Biosafety Manual For Tennessee State University. Revised October 2006

Transcription

1 Biosafety Manual For Tennessee State University Revised October 2006

2 Table of Contents: Important Telephone Numbers Policy Statement Classification of Potentially Infectious Agents Microorganisms Genetically Engineered Microorganisms Human Clinical Materials Plant and Animal Pathogens Select Agents Biosafety Containment Levels Biosafety Level 1 (BL1) Biosafety Level 2 (BL2) Biosafety Level 3 (BL3) Biosafety Level 4 (BL4) Summary of Recommended Biosafety Levels Emergency Procedures Biological Spills Injury Involving Biological Materials Fires Involving Biological Materials Decontamination and Disposal Chemical Disinfectants Disinfectants Commonly Used in the Laboratory Dilution of Disinfectants Autoclaving Procedures Autoclave Safety Use and Disposal of Sharps Biological Waste Disposal Procedures Biosafety Equipment Biological Safety Cabinets Operation of Class II Biological Safety Cabinets Centrifuge Containment Protection of Vacuum Lines Shipment of Biological Materials General Information Permits Packaging Genetically Modified Microorganisms Human Clinical Materials On-Campus Transport

3 Appendix 1: Appendix 2: Appendix 3: Appendix 4: Appendix 5: Biosafety Level 1 Practices and Facilities Biosafety Level 2 Practices and Facilities Biosafety Level 3 Practices and Facilities Select Agents and Toxins Department of Commerce List of Select Microorganisms and Toxins Important Telephone Numbers Emergency Telephone Numbers: Campus Police: Fire, Police, Rescue Biosafety Committee Chair: Dr. Mohammad Karim Other Important Numbers: University Health center: Facilities Management: Radiation Safety Officer: Useful Websites: NIH Guidelines: BMBL: NIH Office of Biotechnology Activities: CDC Select Agents Program: USDA/APHIS Select Agents Program: CDC Permit to Import or Transport Etiologic Agents: USDA/APHIS Permit to Import or Transport Livestock Pathogens:

4 USDA/APHIS Permit to Field Test, Import, or Transport Genetically Modified Organisms: University of Maryland Form for Registration of Biological Materials: Selection, Installation, and Use of Biological Safety Cabinets: Policy Statement I. Purpose: The purpose of the manual is to establish the process for compliance with the following documents: A. NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines): B. Biosafety in Microbiological and Biomedical Laboratories (BMBL) II. Policy: Tennessee State University is committed to preserving the health and safety of its students, faculty and staff. The University is also committed to protecting the environment and the community. It is recognized that the use of recombinant DNA or other potentially harmful pathogenic microorganisms is necessary in many research and teaching laboratories at the University. The University requires the compliance with the NIH guidelines and with the recommendations in BMBL to ensure the safe handling of these organisms. Compliance with other applicable Federal, State, and Local regulations is also required. III. Responsibilities: The Principal Investigator (PI) is directly and primarily responsible for the safe operation of the laboratory. His/her knowledge and judgment are critical in assessing risks and appropriately applying the recommendations in this manual. However, safety is a shared responsibility among all of the laboratory staff. Institutional Biosafety Committee (IBC) is to assist the PI with these responsibilities. A. The University Biosafety Committee shall: 1. Prepare the Biosafety Manual, with revisions as necessary; 2. Distribute the Manual to each faculty member who works with biological materials; 3. Investigate accidents involving infectious agents; 4. Provide or coordinate biosafety training as requested 5. Assist investigators with risk assessment 6. Administer all elements of the Biosafety Program, assist faculty with submission of registrations to the IBC, and maintain registration files 7. Review rdna research conducted at or sponsored by the university for compliance with the NIH Guidelines, and approve those research projects that are found to conform with the NIH Guidelines;

5 8. Review research involving infectious agents conducted at or sponsored by the university for compliance with the guidelines in Biosafety in Microbiological and Biomedical Laboratories (BMBL), and approve those research projects that are found to conform with the recommendations in BMBL; 9. Notify the PI of the results of the IBC's review and approval; 10. Report any significant problems with or violations of the NIH Guidelines and any significant research-related accidents or illness to the appropriate Institutional official and to the NIH Office of Biotechnology Activities (OBA) within 30 days; and 11. Follow the guidelines for membership defined by NIH, with the additional requirement of one representative from the University of Maryland Animal Care and Use Committee, and a plant pathologist from USDA as appropriate. B. PIs shall: 1. Assess the risks of their experiments; 2. Ensure the safe operation of their laboratory; 3. Train laboratory personnel in safe work practices; 4. Comply with all applicable state and federal regulations and guidelines; 5. Register the following experiments with the IBC, as required: a. recombinant DNA activities; b. work with infectious agents; c. experiments involving the use of human blood or other potentially infectious materials, such as unfixed human tissues, primary human cell lines, and certain body fluids; and d. animal and plant pathogens. C. The University Health Center (UHC) shall: 1. Provide medical surveillance, as required by the OSHA Bloodborne Pathogens Standard (CFR ), and as recommended in the BMBL and NIH Guidelines; and 2. Provide vaccinations, as required. D. Laboratory personnel shall: 1. Comply with safety recommendations for the work being performed; and 2. Report accidents or injuries to the PI.

6 Classification of Potentially Infectious Agents Procedures and facilities involved in protecting laboratory workers, the public, and the environment from laboratory biological hazards are governed by federal and state regulations and guidelines. Many granting agencies require that grant recipients certify that they adhere to both the guidelines and the regulations. Microorganisms The National Institutes of Health (NIH) and the Centers for Disease Control and Prevention (CDC) publish guidelines for work with infectious microorganisms. The publication, entitled Biosafety in Microbiological and Biomedical Laboratories (BMBL), recommends that work be done using one of four levels of containment: Biosafety Level 1 (BSL1), BSL2, BSL3 and BSL4 (see next chapter). The NIH Guidelines (Appendix B) classify pathogenic agents into one of four risk groups according to specific criteria. It is Tennessee State University policy that all laboratories adhere to these NIH/CDC guidelines. Microorganisms capable of causing infection in humans Investigators must register any project involving a pathogenic agent with the IBC and receive its approval before work is begun. Following receipt of the completed Registration Document by IBC, the laboratory will be surveyed by the Institutional Biosafety Committee (IBC) to ascertain that it meets the containment requirements listed in BMBL for the agent being studied. If the lab meets the requirements, the work will be reviewed and approved or disapproved by the IBC. Genetically Engineered Microorganisms Work with all genetically engineered organisms must comply with the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines-Recent Report April 2002). These guidelines classify recombinant DNA experiments into four levels of containment (BSL1, BSL2, BSL3, and BSL4) based on the hazard of the microorganism and the procedures and quantities being used. Additionally, the United States Department of Agriculture (USDA) requires permits for field testing of genetically engineered plants. It is Tennessee State University policy that all laboratories follow these guidelines. Registration Each PI is responsible for registering all recombinant DNA experiments with the IBC, including those exempt from the NIH Guidelines. The IBC audits all laboratories where BSL2 or BSL3 containment is required. BSL1 laboratories are audited on request of the PI. Review and Approval of Experiments The IBC, which oversees recombinant DNA research at Tennessee State University, will review the registration. a. Experiments covered by the NIH Guidelines Many experiments involving rdna molecules require registration and approval by the IBC before work may be initiated. Experiments that require IBC approval before initiation include those that involve: o Risk Group 2, 3, 4, or research involving Select Agents. o Cloning DNA from Risk Group 2, 3, 4, or Select Agents.

7 o o o Infectious virus, or defective virus in the presence of helper virus in tissue culture systems. Whole plants or animals. More than 10 liters of culture. Experiments that must be registered at the time of initiation include those that involve: o o o the formation of recombinant DNA molecules containing no more than 2/3 of the genome of any eukaryotic virus propagated in tissue culture. recombinant DNA-modified whole plants, and/or recombinant DNA-modified organisms associated with whole plants, except those that fall under Section III-A, III-B, III-C, or III-E of the Guidelines. the generation of transgenic rodents that require BSL1 containment. b. Experiments exempt from the NIH Guidelines Experiments exempt from the NIH Guidelines, although requiring registration with the IBC, may be initiated immediately. The Chair of the IBC or the BSO will review the registration and confirm that the work is classified correctly according to the NIH Guidelines. Exempt experiments are those that: o use rdna molecules that are not in organisms or viruses. o consist entirely of DNA segments from a single nonchromosomal or viral DNA source, though one or more of the segments may be a synthetic equivalent. o consist entirely of DNA from a prokaryotic host including its indigenous plasmids or viruses when propagated only in that host (or a closely related strain of the same species), or when transferred to another host by well established physiological means. o consist entirely of DNA from an eukaryotic host including its chloroplasts, mitochondria, or plasmids (but excluding viruses) when propagated only in that host (or a closely related strain of the same species). o consist entirely of DNA segments from different species that exchange DNA by known physiological processes, though one or more of the segments may be a synthetic equivalent. o do not present a significant risk to health or the environment as determined by the NIH Director. o o o contain less than one-half of any eukaryotic viral genome propagated in cell culture. use E. coli K12, Saccharomyces cerevisiae, or Bacillus subtilis host - vector systems, unless genes from Risk Group 3 or 4 pathogens or restricted animal pathogens are cloned into these hosts. involve the purchase or transfer of transgenic rodents for experiments that require BSL1 containment. Human Blood, Unfixed Tissue, and Cell Culture Please refer to the Bloodborne Pathogens Exposure Control Plan Appendix 6 for detailed information on handling human clinical material.

8 Work with human material is regulated by the Occupational Safety and Health Administration (OSHA) Bloodborne Pathogens Standard, 29 CFR, Part Human blood, unfixed tissue, cell culture, and certain other body fluids are considered potentially infectious for bloodborne pathogens such as hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). All human clinical material should be presumed infectious and handled using BSL2 work practices. This concept is called Universal Precautions. Investigators are responsible for notifying IBC of their use of human materials. Training and immunization are required by OSHA. Plant and Animal Pathogens The IBC requires investigators to register their campus use of plant pathogens. The registration form for animal pathogens is available at the web site: Registration of plant pathogens may be completed by forwarding a copy to the Biosafety Office. Select Agents Select Agents are microorganisms and toxins that have potential for use by terrorists. The Public Health Security and Bioterrorism Preparedness and Response Act of 2002 restricts their possession and use, and requires the University to collect and maintain information on the location and use on campus of any select agents or toxins. Please contact the Biosafety Office immediately if you currently possess or plan to acquire any of the listed agents and have not yet reported that fact. Failure to provide notice may result in civil and criminal liability for individual researchers and/or the University. If you have questions, you may contact the Biosafety Office, or visit CDC's Select Agent Program web site, which provides links to select agent program information. Biosafety Containment Levels Four levels of Biosafety are defined in the publication Biosafety in Microbiological and Biomedical Laboratories (BMBL), published by the CDC and NIH. The levels, designated in ascending order by degree of protection provided to personnel, the environment, and the community, are combinations of laboratory practices, safety equipment, and laboratory facilities (see Appendices). Most microbiological work Tennessee State University is conducted at BSL1 or BSL2 containment. There are no BSL4 laboratories at the university. Biosafety Level 1 BSL1 is appropriate for undergraduate and secondary educational training and teaching laboratories, and for other facilities in which work is done with well-characterized agents not known to cause disease in healthy adult humans. The laboratory is not necessarily separated from the general traffic patterns in the building. BSL1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for hand washing. The following Standard Microbiological Practices apply to all Biosafety Levels. Additional practices recommended for BSL2 are in Appendix 2, and for BSL3 in Appendix 3.

9 Standard Microbiological Practices: 1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress. 2. Persons wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the laboratory. 3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas where there is reasonable likelihood of exposure to potentially infectious materials. 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. All procedures are performed carefully to minimize the creation of splashes or aerosols. 6. Work surfaces are decontaminated at least once a day and after any spill of viable material. 7. 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 for transport from the laboratory. Materials to be decontaminated off-site are packaged in accordance with applicable state and federal regulations before removal from the facility. 8. An insect and rodent control program is in effect. Biosafety Level 2 BSL2 is similar to Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs 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. With good microbiological techniques, work at BSL2 can be conducted safely on the open bench, provided the potential for producing splashes or aerosols is low. Primary hazards to personnel working with BSL2 agents relate to accidental percutaneous or mucous membrane exposures, or ingestion of infectious materials. BSL2 is appropriate when work is done with any human-derived blood, body fluids, or tissues where the presence of an infectious agent may be unknown. See Appendix 2 for a complete list of BSL2 criteria. Biosafety Level 3 BSL3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents. Primary hazards to personnel working at BSL3 relate to autoinoculation, ingestion, and exposure to infectious aerosols. See Appendix 3 for a complete list of BSL3 criteria.

10 Biosafety Level 4 BSL4 is required for work with dangerous and exotic agents which pose a high individual risk of aerosoltransmitted laboratory infections and life-threatening disease. Agents with a close or identical antigenic relationship to BSL4 agents are handled at this level until sufficient data are obtained either to confirm continued work at this level, or to work with them at a lower level. Members of the laboratory staff have specific and thorough training in handling extremely hazardous infectious agents; and they understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics. They are supervised by competent scientists who are trained and experienced in working with these agents. Access to the laboratory is strictly controlled by the laboratory director. The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. A specific facility operations manual is prepared or adopted. Within work areas of the facility, all activities are confined to Class III biological safety cabinets, or Class II biological safety cabinets used with one-piece positive pressure personnel suits ventilated by a life support system. The BSL4 laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment. 1 From: Biosafety in Microbiological and Biomedical Laboratories. Centers for Disease Control and Prevention, and National Institutes of Health, Biosafety Level BSL1 Basic Laboratory BSL2 Basic Laboratory with biosafety cabinets and other physical containment devices as required BSL3 Containment Laboratory with special engineering and design features Risk Assessment Individual risk: Low Community risk: Low Individual risk: Moderate Community risk: Low Individual risk: High Community risk: Low Practices and Techniques Standard Microbiological Practices. Level 1 practices plus: lab coats; autoclaving all biological waste preferred; limited access; biohazard warning signs on doors and equipment. Level 2 practices plus: special protective clothing; controlled access through entrance room; biological waste must be autoclaved, preferably within the Safety Equipment None: primary containment provided by adherence to standard lab practices during open bench operations. Partial containment (i.e., Class I or II biosafety cabinets) for procedures which produce aerosols. Partial containment equipment used for all manipulations of infectious materials; directional airflow. Examples E. coli K12; S. cerevisiae; short term, long term culture of most non-primate mammalian cells. E. coli O157; Hepatitis B virus; Salmonella typhimurium; human blood; Neisseria gonorrhoeae; culture of lymphoid lines carrying inducible EB; many common human pathogens. Yellow fever virus; M. tuberculosis; Industrial scale volumes of HIV.

11 BSL4 Maximum Containment Laboratory Biological Spills Individual risk: High Community risk: High laboratory. Level 3 practices plus: entrance through change room; complete change of clothing from street to laboratory gear; shower at exit; all wastes decontaminated on exit from facility. Emergency Procedures Maximum containment equipment (i.e., Class III biosafety cabinet or partial containment in combination with fullbody, air-supplied positive-pressure personnel suit) used for all procedures and activities. Ebola virus; Marburg virus; propagation of herpes virus simiae. A spill kit should be kept in each laboratory where work with microorganisms is conducted. Basic equipment is: concentrated disinfectant (such as chlorine bleach), a package of paper towels, household rubber gloves, autoclave bags, sharps container, and forceps to pick up broken glass General Spill Cleanup Guidelines Wear gloves and lab coat. Use forceps to pick up broken glass and discard into sharps container. Cover spilled material with paper towels. Add diluted disinfectant in sufficient quantity to ensure effective microbial inactivation. Dispose of towels in biohazard waste container. Wipe spill area with diluted disinfectant. Wash hands with soap and water when finished. Specific Spill Cleanup Guidelines 1. Spill of BSL1 material a. Wearing gloves and a lab coat, pick up broken glass with forceps and place in sharps container. b. Absorb the spill with paper towels or other absorbent material. c. Discard these contaminated materials into biohazard waste container. d. Wipe the spill area with the appropriate dilution of a disinfectant effective against the organism. e. Autoclave all towels, gloves, and other materials worn or used to clean up the spill. f. Wash hands with soap and water. 2. Spill of Human Blood a. Wear gloves and lab coat to clean up spill. b. If broken glass is present, use forceps to pick up and place in sharps container. c. Absorb blood with paper towels and discard in biohazard waste container. d. Using a detergent solution, clean the spill site of all visible blood.

12 e. Wipe the spill site with paper towels soaked in a disinfectant such as bleach diluted 1:10 (vol/vol). f. Discard all contaminated materials into biohazard waste container. g. Wash hands with soap and water. 3. Spill of BSL2 material a. Keep other workers out of the area to prevent spreading spilled material. Post warning sign, if needed. b. Remove contaminated clothing and put into a biohazard bag for decontamination later. c. Wash hands and exposed skin and inform the PI of the spill. Call the Biosafety Office at for assistance, if necessary. d. Put on protective clothing (lab coat, gloves and if needed, face protection and shoe covers) and assemble clean-up materials (disinfectant, autoclavable container or bag, forceps, sharps container, and paper towels). e. Pick up broken glass with forceps and dispose into Sharps container. f. Cover the spill with paper towels and add appropriately diluted disinfectant. g. After at least 20 minutes contact time, pick up the paper towels and re-wipe the spill area with diluted disinfectant. h. Collect all contaminated materials into biohazard waste container and autoclave. i. Wash hands with soap and water. 4. Spill of a BSL3 material a. Stop work immediately. b. Avoid inhaling airborne material while quickly leaving the room. Notify others to leave. Close door, and post with warning sign. c. Remove contaminated clothing, turn exposed area inward, and place in a biohazard bag. Wash hands with soap and water. d. Notify the PI. Call the Biosafety Office at (after hours and weekends call 911) for assistance if necessary. e. Allow 30 minutes for aerosols to disperse before re-entering the laboratory to begin cleanup. f. Put on personal protective equipment (PPE) (HEPA filtered respirator, gown, gloves, and shoe covers) and assemble clean-up materials (disinfectant, autoclavable container or bag, forceps, sharps container, and paper towels). g. Contain the spill with absorbent paper towels or disposable pads. Carefully add 10% chlorine bleach to the spill; avoid creating aerosols when pouring the disinfectant. Leave the room and allow 30 minutes for the bleach to inactivate the material. h. Pick up broken glass with forceps and discard in Sharps container. i. Clean up liquid with paper towels and collect all contaminated materials into biohazard bag or container. Remove all spilled materials and decontaminate the area again with an appropriate disinfectant. j. Autoclave (or soak in 10% bleach solution) lab coat, gloves, and other protective equipment that was worn for clean up. k. Wash hands thoroughly with soap and water. 5. Spill in a Biological Safety Cabinet a. Leave the cabinet fan running. b. Wearing gloves and lab coat, spray or wipe cabinet walls, work surfaces, and equipment with disinfectant such as 70% ethanol. If necessary, flood work surface, as well as drain

13 pans and catch basins below the work surface, with disinfectant. Allow at least 20 minutes contact time. c. Soak up the disinfectant and spill with paper towels, and drain catch basin into a container. Lift front exhaust grille and tray, and wipe all surfaces. Ensure that no paper towels or solid debris are blown into area below the grille. d. Surface disinfect all items that may have been spattered before removing them from the cabinet. e. Discard all clean-up materials into biohazard waste container. Wash hands and exposed skin areas with soap and water. f. The Biosafety Office should be notified if the spill overflows into the interior of the cabinet. It may be necessary to do a more extensive decontamination of the cabinet. 6. Spill of Biological Radioactive Material A spill involving both radioactive and biological materials requires emergency procedures that are different from the procedures used for either material alone. As a general rule, disinfect the microorganism using a chemical disinfectant, then dispose of all clean-up materials in a separate bag/container labeled to indicate that the radioisotope is mixed with a chemically disinfected microorganism. Do not use bleach solutions as a disinfectant on materials that contain iodinated compounds, because radioactive iodine gas may be released. Be sure to use procedures to protect yourself from the radionuclide while you disinfect the biological material. Before any clean-up, consider the type of radionuclide, the characteristics of the microorganism, and the volume of the spill. Contact the Radiation Safety Officer (RSO) at for specific radioisotope clean-up procedures. Preparation for Clean-up a. Avoid inhaling airborne material, while quickly leaving the room. Notify others to leave. Close door, and post with warning sign. b. Remove contaminated clothing, turn exposed area inward, and place in a biohazard bag. c. Wash all exposed skin with soap or handwashing antiseptic, followed by a three minute water rinse. d. Inform the PI and the RSO at (40) of the spill, and monitor all exposed personnel for radiation. e. Allow aerosols to disperse for at least 30 minutes before reentering the laboratory. Assemble clean-up materials (disinfectant, autoclavable containers, forceps, paper towels, sharps container). f. Confirm with the RSO that it is safe to enter the lab. Clean-up of a Biological Radioactive Spill a. Put on protective clothing (lab coat, surgical mask, gloves, and shoe covers). Depending on the nature of the spill, it may be advisable to wear a HEPA filtered respirator instead of a surgical mask. In setting up your spill plan, contact DES for advice since the use of many types of respirators requires prior training, fit-testing, and medical approval. b. Pick up any sharp objects with forceps and put in Sharps container labeled according to Radiation Safety guidelines. c. Cover the area with paper towels, and carefully pour diluted disinfectant around and into the spill. Avoid enlarging the contaminated area. Use additional disinfectant as it becomes diluted by the

14 spill. Allow at least 20 minutes contact time. Do not use bleach solutions on iodinated materials: radioiodine gas may be released. Instead, use an alternative disinfectant such as an iodophor. d. Wipe surrounding areas where the spill may have splashed with disinfectant. e. Absorb the disinfectant and spill materials with additional paper towels, and place into an approved radioactive waste container. Keep separate from other radioactive waste. Do not autoclave contaminated waste unless approved by the rso. f. Disinfect contaminated protective clothing prior to disposal as radioactive waste. i. Place contaminated item(s) on absorbent paper and scan for radioactivity. If none is detected, dispose of these items as biohazard waste. ii. If radioactive, spray with disinfectant and allow a 20 minute contact time. iii. Wrap the item(s) inside the absorbent paper and dispose of as radioactive waste. g. Wash hands and exposed skin areas with soap and water, and monitor personnel and spill area for residual radioactive contamination. If skin contamination is detected, repeat decontamination procedures under the direction of the RSO. If spill area has residual activity, determine if it is fixed or removable and handle it accordingly. For Severe Injuries Injury Involving Biological Materials Call 911 for assistance and transportation to the nearest emergency room. Accompany the injured person to the medical facility and provide information to personnel about the accident/exposure. Report accident to the PI and Biosafety Office. For Splash To The Eye Immediately flush the eye with a gentle stream of clean, temperate water for 15 minutes. Hold the eyelid open. Be careful not to wash the contaminant into the other eye. Use an emergency eyewash if one is accessible. Contact the University Health Center (UHC) to obtain care. If UHC is closed, go to the emergency room at the most convenient local emergency hospital. Report the accident to the PI and Biosafety Office, and seek additional medical assistance if necessary. For Contamination To The Body Immediately remove contaminated clothing and drench skin with water. Wash with soap and water, and flush the area for 15 minutes. Contact the UHC at to obtain care. If UHC is closed, go to the emergency room at a local hospital. Report the injury to the PI and to Biosafety Office, and seek additional medical assistance if necessary. Fires Involving Biological Materials

15 Without placing yourself in danger, put biological materials in secure location, such as incubator or freezer. Activate the building fire alarm. Leave the building at once. Call the fire department from a safe location. Meet the fire department outside and direct them to the fire. Decontamination and Disposal of Biological Organisms Sterilization, disinfection, and antisepsis are all forms of decontamination. Sterilization implies the killing of all living organisms. Disinfection refers to the use of antimicrobial agents on inanimate objects; its purpose is to destroy all non-spore forming organisms. Antisepsis is the application of a liquid antimicrobial chemical to living tissue. Chemical Disinfectants Chemical disinfectants are used to render a contaminated material safe for further handling, whether it is a material to be disposed of as waste, or a laboratory bench on which a spill has occurred. It is important to choose a disinfectant that has been proven effective against the organism being used. Chemical disinfectants are registered by the EPA under the following categories: 1. Sterilizer or Sterilant - will destroy all microorganisms including bacterial and fungal spores on inanimate surfaces. 2. Disinfectant - will destroy or irreversibly inactivate specific viruses, bacteria, and pathogenic fungi, but not bacterial spores. 3. Hospital Disinfectant - agent shown to be effective against S. aureus, S. choleresis and P. aeruginosa. It may be effective against M. tuberculosis, pathogenic fungi or specifically named viruses. 4. Antiseptic - agent formulated to be used on skin or tissue - not a disinfectant. Disinfectants Commonly Used in the Laboratory 1. Iodophors o Recommended dilution is 75 ppm, or approximately 4.5 ml/liter water. o Effective against vegetative bacteria, fungi, and viruses. o Effectiveness reduced by organic matter (but not as much as with hypochlorites). o Stable in storage if kept cool and tightly covered. o Built-in color indicator; if solution is brown or yellow, it is still active. o Relatively harmless to humans. 2. Hypochlorites (bleach) o Working dilution is 1:10 to 1:100 in water. o Effective against vegetative bacteria, fungi, most viruses at 1:100 dilution. o Effective against bacterial spores at 1:10 dilution. o Very corrosive. o Rapidly inactivated by organic matter. o Solutions decompose rapidly; fresh solutions should be made daily. 3. Alcohols (ethanol, isopropanol)

16 o The effective dilution is 70-85%. o Effective against a broad spectrum of bacteria and many viruses. o Fast acting. o Leaves no residue. o Non-corrosive. o Not effective against bacterial spores. Important Characteristics of Disinfectants Hypochlorites "Bleach" Iodoform "Wescodyne" Ethyl Alcohol Shelf-life > 1 wk X X Corrosive X X Residue X X Inactivation by Organic X X Matter Skin Irritant X X Respiratory Irritant X Eye Irritant X X X Toxic X X X Dilution of Disinfectants 1. Chlorine compounds (Household Bleach) Dilution in water Available chlorine % Available chlorine ppm Not diluted ,000 1/ ,000 1/ Bleach solutions decompose at room temperature and should be made fresh daily. However, if stored in tightly closed brown bottles, bleach solutions retain activity for 30 days. The use concentration is dependent on the organic load of the material to be decontaminated. Use a 1% solution to disinfect clean surfaces, and 10% solution to disinfect surfaces contaminated with a heavy organic load. To disinfect liquid biological waste before disposal, add concentrated bleach to a final concentration of 1%. 2. Iodophor Manufacturer's recommended dilution is 3 ounces (90 ml) into 5 gallons water, or approximately 4.5 ml/liter. For porous surfaces, use 6 ounces into 5 gallons water.

17 3. Alcohols Ethyl alcohol and isopropyl alcohol diluted to 70-85% in water are useful for surface disinfection of materials that may be corroded by a halogen or other chemical disinfectant. Autoclaving Procedures Autoclaves use pressurized steam to destroy microorganisms, and are the most dependable system available for the decontamination of laboratory waste and the sterilization of laboratory glassware, media, and reagents. For efficient heat transfer, steam must flush the air out of the autoclave chamber. Before using the autoclave, check the drain screen at the bottom of the chamber and clean if blocked. If the sieve is blocked with debris, a layer of air may form at the bottom of the autoclave, preventing efficient operation. Container Selection Polypropylene bags. Commonly called biohazard or autoclave bags, these bags are able to withstand autoclaving and are tear resistant, but can be punctured or burst in the autoclave. Therefore, place bags in a rigid container such as a polypropylene or stainless steel pan during autoclaving. Bags are available in a variety of sizes, and some are printed with an indicator that changes color when processed. Polypropylene bags are impermeable to steam, and for this reason should not be twisted and taped shut, but gathered loosely at the top and secured with a large rubber band or autoclave tape. This will create an opening through which steam can penetrate. Polypropylene containers and pans. Polypropylene is a plastic capable of withstanding autoclaving, but resistant to heat transfer. Therefore, materials contained in a polypropylene pan will take longer to autoclave than the same materials in a stainless steel pan. To decrease the time required to sterilize material in these containers, o remove the lid (if applicable). o turn the container on its side when possible. o select a container with the lowest sides and widest diameter possible for the autoclave. Stainless steel containers and pans. Stainless steel is an efficient conductor of heat and is less likely to increase sterilizing time, though is more expensive than polypropylene. Preparation and Loading of Materials Fill liquid containers only half full. Loosen caps, or use vented closures. Always put bags of biological waste into pans to catch spills. Position biohazard bags on their sides, with the bag neck taped loosely. Leave space between items to allow steam circulation. Household dishpans melt in the autoclave. Use autoclavable polypropylene or stainless steel pans. Cycle Selection Use liquid cycle (slow exhaust) when autoclaving liquids, to prevent contents from boiling over. Select fast exhaust cycle for glassware.

18 Use fast exhaust and dry cycle for wrapped items. Time Selection Take into account the size of the articles to be autoclaved. A 2-liter flask containing 1 liter of liquid takes longer to sterilize than four 500 ml flasks each containing 250 ml of liquid. Material with a high insulating capacity (animal bedding, high-sided polyethylene containers) increases the time needed for the load to reach sterilizing temperatures. Bags of biological waste should be autoclaved for 50 minutes to assure decontamination. Removing the Load Check that the chamber pressure is zero. Wear lab coat, eye protection, heat insulating gloves, and closed-toe shoes. Stand behind door when opening it. Slowly open door only a crack. Beware of rush of steam. After the slow exhaust cycle, open autoclave door and allow liquids to cool for 20 minutes before removing. Monitoring Autoclaves used to decontaminate laboratory waste should be tested periodically to assure effectiveness. Two types of tests are used: 1) a chemical indicator that fuses when the temperature reaches 121 C, and 2) heat-resistant spores (Bacillus stearothermophilis) that are killed by exposure to 121 C for approximately 15 minutes. Both types of tests should be placed well down in the center of the bag or container of waste, at the point slowest to heat. The chemical test should be used first to determine that the temperature in the center of the container reaches 121 C. Ampules of heat-resistant spores should be used in subsequent test runs to determine the amount of time necessary to achieve sterilization. If you need assistance, please contact the Biosafety Office Autoclave Safety Caution - Autoclaves May Cause Serious Burns To Prevent Injury: Loosen screw caps on bottles and tubes of liquids before autoclaving. Check that chamber pressure has returned to zero before opening door. Wear eye and face protection. Stand behind door when opening it. Slowly open door only a crack. Beware rush of steam. Keep face away from door as it opens. Escaping steam may burn face. Wait 5 minutes after opening door before removing liquids. Liquids removed too soon may boil up and out of container, burning operator.

19 Use and Disposal of Sharps To prevent needle stick injuries: Avoid using needles whenever possible. Do not bend, break, or otherwise manipulate needles by hand. Do not recap needles by hand. Do not remove needles from syringes by hand. Immediately after use, discard needle and syringe (whether contaminated or not) into puncture resistant sharps containers. Never discard sharps into regular trash. Never discard sharps into bags of biological waste. Use care and caution when cleaning up after procedures that require the use of syringes and needles. Do not overfill the sharps containers. Close completely when they are 3/4 full. Locate sharps containers in areas in which needles are commonly used. Make containers easily accessible. Sharps containers may be purchased from VWR and Fisher Scientific and other scientific supply vendors. In the event of a needle stick injury: Wash thoroughly with soap and water. Notify supervisor and go immediately to Urgent Care Clinic at UHC. If UHC is closed, go to the emergency room at the most convenient local emergency room. Biological Waste Disposal Procedures Please read and follow the Waste Disposal Guidelines wall chart. I. Biological Waste A. All biological waste from BSL1, BSL2, and BSL3 laboratories must be decontaminated prior to disposal. B. Decontamination and disposal are the responsibility of the person/laboratory generating the waste. 1. Collect disposable, solid materials contaminated by an infectious agent, excluding sharps, or broken or unbroken glass, into an autoclave bag within a sturdy container. When full, these bags are autoclaved, cooled, and then placed in the building's dumpster. 2. Decontaminated liquids containing a biological agent by the addition of a chemical disinfectant such as sodium hypochlorite (household bleach) or an iodophor, or by autoclaving, then dispose of by pouring down the sink. It is not necessary to autoclave liquids that have been chemically disinfected. However, if bleach has been used in the tray used to collect labware that will later be autoclaved, sodium thiosulfate must be added to the bleach to prevent the release of chlorine gas during autoclaving.

20 II. Reusable Labware Items such as culture flasks and centrifuge bottles are decontaminated by lab personnel before washing by one of two methods. 1. Autoclave items that have been collected in autoclavable container. 2. Chemically disinfect items by soaking in diluted disinfectant for one hour before washing. III. Disposal of Blood Products and Body Fluids A. All human blood and other potentially infectious materials should be handled using Universal Precautions. B. Discard disposable items contaminated with human blood or body fluids (excluding sharps and glassware) into the incinerator boxes. Do not overfill boxes or use without the plastic liners provided with them. These boxes may be used for temporary storage and accumulation of waste. When full, close and seal the plastic liner and box. C. Biological waste pickup request forms may be filled out and submitted electronically from the Biosafety Webpage at Biosafety Office will contact you and make the necessary action for disposal. IV. Disposal of Sharps and Disposable Glassware A. Discard all needles, needle and syringe units, scalpels, and razor blades, whether contaminated or not, directly into rigid, red, labeled sharps containers. Do not recap, bend, remove or clip needles. Sharps containers should not be overfilled. To request pickup of sharps containers, fill out and submit a biological waste pickup request form from the Biosafety Office web page at: Alternatively, closed sharps containers may be packaged in incinerator boxes (Section III above). Sharps containers may be purchased from VWR and Fisher Scientific, and other scientific supply vendors. B. Uncontaminated pasteur pipets and broken or unbroken glassware are discarded into containers specifically designed for broken glass disposal, or into heavy-duty cardboard boxes that are closeable. When boxes are full, tape closed and place in the building's dumpster. C. Contaminated pasteur pipets, and broken or unbroken glassware may be treated in one of two ways: 1. Discarded into approved sharps containers, as in Section A above, or 2. Decontaminated by autoclaving or chemical disinfection, then discarded into glass disposal boxes as in Section B above. D. Sharps that are contaminated with radioactive materials or hazardous chemicals should be discarded into separate sharps containers labeled with the name of the isotope or chemical. Contact Biosafety Office for disposal information. V. Multi-hazard or Mixed Waste A. Avoid generating mixed waste if possible. Keep volume to minimum. B. Do not autoclave mixed waste.

21 C. When discarding waste containing an infectious agent and radioactive material, inactivate the infectious agent first, then dispose of as radioactive waste. Seek advice from the RSO at before beginning inactivation procedures. D. When discarding waste containing an infectious agent and a hazardous chemical, inactivate the infectious agent first, then dispose of as chemical waste. Seek advice before beginning inactivation procedures. Contact Biosafety Office at for instructions. VI. Disposal of Animal Tissues, Carcasses and Bedding A. Disposal of animal carcasses/tissues is coordinated through the Central Facilities Management. 1. Place animal carcasses/tissues into plastic bag. Double-bag when carcass contains zoonotic agent (transmissible from animals to humans). 2. Place bag in freezer until pick-up. 3. Call Facilities Management at for pick-up. B. Disposal of animal carcasses/tissues that are contaminated with radioactive materials or biohazardous chemicals is through Biosafety Office. VII. Disposal Containers Each laboratory is responsible for purchasing containers for the disposal of biological waste. The following types of containers are available: A. Sharps containers may be purchased from local sources (including Chemistry Stores) as well as from laboratory product distributors. They are available in various sizes, and should be puncture resistant, red, labeled as "Sharps," and have a tightly closing lid. Do not purchase "needle-cutter" devices, which may produce aerosols when used. B. Biohazard Autoclave Bags may be purchased from various laboratory product distributors, such as Fisher Scientific, VWR, and Baxter. Be sure to select polypropylene bags which are able to withstand autoclaving. They should be placed inside a rigid container with lid while waste is being collected. C. Incinerator Boxes must be used. A plastic liner must be used to prevent contamination of the box. D. Glass Disposal Boxes may be purchased from Chemistry Stores and various laboratory product distributors. Alternatively, heavy-duty, closeable cardboard boxes may be used for disposal of broken glass. VIII. What to do with Filled Waste Containers A. Sharps containers and incinerator boxes - To request pickup, facilities management B. Biohazard autoclave bags and glass disposal boxes - close and autoclave bags, tape boxes closed; put both in building dumpster.

22 Instructions Biological, Pathological or Medical Waste Removal Request Form Biological Safety Cabinets Biosafety Equipment The biological safety cabinet (BSC) is designed to provide protection to the product, the user, and the environment when appropriate practices and procedures are followed. Three types of BSCs (Class I, II, III) and the horizontal laminar flow cabinet are described below. The common element to all classes of BSCs is the high efficiency particulate air (HEPA) filter. This filter removes particulates of 0.3 microns with an efficiency of 99.97%. However, it does not remove vapors or gases. The biosafety cabinet requires regular maintenance and certification by a professional technician to assure that it protects you, your experiments, and the environment. Each cabinet should be certified when it is installed, each time it is moved or repaired, and at least annually. Contact Biosafety Office at to confirm that your cabinet is included in this program. 1. Class I cabinets protect personnel and the environment, but not research materials. They provide an inward flow of unfiltered air, similar to a chemical fume hood, which protects the worker from the material in the cabinet. The environment is protected by HEPA filtration of the exhaust air before it is discharged into the laboratory or to the outside via the building exhaust. 2. Class II (Types A1, A2, B1, B2, and B3) BSCs provide personnel, environment, and product protection. Air is drawn around the operator into the front grille of the cabinet, which provides personnel protection. In addition, the downward laminar flow of HEPA-filtered air within the cabinet provides product protection by minimizing the chance of cross-contamination along the work surface of the cabinet. Because cabinet air passes through the exhaust HEPA filter, it is contaminant-free (environmental protection), and may be recirculated back into the laboratory (Type A) or ducted out of the building (Type B). 3. Class III cabinets (sometimes called Class III glove boxes) were designed for work with infectious agents that require BSL4 containment, and provide maximum protection to the environment and the worker. The cabinet is gas-tight with a non-opening view window, and has rubber gloves attached to ports in the cabinet that allow for manipulation of materials in the cabinet. Air is filtered through one HEPA filter as it enters the cabinet, and through 2 HEPA filters before it is exhausted to the outdoors. This type of cabinet provides the highest level of product, environmental, and personnel protection. 4. Horizontal laminar flow "clean air benches" are not BSCs. They discharge HEPA-filtered air across the work surface and toward the user, providing only product protection. They can be used for certain clean activities, such as dust-free assembly of sterile equipment or electronic devices. However, they should never be used when handling cell culture materials or potentially infectious materials, or as a substitute for a BSC in research laboratories.