according to any local, state, or federal regulations.
   9. Cultures, tissues, specimens of body fluids, or potentially infectious wastes are placed in a container with a cover that prevents leakage during collection, handling, processing, storage, transport, or shipping.
   10. Laboratory equipment and work surfaces should be decontaminated with an effective disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated according to any local, state, or federal regulations before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, state, or federal regulations, before removal from the facility.
   11. Spills and accidents that result in overt exposures to infectious materials are immediately reported to the laboratory director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.
   12. Animals not involved in the work being performed are not permitted in the lab.
3. Safety Equipment (Primary Barriers)
   
   
   1. Properly maintained biological safety cabinets, preferably Class II, or other appropriate personal protective equipment or physical containment devices are used whenever:
      
      
      • Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals or embryonate eggs.
      • High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed rotor heads or centrifuge safety cups are used, and if these rotors or safety cups are opened only in a biological safety cabinet.
   2. Face protection (goggles, mask, face shield or other splatter guard) is used for anticipated splashes or sprays of infectious or other hazardous materials to the face when the microorganisms must be manipulated outside the biological safety cabinet.
   3. Protective laboratory coats, gowns, smocks, or uniforms designated for lab use are worn, while in the laboratory. This protective clothing is removed and left in the laboratory before leaving for non laboratory areas (e.g., cafeteria, library, and administrative offices). All protective clothing is either disposed of in the laboratory or laundered by the institution; personnel should never take it home
   4. Gloves are worn when hands may contact potentially infectious materials, contaminated surfaces or equipment. Wearing two pairs of gloves may appropriate. Gloves are disposed of when overtly contaminated, and removed when work with infectious materials is completed or when the integrity of the glove is compromised. Disposable gloves are not washed, reused, or used for touching “clean” surfaces (keyboards, telephones, etc.), and they should not be worn outside the lab. Alternatives to powdered latex gloves should be available. Hands are washed following removal of gloves.
4. Laboratory Facilities (Secondary Barriers)
   
   
   1. Provide lockable doors for facilities that house restricted agents (as defined in 42 CFR 72.6).
   2. Consider locating new laboratories away from public areas.
   3. Each laboratory contains a sink for handwashing. Foot, knee, or automatically operated sinks are recommended.
   4. The laboratory is designed so that it can be easily cleaned. Carpets and rugs in laboratories are inappropriate.
   5. Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.
   6. Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning. Chairs and other furniture used in laboratory work should be covered with a non fabric material that can be easily decontaminated.
   7. Install biological safety cabinets in such a manner that fluctuations of the room supply and exhaust air do not cause the biological safety cabinets to operate outside their parameters for containment. Locate biological safety cabinets away from doors, from windows that can be opened, from heavily traveled laboratory areas, and from other potentially disruptive equipment so as to maintain the biological safety cabinets’ air flow parameters for containment.
   8. An eyewash station is readily available.
   9. Illumination is adequate for all activities, avoiding reflections and glare that could impede vision.
   10. There are no specific ventilation requirements. However, planning of new facilities should consider mechanical ventilation systems that provide an inward flow of air without re-circulation to spaces outside of the laboratory. If the laboratory has windows that open they are fitted with fly screens.

Certification:

I have received and understand the biological safety training as outlined in this document.

Proper treatment, handling and disposal of cultures and items contaminated by potentially biohazardous agents is a vital step toward protection of laboratory and physical plant personnel from infectious disease. This waste handling process is also necessary to prevent the release of potentially infectious agents into the community at large. Treatment and disposal of biological waste is regulated by several federal and state agencies. General guidelines for handling this waste stream are provided below. Additional information regarding these procedures can be found at: http://www.uwm.edu/Dept/EHSRM/BIO/index.html

Proper segregation, storage, treatment and disposal of biohazardous waste is essential not only to comply with waste regulations, but more importantly to reduce personnel exposure to potentially infectious materials. An exposure occurs when potentially infectious materials are permitted to enter a person’s bloodstream through a break in the skin or contact with the eyes, nose, or mouth. Examples of exposure related to biohazardous waste handling include incidents such as:

• Splashing liquid biological waste into the eye during pour-off for disposal
• Puncturing the skin with a biologically contaminated needle
• Spilling liquids from a ruptured biohazardous bag onto broken, unprotected skin.

In the event of an exposure to potentially infectious materials, take the following actions:

• Wash the exposed skin or flush the mucous membrane for 10-15 minutes.
• Notify your lab director or supervisor and University Safety (229-6339).
• Contact your health care provider to determine the need for evaluation and/or possible treatment.

Sharps

Sharps are generally agreed to be the most hazardous items in the potentially infectious waste stream. A high degree of caution should always be used when handling any sharp object, contaminated or not. All disposable sharps (hypodermic, intravenous or other medical needles and syringes; pasteur pipettes; scapel or razor blades; blood vials; glass test tubes and centrifuge tubes; microscope slides and coverslips; and any other laboratory glassware that has had contact with infectious agents) must be placed into an approved sharps container immediately following use to reduce puncture risk. An approved sharps container is one that is leak-proof, puncture-resistant, closeable and bears the biohazard symbol. Once filled, the container should be permanently closed and disposed of through the UWM Environmental Affairs Program.

Solid Non-Sharps Biohazardous Waste

All non-sharp laboratory materials utilized in experiments with biological materials (e.g., microorganisms, recombinant DNA, cell cultures, etc) must be treated prior to disposal by an approved decontamination method such as autoclaving. These wastes should be stored in bags bearing the biohazard symbol prior to decontamination. While in use for waste storage, biohazard bags must be secured in a manner that will eliminate spillage. If a bag is used primarily for disposal items that are not likely to release liquids (i.e., pipette tips, kim wipes, etc.), a wire bag rack or rigid container is an acceptable means of securing the bag to eliminate spillage. If the bag is used for storage of items that are likely to release liquids and possible result in leakage, the bag should be stored in a leak proof container such as a trash can with a lid that is also labeled with the biohazard symbol.

When transporting waste bags to the autoclave for treatment, secure the bags closed with a rubber band, twist tie or other closure device that can be easily removed and place the bags in secondary container such as a pan or bucket. Always use a cart to move the bags if possible.

After autoclaving, any bag displaying the biohazard symbol should be placed in a non-transparent plastic bag or other secondary non-transparent container (i.e., closed cardboard box, dark colored trash bag) prior to disposal into the normal trash. Bags with the biohazard symbol, regardless of use, must not be placed into the regular waste stream without defacing the symbol or over-bagging.

Liquid Biohazardous Waste

Human or animal blood and body fluids can be flushed into the sanitary sewer without prior treatment. However, chemical disinfection is recommended prior to disposal if feasible. All other potentially infectious liquids (i.e., media with growth, cell line waste, etc.) must be autoclaved or chemically disinfected before disposal into the sanitary sewer. Do not autoclave waste that are chemically treated as this action may create a chemical exposure hazard.

If using a chemical disinfectant, follow the manufacturer’s label instructions regarding concentration, and contact time. Also note that disinfectants are hazardous materials that may require the use of additional personal protective equipment to control chemical exposure. Refer to your product’s material safety data sheet for further information or contact University Safety for assistance.

Animal Carcasses

Carcasses and body parts from animals used in infectious disease or recombinant DNA research must be incinerated as pathological waste. These carcass wastes need to be packaged and handled separately from normal carcass waste generated on campus. Please consult with the Animal Care Program (229-6016) in advance of your project to arrange for special disposal of these carcasses.

This appendix includes those biological agents known to infect humans as well as selected animal agents that may pose theoretical risks if inoculated into humans. Included are lists of representative genera and species known to be pathogenic. Mutated, recombined, and non pathogenic species and strains are not considered. Non infectious life cycle stages of parasites are excluded.

This appendix reflects the current state of knowledge and should be considered a resource document. The list includes the more commonly encountered agents and is not meant to be all-inclusive. Information on agent risk assessment may be found in the Agent Summary Statements of the CDC/NIH Biosafety in the Microbiological and Biomedical Laboratories. Further guidance on agents not listed in this Appendix may be obtained through: Centers for Disease Control and Prevention, Biosafety Branch, Atlanta, Georgia 30333, Phone: (404) 639 3883, Fax: (404) 639 2294; National Institutes of Health, Division of Safety, Bethesda, Maryland 20892, Phone: (301) 496 1357; National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 50010, Phone: (515) 862 8258.

Risk Group 1 (RG1) Agents

RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents include asporogenic Bacillus subtilis or Bacillus licheniformis.

Those agents not listed in Risk Groups (RGs) 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

RG2 agents are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available.