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Biosafety Manual

2015

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Policy Statement

It is the policy of Northern Arizona University (NAU) to provide a safe working environment. The primary responsibility for insuring safe conduct and conditions in the laboratory resides with the principal investigator.

The Biological Safety Office is committed to providing up-to-date information, training, and monitoring to the research and biomedical community concerning the safe conduct of biological, recombinant, and acute toxin research and the handling of biological materials in accordance with all pertinent local, state and federal regulations, guidelines, and laws. To that end, this manual is a resource, to be used in conjunction with the CDC and NIH guidelines, the NAU Select Agent Program, Biosafety in Microbiological and Biomedical Laboratories (BMBL), and other resource materials.

Introduction

This Biological Safety Manual is intended for use as a guidance document for researchers and clinicians who work with biological materials. It should be used in conjunction with the Laboratory-Specific Safety Manual, which provides more general safety information. These manuals describe policies and procedures that are required for the safe conduct of research at NAU. The NAU Personnel Policy on Safety 5.03 also provides guidance for safety in the workplace.

Responsibilities

In the academic research/teaching setting, the principal investigator (PI) is responsible for ensuring that all members of the laboratory are familiar with safe research practices. In the clinical laboratory setting, the faculty member who supervises the laboratory is responsible for safety practices.

Lab managers, supervisors, technicians and others who provide supervisory roles in laboratories and clinical settings are responsible for overseeing the safety practices in laboratories and reporting any problems, accidents, and spills to the appropriate faculty member.

Employees who work with biological materials are responsible for reading this manual and carrying out the safety practices outlined here.

The Biosafety Office will provide guidance, information, review, monitoring, and training regarding biological safety programs, when appropriate. This includes implementing registration activities for certain research projects, acting as a consultant for departments regarding implementation and enforcement of biological safety programs, evaluating work practices and personal protective equipment, providing educational materials, tracking employee training, and medical monitoring.

The mission of the EH&S Biosafety Office is to minimize injury to faculty, staff, students and visitors and to minimize damage to University property. Inherent in this mission is the charge to provide a safe and healthy environment in which the University’s activities can be pursued.

All applicable federal and state safety laws, rules and regulations are adopted by the University. In order to carry out its duties and responsibilities, the EH&S Biosafety Office will adhere to standards or codes related to biosafety which have been adopted and promulgated by nationally recognized standards-setting organizations. The interpretation of biosafety regulations and guidelines is the responsibility of the EH&S Biosafety Office.In order to assure an effective Biosafety Program for the Northern Arizona University, it is imperative that all individuals associated with the University comply fully with the policies and procedures set forth in the manual.

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Emergency Phone Numbers

General

University Police Department 523-3000

Flagstaff Fire Department 911

Gas leak 911

Spills/Accidents

Biological or Recombinant Materials 480-248-0741

Chemicals (laboratory) 928-607-9995

Radioactive Materials 928-220-1388

Select Agents 480-248-0741

Incidents involving Air Monitoring 928-220-1728

Medical Emergency

Campus Health Services 523-2131

Flagstaff Medical Center Emergency 779-3366

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Table of Contents 1 – Biological Safety............................................................................................................. 10

Principles of Biological Safety........................................................................................10Laboratory Practice and Technique...............................................................................10Safety Equipment (Primary Barrier)..............................................................................11Facility Design (Secondary Barrier)..............................................................................11Biosafety Levels............................................................................................................ 12Animal Facilities........................................................................................................... 14Clinical Laboratories..................................................................................................... 14Importation and Interstate Shipment of Certain Biomedical Materials...........................15

Biological Safety Levels................................................................................................... 15Biosafety Level 1.......................................................................................................... 15Biosafety Level 2.......................................................................................................... 17Biosafety Level 3.......................................................................................................... 20Animal Biosafety Level 1 (ABSL-1).............................................................................25Animal Biosafety Level 2 (ABSL-2).............................................................................27Animal Biosafety Level 3 (ABSL-3).............................................................................30

Agents List....................................................................................................................... 37Biological Safety Level 1 (BSL-1)................................................................................37Biological Safety Level 2 (BSL-2)................................................................................37

BSL-2 - Bacterial Agents Including Chlamydia.....................................................38BSL-2 - Fungal Agents..........................................................................................39BSL-2 - Parasitic Agents........................................................................................ 39BSL-2 - Viruses..................................................................................................... 40

Biological Safety Level 3 (BSL-3)................................................................................42BSL-3 - Bacterial Agents Including Rickettsia.......................................................42BSL-3 - Fungal Agents..........................................................................................42BSL-3 - Parasitic Agents........................................................................................ 42BSL-3 - Viruses and Prions....................................................................................43

Biological Safety Level 4 (BSL-4)................................................................................44BSL-4 - Bacterial Agents.......................................................................................44BSL-4 - Fungal Agents..........................................................................................44BSL-4 - Parasitic Agents........................................................................................ 44BSL-4 - Viral Agents.............................................................................................44

2 - Information for Researchers........................................................................................... 45

Project Registration........................................................................................................ 45Biological Agent (BA) Registration...............................................................................45Recombinant DNA (R-DNA) Registration....................................................................45Acute Toxins (AT) Registration....................................................................................46Regulated Biological Materials......................................................................................46Project Amendment....................................................................................................... 46

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Typical changes include:............................................................................................... 46Table 3: Toxin Table..................................................................................................... 47Select Agents................................................................................................................. 48

Minors in Research Laboratories or Animal Facilities.................................................50

Responsibilities of the Principle Investigator during the Conduct of the Research... .51Reportable Incidents and Violations..............................................................................52Institutional Reporting Responsibilities.........................................................................52

Biological Waste Disposal Policy.........................................................................................53

Categories........................................................................................................................ 531) Infectious/potentially infectious/R-DNA................................................................532) Non-infectious waste.............................................................................................53

3) Mixed radioactive/biohazardous waste...............................................................534) Mixed chemical/biohazardous waste..................................................................545) Animal carcasses and materials..........................................................................546) Human remains.................................................................................................. 54

Packaging...................................................................................................................... 541) Biohazard bags................................................................................................... 542) Sharps................................................................................................................ 543) Containers........................................................................................................ 54

Labeling........................................................................................................................ 541) Date................................................................................................................... 542) Name/Location/Phone Number..........................................................................553) Biohazard sign................................................................................................... 55

Transport....................................................................................................................... 55Training......................................................................................................................... 55

Biological Waste Disposal Containers.................................................................................. 55Bulk Biohazard Receptacles..........................................................................................56Sharps Boxes................................................................................................................. 56Biohazard Bags.............................................................................................................. 56

Autoclave Use and Testing................................................................................................... 57Autoclave Testing.......................................................................................................... 57Record-Keeping............................................................................................................. 58Autoclave Operating Procedures.................................................................................... 58Autoclave Operation and Safety Training......................................................................59Autoclave Guidelines....................................................................................................60

Disinfectants......................................................................................................................... 61Liquids.......................................................................................................................... 61

Alcohols................................................................................................................. 61Chlorine compounds.............................................................................................. 62Formaldehyde........................................................................................................ 62Glutaraldehyde....................................................................................................... 62Hydrogen peroxide................................................................................................. 62Iodine and Iodophors.............................................................................................. 62Phenol and phenolic compounds............................................................................63

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Quatenary ammonium compounds.........................................................................63Gases............................................................................................................................. 63

Ethylene Oxide (Not currently allowed at NAU)....................................................63Vapor Phase Hydrogen Peroxide............................................................................63Chlorine Dioxide gas.............................................................................................. 63Ozone..................................................................................................................... 63Formaldehyde Gas (from heating paraformaldehyde).............................................64

Irradiation...................................................................................................................... 64Ultraviolet, UV radiation........................................................................................ 64Ionizing radiation................................................................................................... 64Electron Beam........................................................................................................ 64Microwaves............................................................................................................ 64

Disinfectants Bibliography............................................................................................ 64Shipment of Biological Materials......................................................................................... 69Laboratory Spills.................................................................................................................. 70

Spill in the Open Laboratory.........................................................................................70Spill in a Biological Safety Cabinet...............................................................................71

Biological Safety Cabinets.................................................................................................... 72Biological Safety Cabinets (BSCs)................................................................................72

The Class I BSC..................................................................................................... 72The Class II BSC.................................................................................................... 72The Class II, Type A BSC...................................................................................... 73The Class II, Type B1 BSC....................................................................................73The Class II, Type B2 BSC....................................................................................73The Class II, Type B3 BSC....................................................................................74The Class III BSC.................................................................................................. 74

Horizontal Laminar Flow “Clean Bench”......................................................................74Vertical Laminar Flow “Clean Bench”..........................................................................74Operations within a Class II BSC..................................................................................75

Laboratory Hazards................................................................................................ 75Decontamination........................................................................................................... 76

Surface Decontamination.......................................................................................76Gas Decontamination.............................................................................................77

Engineering Requirements............................................................................................. 77Ultraviolet Lamps..................................................................................................77BSC Placement...................................................................................................... 77HEPA Filters.......................................................................................................... 77Certification of BSCs.............................................................................................77

3 – Programs........................................................................................................................ 78Animal Contact Medical Monitoring Program....................................................................78

NAU Bloodborne Pathogen Program.............................................................................80BBP Exposure Information....................................................................................80

4 -Medical Surveillance........................................................................................................ 81

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HIV Research Laboratory Occupational Medicine Policy............................................82Pre-employment............................................................................................................ 82Continuing employment................................................................................................ 82Post-exposure prophylaxis............................................................................................. 82References..................................................................................................................... 83

Immunoprophylaxis........................................................................................................ 84Recommendations for Prophylactic Immunization of Laboratory Personnel Working with Infectious Agents........................................................................................................... 85

Bacterial agents...................................................................................................... 85Rickettsial agents................................................................................................... 86Viral agents............................................................................................................ 86

Vaccinia Immunization Policy........................................................................................ 87Blood Serum Sampling.................................................................................................. 88Health Assessments....................................................................................................... 88Exposure to Mycobacterium Tuberculosis.....................................................................88

5 – Appendices - Information for Researchers.....................................................................89

Transgenic Animals......................................................................................................... 89

List of Tables

Table 1: Summary of Recommended Biosafety Levels for Infectious Agents........................35Table 2: Summary of Recommended Biosafety Levels for Activities in Which

Experimentally or Naturally Infected Vertebrate Animals Are Used..............................36Table 3: Toxin Table............................................................................................................. 48Table 4: Dilutions of Household Bleach................................................................................62Table 5: Summary and Comparison of Liquid Disinfectants (Page 1)....................................65Table 6: Summary of Practical Disinfectants.........................................................................67Table 7: Reprocessing Methods for Equipment Used in the Health Care Setting...................68

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1 – Biological Safety

Principles of Biological SafetyThe following is from Biosafety in Microbiological and Biomedical Laboratories, 2009, HHS publication No. (CDC) 21-1112, Centers for Disease Control & Prevention/National Institutes of Health

The term “containment” is used in describing safe methods for managing infectious agents in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents.

Both good microbiological technique and the use of appropriate safety equipment provide primary containment, the protection of personnel and the immediate laboratory environment from exposure to infectious agents. The use of vaccines may provide an increased level of personal protection. Secondary containment, the protection of the environment external to the laboratory from exposure to infectious materials, is provided by a combination of facility design and operational practices. Therefore, the three elements of containment include laboratory practice and technique, safety equipment, and facility design. The risk assessment of the work to be done with a specific agent will determine the appropriate combination of these elements.

Laboratory Practice and TechniqueThe most important element of containment is strict adherence to standard microbiological practices and techniques. Persons working with infectious agents or potentially infectious materials must be aware of potential hazards, and must be trained and proficient in the practices and techniques required for handling such material safely. The director or person in charge of the laboratory is responsible for providing or arranging for appropriate training of personnel.

Each laboratory should develop or adopt a biosafety or operations manual that identifies the hazard that will or may be encountered, and which specifies practices and procedures designed to minimize or eliminate risks. Personnel should be advised of special hazards and should be required to read and to follow the required practices and procedures. A scientist trained and knowledgeable in appropriate laboratory techniques, safety procedures, and hazards associated with handling infectious agents must direct laboratory activities.

When standard laboratory practices are not sufficient to control the hazard associated with a particular agent or laboratory procedure, additional measures may be needed. The laboratory director is responsible for selecting additional safety practices, which must be in keeping with the hazard associated with the agent or procedure.

Laboratory personnel, safety practices, and techniques must be supplemented by appropriate facility design and engineering features, safety equipment, and management practices.

Safety Equipment (Primary Barrier)Safety equipment includes biological safety cabinets (BSCs), enclosed containers, and other

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engineering controls designed to remove or minimize exposures to hazardous biological materials. The biological safety cabinet (BSC) is the principal device used to provide containment of infectious splashes or aerosols generated by many microbiological procedures. Three types of BSCs (Class I, II, III) used in microbiological laboratories are described in Section 2. Open-fronted Class I and Class II BSCs are primary barriers which offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques. The Class II BSC also provides protection from external contamination of the materials (e.g., cell cultures, microbiological stocks) being manipulated inside the cabinet. The gas-tight Class III BSC provides the highest attainable level of protection to personnel and the environment.

An example of another primary barrier is the safety centrifuge cup, an enclosed container designed to prevent aerosols from being released during centrifugation. To minimize this hazard, containment controls such as BSC's or centrifuge cups must be used for handling infectious agents that can be transmitted through the aerosol route of exposure.

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

Facility Design (Secondary Barrier)The design of the facility is important in providing a barrier to protect persons working inside and outside of the laboratory within the facility, and to protect persons or animals in the community from infectious agents that may be accidentally released from the laboratory. Laboratory management is responsible for providing facilities commensurate with the laboratory's function and the recommended biosafety level for the agents being manipulated.

The recommended secondary barrier(s) will depend on the risk of transmission of specific agents. For example, the exposure risks for most laboratory work in Biosafety Level 1 and 2 facilities will be direct contact with the agents, or inadvertent contact exposures through contaminated work environments. Secondary barriers in these laboratories may include separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and hand washing facilities.

As the risk for aerosol transmission increases, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Such design features could include specialized ventilation systems to assure directional air flow, air treatment systems to decontaminate or remove agents from exhaust air, controlled access zones, airlocks as laboratory entrances, or separate buildings or modules for isolation of the laboratory. Design engineers for laboratories may refer to specific ventilation

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recommendations as found in the Applications Handbook for Heating, Ventilation, and Air-conditioning (HVAC) published by the American Society of Heating, Refrigerating, and Air-conditioning Engineers (ASHRAE).

Biosafety LevelsFour biosafety levels (BSLs) are described which consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities. Each combination is specifically appropriate for the operations performed, the documented or suspected routes of transmission of the infectious agents, and for the laboratory function or activity.

The recommended BSLs for the organisms represent those conditions under which the agent can ordinarily be safely handled. The Principle Investigator (PI) is specifically and primarily responsible for assessing risks and for appropriately applying the recommended biosafety levels. Generally, work with known agents should be conducted at the biosafety level recommended. When specific information is available to suggest that virulence, pathogenicity, antibiotic resistance patterns, vaccine and treatment availability, or other factors are significantly altered, more (or less) stringent practices may be specified.

Biosafety Level 1 practices, safety equipment, and facilities are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other facilities in which work is done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans. Bacillus subtilis, Naegleria gruberi, and infectious canine hepatitis virus are representative of those microorganisms meeting these criteria. Many agents not ordinarily associated with disease processes in humans are, however, opportunistic pathogens and may cause infection in the young, the aged, and immunodeficient or immunosuppressed individuals. Vaccine strains which have undergone multiple in vivo passages should not be considered avirulent simply because they are vaccine strains.

Biosafety Level 1 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.

Biosafety Level 2 practices, equipment, and facilities are applicable to clinical, diagnostic, teaching, and other facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Salmonellae, clinical samples of Hepatitis B, and Toxoplasma spp. are representative of microorganisms assigned to this containment level. Biosafety Level 2 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. (Laboratory personnel working with human-derived materials should refer to the Bloodborne Pathogen Standard for specific, required precautions).

Primary hazards to personnel working with these agents relate to

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accidental percutaneous or mucous membrane exposures, or ingestion of infectious materials. Extreme precaution with contaminated needles or sharp instruments must be emphasized. Even though organisms routinely manipulated at BSL-2 are not known to be transmissible by the aerosol route, procedures with aerosol or high splash potential that may increase the risk of such personnel exposure must be conducted in primary containment equipment, or devices such as a BSC or safety centrifuge cups. Other primary barriers should be used, as appropriate, such as splash shields, face protection, gowns, and gloves.

Secondary barriers such as hand washing and waste decontamination facilities must be available to reduce potential environmental contamination.

Biosafety Level 3 practices, safety equipment, and facilities are applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with a potential for respiratory transmission, and which may cause serious and potentially lethal infection. Hepatitis B. (propagation of high concentrations only), Mycobacterium tuberculosis, St. Louis encephalitis virus, and Coxiella burnetii are representative of microorganisms assigned to this level. Primary hazards to personnel working with these agents relate to autoinoculation, ingestion, and exposure to infectious aerosols.

At Biosafety Level 3, more emphasis is placed on primary and secondary barriers to protect personnel in contiguous areas, the community, and the environment from exposure to potentially infectious aerosols. For example, all laboratory manipulations should be performed in a BSC or other enclosed equipment, such as a gas-tight aerosol generation chamber. Secondary barriers for this level include controlled access to the laboratory and a specialized ventilation system that minimizes the release of infectious aerosols from the laboratory.

Biosafety Level 4 practices, safety equipment, and facilities are applicable for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, which may be transmitted via the aerosol route, and for which there is no available vaccine or therapy. Additionally, agents with a close or identical antigenic relationship to Biosafety Level 4 agents should also be handled at this level. When sufficient data are obtained, work with these agents may continue at this level or at a lower level. Viruses such as Marburg or Congo-Crimean hemorrhagic fever are manipulated at Biosafety Level 4.

The primary hazards to personnel working with Biosafety Level 4 agents are respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets, and autoinoculation. All manipulations of potentially infectious diagnostic materials, isolates, and naturally or experimentally infected animals pose a high risk of exposure and infection to laboratory personnel, the community, and the environment.

The laboratory worker's complete isolation of aerosolized infectious materials is accomplished primarily by working in a Class III BSC or a full-body, air-supplied positive-pressure personnel suit. The Biosafety Level 4 facility itself is generally a separate building or completely isolated zone with complex, specialized ventilation and waste management systems to prevent release of viable agents to the environment.

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The laboratory director is specifically and primarily responsible for the safe operation of the laboratory. His/her knowledge and judgment are critical in assessing risks and appropriately applying these recommendations. The recommended biosafety level represents those conditions under which the agent can ordinarily be safely handled. Special characteristics of the agents used, the training and experience of personnel, and the nature or function of the laboratory may further influence the director in applying these recommendations.

Animal FacilitiesFour biosafety levels are also described for activities involving infectious disease work with experimental mammals. These four combinations of practices, safety equipment, and facilities are designated Animal Biosafety Levels 1, 2, 3, and 4, and provide increasing levels of protection to personnel and the environment.

Clinical LaboratoriesClinical laboratories, especially those in health care facilities, receive clinical specimens with requests for a variety of diagnostic and clinical support services. Typically, the infectious nature of clinical material is unknown, and specimens are often submitted with a broad request for microbiological examination for multiple agents (e.g., sputa submitted for “routine,” acid-fast, and fungal cultures). It is the responsibility of the laboratory director to establish standard procedures in the laboratory that realistically address the issue of the infectious hazard of clinical specimens.

Except in extraordinary circumstances (e.g., suspected hemorrhagic fever), the initial processing of clinical specimens and identification of isolates can be done safely at Biosafety Level 2, the recommended level for work with bloodborne pathogens such as hepatitis B virus and HIV. The containment elements described in Biosafety Level 2 are consistent with the Occupational Exposure to Bloodborne Pathogens Standard from the Occupational Safety and Health Administration (OSHA), that requires the use of specific precautions with all clinical specimens of blood or other potentially infectious material (Universal Precautions). Additionally, other recommendations specific for clinical laboratories may be obtained from the National Committee for Clinical Laboratory Standards.

Biosafety Level 2 recommendations and OSHA requirements focus on the prevention of percutaneous and mucous membrane exposures to clinical material. Primary barriers such as biological safety cabinets (Class I or II) should be used when performing procedures that might cause splashing, spraying, or splattering of droplets. Biological safety cabinets should also be used for the initial processing of clinical specimens when the nature of the test requested or other information is suggestive that an agent readily transmissible by infectious aerosols is likely to be present (e.g., M. tuberculosis), or when the use of a biological safety cabinet (Class II) is indicated to protect the integrity of the specimen.

The segregation of clinical laboratory functions and limiting or restricting access to such areas is the responsibility of the laboratory director. It is also the director's responsibility to establish standard, written procedures that address the potential hazards and the required precautions to be

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implemented.

Importation and Interstate Shipment of Certain Biomedical MaterialsThe importation of etiologic agents and vectors of human diseases is subject to the requirements of the Public Health Service Foreign Quarantine regulations. Companion regulations of the Public Health Service and the Department of Transportation specify packaging, labeling, and shipping requirements for etiologic agents and diagnostic specimens shipped in interstate commerce.

The U. S. Department of Agriculture regulates the importation and interstate shipment of animal pathogens and prohibits the importation, possession, or use of certain exotic animal disease agents that pose a serious disease threat to domestic livestock and poultry.

USDA/APHIS permits are required for both importation and within USA transport of many hazardous biological agents. EH&S maintains all permits and shall be contacted for information related to any transport to and from NAU. Please see the section entitled “Shipment of Biological Materials” for more information.

Biological Safety LevelsThe following is from Biosafety in Microbiological and Biomedical Laboratories, 2009, HHS publication No. (CDC) 21-1112, Centers for Disease Control & Prevention/National Institutes of Health

Biosafety Level 1Biosafety Level 1 is suitable for work involving well-characterized agents not known to 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 not 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 the handling of agents assigned to Biosafety Level 1:

A. Standard Microbiological Practices1. 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.

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3. Eating, drinking, use of tobacco products, 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 that 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 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 from the laboratory are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility.

8. An insect and rodent control program is in effect.

B. Special Practices: None

C. 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 exists.4. Protective eyewear should be worn for anticipated splashes of microorganisms or

other hazardous materials to the face.

D. Laboratory Facilities (Secondary Barriers)1. Each laboratory contains a sink for hand washing.2. The laboratory is designed so that it can be easily cleaned. Walls and floors must

be constructed of water impervious materials that will stand up to hard disinfectants. Carpeting is not allowed in laboratories.

3. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

4. Laboratory furniture is sturdy and should not be cloth upholstered. Spaces between benches, cabinets, and equipment are accessible for cleaning.

5. If the laboratory has windows that open, they are fitted with fly screens.

Biosafety Level 2Biosafety Level 2 is similar to Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs in four ways.

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1. Laboratory personnel must have specific training in handling pathogenic agents and should be directed by competent scientists.

2. Access to the laboratory must be limited when work is being conducted. 3. Extreme precautions must be taken with contaminated sharp items. 4. Certain procedures in which infectious aerosols or splashes may be created must be

conducted in biological safety cabinets or other physical containment equipment.

The following standard and special practices, safety equipment, and facilities apply to the use of agents assigned to Biosafety Level 2:


director when experiments 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, the use of tobacco products, handling contact lenses, and

applying cosmetics 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 for that 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 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 from the laboratory are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility.


B. Special Practices1. Access to the laboratory is limited or restricted by the laboratory director when

work with infectious agents is in progress. In general, persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. The laboratory director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.

2. The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet specific entry requirements (e.g., immunization) enter the laboratory or animal rooms.

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3. When the infectious agent(s) in use in the laboratory require special provisions or entry (e.g., immunization), a hazard warning sign incorporating the universal biohazard symbol is posted on the access door to the laboratory work area. The hazard warning sign identifies the infectious agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates the special requirement(s) for entering 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. A Biosafety Manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.

7. Laboratory 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 scalpels. 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. Plasticware should be substituted for glassware whenever possible.a. 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, preferable by autoclaving.

b. Syringes that re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

c. 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, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.

10. Laboratory equipment and work surfaces should be decontaminated with an appropriate 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. Equipment must also be

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decontaminated before removal from the facility when it must be packaged for transport. Packaging and shipment shall be in accordance with applicable local, state, or federal regulations.

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 work being performed aren’t permitted in the lab.

C. 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 under the following conditions. a. Use Biological Safety Cabinets 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 eggs.

b. Use Biological Safety Cabinets whenever 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 guards) is required to prevent splashes or sprays of infectious or other hazardous materials to the face, when the microorganisms must be manipulated outside the BSC.

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 required when handling infected animals and when hands may contact infectious materials, contaminated surfaces or equipment. Wearing two pairs of gloves may be appropriate; if a spill or splatter occurs, the hand will be protected after the contaminated glove is removed. Gloves are disposed of when contaminated, removed when work with infectious materials is completed, and are never worn outside the laboratory. Disposable gloves are not washed or reused.

D. Laboratory Facilities (Secondary Barriers)1. Each laboratory contains a sink for hand washing.2. The laboratory is designed so that it can be easily cleaned. Walls and floors

should be smooth, hard and constructed of water impervious material that will stand up to harsh disinfectants. Carpeting is not allowed in laboratory facilities.

3. Bench tops should be smooth, hard and impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

Biosafety Manual 19

4. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning. No cloth or fabric seating is permitted.

5. If the laboratory has windows that open, they are fitted with fly screens.6. A method for decontamination of infectious or regulated laboratory wastes is

available (e.g., autoclave, chemical disinfection, incinerator, or other approved decontamination system).

7. An eyewash/safety shower facility is readily available.8. All laboratories require single pass air that is not then recirculated to any other

area of the facility. Laboratories should be negative pressure to surrounding areas to prevent accidental spread of potentially infectious or recombinant agents.

9. 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.

Biosafety Level 3Biosafety Level 3 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.

All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features.

It is recognized, however, that many existing facilities may not have all the facility safeguards recommended for Biosafety Level 3 (e.g., access zone, sealed penetrations, directional airflow, etc.). In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g. diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in Biosafety Level 2 facilities. However, the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 must be rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made only by the lab director. The following standard and special safety practices, equipment, and facilities apply to the use of agents assigned to Biosafety Level 3:


director when experiments are in progress.

Biosafety Manual20

2. Persons wash their hands after handling infectious materials and animals, after removing gloves, and when they leave the laboratory.

3. Eating, drinking, the use of tobacco products, handling contact lenses and applying cosmetics or lip balm are not permitted in the laboratory. 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 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 aerosols.7. Work surfaces are decontaminated at least once a day and immediately after any

spill of viable material.8. All cultures, stocks, and 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. Infectious wastes from BSL-3 laboratories must be decontaminated before removal for off-site disposal.

9. An insect and rodent control program is in effect.10. Policies for the safe handling of sharps are instituted.

B. Special Practices1. Laboratory doors are kept closed at all times.2. The laboratory director controls access to the laboratory and restricts access to

persons whose presence is required for program or support purposes. For example, persons who are immunocompromised or immunosuppressed, or for whom infection may be unusually hazardous, are not allowed in the laboratory or animal rooms. The director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. No minors are allowed in level 3 laboratories.

3. The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential biohazard, who meet any specific entry requirements (e.g., immunizations), and who comply with all entry and exit procedures, enter the laboratory or animal rooms.

4. When infectious materials or infected animals are present in the laboratory or containment module, a hazard warning sign incorporating the universal biohazard symbol, is posted on all laboratory and animal room access doors. The hazard warning sign identifies the agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates any special requirements for entering the laboratory, such as the need for immunizations, respirators, or other personal protective measures.

5. Laboratory personnel receive the appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing and periodic testing).

Biosafety Manual 21

6. Baseline serum samples are collected and stored for all laboratory and other at-risk personnel. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory.

7. A Biosafety Manual specific to the laboratory is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures. An SOP manual is prepared to cover all procedures and activities.

8. Laboratory 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 changes.

9. The laboratory director is responsible for insuring that all personnel demonstrate proficiency in standard microbiological practices and techniques, and in the practice and operations specific to the laboratory facility, before working with organisms at Biosafety Level 3. This might include prior experience in handling human pathogens or cell cultures, or a specific training program provided by the laboratory director or other competent scientist proficient in safe microbiological practices and techniques.

10. A high degree of precaution must always be taken with all contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. 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 animal and diaphragm bottles. Plastic ware should be substituted for glassware whenever possible.a. 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.


c. 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 should be decontaminated before disposal, in accordance with any local, state, or federal regulations.

11. All manipulations involving infectious materials are conducted in biological safety cabinets or other physical containment devices within the containment module. No work in open vessels is conducted on the open bench.

12. Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is

Biosafety Manual22

finished. It must be cleaned and decontaminated after overt spills, splashes, or other contamination with infectious materials. Contaminated equipment should also be decontaminated before it is sent for repair or maintenance. An Equipment Release Form, available on the EH&S website, should be used to assure that equipment has been properly decontaminated prior to servicing. In addition, it must be packaged for transport in accordance with applicable local, state, or federal regulations, before removal from the facility. Plastic-backed paper toweling can be used on non-perforated work surfaces within biological safety cabinets to facilitate clean up.

13. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.

14. All potentially contaminated materials (e.g., gloves, lab coats, etc.) from laboratories or animal rooms are decontaminated before disposal or reuse.

15. Spills of infectious materials are decontaminated, contained and cleaned up by appropriate professional staff, or others properly trained and equipped to work with concentrated infectious material.

16. Spills and accidents that result in overt or potential exposures to infectious materials are immediately reported to the laboratory director. Appropriate medical evaluation, surveillance, and treatment are provided and written records are maintained.

17. Animals and plants not related to the work being conducted are not permitted in the laboratory.

C. Safety Equipment (Primary Barriers)1. Properly maintained biological safety cabinets are used (Class II or III) for all

manipulation of infectious materials. Biological Safety Cabinets (BSC) should be located away from doors, air supplies and other heavily traveled areas. Biosafety Cabinets must be certified at least yearly.

2. Outside of a BSC, appropriate combinations of personal protective equipment are used (e.g., special protective clothing, masks, gloves, face protection, or respirators) in combination with physical containment devices (e.g., centrifuge safety cups, sealed centrifuge rotors, or containment caging for animals).

3. Biological Safety Cabinets must be used for manipulations of cultures and clinical or environmental materials that may be a source of infectious aerosols. The aerosol challenge of experimental animals; harvesting of tissues or fluids from infected animals, and embryonated eggs, and necropsy of infected animals also require the use of BSCs.

4. Face protection (goggles and mask, or face shield) is worn for manipulations of infectious materials outside of a biological safety cabinet.

5. Respiratory protection is worn when aerosols cannot be safety contained (e.g., outside of a biological safety cabinet), and in rooms containing infected animals.

6. Protective laboratory clothing such as solid-front or wrap-around gowns, scrub suits, or coveralls must be worn in, and not worn outside, the laboratory. Reusable laboratory clothing is to be decontaminated before being laundered. Change protective clothing immediately if contaminated.

Biosafety Manual 23

7. Gloves must be worn when handling infected animals and when hands may contact infectious materials and contaminated surfaces or equipment. Disposable gloves should be discarded when contaminated, and never washed for reuse. Always wash hands between glove changes.

D. Laboratory Facilities (Secondary Barriers)1. The laboratory is separated from areas that are open to unrestricted traffic flow

within the building. Passage through two sets of self-closing doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. A clothes change room (shower optional) may be included in the passageway.

2. Each laboratory contains a sink for hand washing. The sink is foot, elbow, or automatically operated and is located near the laboratory exit door.

3. The interior surfaces of walls, floors, and ceilings are smooth, hard, impervious, and water-resistant so that they can be easily cleaned and capable of disinfection. Penetrations around these surfaces are sealed or capable of being sealed to facilitate decontamination. Floors should be monolithic and coved to the walls.

4. Bench tops are smooth, hard and impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

5. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning. No fabric materials are allowed.

6. Windows in the laboratory are closed and sealed.7. A method for decontaminating all laboratory wastes is available, preferably

within the laboratory (i.e., autoclave, chemical disinfection, incineration, or other approved decontamination method).

8. A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air from “clean” areas into the laboratory toward “contaminated” areas. The exhaust air is not recirculated to any other area of the building, and, depending on risk assessment, discharged to the outside through a HEPA filtration system. The outside exhaust must be dispersed away from occupied areas and air intakes. Laboratory personnel must verify that the direction of the airflow (into the laboratory) is proper. Visual monitoring devices, such as digital readout or maghihelic gauges are recommended.

9. The High Efficiency Particulate Air (HEPA)-filtered exhaust air from Class II or Class III biological safety cabinets is discharged directly to the outside or through the building exhaust system. If the HEPA-filtered exhaust air from Class II Type A biological safety cabinets is to be discharged to the outside through the building exhaust air system, it is connected to this system in a manner (e.g., thimble unit connection) that avoids any interference with the air balance of the cabinets or building exhaust system. Exhaust air from Class II type A biological safety cabinets may be exhausted into the laboratory if the cabinet is tested and certified at least every twelve months. Class III biosafety cabinets must be hard ducted to the building exhaust system.

10. Continuous flow centrifuges or other equipment that may produce aerosols are contained in devices that exhaust air through HEPA filters before discharge into the laboratory.

Biosafety Manual24

11. Vacuum lines are protected with liquid disinfectant traps or HEPA filters, or their equivalent, which are routinely maintained and replaced as needed.

12. An eyewash/safety shower is readily available within the BSL-3 suite.13. Biosafety Level 3 facility design and operational procedures must be documented.

The facility must be tested for verification that the design and operational parameters have been met prior to operation. Facilities should be re-verified/certified at least annually (HEPAs and air systems).

14. Additional environmental protection (e.g. personnel showers, containment of other piped services and the provision for effluent decontamination) should be added when recommended by the agent summary statement, as determined by risk assessment, the site conditions or other applicable federal, state, or local regulations.

15. Level 3 laboratories and animal areas are audited by The EH&S Biosafety Office quarterly.

16. Openings such as around ducts, spaces between doors and frames and other penetrations must be capable of being sealed to facilitate decontamination.

A. Standard Practices1. The animal facility director establishes polices, procedures, and protocols for

emergency situations. Each project is subject to pre-approval by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC). Any special practices are approved at this time.

2. Only those persons required for program or support purposes are authorized to enter the facility. Before entering, persons are advised of the potential biohazards and are instructed on the appropriate safeguards.

3. An appropriate medical surveillance program is in place.4. A safety manual is prepared or adopted. Personnel are advised of special hazards,

and are required to read and follow instructions on practices and procedures.5. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and

storing food for human use should only be done in designated areas and are not permitted in animal or procedure rooms.

6. All procedures are carefully performed to minimize the creation of aerosols or splatters.

Biosafety Manual

Animal Biosafety Level 1 (ABSL-1)

Animal Biosafety Level 1 (ABSL-1) is suitable for work involving well characterized agents that are not known to cause disease in healthy adult humans, and that are of minimal potential hazard to laboratory personnel and the environment.

25

7. Work surfaces are decontaminated after use or after any spill of viable materials.8. All wastes from the animal room (including animal tissues, carcasses, and

contaminated bedding) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional or local requirements. As a means of incineration is not available on the NAU Campus, all animal wastes must be packaged and shrouded and delivered to the Vivarium Manager at the Biological Sciences Annex.

9. Policies for the safe handling of sharps are instituted.10. Personnel wash their hands after handling cultures and animals, after removing

gloves, and before leaving the animal facility. 11. A biohazard sign must be posted on the entrance to the animal room whenever

infectious agents are present. The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements for entering the animal room (e.g., the need for immunizations and respirators).

12. An insect and rodent control program is in effect.B. Special Practices: NoneC. Safety Equipment (Primary Barriers):

1. The wearing of laboratory coats, gowns, and/or uniforms in the facility is recommended. Laboratory coats remain in the animal room. Gowns and uniforms are not worn outside the facility.

2. Persons having contact with non-human primates should assess their risk of mucous membrane exposure and wear appropriate eye and face protection.

D. Facilities (Secondary Barriers)1. The animal facility is separated from areas that are open to unrestricted personnel

traffic within the building.2. External facility doors are self-closing and self-locking. Doors to animal rooms

open inward, are self-closing, and are kept closed when experimental animals are present. Cubicle room inner doors may open outward or be horizontal or vertical sliding.

3. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are water resistant.

4. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas.

5. Windows are not recommended. Any windows must be resistant to breakage. Where possible, windows should be sealed. If the animal facility has windows that open, they are fitted with fly screens.

6. If floor drains are provided, the traps are always filled with water and/or an appropriate disinfectant.

7. Ventilation should be provided in accordance with the Guide for Care and Use of Laboratory Animals, latest edition. No recirculation of exhaust air should occur. It is recommended that animal rooms maintain negative pressure compared to adjoining hallways.

Biosafety Manual26

8. The facility has a hand washing sink.9. Cages are washed manually or in a cage washer. The mechanical cage washer

should have a final rinse temperature of at least 180F.10. Illumination is adequate for all activities, avoiding reflections and glare that could

impede vision.

Animal Biosafety Level 2 (ABSL-2)

Animal Biosafety Level 2 involves practices for work with those agents associated with human disease. It addresses hazards from ingestion as well as from percutaneous and mucous membrane exposure. ABSL-2 builds upon the practices, procedures, containment equipment, and facility requirements of ABSL-1.

A. Standard Practices1. Aside from the standard policies, procedures, and protocols for emergency

situations established by the facility director, appropriate special policies and procedures should be developed as needed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC).

2. Access to the animal room is limited to the fewest number of individuals possible. Personnel who must enter the room for program or service purposes when work is in progress are advised of the potential hazard.

3. An appropriate medical surveillance program is in place. All personnel receive appropriate immunizations or tests for the agents handled or potentially present (e.g., hepatitis B vaccine, TB skin testing). When appropriate, a serum surveillance system should be implemented.

4. A Biosafety Manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and follow instructions on practices and procedures.

5. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use should only be done in designated areas and are not permitted in animal or procedure rooms.


7. Equipment and work surfaces in the room are routinely decontaminated with an effective disinfectant after work with the infectious agent, and especially after overt spills, splashes, or other contamination by infectious materials.

8. All infectious samples are collected, labeled, transported, and processed in a manner that contains and prevents transmission of the agent(s). All wastes from the animal room (including animal tissues, carcasses, contaminated bedding,

Biosafety Manual 27

unused feed, sharps, and other refuse) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional or local requirements. The outer surface of the containers is disinfected prior to moving the material. As a means of incineration is not available on the NAU Campus, all animal wastes must be packaged and shrouded and delivered to the Vivarium Manager at the Biological Sciences Annex.

9. Policies for the safe handling of sharps are instituted:a. Needles and syringes or other sharp instruments are restricted for

use in the animal facility only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles.


c. Plastic-ware should be substituted for glassware whenever possible.

10. Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility.

11. A biohazard sign must be posted on the entrance to the animal room whenever infectious agents are present. The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements (e.g., the need for immunizations and respirators) for entering the animal room.


B. Special Practices1. Animal care 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. Records of all training provided are maintained. In general, persons who may be at increased risk of acquiring infection, or for whom infection might be unusually hazardous, are not allowed in the animal facility unless special procedures can eliminate the extra risk.

2. Only animals used for the experiment(s) are allowed in the room.3. All equipment must be appropriately decontaminated prior to removal from the

room.4. Spills and accidents which result in overt exposures to infectious materials must

be immediately reported to the facility director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.

C. Safety Equipment (Primary Barriers)1. Gowns, uniforms, or laboratory coats are worn while in the animal room. The

laboratory coat is removed and left in the animal room. Gowns, uniforms, and laboratory coats are removed before leaving the animal facility. Gloves are worn when handling infected animals and when skin contact with infectious materials is unavoidable.

Biosafety Manual28

2. Personal protective equipment is used based on risk assessment determinations. Appropriate face/eye and respiratory protection is worn by all personnel entering animal rooms that house nonhuman primates.

3. Biological safety cabinets, other physical containment devices, and/or personal protective equipment (e.g., respirators, face shields) are used whenever conducting procedures with a high potential for creating aerosols. These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, or intranasal inoculation of animals.

4. When needed, animals are housed in primary biosafety containment equipment appropriate for the animal species. Filter top cages are always handled in properly designed and operating animal bio-containment cabinets recommended for rodents.


traffic within the building.2. Access to the facility is limited by secure locked doors. External doors are self-

closing and self-locking. Doors to animal rooms open inward, are self-closing, and are kept closed when experimental animals are present. Cubicle room inner doors may open outward or be horizontal or vertical sliding.

3. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are smooth, hard and impervious.


5. Any windows must be resistant to breakage. Where possible, windows should be sealed. If the animal facility has windows that open, they are fitted with fly screens.

6. If floor drains are provided, the traps are always filled with an appropriate disinfectant.

7. Exhaust air is discharged to the outside without being recirculated to other rooms. Ventilation should be provided in accordance with criteria from Guide for Care and Use of Laboratory Animals, latest edition. The direction of airflow in the animal facility is inward; animal rooms should maintain negative pressure compared to adjoining hallways.

8. Cages are washed manually or in an appropriate cage washer. The mechanical cage washer should have a final rinse temperature of at least 180F.

9. An autoclave is available in the animal facility to decontaminate infectious waste.10. A hand washing sink is in the animal room where infected animals are housed, as

well as elsewhere in the facility.11. Illumination is adequate for all activities, avoiding reflections and glare that could

impede vision.

Biosafety Manual 29

Animal Biosafety Level 3 (ABSL-3)Animal Biosafety Level 3 involves practices suitable for work with animals infected with indigenous or exotic agents that present the potential of aerosol transmission and of causing serious or potentially lethal disease. ABSL-3 builds upon the standard practices, procedures, containment equipment, and facility requirements of ABSL-2.

A. Standard Practices1. Aside from the standard policies, procedures, and protocols for emergency

situations established by the facility director, appropriate special policies and procedures should be developed as needed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC).

2. The laboratory or animal facility director limits access to the animal room to the fewest number of individuals possible. Personnel who must enter the room for program or service purposes when work is in progress are advised of the potential hazard.

3. An appropriate medical surveillance program is in place. All personnel receive appropriate immunizations or tests for the agents handled or potentially present (e.g., hepatitis B vaccine, TB skin testing). When appropriate, a serum surveillance system should be implemented. In general, persons who may be at increased risk of acquiring infection, or for whom infection might have serious consequences, are not allowed in the animal facility unless special procedures can eliminate the extra risk. Assessment should be made by the occupational health physician.

4. A Biosafety Manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and follow instructions on practices and procedures.

5. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use should be done only in designated areas and are not permitted in animal or procedure rooms.


7. Equipment and work surfaces in the room are routinely decontaminated with an effective disinfectant after work with the infectious agent, and especially after overt spills, splashes, or other contamination by infectious materials.

8. All wastes from the animal room (including animal tissues, carcasses, contaminated bedding, unused feed, sharps, and other refuse animal tissues) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional or local requirements. The outer surface of the containers is disinfected prior to moving the material. As a means of incineration is not available on the NAU Campus, all animal wastes must be packaged and shrouded and delivered to the Vivarium Manager at the Biological Sciences Annex. (see Special Practices #3 below).

9. Policies for the safe handling of sharps are instituted:

Biosafety Manual30

a. Needles and syringes or other sharp instruments are restricted in the animal facility for use only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles.

b. Syringes that re-sheathe the needle, needle-less systems, and other safety devices should be used when appropriate.

c. Plastic ware should be substituted for glassware whenever possible.10. Personnel wash their hands after handling cultures and animals, after removing

gloves, and before leaving the animal facility.11. A biohazard sign must be posted on the entrance to the animal room whenever

infectious agents are present. The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the responsible person(s), and indicates the special requirements for entering the animal room (e.g., the need for immunizations and respirators).

12. All infectious samples are collected, labeled, transported, and processed in a manner that contains and prevents transmission of the agent(s).

13. 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. As necessary, personnel receive updates and/or additional training on procedural or policy changes. Records of all training provided are maintained.


B. Special Practices1. Cages are autoclaved or thoroughly decontaminated before bedding is removed

and before they are cleaned and washed. Equipment must be decontaminated according to any local, state, or federal regulations before being packaged for transport or removal from the facility for repair or maintenance.

2. A spill procedure is developed and posted. Only personnel properly trained and equipped to work with infectious materials are to clean up spills. Spills and accidents that result in overt exposures to infectious materials must be immediately reported to the facility director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.

3. Materials not related to the experiment (e.g., plants, animals) are not permitted in the animal room.

C. Safety Equipment (Primary Barriers)1. Uniforms or scrub suits are worn by personnel entering

the animal room. Wrap-around or solid-front gowns should be worn over this clothing. Front-button laboratory coats are unsuitable. The gown must be removed and left in the animal room. Before leaving the animal facility, scrub suits and uniforms are removed and appropriately contained and decontaminated prior to laundering or disposal.

Biosafety Manual 31

2. Personal protective equipment used is based on risk assessment determinations. a. Personal protective equipment is used for all activities involving

manipulations of infectious material or infected animals. b. Personnel wear gloves when handling infected animals. Gloves are

removed aseptically and autoclaved with other animal room wastes before disposal.

c. Appropriate face/eye and respiratory protection (e.g., respirators and face shields) is worn by all personnel entering animal rooms.

d. Boots, shoe covers, or other protective footwear, and disinfectant foot baths are available and used where indicated.

3. The risk of infectious aerosols from infected animals or their bedding also can be reduced if animals are housed in containment caging systems, such as open cages placed in inward flow ventilated enclosures (e.g., laminar flow cabinets), solid wall and bottom cages covered with filter bonnets, or other equivalent primary containment systems.

4. Biological safety cabinets and other physical containment devices are used whenever conducting procedures with a potential for creating aerosols. These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, or intranasal inoculation of animals. At ABSL-3, all work should be done in a primary barrier; otherwise respirators should be worn by personnel in the room.


traffic within the building. 2. Access to the facility is limited by a self-closing and self-locking door. This

exterior entry door may be controlled by a key lock, card key, biometrics, or proximity reader. Entry into the animal room is via a double-door entry which includes a change room and shower(s). An additional double-door access (air-lock) or double-doored autoclave may be provided for movement of supplies and wastes into and out of the facility, respectively. Doors to animal rooms open inward and are self-closing. Doors to cubicles inside an animal room may open outward or slide horizontally or vertically.

3. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior surfaces (walls, floors, and ceilings) are smooth, hard, impervious and capable of harsh disinfection. Penetrations in floors, walls and ceiling surfaces are sealed and openings around ducts and the spaces between doors and frames are capable of being sealed to facilitate decontamination.

4. A hands-free or automatically operated hand washing sink is provided in each animal room near the exit door. The sink trap is filled with an appropriate disinfectant after each use.


6. Windows are not recommended. Any windows must be resistant to breakage and must be sealed. Direct viewing of animals is prohibited.

Biosafety Manual32

7. If floor drains are provided, they are always filled with an appropriate disinfectant.

8. Ventilation should be provided in accordance with criteria from the Guide for Care and Use of Laboratory Animals, latest edition. A ducted exhaust air ventilation system is provided. This system creates directional airflow which draws air into the laboratory from "clean" areas and toward "contaminated" areas. The exhaust air is not recirculated to any other area of the building. Filtration and other treatments of the exhaust air may not be required, but should be considered based on site requirements, and specific agent manipulations and use conditions. The exhaust must be dispersed away from occupied areas and air intakes, or the exhaust must be HEPA-filtered. Personnel must verify that the direction of the airflow (into the animal areas) is proper. It is recommended that a visual monitoring device that indicates and confirms directional inward airflow be provided at the animal room entry. Consideration should be given to installing an HVAC control system to prevent sustained positive pressurization of the animal spaces. Audible alarms should be considered to notify personnel of HVAC system failure.

9. HEPA-filtered exhaust air from a Class II type A biological safety cabinet can be exhausted into the animal room if the cabinet is tested and certified at least annually. When exhaust air from Class II type A safety cabinets is to be discharged to the outside through the building exhaust air system, the cabinets must be connected in a manner that avoids any interference with the air balance of the cabinets or the building exhaust system (e.g., a thimble unit, or an air gap between the cabinet exhaust and the exhaust duct). When Class III biological safety cabinets are used, they should be directly connected (hard ducted) to the exhaust system

10. Cages are washed in a cage washer. The mechanical cage washer has a final rinse temperature of at least 180F.

11. An autoclave is available which is convenient to (preferably within) the animal rooms where the biohazard is contained. The autoclave is utilized to decontaminate infectious waste before moving it to other areas of the facility. A Pass Through autoclave is recommended.

12. If vacuum service (i.e., central or local) is provided, each service connection should be fitted with liquid disinfectant traps and an in-line HEPA filter, placed as near as practicable to each use point or service cock. Filters are installed to permit in-place decontamination and replacement.

13. Illumination is adequate for all activities, avoiding reflections and glare that could impede vision.

14. The completed Biosafety Level 3 facility design and operational procedures must be documented. The facility must be tested for verification that the design and operational parameters have been met prior to operation. Facilities should be re-verified at least annually against these procedures as modified by operational experience.

15. Additional environmental protection (e.g., personnel showers, HEPA filtration of exhaust air, containment of other piped services, and the provision of effluent decontamination) should be considered if recommended by the agent summary

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statement, as determined by risk assessment of the site conditions, or other applicable federal, state, or local regulations. If USDA oversight or funding is involved, HEPA filtered exhaust, personnel shower in/shower out and a Pass Through autoclave will be required.

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Table 1: Summary of Recommended Biosafety Levels for Infectious Agents

BSL Agents Practices Safety Equipment (Primary Barriers)

Facilities (Secondary Barriers)

1 Not known to consistently cause disease in healthy adults

Standard Microbiological Practices None required Open bench top Sink for hand washing

2 Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure

BSL-1 practice plus: Limited access Biohazard warning signs “Sharps” precautions Biosafety manual defining any

needed waste decontamination or medical surveillance policies

Primary barriers = Class I or II BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials; PPE; laboratory coats; gloves; face protection as needed

BSL-1 plus: Autoclave available Single pass air with no

recirculation Hard, smooth, impervious

floor, walls, and countertops

3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences

BSL-2 practice plus: Controlled access Decontamination of all waste Decontamination of lab

clothing before laundering Baseline serum Decontamination of all effluent

Primary barriers = Class I or II BSCs or other physical containment devices for all open manipulations of agents; PPE; protective lab clothing; gloves; respiratory protection as needed

BSL-2 plus: Physical separation from

access corridors Self-closing, double-door

access Exhausted air not re-

circulated Negative airflow into

laboratory (single pass) No floor drains Hard, smooth, impervious

ceilings4 Dangerous/exotic agents

which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission

BSL-3 practice plus: Clothing change before

entering Shower on exit All material decontaminated on

exit from facility

Primary barriers = All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body air-supplied, positive pressure personnel suit

BSL-3 plus: Separate building or

isolated zone Dedicated supply and

exhaust, vacuum, and decontamination systems

Other requirements outlined in the text

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Table 2: Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally Infected Vertebrate Animals Are Used

ABSL Agents Practices Safety Equipment (Primary Barriers)

Facilities (Secondary Barriers)

1 Not known to consistently cause disease in healthy adults

Standard animal care and management practices, including appropriate medical surveillance programs

As required for normal care of each species

Standard animal facility No recirculation of exhaust air Directional air flow recommended Hand washing sink recommended

2 Associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure

ABSL-1 practice plus: Limited access Biohazard warning signs “Sharps” precautions Decontamination of all

infectious wastes and of animal cages prior to washing

ABSL-1 equipment plus barriers: containment equipment appropriate for animal species; PPE; laboratory coats, gloves, face and respiratory protection as needed.

ABSL-1 facility plus: Autoclave available Hand washing sink available in

animal room Mechanical cage washer used Hard, smooth, impervious walls,

floors, ceilings and countertops

3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences

ABSL-2 practice plus: Controlled access Decontamination of all waste Cages decontaminated before

bedding removed Disinfectant foot bath as

needed Decontamination of all effluent

ABSL-2 equipment plus: Containment equipment for

housing animals and cage dumping activities

Class I or II BSCs available for manipulative procedures(inoculation, necropsy) that may create infectious aerosols. PPE: appropriate respiratory protection

ABSL-2 facility plus: Physical separation from access

corridors Self-closing, double-door access Sealed penetrations Sealed windows Autoclave available in facility

4 Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission

ABSL-3 practice plus: Entrance through change room

where personal clothing is removed and laboratory clothing is put on; shower on exit

All wastes are decontaminated before removal from facility

ABSL-3 equipment plus maximum containment equipment (i.e. Class III BSC or partial containment equipment in combination with full body, air-supplied positive-pressure personnel suit) used for all procedures and activities

ABSL-3 facility plus: Separate building or isolated zone Dedicated air supply and exhaust,

vacuum and decontamination systems

Other requirements outlined in the text

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Agents List

The following agents have been listed according to the most appropriate Biological Safety Level to be used. The lists presented on the following pages are based upon the most recent information available in the “Risk Group Classifications for Infectious Agents” section of the American Biological Safety Association (ABSA) Website. Every attempt to provide accurate classification for the agents listed has been made. As this is not a complete list of infectious agents, and these classifications are subject to change, please refer to the following website: http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php, for classification of other agents, and the most up to date information for those listed.

Please note that Biological Safety Levels are not inherent to an agent but are performance recommendations and should be chosen after a risk assessment is completed. A proper risk assessment takes into account the characteristics of the agent involved, the activities to be performed, and the environment in which the work will be completed. Therefore, certain agents may be used at different Biological Safety Levels depending upon the circumstances. For instance, human clinical samples from HIV-positive patients may be safely handled at BSL-2. Growth of HIV in culture should be performed under BSL-3 containment. Biological Safety Levels may be higher or lower than what is given below for a particular agent depending upon the circumstances of its use.

The EH&S Biological Safety Officer (BSO) reviews all projects involving recombinant DNA, infectious disease agents, and agents of concern to livestock and agriculture and will assist you in the risk assessment process. Once the Institutional Biosafety Committee (IBC) and/or the EH&S BSO assigns a Biological Safety Level, it must be adhered to unless new information to warrant a change, in most cases from peer-reviewed literature, is provided. The IBC and/or BSO will review the literature and make an adjustment, if warranted.

Biological Safety Level 1 (BSL-1)Agents that are not associated with disease in healthy adult humans, are of minimal potential hazard to laboratory personnel, and of minimal potential hazard to the environment may be used at BSL-1. Agents that may be used at BSL-1 include Lactobacillus spp., asporogenic Bacillus subtilis or Bacillus licheniformis, Escherichia coli-K12 (cloning strains), Baculoviruses, and adeno-associated virus types 1 through 4 in low concentrations (<109 IP/ml)

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http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php

Those agents not listed under Biological Safety Levels 2, 3 and 4 are not automatically or implicitly classified as BSL-1; a risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed.

Biological Safety Level 2 (BSL-2)Agents to be used at BSL-2 are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available. They are of moderate potential hazard to laboratory personnel and/or the environment.

BSL-2 - Bacterial Agents Including Chlamydia Acinetobacter baumannii (formerly Acinetobacter calcoaceticus) Actinobacillus Actinomyces pyogenes (formerly Corynebacterium pyogenes) Aeromonas hydrophila Amycolata autotrophica Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum) Arizona hinshawii - all serotypes Bartonella henselae, B. quintana, B. vinsonii Bordetella including B. pertussis Borrelia recurrentis, B. burgdorferi Burkholderia (formerly Pseudomonas species) except those listed under BSL-3 Campylobacter coli, C. fetus, C. jejuni Chlamydia psittaci (non-avian strains), trachomatis, C. pneumoniae Clostridium haemolyticum, Cl. histolyticum, Cl. novyi, Cl. septicum, Cl. tetani Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale Dermatophilus congolensis Edwardsiella tarda Erysipelothrix rhusiopathiae Escherichia coli - all enteropathogenic, enterotoxigenic, enteroinvasive and strains bearing K1 antigen, including E. coli

O157:H7 Haemophilus ducreyi, H. influenzae Helicobacter pylori Klebsiella Legionella including L. pneumophila

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Leptospira interrogans - all serotypes Listeria Moraxella Mycobacterium (except those listed under BSL-3) including M. avium complex, M. asiaticum, M. bovis BCG vaccine

strain, M. chelonei, M. fortuitum, M. kansasii, M. malmoense, M. marinum, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi

Neisseria gonorrhoea, N. meningitidis Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. transvalensis Rhodococcus equi Salmonella including S. arizonae, S. cholerasuis, S. enteritidis, S. gallinarum-pullorum, S. meleagridis, S. paratyphi, A, B,

C, S. typhi, S. typhimurium Shigella including S. boydii, S. dysenteriae, type 1, S. flexneri, S. sonnei Sphaerophorus necrophorus Staphylococcus aureus Streptobacillus moniliformis Streptococcus including S. pneumoniae, S. pyogenes Treponema pallidum, T. carateum Vibrio cholerae, V. parahemolyticus, V. vulnificus Yersinia enterocolitica

BSL-2 - Fungal Agents Cryptococcus neoformans Dactylaria galopava (Ochroconis gallopavum) Epidermophyton Exophiala (Wangiella) dermatitidis Fonsecaea pedrosoi Microsporum Penicillium marneffei Sporothrix schenckii Trichophyton

BSL-2 - Parasitic Agents Ancylostoma human hookworms including A. duodenale, A. ceylanicum Ascaris including Ascaris lumbricoides suum

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Babesia including B. divergens, B. microti Brugia filaria worms including B. malayi, B. timori Coccidia Cryptosporidium including C. parvum Cysticercus cellulosae (hydatid cyst, larva of T. solium) Entamoeba histolytica Enterobius Fasciola including F. gigantica, F. hepatica Giardia including G. lamblia Heterophyes Hymenolepis including H. diminuta, H. nana Isospora Leishmania ethiopia, L. major, L. mexicana, L. peruvania, L. tropica Loa loa filaria worms Microsporidium Necator human hookworms including N. americanus Onchoerca filaria worms including, O. volvulus Plasmodium including simian species, P. cynomologi, P. falciparum, P. malariae, P. ovale, P. vivax Sarcocystis including S. sui hominis Schistosoma including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi Strongyloides including S. stercoralis Toxocara including T. canis Toxoplasma including T. gondii Trichinella spiralis Trypanosoma including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cruzi Wuchereria bancrofti filaria worms

BSL-2 - Viruses Adenoviruses, human - all types except African Swine Fever virus

Arenaviruses Lymphocytic choriomeningitis virus (non-neurotropic strains) Other viruses as listed by ABSA/CDC

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Bunyaviruses Bunyamwera virus Rift Valley fever virus vaccine strain MP-12 only Other viruses as listed by ABSA/CDC

Calciviruses Norovirus Sapovirus

Coronaviruses except SARS

Flaviviruses (Togaviruses) - Group B Arboviruses Yellow fever virus vaccine strain 17D Other viruses as listed by ABSA/CDC

Hepatitis A, B, C, D, and E viruses

Herpesviruses - except Herpesvirus simiae (Monkey B virus), BSL-4 Cytomegalovirus Epstein Barr virus Herpesvirus ateles Herpesvirus saimiri Herpes simplex types 1 and 2 Herpes zoster Human herpesvirus types 6 and 7

Marek's disease virusMurine cytomegalovirusPseudorabies virus

Orthomyxoviruses Influenza viruses types A, B, and C Other tick-borne orthomyxoviruses as listed by ABSA/CDC

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Papovaviruses All human papilloma viruses Bovine papilloma virus Polyoma virus Shope papilloma virus Simian virus 40 (SV40)

Paramyxoviruses Newcastle disease virus (non-exotic, non-virulent strains) Measles virus Mumps virus Parainfluenza viruses types 1, 2, 3, and 4 Respiratory syncytial virus

Parvoviruses Human parvovirus (B19) Canine Parvovirus (CPV)

Picornaviruses Coxsackie viruses types A and B Echoviruses - all types Polioviruses - all types, wild and attenuated Rhinoviruses - all types

Poxviruses Vaccinia - all types except Monkeypox virus (BSL-3) and restricted poxviruses including Alastrim, Camelpox, Goatpox,

Smallpox, and Whitepox (restricted to the CDC, Atlanta, GA)

Reoviruses - Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus)

Retroviruses Bovine leukemia virus Clinical samples from HIV-positive patients Feline immunodeficiency virus Feline leukemia virus

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Feline sarcoma virus Gibbon leukemia virus Mason-Pfizer monkey virus Mouse mammary tumor virus Murine leukemia virus Murine sarcoma virus Rat leukemia virus

NOTE: Containment requirements for research involving rDNA Experiments involving recombinant DNA lend themselves the application of highly specific biological barriers. The containment levels required for research involving recombinant DNA associated with plants or animals is based on classification of experiments in Section III, of the NIH document “Experiments Covered by the NIH Guidelines” found at www4.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_SECTION_III._EXPERIMENTS. Consult this document for containment requirements specific to your research.

NOTE: Murine Retroviral VectorsMurine retroviral vectors to be used for human transfer experiments (less than 10 liters) that contain less than 50% of their respective parental viral genome and that have been demonstrated to be free of detectable replication competent retrovirus can be maintained, handled, and administered, under BL1 containment.

Rhabdoviruses Mokola Virus Vertebrate and Invertebrate lyssaviruses

Togaviruses (see Alphaviruses and Flaviviruses) Rubivirus (rubella) Ross River Virus

Biological Safety Level 3 (BSL-3)Agents to be used at BSL-3 are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available.

BSL-3 - Bacterial Agents Including Rickettsia Bacillus anthracis Bartonella

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http://www4.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm#_SECTION_III._EXPERIMENTS

Brucella including B. abortus, B. canis, B. suis Burkholderia (Pseudomonas) mallei, B. pseudomallei Clostridium botulinum, Cl. chauvoei Coxiella burnetii Francisella tularensis (Types A and B) Mycobacterium bovis (except BCG strain, BSL-2), M. tuberculosis, M. leprae Mycoplasma, except M. mycoides and M. agalactiae which are restricted animal pathogens Pasteurella multocida type B -“buffalo” and other virulent strains Rickettsia akari, R. australis, R. canada, R. conorii, R. prowazekii, R. rickettsii, R, siberica, R. tsutsugamushi, R. typhi (R.

mooseri) Yersinia pestis

BSL-3 - Fungal Agents Blastomyces dermatitidis Cladosporium bantianum, C. (Xylohypha) trichoides Coccidioides immitis (sporulating cultures; contaminated soil) Histoplasma capsulatum, H. capsulatum var.. duboisii Paracoccidioides braziliensis

BSL-3 - Parasitic Agents Echinococcus including E. granulosis, E. multilocularis, E. vogeli Leishmania including L. braziliensis, L. donovani Naegleria fowleri Plasmodium falciparum Taenia solium

BSL-3 - Viruses and Prions Alphaviruses (Togaviruses) - Group A Arboviruses Eastern equine encephalomyelitis virus Venezuelan equine encephalomyelitis vaccine strain TC-83 Western equine encephalomyelitis virus Semliki Forest virus

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Venezuelan equine encephalomyelitis virus (except the vaccine strain TC-83 is BSL-2) Other viruses as listed by ABSA/CDC

Arenaviruses Lymphocytic choriomeningitis virus (LCM) (neurotropic strains) Flexal

Bunyaviruses Hantaviruses including Hantaan virus Rift Valley fever virus Other viruses as listed by ABSA/CDC

Flaviviruses (Togaviruses) - Group B Arboviruses Dengue virus serotypes 1, 2, 3, and 4 Japanese encephalitis virus St. Louis encephalitis virus Yellow fever virus – wild type Other viruses as listed by ABSA/CDC

Poxviruses Monkeypox virus

Prions Transmissible spongioform encephalopathies (TME) agents, Creutzfeldt-Jacob disease and kuru agents (see BMBL

for specific containment instruction)

Retroviruses Avian leukosis virus Avian sarcoma virus Human immunodeficiency virus (HIV) types 1 and 2 Human T cell lymphotropic virus (HTLV) types 1 and 2 Simian immunodeficiency virus (SIV)

Rhabdoviruses Rabies virus

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Vesicular stomatitis virus - laboratory adapted strains ONLY including VSV-Indiana, San Juan, and Glasgow

Biological Safety Level 4 (BSL-4)Agents to be used at BSL-4 are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available.

BSL-4 - Bacterial Agents None

BSL-4 - Fungal Agents None

BSL-4 - Parasitic Agents None

BSL-4 - Viral Agents Arenaviruses (Togaviruses) - Group A Arboviruses Guanarito virus Lassa virus Junin virus Machupo virus Sabia virus

Bunyaviruses (Nairovirus) Crimean-Congo hemorrhagic fever virus

Filoviruses Ebola virus Marburg virus

Flaviruses (Togaviruses) - Group B Arboviruses Tick-borne encephalitis virus complex including Absetterov, Central European encephalitis, Hanzalova, Hypr, Kumlinge,

Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer encephalitis viruses

Herpesviruses (alpha) Herpesvirus simiae (Herpes B or Monkey B virus)

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Paramyxiviruses Equine morbillivirus

Hemorrhagic fever agents and viruses as yet undefined.

2 - Information for Researchers

Project RegistrationNorthern Arizona University Insitutional Biosafety Committee

Many research projects involve work with potentially hazardous biological agents, known infectious disease agents, or biological materials regulated by the federal or state government. Granting agencies require that the university monitor the use of biological hazards, infectious disease agents, and recombinant DNA in order for them to release funds to investigators. Therefore, we have developed a registration system to ensure that all biological materials are handled properly and disposed of appropriately. The EH&S Biological Safety Office administers four registration programs for research projects.

The NAU Institutional Biosafety Committee (IBC) reviews all registrations. Approval by the NAU IBC, in writing, is required before ordering or working with any agents. Please contact the EH&S BSO for more information.

Fillable registration forms are available on the EH&S website at: http://nau.edu/Research/Compliance/Environmental-Health-and-Safety/Forms/

Biosafety Manual 47

http://nau.edu/Research/Compliance/Environmental-Health-and-Safety/Forms/

http://nau.edu/Research/Compliance/Environmental-Health-and-Safety/Forms/

Biological Agent (BA) RegistrationUse of the following materials requires that the Principal Investigator completes and submits the bio-agent registration document for approval by the EH&S Biological Safety Officer.

Agents characterized at Biosafety Level 1 require registration. These will be expedited and administratively approved barring any complications.

Agents to be used at Biosafety Level 2 (BSL-2) or Biosafety Level 3 (BSL-3):1. All human, animal, or plant pathogens that require BSL-2 or BSL-3 containment and handling (see previous section:

“Agents List”) must be registered. Please note that BSL-4 agents may not be stored or used at NAU. 2. Unknown human and animal pathogens must be registered. These are considered BSL-2 until identified.3. Cell lines or cultures that

1) have been immortalized with a virus (such as EBV or a retrovirus), 2) are known to be tumorigenic in primates (including humans), or3) are primary human tumor cells.

These are considered BSL-2 (or higher in many cases).4. Human blood or other tissues, when used in research, must be registered.5. Human blood or other tissue known to be HIV positive or known to contain any human disease causing agent may

require higher containment depending upon the IBC evaluation.

Recombinant DNA (R-DNA) RegistrationAll R-DNA projects that involve a recombinant organism (this excludes projects that involve DNA only, i.e. PCR) require registration with the EH&S Biological Safety Office. A subset of R-DNA projects requires review and approval from the Institutional Biosafety Committee (IBC). The NAU IBC oversees all research projects and issues involving R-DNA at NAU. Use of the following requires that the principal investigator completes and submits an R-DNA registration document. 1. All R-DNA projects, including the growth of recombinant bacteria for probe isolation (plasmid or phage preparations)

require registration. Projects must be registered regardless of where the material came from or who originally constructed it.

2. Projects that are exempt from the NIH Guidelines must also be registered.3. The development of transgenic animals and plants requires registration.

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R-DNA projects are performed at BSL-1, BSL-2, BSL-3 or the corresponding levels for whole plant (BSL-1P, BSL-2P, BSL-3P) or whole animal (BSL-1N, BSL-2N, BSL-3N) work. The EH&S Biological Safety Officer, in conjunction with the IBC, will make the final determination.

Acute Toxins (AT) RegistrationThe use and storage of chemicals with a mammalian LD50 of < 100 g/kg require registration. For a partial list, see the Toxins Table that follows. Acute toxins may only be ordered following written approval by the BSO or IBC.

Regulated Biological MaterialsAgents, such as plant pathogens or exotic microorganisms, that are regulated by federal or state agencies (CDC, HHS, DOC, USDA/APHIS, EPA, FDA, DPI, etc.) shall be registered with the EH&S Biological Safety Office by submission of a biological agent registration form. All permits for transport, transfer, import or export of these regulated agents are the responsibility of the Principal Investigator. The NAU Responsible Official (RO) will be responsible for all Select Agent permits. Please allow a minimum of 6 weeks to obtain new federal permits.

Project AmendmentChanges to an existing registration can be done on an amendment unless said changes result in a dramatic change to the overall project or the containment level.

Typical changes include:

Addition or deletion of new personnel Addition or deletion of new agents (same containment level) Minor modifications to the protocol or procedures ( final decision to be made by the IBC)

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Table 3: Toxin TableToxins with a mammalian LD50 of < 100 g/kg must be registered with the EH&S Biological Safety Office. Therefore, use of the following toxins requires registration. If a toxin is not on the list, it still may require registration, depending upon the LD50. For more information, please contact the EH&S Biological Safety Office.

ToxicityLD50 (g/kg)*

Abrin 0.7Aerolysin 7.0Botulinin toxin A 0.0012Botulinin toxin B 0.0012Botulinin toxin C1 0.0011Botulinin toxin C2 0.0012Botulinin toxin D 0.0004Botulinin toxin E 0.0011Botulinin toxin F 0.0025-bungarotoxin 14.0Caeruleotoxin 53Cereolysin 40-80Cholera toxin 250Clostridium difficile enterotoxin A 0.5Clostridium difficile cytotoxin B 220Clostridium perfringens lecithinase 3Clostridium perfringens kappa toxin 1500Clostridium perfringens perfringolysin O 13-16Clostridium perfringens enterotoxin 81Clostridium perfringens beta toxin 400Clostridium perfringens delta toxin 5Clostridium perfringens epsilon toxin 0.1Conotoxin 12-30

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Crotoxin 82Diphtheria toxin 0.1Listeriolysin 3-12Leucocidin 50Modeccin 1-10Nematocyst toxins 33-70Notexin 25Pertussis toxin 15

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ToxicityLD50 (g/kg)*

Pneumolysin 1.5Pseudomonas aeruginosa toxin A 3Ricin 2.7Saxitoxin 8Shiga toxin 0.250Shigella dysenteriae neurotoxin 1.3Streptolysin O 8Staphylococcus enterotoxin B 25Staphylococcus enterotoxin F 2-10Streptolysin S 25Taipoxin 2Tetanus toxin 0.001Tetrodotoxin 8Viscumin 2.4-80Volkensin 1.4Yersinia pestis murine toxin 10

*Please note that the LD50 values are from a number of sources (see below). For specifics on route of application (i.v., i.p., s.c.), animal used, and variations on the listed toxins, please go to the references listed below.

Reference:1. Gill, D. Michael; 1982; Bacterial toxins: a table of lethal amounts; Microbiological Reviews; 46: 86-942. Stirpe, F.; Luigi Barbieri; Maria Giulia Battelli, Marco Soria and Douglas A. Lappi; 1992; Ribosome-inactivating

proteins from plants: present status and future prospects; Biotechnology; 10: 405-4123. Registry of toxic effects of chemical substances (RTECS): comprehensive guide to the RTECS. 1997. Doris V. Sweet,

ed., U.S. Dept of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health; Cincinnati, Ohio

Select Agents

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The following lists of agents and toxins are classified by the Federal government as Select Agents. Any possession, use, transfer or shipment of these materials is strictly controlled by regulation.

Researchers considering work with any of these materials must first contact the NAU Responsible Official for the approvals, permits, clearances and other necessary paperwork. Be aware that government clearance can take as much as 6 months to complete in advance of any project.

Failure to comply with these Federal Regulations is punishable by both fines and imprisonment.

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HHS and USDA Select Agents and Toxins7CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73

HHS SELECT AGENTS AND TOXINSAbrinBotulinum neurotoxins* Botulinum neurotoxin producing species of Clostridium*Conotoxins (Short, paralytic alpha conotoxinscontaining the following amino acid sequenceX1CCX2PACGX3X4X5X6CX7)1

Coxiella burnetii Crimean-Congo haemorrhagic fever virus DiacetoxyscirpenolEastern Equine Encephalitis virus3 Ebola virus*Francisella tularensis*Lassa fever virusLujo virus Marburg virus*Monkeypox virus3

Reconstructed replication competent forms of the1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (Reconstructed 1918 Influenza virus)RicinRickettsia prowazekii SARS-associated coronavirus (SARS-CoV)Saxitoxin South American Haemorrhagic Fever viruses: Chapare Guanarito

OVERLAP SELECT AGENTS AND TOXINSBacillus anthracis*Bacillus anthracis Pasteur strain Brucella abortusBrucella melitensisBrucella suisBurkholderia mallei*Burkholderia pseudomallei*Hendra virus Nipah virus Rift Valley fever virus Venezuelan equine encephalitis virus3

USDA SELECT AGENTS AND TOXINS African horse sickness virus African swine fever virus Avian influenza virus3

Classical swine fever virusFoot-and-mouth disease virus* Goat pox virus Lumpy skin disease virus Mycoplasma capricolum3

Mycoplasma mycoides3

Newcastle disease virus2,3

Peste des petits ruminants virus Rinderpest virus*Sheep pox virusSwine vesicular disease virus

USDA PLANT PROTECTION AND QUARANTINE (PPQ)

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Junin Machupo SabiaStaphylococcal enterotoxins A,B,C,D,E subtypes T-2 toxinTetrodotoxinTick-borne encephalitis complex (flavi) viruses: Far Eastern subtype Siberian subtypeKyasanur Forest disease virus Omsk hemorrhagic fever virus Variola major virus (Smallpox virus)*Variola minor virus (Alastrim)*Yersinia pestis*

SELECT AGENTS AND TOXINSPeronosclerospora philippinensis (Peronosclerospora sacchari) Phoma glycinicola (formerly Pyrenochaeta glycines)Ralstonia solanacearumRathayibacter toxicusSclerophthora rayssiaeSynchytrium endobioticumXanthomonas oryzae

*Denotes Tier 1 Agent

1 C = Cysteine residues are all present as disulfides, with the 1st and 3rd Cysteine, and the 2nd and 4th Cysteine forming specific disulfide bridges; The consensus sequence includes known toxins α-MI and α-GI (shown above) as well as α-GIA, Ac1.1a, α-CnIA, α-CnIB; X1 = any amino acid(s) or Des-X; X2 = Asparagine or Histidine; P = Proline; A = Alanine; G = Glycine; X3 = Arginine or Lysine; X4 = Asparagine, Histidine, Lysine, Arginine, Tyrosine, Phenylalanine or Tryptophan; X5 = Tyrosine, Phenylalanine, or Tryptophan; X6 = Serine, Threonine, Glutamate, Aspartate, Glutamine, or Asparagine; X7 = Any amino acid(s) or Des X and; “Des X” = “an amino acid does not have to be present at this position.” For example if a peptide sequence were XCCHPA then the related peptide CCHPA would be designated as Des-X.2 A virulent Newcastle disease virus (avian paramyxovirus serotype 1) has an intracerebral pathogenicity index in day-old chicks (Gallus gallus) of 0.7 or greater or has an amino acid sequence at the fusion (F) protein cleavage site that is consistent with virulent strains of Newcastle disease virus. A failure to detect a cleavage site that is consistent with virulent strains does not confirm the absence of a virulent virus.3 Select agents that meet any of the following criteria are excluded from the requirements of this part: Any low pathogenic strains of avian influenza virus, South American genotype of eastern equine encephalitis virus , west African clade of Monkeypox viruses, any strain of Newcastle disease virus which does not meet the criteria for virulent Newcastle disease virus, all subspecies Mycoplasma capricolum except subspecies capripneumoniae (contagious

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caprine pleuropneumonia), all subspecies Mycoplasma mycoides except subspecies mycoides small colony (Mmm SC) (contagious bovine pleuropneumonia), and any subtypes of Venezuelan equine encephalitis virus except for Subtypes IAB or IC, provided that the individual or entity can verify that the agent is within the exclusion category. 9/10/13

Minors in Research Laboratories or Animal Facilities

Unless enrolled as a Northern Arizona University student, minors are not allowed to work or conduct research in Northern Arizona University research laboratories, greenhouses or animal facilities except as identified specifically below. In addition, minors are prohibited from operating farm machinery or state vehicles and from working in machine shops.

1. All Minors are prohibited from working or conducting research in the following:a. Any laboratory or facility designated as BSL-3, ABSL-3 or higher for recombinant or infectious organisms.b. Any laboratory where select agents or explosives are used or stored.c. Any Animal Care Services (ACS) housing or procedure area/lab/facility.

2. Minors are prohibited from working with any of the following materials.a. Radioactive materials or radiation (X-rays)b. Acute Toxins

3. Minors are allowed to work or conduct research in laboratories (not listed in #1 or #2 above) if the following requirements are met in full:

a. Northern Arizona University EH&S Biosafety Office Policy titled; Minors In Research Laboratories Or Animal Facilities (www4.nau.edu/ovp/regulatorycompliance/safety/MinorsResearchPolicy.asp.) has been read and understood. The Potential Hazards Information & Signature Sheet (www4.nau.edu/ovp/regulatorycompliance/safety/Potentialhazardssheet.asp.) has been reviewed, signed by the parent/legal guardian and returned to the EH&S Biosafety Office by mail or by fax.

b. A Minor’s Research Proposal Registration Form (www4.nau.edu/ovp/regulatorycompliance/biosafety/forms/minorsregistrationform.doc.) is submitted to and approved by Northern Arizona University Institutional Biosafety Committee which

includes:i. A description of the project and all materials

ii. A parental consent formiii. A sponsor consent form

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c. Hazard specific safety training is completed by the Principal Investigator/Sponsor with the minor as approved by EH&S BSO.

d. Personal protective equipment, specific to the hazard, is provided to the minor with instructions for use and disposal.

e. The minor is supervised at all times while in the laboratory and never left alone.f. Hours of work comply with Federal Regulation 29 CFR 570.35.

The laboratory is in full compliance with all applicable Northern Arizona University safety programs and regulations.

Responsibilities of the Principle Investigator during the Conduct of the Research

The Principal Investigator shall:

1. Supervise the safety performance of the laboratory staff to ensure that the required safety practices and techniques are employed.

2. Report any significant incident, violation of the NIH Guidelines, or any significant, research-related accidents and illnesses immediately by contacting the Biosafety Officer. Examples of incidents and violations include:

a. Overt exposures (exposures that result in direct personnel exposure to biohazards such as injection, spills, splashes or aerosol inhalation)

b. Potential exposures (exposures that have a high risk of exposing personnel to biohazards such as spills, containment failure while working with the agent or equipment failure that may produce aerosols)

c. Any exposure (overt or potential) in a BSL-3 laboratory d. Overt exposure in BSL1 or BSL-2 laboratoriese. Any illness that may be caused by the agents used in the laboratoryf. Incidents involving the improper disposal of biohazards.

3. Ensure the integrity of the physical containment (e.g., biological safety cabinets) and the biological containment (e.g., purity and genotypic and phenotypic characteristics) and correct procedures or conditions that might result in release of or exposure to biohazards.

4. Limit or restrict access to the laboratory when work with biohazards is in progress; this includes making a determination of who may be at increased risk.

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5. Establish policies and procedures to limit access exclusively to those individuals who have been advised of the potential hazards and meet specific entry requirements.

6. Ensure that laboratory personnel are offered, at no cost, appropriate immunizations or tests for the infectious agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine, tuberculosis skin testing).

Reportable Incidents and ViolationsIncidents/problems involving biohazards must be immediately reported to the Biosafety Officer. Examples of reportable significant incidents include but are not limited to:

1. Any overt exposure, such as a needle stick, splash, and contamination due to equipment failure.2. Any potential exposure in a BSL-3 facility.

A significant event may also occur from a containment breach, which may be subsequently determined to pose either an overt or potential exposure to individuals or the environment. It should be noted that waste from recombinant or synthetic nucleic acid research is also considered biohazardous and incidents involving improper disposal of recombinant or synthetic nucleic acids must also be reported. Questions regarding reportable incidents should be directed to the Biosafety Officer.

Failure by research personnel to follow federal and institutional regulations, guidelines, policies and/or procedures may also require reporting to the appropriate institutional, local, state and/or federal agencies. Violations may include but are not limited to conduct of new or ongoing research without appropriate federal or institutional registration, review, approval or oversight.

Institutional Reporting ResponsibilitiesThe Institutional Biosafety Committee is required, by the NIH Guidelines, to report to the appropriate University official and to the NIH/OBA within thirty days any significant incidents, violations of the NIH Guidelines, or any significant findings of research-related accidents and illnesses. The IBC will be responsible to determine what actions, if any, are necessary. For example, the IBC may determine the need to make changes to the frequency of laboratory inspections or biosafety containment level of the research, based on results of the incident.

Other IBC reporting requirements (to OBA and other agencies) include but are not limited to: 1. Research involving biohazards conducted without prior IBC approval

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2. Lax security, unsafe procedures used in a laboratory setting, improper disposal of recombinant or synthetic nucleic acid waste

3. Changes to research risk that have been initiated without prior approval by IBC

Some incidents must be reported to OBA on an expedited basis. Spills or accidents in BSL-2 laboratories involving recombinant or synthetic nucleic acids that result in an overt exposure must be immediately reported to OBA. In addition, spills or accidents involving recombinant or synthetic nucleic acids occurring in high containment (BSL-3 or higher) laboratories resulting in an overt or potential exposure must be immediately reported to OBA. The IBC working through the IBC Chair and the BSO will report to the Institutional Official, who, in turn will oversee the report to OBA, any of the above-described incidents.

Institutional violations that will also be reported to the appropriate College or department head may include but are not limited to:

1. Lapses in disclosure approval 2. Failure to comply with institutional and federal regulations, guidelines, and policies3. Unsafe work practices

Biological Waste Disposal PolicyThis policy is intended to provide guidance and insure compliance with the NIH/CDC guidelines, the State of Arizona Administrative Code, and restrictions of the Coconino County Landfill.

Categories

1) Infectious/potentially infectious/R-DNAa) human pathogensb) animal pathogensc) plant pathogensd) recombinant DNAe) human and primate blood, blood products and other body fluidsf) human and primate tissueg) any material containing or contaminated with any of the above (test tubes, needles*, syringes, tubing, culture dishes,

flasks, etc.)

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This waste must be inactivated prior to disposal. The preferred method is steam sterilization (autoclaving), although chemical inactivation or incineration may be appropriate in some cases. Per NAU policy, even autoclaved or otherwise inactivated wastes must be disposed of as biohazardous waste. Storage of non-inactivated waste is restricted to within the generating laboratory. The material may not be stored longer than 24 hours prior to inactivation.

2) Non-infectious wasteThis category includes waste that is not contaminated with any of the biological materials listed in category 1. It includes solid waste and sharps generated in clinical or laboratory settings. Sterile or unopened biomedical materials that require disposal are also considered biological waste, and must be treated as such.

IV packs test tubes Petri dishesneedles* razor blades* tissue culture flaskssyringes culture dishesscalpels* flasksbroken glass and plastic ware** pipettes

This material does not require sterilization prior to disposal. *must be packaged in plastic sharps boxes.**must be within a box or other puncture proof container before adding to waste.

3) Mixed radioactive/biohazardous waste The biohazardous component of mixed radioactive/biohazardous waste shall be inactivated prior to its release to Radiation Safety for disposal as radioactive waste. Steam-sterilization or chemical inactivation shall be employed as above. Although some radioactive materials can be autoclaved safely, please check with the EH&S Radiation Safety Officer regarding the best method to employ with any given radionuclide.

4) Mixed chemical/biohazardous waste The biohazardous component of mixed chemical/biohazardous waste shall be inactivated prior to its release for chemical disposal. Precautions should be taken to prevent the generation and release of toxic chemicals during the inactivation process. In general, autoclaving is not recommended because flammable or reactive compounds should not be autoclaved due to the explosion hazard. Please check with the NAU Chemical Hygiene Officer for guidance regarding particular chemicals.

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5) Animal carcasses and materials As there are no tissue digesters or incinerators present on the NAU Campus, all animal materials must be packaged, shrouded and delivered to the Vivarium Manager. No animal bodies or material shall be disposed of as regular trash or through the biomedical waste receptacle.

6) Human remains Please contact the State Anatomical Review Board for information regarding the final disposition of human remains and body parts.

Packaging

1) Biohazard bags These are used for the initial collection of certain biological wastes. All biohazard bags must meet impact resistance (165 grams), tearing resistance (480 grams), and heavy metal concentration (<100 PPM total of lead, mercury, chromium and cadmium) requirements. Written documentation (a test report) from the manufacturer regarding these requirements must be on file. Individual departments are responsible for obtaining the appropriate biohazard bags. These bags must be placed in bulk biohazard containers as described within this section.

2) Sharps Needles, scalpels and razor blades must be containerized in red plastic sharps containers. NAU departments are responsible for obtaining their own sharps containers. All other sharps (broken glass and plastic ware, pipettes, etc.) must be containerized in puncture-resistant cardboard boxes (see #3, below).

3) Containers All biological waste must be containerized in rigid, leak proof, puncture resistant boxes or plastic tubs/barrels as the terminal receptacle. NAU Departments are responsible for obtaining the appropriate containers from the EH&S Hazardous Waste Coordinator.

LabelingAll packages containing biological waste shall be labeled with indelible ink marker (i.e., Sharpie®) as follows:

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1) Date Biohazard bags shall be labeled with the date they were put into use. Please note that biohazard bags must be labeled even though they will be placed inside a secondary container for final disposal.Sharps containers shall be labeled with the date the container is full.

2) Name/Location/Phone Number Generator’s (principal investigator’s) name, lab location (room number) and phone number will be clearly printed on each container.

3) Biohazard sign Only manufactured containers with the preprinted universal biohazard symbol and the words “biomedical”, “biohazardous”, or “infectious” shall be used.

TransportThe transport of biohazardous waste outside of the laboratory (i.e., to an autoclave or incinerator) must be in a closed, leak-proof container that is labeled “biohazard”. Only trained personnel may transport biomedical waste. Labeling may be accomplished by use of a red biohazard bag with the universal biohazard symbol. Only biomedical bags and red plastic sharps containers may be used to transport biological waste to the biomedical waste receptacle. Waste receptacle personnel are instructed not to accept any other type of containers.

Transportation of red-bagged waste must be in closed, leak-proof containers, properly labeled as “Biohazards.” Movement of regulated/biological waste through public corridors, along carpeted hallways, and on public elevators should be avoided whenever possible. Any leakage/spills from these containers must be immediately reported to the NAU Biosafety Office. Signs must be displayed to prevent tracking of the spills to other areas.

TrainingAll employees who handle biological waste shall be trained annually regarding its proper handling. All new employees shall be trained before they are allowed to handle biological waste.

Training may be accomplished through the NAU Bloodborne Pathogen Training Program, informally in the lab setting, or through formal training programs set up by individual departments or divisions. For assistance, please call the NAU Biological Safety Officer or Hazardous Waste Coordinator.

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According to Arizona Statute (Ch. 64E-16 F.A.C.), records of the training session shall be maintained for each employee, along with an outline of the training program. Training records shall be retained for a period of three (3) years.

Biological Waste Disposal ContainersThe following waste disposal containers for biohazardous or biomedical waste are to be used for teaching and research purposes at Northern Arizona University:

ALL BIOHAZARDOUS WASTE MUST BE TREATED BEFORE DISPOSAL.

Bulk Biohazard ReceptaclesThe EH&S currently provides departments who generate biohazardous and biomedical waste with large receptacles for the disposal of full biohazard bags and sharps containers. Individual departments can contact the EH&S Hazardous Waste Coordinator for pickup when those containers are full.

PLEASE NOTE: PER NAU POLICY, EVEN AUTOCLAVED WASTES MUST BE TREATED AS BIOHAZARDOUS WASTES.

Disposal of biomedical waste in the regular trash or dumpster is prohibited. The Coconino County Landfill does not accept any items that resemble “biomedical devices” and will reject the entire load. In some cases, the individual principal investigator has been made responsible for the costs incurred for sorting and disposal of improperly disposed waste.

Sharps BoxesNAU is subject to the requirements of Arizona Administrative Code Rule 18-13-1419, Medical Sharps. This regulation requires that a generator who treats bio-hazardous medical waste on site shall place medical sharps in a sharps container after rendering them incapable of creating a stick hazard by using an encapsulation agent or any other process that prevents a stick hazard. Red plastic sharps boxes are used for disposal of needles, razor blades, scalpels, and small Pasteur pipettes. Sharps boxes that contain infectious materials must be inactivated by autoclaving the closed box. These boxes shall be labeled (with indelible ink marker as above) with the principal investigator’s name, phone number, date the sharps box is full, and lab location (room

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number). Only trained staff/research staff shall handle the full sharps boxes. Full sharps containers can be disposed of in the bulk biohazard receptacles described above.

Biohazard BagsRed plastic biohazard bags shall be used for collection of biohazardous tissue culture items, petri dishes, and other non-sharp items. These biohazard bags shall be immediately labeled with an indelible ink marker (e.g. Sharpie) when the bag is first put into use. Labels shall include the principal investigator’s name, phone number, the date the first biohazard item is placed into the bag, and lab location (room number). The biohazardous waste items must be inactivated by autoclaving in biohazard autoclave bags or treatment with bleach within 30 days of accumulation (Ch. 64E-16 F.A.C.). After treatment, the bags shall be tightly sealed and placed in the bulk containers described above. They may be transported to the biomedical waste receptacle by trained laboratory personnel or by trained staff. Biohazard bags should never be handled by non-research staff or placed in the hallway. Double bagging may be required to prevent leakage, however, absorbent material must be added to prevent the presence of any liquid or fluid. Full, untreated biohazard bags shall be stored only in the lab where accumulation occurred. Full biohazard bags must be treated within 24 hours. As previously noted, even autoclaved wastes must be disposed of as biohazardous waste.

Autoclave Use and TestingRules governing the use and testing of autoclaves are based on Arizona Administrative Code, Rule 18-13-1405, Biohazardous Medical Waste Treated on Site. The following summarize the requirements set by this administrative rule.

A generator who autoclaves bio-hazardous medical waste on site shall:

1) Further process by grinding, shredding, or any other process, any recognizable animals and human tissue, organs, or body parts, to render such wasted non- recognizable and ensure effective treatment.

2) Operate the autoclave at the manufacturer's specifications appropriate for the quantity and density of the load.

3.) Keep records of operational performance levels for six months after each treatment cycle. Operational performance level recordkeeping includes all of the following:

a. Duration of time for each treatment cycle.b. The temperature and pressure maintained in the treatment unit during each cycle.c. The method used to determine treatment parameters in the manufacturer's specifications.d. The method in manufacturer's specifications used to confirm microbial inactivation

and the test results.

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e. Any other operating parameters in the manufacturer's specifications for each treatment cycle.

4) Keep records of equipment maintenance for the duration of equipment use that include the date and result of all equipment calibration and maintenance.

A generator shall also do the following:

1) Package the treated medical waste according to the waste collection agency's requirements;2) Attach to the package or container a label, placard, or tag with the following words: "This

medical waste has been treated as required by the Arizona Department of Environmental Quality standards" before placing the treated medical waste out for collection as a general solid waste. The generator shall ensure that the treated medical waste meets the standards of R18-13-1415.

3) Upon request of the solid waste collection agency or municipal solid waste landfill, provide a certification that the treated medical waste meets the standards of R18-13- 1415.

4) Make treatment records available for Departmental inspection upon request.

A generator of medical sharps shall handle medical sharps as prescribed in R18-13-1419.

A generator of chemotherapy waste, cultures and stocks, or animal waste shall handle that waste asprescribed in R18-13-1420.

Autoclave TestingAutoclaves shall be tested before being placed into service and then periodically for effectiveness. 1) Periodicity

a) Every 40 hours of useRequired for autoclaves that are used to inactivate human or non-human primate blood, tissues, clinical samples, or human pathogens.

b) AnnuallyRequired for autoclaves that are used to inactivate other material.

2) MethodA commercially available test indicator kit that uses bacterial spores (Bacillus stearothermophilus) is the approved method of testing autoclave efficiency. Most spore vial test kits require 56 to 60 °C incubation of the autoclaved test vial along with a non-autoclaved control vial. Incubation causes surviving spores to grow.a) New autoclaves

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Before placing an autoclave into service, a test load approximating the weight and density of the type of waste generated shall be autoclaved with test spore vials. The spore vials should be placed at the bottom, top, front, rear, and center of the autoclave chamber. This can be achieved by either:-placing vials at those positions within one large test load, OR -making several smaller test packs with 1 vial at the center of each and placing the packs at those locations within the chamber.

The appropriate parameters for sterilization including temperature, pressure, and treatment time shall be determined in this way.

b) Autoclaves already in useFor periodic testing, place a spore vial in the very center of a test load prior to autoclaving.

3) Storage InformationPlease read the product information sheet for appropriate storage information. Spore vials should not be frozen. Each batch of vials has an expiration date. Vials should not be used after their expiration date.

Record-KeepingThe following records regarding autoclave use must be kept:1) On-site maintenance records2) Autoclave use log – must be available near the autoclave.

Each load of material inactivated shall be logged as follows:a) Date, time, and operator's nameb) Contact Information: Lab, room number, phone numberc) Is this biohazardous material? d) Confirmation of sterilization:

i) Record the temperature, pressure, and length of time the load is sterilized. Please note that temperature sensitive autoclave tape is not sufficient to indicate that the load reached sterilization conditions because the tape will change color at lower temperatures.

ii) Save the autoclave printout, if the autoclave has a working printer.

Autoclave Operating ProceduresA written sterilization procedure shall be in place for each workplace. This shall include the following:

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1) ParametersAppropriate parameters for sterilization shall be determined from the testing with spore vials. The time it takes to sterilize a load will change depending upon the load density and the sterilization cycle one chooses. Tests have been performed which imitate these various situations. Please follow the established guidelines.

2) ProtocolIdentification of standard treatment containers and proper load placement shall be made.

3) CleaningThe autoclave and work areas shall be cleaned after every use and the work area shall be disinfected, as needed.

Autoclave Operation and Safety TrainingAutoclave training is available from the EH&S Biological Safety Office upon request. The training is geared toward the research staff and goes over proper use of autoclaves and how they may be maintained and used properly. Safety training is also given.

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Autoclave Guidelines

AUTOCLAVE GUIDELINESSTERILIZATION TIMES (Drying Time Not Included)

121 C / 250 F and 15 p.s.i.

BIO WASTE IN AUTOCLAVE BAGS, LOOSELY TIED

MULTIPLE BAGS --- 100 MINUTES OR LONGER

SINGLE BAG (FULL) --- 90 MINUTES

PARTIAL BAG --- 60 MINUTES

DRY GOODS

GLASSWARE, EMPTY, INVERTED --- 15 MINUTES

INSTRUMENTS, WRAPPED --- 30 MINUTES

UTENSILS, WRAPPED --- 30 MINUTES

LIQUIDS (Bottles with vented caps ½ FULL)

75ml --- 25 MINUTES

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250ml --- 30 MINUTES500ml --- 40 MINUTES1000ml --- 45 MINUTES1500ml --- 50 MINUTES2000ml --- 55 MINUTES

The above times should be used as a guide in determining the length of time items should be autoclaved in order to achieve sterilization.

Disinfectants “Disinfectant” refers to an agent that is applied to treat (usually) inanimate objects to render them free of pathogenic or infectious microorganisms. In contrast, the term, “sterilant”, refers to an agent that renders items free of all microorganisms. The two are not the same and should not be confused.

Disinfectants are used in laboratory and chemical settings to 1) treat a surface or an item before or after routine use, or 2) to treat a surface or an item after a spill or “contaminating event”.

Because disinfectants are antimicrobial, they may, by their nature, also have a toxic effect to the user. Therefore, Material Safety Data Sheets and other manufacturer’s product information should be available and thoroughly reviewed before using these products.

There are many different types and formulations of disinfectants. The researcher or clinician should ensure that the proper product, one that is effective against the specific microorganism being studied, is used.

The FDA regulates those products that are marketed as sterilants or sanitizing agents on medical devices. They have published a list of products currently on the market that are labeled as sterilants. (http://www.epa.gov/spdpublc/snap/sterilants/index.html)

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The EPA regulates moderate and low level disinfectants as “chemical germicides” under their pesticide regulations. They have published a list of 1) those products that are effective against Mycobacterium spp. (tubercle bacilli), and 2) those products that are effective against HIV-1 (human immunodeficiency virus).

Please contact the Biological Safety Office for information about any of these lists or for a list of manufacturers. Be aware that most disinfectants assume pre-cleaning to remove gross organic/protein prior to use.

Whenever a disinfectant or sterilant is used, proper safety precautions must be followed. Appropriate clothing (gloves, safety goggles, aprons) must be worn. In addition, these compounds must be used in well-ventilated areas.

Following is a discussion of general categories of disinfectants. Please note that there are several different products and different formulations in each category.

Liquids

Alcohols The most commonly used alcohols, ethanol and isopropanol, are most effective at concentrations of 70% in water. Both higher and lower concentrations are less effective. Alcohols are active against vegetative bacteria, fungi, and lipid viruses but not against spores. They are only moderately effective against nonlipid viruses. Alcohols are difficult to use as contact disinfectants because they evaporate rapidly and do not penetrate organic matter well. When using alcohols, it is best to clean an object, then submerge it in alcohol for the appropriate time. Alcohols are often used in concert with other disinfectants such as formaldehyde (but see toxicity warning below) or chlorine (2000 ppm chlorine in alcohol). Alcohol is NOT a registered tuberculocidal or HIV listed disinfectant.

Chlorine compounds The most commonly used and generally effective disinfectant is sodium hypochlorite (common household bleach). It is a strong oxidizing agent and therefore can be corrosive to metal. A 1:50 dilution, supplying 1000 ppm available chlorine, of the common household product (e.g. chlorox) is very effective as a general laboratory disinfectant and a 1:10 dilution supplying 5000 ppm available chlorine is effective against spills involving blood or other organic material. Please note that the presence of high concentrations of protein can inactivate the action of chlorine products. Dilute hypochlorite solution must be prepared daily to be maximally effective. There are more concentrated sodium hypochlorite solutions available for industrial use, so please read the product information carefully to determine the proper dilution.

Table 4: Dilutions of Household Bleach

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Volume Volume Dilution Sodium AvailableOf Bleach of Water Ratio Hypochlorite % Chlorine (mg/L)Undiluted 0 1:1 5.25 50,000

1 9 1:10 0.5 5,0001 99 1:100 0.05 500

Formaldehyde Formaldehyde is a gas that is available either dissolved in water and methanol as a 37% formaldehyde solution, known as formalin, or as a solid, paraformaldehyde, that may be melted to release the gas. Formaldehyde gas is very active against a variety of microorganisms and is used for space decontamination and to decontaminate biological safety cabinets. Formaldehyde dissolved in water is active at 1-8% solutions and can be used to decontaminate hard surfaces. However, because formaldehyde is very irritating at low concentrations (0.1 to 5 ppm) and a known carcinogen, its use as a hard surface disinfectant is limited to situations in which it is particularly needed. Due to its toxic effects, there are no EPA-registered disinfectants that contain formaldehyde.

Glutaraldehyde Glutaraldehyde is usually supplied as a 20% solution and requires activation by the addition of an alkaline agent prior to use. The activated product may be kept for about two weeks and should be discarded when turbid. Glutaraldehyde is active against almost all microorganisms, but is toxic, irritating, and mutagenic and should be used only when necessary. Please follow the manufacturer’s guidance when using glutaraldehyde-based products because there are many different formulations that have been designed for specific uses.

Hydrogen peroxide Hydrogen peroxide is usually available as a 30% solution. It may be diluted 1:5 for use as a disinfectant. It is active against a wide array of microorganisms. However, it is an oxidizing agent and should not be used on aluminum, copper, zinc, or brass. Hydrogen peroxide is unstable at high temperatures and in light.

Iodine and Iodophors Iodine and iodophors, compounds in which the iodine is combined with a solubilizing or carrier agent, are general, all-purpose disinfectants with an action similar to that of chlorine products. The appropriate concentration for iodine-containing products is 75 ppm available iodine for disinfecting work surfaces. Concentrations may be much higher for other purposes. Like chlorine compounds, the effectiveness of iodine compounds may be diminished in the presence of protein/organic material. Iodophor compounds that are used for antisepsis (germicide applied to tissue or skin) are not appropriate for use as hard surface disinfectants and vice versa. Please read the product material for appropriate dilutions and applications.

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Phenol and phenolic compounds Phenolic compounds are active at 0.2 to 3% concentrations against all forms of vegetative microorganisms but not against spores. They have only limited effectiveness against nonlipid viruses. There are many common disinfectants based on phenol and they should be used according to the manufacturer’s recommendations. Phenol is a hazardous chemical and requires hazard assessment as well as proper PPE selection and use.

Quatenary ammonium compounds Compounds in this class are active at concentrations of 0.1 - 2%. They are active against vegetative bacteria, lipid viruses, but not against bacterial spores, non-lipid viruses, or tubercle bacilli. These compounds should be used only when a low-level disinfectant is required.

Gases

Ethylene Oxide (Not currently allowed at NAU) Inactivates micro-organisms, cellular disruptionTemp 50-60 C, Humidity 30-40%Conc 400-500 mg/literTime 2 hrs at 60 C or 24 hrs at 20 CAeration required after cycle for 8 hrsBiological indicators needed to confirm killSuspected carcinogen with explosive properties

Vapor Phase Hydrogen Peroxide Temp 4-60 C,> temp results in > activityConc 30%, less than 10 mg/literNon-toxic end products of water and oxygenLimited to surfaces, no penetrationCorrosive to some materialsDegrades natural rubber and nylon

Chlorine Dioxide gas Dilute chlorine gas and sodium chlorite, less than 25 mg/literTemp 25-30 C, prehumidification requiredLimited to surfaces, no penetration

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Corrosive to some materialsMucous membrane irritant

Ozone Oxidizing properties, inhibits bacterial growth, reacts with amino acids, RNA/DNATemp 25 CConc 2-5 mg/literSystems convert oxygen to ozoneLimited penetration

Formaldehyde Gas (from heating paraformaldehyde) Temp 20-22 C, humidity 60-85%Conc 0.3 gm/cu ft of volumeTime 6-8 hoursToxic irritant and suspected carcinogenLimited penetration, primarily surface actionRequires aeration and time for formaldehyde to off-gas, usually 8 hours

Irradiation

Ultraviolet, UV radiation Virucidal action correlates with shorter wavelengths of the UV spectrum,

250-260 nm.Mechanism of UV radiation injury attributed to absorption by and resultant damage to nucleic acidsDue to low energy, the power of penetration is poor.Dust and thin layers of proteins on surfaces reduce the virucidal activityDo not rely on Biosafety Cabinet UV lights to disinfect surfaces.

Ionizing radiation Penetrates products and micro-organismsReleases high energy, disrupts cells, DNA & RNA and results in killDamage done to cell membrane and other cellular structuresCobalt 60 and Cesium 137

Electron Beam Limited penetrationAccelerator generates high energy electrons

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Microwaves Thermal and non-thermal activityStill under study for the most part

Disinfectants BibliographyThe following materials were consulted or used in this chapter:Disinfection, Sterilization, and Preservation. Fourth edition. 1991. Seymour S. Block ed., Lea & Febiger, PhilidelphiaLaboratory Biosafety Manual. Second edition. 1993. World Health Organization, Geneva.Laboratory Safety: Principles and practices. Second edition. 1995. Diane O. Fleming et al. eds. ASM Press, Washington DCManual of Clinical Microbiology. Fifth edition. 1991. Albert Balows ed., ASM, Washington DCPrudent Practices in the Laboratory: Handling and disposal of chemicals. 1995 National Research Council. National Academy Press, Washington

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Table 5: Summary and Comparison of Liquid Disinfectants (Page 1)

Commonly used disinfectants, recommended when appropriate.

Class Recommended Use How They Work Advantages Disadvantages Comments/Hazards Examples70% Isopropyl alcohol solution

Cleaning some instruments Cleaning skin

Changes protein structure of microorganism Presence of water assists with killing action

Fairly inexpensive <50% solution not very effective Not active when organic matter present Not active against certain types of viruses Evaporates quickly - contact time not sufficient for killing

FlammableEye IrritantToxic

Chlorine compounds

Spills of human body fluids Bactericidal - Good Fungicidal - Good Sporicidal - Good at >1000 ppm Sodium Hypochlorite

Free available chlorine combines with contents within microorganism - reaction byproducts cause its death Need 500 to 5000 ppm Produce chemical combination with cell substances Depend upon release of hypochlorous acid

Kill hardy viruses (e.g., hepatitis) Kill a wide range of organisms Inexpensive Penetrates well Relatively quick microbial kill May be used on food prep surfaces

Corrode metals such as stainless, aluminum Organics may reduce activity Increase in alkalinity decreases bactericidal property Unpleasant taste and odor Tuberculocidal - with extended contact time

Follow spill procedure and dilution instructions Make fresh solutions before use Eye, skin, and respiratory irritantCorrosiveToxic

Bleach solutions (sodium hypochlorite) Clorox Cryosan Purex

Gluteraldehyde

Bactericidal - Good Fungicidal - Good Tuberculocidal - Excellent Virucidal - Good Sporicidal - Good

Coagulates cellular proteins

Non-staining, relatively non-corrosive Useable as a sterilant on plastics, rubber, lenses, stainless steel, and other items that can’t be autoclaved

Not stable in solution Has to be in alkaline solution Inactivated by organic material

Eye, skin and respiratory irritant.SensitizerToxic

Calgocide 14 Cidex Vespore

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Table 5: Summary and Comparison of Liquid Disinfectants (Page 2)Class Recommended

UseHow They Work Advantages Disadvantages Comments/Hazards Examples

Iodophors (iodine with carrier)

Disinfecting some semi-critical medical equipment Bactericidal - Very Good Fungicidal - Excellent Virucidal - Excellent

Free iodine enters microorganism and binds with its cellular components Carrier helps penetrate soil/fat Need 30 to 50 ppm Probably by disorder of protein synthesis due to hindrance and/or blocking of hydrogen bonding

Kill broad range of organisms Highly reactive Low tissue toxicity Kill immediately rather than by prolonged period of stasis Not affected by hard water May be used on food prep surfaces

May stain plastics or corrode metal May stain skin/laundry Stains most materials Odor Some organic and inorganic substances neutralize effect Tuberculocidal - with extended contact time Sporicidal - some

Dilution critical - follow directions! Use only EPA-registered hard surface iodophor disinfectants Don’t confuse skin antiseptic iodophors for disinfectantsSkin and eye irritantCorrosiveToxic

Bactergent Hy-Sine Ioprep Providone-iodine; betadine Wescodyne

Phenolic Compounds

Bactericidal - Excellent Fungicidal - Excellent Tuberculocidal - Excellent Virucidal - Excellent

Gross protoplasmic poison Disrupts cell walls Precipitates cell proteins Low concentrations inactivate essential enzyme systems

Nonspecific concerning bactericidal and fungicidal action When boiling water would cause rusting, the presence of phenolic substances produces an anti-rusting effect

Unpleasant odor Some areas have disposal restrictions Effectiveness reduced by alkaline pH, natural soap, or organic material Sporicidal - NO

Skin and eye irritantSensitizerCorrosiveToxic

Hil-Phene Lph Metar Vesphene

Quaternary ammonium compounds (QUATS)

Ordinaryhousekeeping(e.g., floors, furniture, walls) Bactericidal - Excellent Fungicidal - Good Virucidal - Good (not as effective as phenols)

Affect proteins and cell membrane of microorganism Release nitrogen and phosphorous from cells

Contain a detergent to help loosen soil Rapid action Colorless, odorless Non-toxic, less corrosive Highly stable May be used on food prep surfaces

Do not eliminate spores, TB bacteria, some viruses Effectiveness influenced by hard water Layer of soap interferes with action

Select from EPA list of hospital disinfectantsSkin and eye irritantToxic

Coverage 258 End-Bac Hi Tor

Barbara Fox Nellis (8-23-96)

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Table 6: Summary of Practical Disinfectants

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Quatenary Ammonium Compounds

Phenolic Compounds

Chlorine Compounds Iodophor Ethyl Alcohol

Isopropyl Alcohol

Formaldehyde Glutaraldehyde

InactivatesVegetative Bacteria + + + + + + + +Lipoviruses + +a + + +a + + +Nonlipid Viruses - - + + - - + +Bacterial Spores - - + + - - + +

Treatment RequirementsUse Dilution 0.1-2.0% 1.0-5.0% 500ppmb 25-1600ppmb 70-85% 70-85% 0.2-0.8% 2%Contact Time-minutes

Lipovirus 10 10 10 10 10 10 10 10Broad Spectrum NE NE 30 30 NE NE 30 30

Important CharacteristicsEffective Shelf Life > 1 weekc

+ + - + + + + +

Corrosive - + + + - - - -Flammable - - - - + + - -Explosion Potential - - - - - - - -Inactivated by organic matter

+ - + + - - - -

Skin Irritant + + + + - - + +Eye Irritant + + + + + + + +Respiratory Irritant - - + - - - + +Toxicd + + + + + + + +

ApplicabilityWaste Liquids - - + - - - - -Dirty Glassware + + + + + + + +Equipment, Surface Decontamination

+ + + + + + + +

Proprietary Productse A-33, CDQ, End-Bac, Hl-Tor, Mikro-Quat

Hil-Phene, Metar, Mikro-

Bac, O-Syl

Chloramine T, Clorox,

Purex

Hy-Sine, Ioprep,

Mikroklene, Wescodyne

Sterac Cidex

Source: Adapted from Laboratory Safety Monograph, A Supplement to the NIH Guidelines for Recombinant DNA Research. National Institutes of Health, Office of Research Safety, National Cancer Institute, and the Special Committee of Safety and Health Experts, Bethesda, Maryland. January 1989: 104-105+ Yes a Variable results depending on virus d By skin or mouth or both- No b Available Halogen e Space limitations preclude listing all products available. Individual listings (or omissions)NE Not Effective c Protected from light and air do not imply endorsement (or rejection) of any product by the National Institutes of Health.

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Table 7: Reprocessing Methods for Equipment Used in the Health Care Setting

Sterilization: Destroys: All forms of microbial life including high numbers of bacterial spores.

Methods:Steam under pressure (autoclave), gas (ethylene oxide), dry heat or immersion in an approved chemical “sterilant” (e.g., US Environmental Protection Agency-approved) for prolonged period of time, e.g., 6-10 hours or according to manufacturer’s instructions. Note: Liquid chemical “sterilants” should be used only on those instruments that are impossible to sterilize or disinfect with heat.

Use:For those instruments or devices that penetrate skin or contact normally sterile areas of the body, e.g., scalpels, needles, etc. Disposable invasive equipment eliminates the need to reprocess these types of items. When indicated, however, arrangements should be made with a health-care facility for reprocessing of reusable invasive instruments.

High-LevelDisinfection:

Destroys: All forms of microbial life except high numbers of bacterial spores.

Methods: Hot water pasteurization (80-100°C, 30 minutes) or exposure to an approved (e.g., US EPA-approved) “sterilant” chemical as above, except for a short exposure time (10-45 minutes or as directed by the manufacturer).

Use: For reusable instruments or devices that come into contact with mucous membranes (e.g., laryngoscope blades, endotracheal tubes, etc.).

Intermediate-Level

Disinfection:

Destroys: Mycobacterium tuberculosis, vegetative bacteria, most viruses and most fungi, but does not kill bacterial spores.

Methods:Approved (e.g. US EPA-approved) “hospital disinfectant” chemical germicides that have a label claim for tuberculocidal activity; commercially available hard-surface germicides or solutions containing at least 500 ppm free available chlorine (a 1:10 dilution of common household bleach- about ¼ cup bleach per gallon of tap water).

Use:For those surfaces that come into contact only with intact skin, e.g., stethoscopes, blood pressure cuffs, splints, etc., and have been visibly contaminated with blood or bloody body fluids. Surfaces must be pre-cleaned of visible material before the germicidal chemical is applied for disinfection.

Low-LevelDisinfection:

Destroys: Most bacteria, some viruses, some fungi, but no Mycobacterium tuberculosis or bacterial spores.

Methods: Approved (e.g., US EPA-approved) “hospital disinfectants” (no label claim for tuberculocidal activity).

Use: Use these excellent cleaning agents for routine housekeeping or removal of soiling in the absence of visible blood contamination.

EnvironmentalDisinfection:

Environmental surfaces that have become soiled should be cleaned and disinfected using any cleaner or disinfectant that is intended for environmental use. Such surfaces include floors, woodwork, ambulance seats, countertops, etc.

Important: To assure effectiveness of any sterilization or disinfection process, equipment and instruments must first be thoroughly cleaned of all visible soil.

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Shipment of Biological MaterialsThe following regulations apply to the packaging and shipment of biological materials:

U.S. Department of Transportation, 49 CFR Parts 171-180 and amendmentsU.S. Public Health Service, 42 CFR Part 72, Interstate Shipment of Etiolgic AgentsU.S. Department of Labor, Occupational Safety and Health Administration, 29 CFR Part

1910.1030, Bloodborne PathogensInternational Air Transport Association (IATA), Dangerous Goods RegulationsU.S. Postal Service, 39 CFR Part 111, Mailability of Etiologic Agents, Mailability of Sharps and

Other Medical Devices, and Publication 52, Acceptance of Hazardous, Restricted or Perishable Matter

International Civil Aviation Organization, Technical Instructions for the Safe Transport of Dangerous Goods by Air

United Nations, Recommendations of the Committee of Experts on the Transportation of Dangerous Goods

All North American airlines and FedEx, the largest shipper of infectious materials, use the IATA regulation (also referred to as the Dangerous Goods Regulation or DGR) as their standard. Meeting the conditions of this standard will ensure meeting the provisions of the other US regulations.

General information is provided below, but please contact the EH&S Biological Safety Office for specific information. Note that for any biological materials for which a state or federal permit or license is required, registration with the EH&S Biological Safety Office is also required.

Any NAU faculty/staff, regardless of job or task, must be trained to package, ship or receive packages of materials/samples classified by regulation as “dangerous goods”. This training is available from EH&S. Regulation requires retraining every other year OR whenever the regulations change.

Laboratory SpillsA spill of biological materials that takes place in the open laboratory may create a serious problem. Every effort should be taken to prevent spilling materials.

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A spill poses less of a problem if it happens inside a biological safety cabinet, provided splattering to the outside of the cabinet does not occur. Direct application of concentrated liquid disinfectant and a thorough wipe down of the internal surfaces of the cabinet will usually be effective for decontaminating the work zone, but gaseous sterilants will be required to disinfect the interior sections of the cabinet and HEPA filter(s).

Spill in the Open LaboratoryAdvance preparation for management of a spill is essential. A “spill kit” may include the following: Leak-proof containers Forceps Paper towels Disinfectant Respirators, if necessary Rubber gloves and other personal protective equipment Absorbent powder or other material Red bag

If potentially hazardous biological material is spilled in the laboratory, avoid inhaling any airborne material by holding your breath and leaving the laboratory. Warn others in the area by posting signs and go directly to wash or change room area. If clothing is known (or suspected) to be contaminated, remove the clothing with care, folding the contaminated area inward. Discard the clothing into a bag or place the clothing directly in an autoclave. Wash all potentially contaminated body areas as well as the arms, face and hands. Shower if facilities are available. Re-entry into the laboratory should be delayed for a period of at least thirty minutes to allow reduction of the aerosol generated by the spill.

Protective clothing should be worn when entering the laboratory to clean the spill area. Rubber gloves, autoclavable or disposable footwear, an outer garment, and a respirator equipped with a HEPA filter should be worn. If the spill was on the floor, do not use a surgical gown that may trail on the floor when bending down. Take the “ spill kit” into the laboratory room, place a discard container near the spill, and transfer large fragments of material into it; replace the cover. Using a freshly prepared hypochlorite containing 5000 ppm (10% by volume) available chlorine, iodophor solution containing 1600 ppm iodine, or other appropriate EPA registered disinfectant, carefully pour the disinfectant around and into the visible spill. Avoid splashing and work from the outside toward the center. Allow 30 minutes' contact time. Use paper or cloth towels to wipe up the disinfectant and spill, working toward the center of the spill. Discard all towels and other clean up materials into a discard container as they are used. Wipe the outside of the discard containers, especially the bottom, with a towel soaked in a disinfectant. Place the discard container and other materials in an autoclave and sterilize. Alternately, place all materials in the appropriate biomedical waste system for incarceration. Remove shoes or

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shoe covers, outer clothing, respirator, and gloves and sterilize by autoclaving. Wash hands, arms and face, or if possible, shower.

If gaseous decontamination of the room is required, contact the Biosafety Office.

Spill in a Biological Safety CabinetA spill that is confined to the interior of the biological safety cabinet should present little or no hazard to personnel in the area. However, chemical disinfection procedures should be initiated at once while the cabinet ventilation system continues to operate to prevent escape of contaminants from the cabinet.

Spray or wipe walls, work surfaces, and equipment with a disinfectant. A disinfectant with a detergent has the advantage of detergent activity that will help clean the surfaces by removing both dirt and microorganisms. A suitable disinfectant is a 3% solution of an iodophor such as Wescondyne or a 1:100 dilution of household bleach (e.g. Chlorox) with 0.7% nonionic detergent. The operator should wear gloves during this procedure. Use sufficient disinfectant solution to ensure that the drain pans and catch basins below the work surface contain the disinfectant. Lift the front exhaust grill and tray and wipe all surfaces. Wipe the catch basin and drain the disinfectant into a container. The disinfectant, gloves, wiping cloth and sponges should be discarded into an autoclave pan and autoclaved.

The above procedure will not disinfect the filters, blower, air ducts, or other interior parts of the cabinet. If the entire interior of the cabinet needs to be sterilized, contact the EH&S Biosafety Office for the name of the current contractor.

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Biological Safety CabinetsThe following is excerpted from Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets , 1995, Centers for Disease Control & Prevention/National Institutes of Health

This chapter presents information on the selection, function, and use of biological safety cabinets (BSCs), which are the primary means of containment developed for working safely with infectious microorganisms. BSCs are designed to provide personnel, environment, and product protection when appropriate practices and procedures are followed.

Three kinds of biological safety cabinets, designated as Class I, II, and III, have been developed to meet varying research and clinical needs.

Biological safety cabinets use high efficiency particulate air (HEPA) filters in their exhaust and/or supply systems.

Biological Safety Cabinets (BSCs)The similarities and differences in protection offered by the various classes of biosafety cabinets are reflected in Table 1.

The Class I BSC This type of cabinet is not for aseptic or sterile technique. The Class I BSC provides personnel and environmental protection, but no product protection. It is similar in air movement to a chemical fume hood, but has a HEPA filter in the exhaust system to protect the environment.

The Class II BSC The Class II (Types A and B) biological safety cabinets provide personnel, environmental, and product protection. Air flow 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 provides product protection by minimizing the chance of cross-contamination along the work surface of the cabinet. Because cabinet air has passed through the exhaust HEPA filter, it is contaminant-free (environmental protection), and may be recirculated back into the laboratory (Type A BSC) or ducted out of the building (Type B BSC).

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The Class II, Type A BSC An unducted Class II Type A BSC is not to be used for work involving volatile or toxic chemicals. The buildup of chemical vapors in the cabinet (by recirculated air) and in the laboratory (from exhaust air) could create health and safety hazards. Generally, BSCs are not for use with chemicals. Small quantities of chemicals and chemotherapeutic agents may be used in ducted, 100% BSCs.

It is possible to duct the exhaust from a Type A cabinet out of the building. However, it must be done in a manner that does not alter the balance of the cabinet exhaust system, thereby disturbing the internal cabinet air flow. The typical method of ducting a Type A cabinet is to use a “thimble”, or canopy unit, which maintains a small opening (usually 1 inch) around the cabinet exhaust filter housing. The volume of the exhaust must be sufficient to maintain the flow of room air into the space between the thimble unit and the filter housing (contact manufacturers for any additional specifications). The thimble must be removable or be designed to allow for operational testing of the cabinet. The performance of a cabinet with this exhaust configuration is unaffected by fluctuations in the building exhaust system.

“Hard-ducting” (i.e., direct connection) of Class II Type A cabinets to the building exhaust system is not recommended. The building exhaust system must be precisely matched to the airflow from the cabinet in both volume and static pressure. However, fluctuations in air volume and pressure that are common to all building exhaust systems make it difficult, if not impossible, to match the airflow requirements of the cabinet.

The Class II, Type B1 BSC Some biomedical research requires the use of small quantities of certain hazardous chemicals, such as carcinogens. The powdered form of these carcinogens should be weighed or manipulated in a chemical fume hood or a static-air glove box. Carcinogens used in cell culture or microbial systems require both biological and chemical containment.

Type B1 cabinets must be hard-ducted to their own dedicated exhaust system. As indicated earlier, blowers on laboratory exhaust systems should be located at the terminal end of the ductwork. A failure in the building exhaust system may not be apparent to the user, as the supply blowers in the cabinet will continue to operate. A pressure-independent monitor should be installed to sound an alarm and shut off the BSC supply fan, should failure in exhaust airflow occur. Since all cabinet manufacturers do not supply this feature, it is prudent to install a sensor in the exhaust system as necessary. To maintain critical operations, laboratories using Type B BSCs should connect the exhaust blower to the emergency power supply.

The Class II, Type B2 BSC This BSC is a total-exhaust cabinet; no air is recirculated within it. This cabinet provides simultaneous primary biological and chemical containment.

Should the building or cabinet exhaust fail, the cabinet will be pressurized, resulting in a flow of air from the work area back into the laboratory. Cabinets built since the early

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1980's usually have an interlock system installed by the manufacturer to prevent the supply blower from operating whenever the exhaust flow is insufficient. Presence of such an interlock system should be verified; systems can be retrofitted if necessary. A pressure-independent device should monitor exhaust air movement.

The Class II, Type B3 BSC This biological safety cabinet is a ducted Type A cabinet. All positive pressure contaminated plenums within the cabinet are surrounded by a negative air pressure plenum. Thus, leakage in a contaminated plenum will be into the cabinet and not into the environment.

The Class III BSC The Class III biological safety cabinet was designed for work with biosafety level 4 microbiological agents, and provides maximum protection to the environment and the worker. It is a gas-tight enclosure with a non-opening view window.

Long, heavy-duty rubber gloves are attached in a gas-tight manner to ports in the cabinet and allow for manipulation of the materials isolated inside. Although these gloves restrict movement, they prevent the user's direct contact with the hazardous materials. The trade-off is clearly on the side of maximizing personal safety. Depending on the design of the cabinet, the supply HEPA filter provides particulate-free, albeit somewhat turbulent, airflow within the work environment.

Horizontal Laminar Flow “Clean Bench”Horizontal laminar flow clean air benches are NOT BSCs. They discharge HEPA-filtered air across the work surface and toward the user. These devices only provide product protection. They can be used for certain clean activities, such as the dust-free assembly of sterile equipment or electronic devices. These benches should never be used when handling cell culture materials or drug formulations, or when manipulating potentially infectious materials. The worker can be exposed to materials (including proteinaceous antigens) being manipulated on the clean bench, which may cause hypersensitivity. Horizontal clean air benches should never be used as a substitute for a biological safety cabinet in research, biomedical or veterinary laboratories and/or applications.

Vertical Laminar Flow “Clean Bench”Vertical laminar flow clean benches also are NOT BSCs. They may be useful, for example, in hospital pharmacies when a clean area is needed for preparation of intravenous drugs. While these units generally have a sash, the air is usually discharged into the room under the sash, resulting in the same potential problems as the horizontal laminar flow clean benches.

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Operations within a Class II BSC

Laboratory Hazards Many common procedures conducted in BSCs may create splatter or aerosols. Good microbiological techniques should always be used when working in a biological safety cabinet. For example, techniques to reduce splatter and aerosol generation will minimize the potential for exposure to personnel from infectious materials manipulated within the cabinet. Class II cabinets are designed so that horizontally nebulized spores will be captured by the downward flowing cabinet air within fourteen inches of travel. Therefore, as a general rule of thumb, keeping clean materials at least one foot away from aerosol-generating activities will minimize the potential for cross-contamination.

The general workflow should be from “clean to contaminated (dirty).” Materials and supplies should be placed in such a way as to limit the movement of “dirty” items over “clean” ones.

Several measures can be taken to reduce the chance for cross-contamination when working in a BSC. Work at least 6” back from the front edge and never cover the front grill. Opened tubes or bottles should not be held in a vertical position. Investigators working with Petri dishes and tissue culture plates should hold the lid above the open sterile surface to minimize direct impact of downward air. Bottle or tube caps should not be placed on the toweling. Items should be recapped or covered as soon as possible.

Open flames are not permitted in the near microbe-free environment of a BSC. On an open bench, flaming the neck of a culture vessel will create an upward air current that prevents microorganisms from falling into the tube or flask. An open flame in a BSC, however, creates turbulence that disrupts the pattern of air supplied to the work surface. When deemed absolutely necessary, touch-plate microburners equipped with a pilot light to provide a flame on demand may be used. Internal cabinet air disturbance and heat buildup will be minimized. The burner must be turned off when work is completed. Small electric “furnaces” are available for decontaminating bacteriological loops and needles and are preferable to an open flame inside the BSC. Disposable sterile loops should be used to eliminate the need for heat or flame.

Aspirator bottles or suction flasks should be connected to an overflow collection flask containing appropriate disinfectant, and to an in-line HEPA or equivalent filter. This combination will provide protection to the central building vacuum system or vacuum pump, as well as to the personnel who service this equipment. Inactivation of aspirated materials can be accomplished by placing sufficient chemical decontamination solution into the flask to kill the microorganisms as they are collected. Once inactivation occurs, liquid materials can be disposed of appropriately as noninfectious waste.

Investigators must determine the appropriate method of decontaminating materials that will be removed from the BSC at the conclusion of the work. When chemical means are appropriate, suitable liquid disinfectant should be placed into the discard pan before work begins. Items should be introduced into the pan with minimum splatter, and allowed

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appropriate contact time as per manufacturer's instructions. Alternatively, liquids can be autoclaved prior to disposal. Contaminated items should be placed into a biohazard bag or discard tray inside the BSC. Water should be added to the bag or tray prior to autoclaving.

When a steam autoclave is to be used, contaminated materials should be placed into a biohazard bag or discard pan containing enough water to ensure steam generation during the autoclave cycle. The bag should be taped shut or the discard pan should be covered in the BSC prior to removal to the autoclave. The bag should be transported and autoclaved in a leak-proof tray or pan.

Decontamination

Surface Decontamination All containers and equipment should be surface decontaminated and removed from the cabinet when work is completed. At the end of the workday, the final surface decontamination of the cabinet should include a wipe-down of the work surface, the cabinet's sides and back, and the interior of the glass. If necessary, the cabinet should also be monitored for radioactivity and decontaminated when necessary. Investigators should remove their gloves and gowns and wash their hands as the final step in safe microbiological practices.

Small spills within the BSC can be handled immediately by removing the contaminated absorbent paper toweling and placing it into the biohazard bag. Any splatter onto items within the cabinet, as well as the cabinet interior, should be immediately wiped with a towel dampened with decontaminating solution. Gloves should be changed after the work surface is decontaminated and before placing clean absorbent toweling in the cabinet. Hands should be washed whenever gloves are changed or removed.

Spills large enough to result in liquids flowing through the front or rear grilles require more extensive decontamination. All items within the cabinet should be surface decontaminated and removed. After ensuring that the drain valve is closed, decontaminating solution can be poured onto the work surface and through the grille(s) into the drain pan.

Thirty minutes is generally considered an appropriate contact time for decontamination, but this varies with the disinfectant and the microbiological agent. Manufacturer's directions should be followed. The spilled fluid and disinfectant solution on the work surface should be absorbed with paper towels and discarded into a biohazard bag. The drain pan should be emptied into a collection vessel containing disinfectant. A flexible tube should be attached to the drain valve and be of sufficient length to allow the open end to be submerged in the disinfectant within the collection vessel. This procedure serves to minimize aerosol generation. The drain pan should be flushed with water and the drain tube removed.

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Should the spilled liquid contain radioactive material, a similar procedure can be followed. The EH&S Radiation Safety Officer should be contacted for specific instructions. See contact information in the front of this manual.

Gas Decontamination BSCs that have been used for work involving infectious materials must be decontaminated before HEPA filters are changed, internal repair work is done or before a BSC is relocated. The most common decontamination method uses formaldehyde gas, although more recently, hydrogen peroxide vapor has been used successfully. This environmentally benign vapor is useful in decontaminating HEPA filters, isolation chambers, and centrifuge enclosures. Call the EH&S Biosafety Office for the current BSC vendor who does the gas decontaminations and certifications. All BSCs must be re-certified following any gas decontamination, maintenance or relocation.

Engineering Requirements

Ultraviolet Lamps Ultraviolet (UV) lamps are not required in BSCs. If installed, UV lamps must be cleaned weekly to remove any dust and dirt that may block the germicidal effectiveness of the ultraviolet light. The lamps should be checked periodically with a meter to ensure that the appropriate intensity of UV light is being emitted. UV lamps must be turned off when the room is occupied to protect eyes and skin from UV exposure, which can burn the cornea and cause skin cancer. Do not depend on UV lamps to disinfect the area.

BSC Placement The ideal location for the biological safety cabinet is remote from the entry (e.g., the rear of the laboratory away from traffic), since people walking parallel to the face of a BSC can disrupt the air curtain. The air curtain created at the front of the cabinet is quite fragile, amounting to a nominal inward and downward velocity of 1 mph. Open windows, air supply registers, or laboratory equipment that creates air movement (e.g., centrifuges, and vacuum pumps) should not be located near the BSC. Similarly, chemical fume hoods must not be located close to BSCs.

HEPA Filters HEPA filters, whether part of a building exhaust system or part of a cabinet, will require replacement when they become so loaded that sufficient air flow can no longer be maintained. Filters must be decontaminated before removal.

Certification of BSCs All BSCs must be certified (according to a National Sanitation Foundation standard) annually according to NAU policy. Please contact the EH&S Biological Safety Office for the name and phone number of the current contractor performing this service. Prices and quality vary widely, so only BSO-approved contractors may be used.

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3 – Programs

Animal Contact Medical Monitoring ProgramThe NAU Animal Contact Medical Monitoring Program is designed to protect employees from occupational exposure to conditions that may result in animal related illnesses. According to the Public Health Service, and occupational health program is required for institutions that employ personnel who have animal contact. The NAU Animal Contact Monitoring Program is overseen by the NAU Institutional Animal Care and Use Committee (IACUC).

Occupational Health and Safety (Animal Contact Medical Monitoring Program)Occupational health and safety is of utmost importance to the NAU IACUC. Regulations require that the IACUC monitor personnel occupational health programs and training related to animal use at NAU. Occupational health programs must include training in basic safety techniques and information about hazards which will be encountered in each particular job. Hazardous materials must be stored safely and in compliance with MSDS and OSHA regulations. Posted signs must warn of hazards present. The NAU program is designed to protect all personnel from occupational exposure to conditions that may result in animal related illnesses.

Responsibilities Under the Program All individuals who engage in animal-related work will participate in the university occupational health program. As the initial step in this program, individuals will complete the "Occupational Health Questionnaire" found at the IACUC website www.nau.edu/iacuc. The University Veterinarian will review the questionnaire, make any comments which may highlight animal work related concerns, and forward the form to health care providers at Fronske Health Center. At the discression of Fronske medical personnel, the individual will be scheduled for a brief interview or examination, where potential health-risks will be discussed relative to the individual's health status and job requirements. All individuals who work with animals will be current on their tetanus vaccine. Serology and/or vaccination for rabies, Hepatitis B, toxoplasmosis, measles, and skin testing for tuberculosis may be required for work with certain species or protocols. Vaccination for rabies will be required to work with wild or random-source bats, skunks, and carnivores. Tuberculosis testing will be required for work with all non-human primates. This program is provided at no cost to the employee.

Principle Investigators are responsible for ensuring that all personnel (including employees, students, colleagues, collaborators, and volunteers) involved with their IACUC-approved project are given this program information and that they receive the appropriate immunizations and tests, etc. Investigators who do not respond to requests for registration may have their animal use approval rescinded by the IACUC.

Supervisors are responsible for providing information to animal care technicians, veterinarians, and associated animal workers (e.g. workers in Animal Care

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http://www.nau.edu/iacuc

Services) who work with animals but are not on a specific research project.

The University Veterinarian and/or health care providers at Fronske will conduct an educational component of the occupational health program as appropriate. Topics to be discussed include zoonoses relative to the protocol, allergy risk, possible risks to immunocompromised individuals, and use of protective equipment and supplies such as gloves, eye protection, masks, lab coats, scrubs, respirators, head covers, boots, and heat resistant gloves chemical resistant gloves. An animal exposure case involves mucous membrane contact with animal fluids or excreta, ingestion of animal fluids or excreta, bites, scratches, etc.. Emergency medical care for minor animal exposure cases is available at Fronske Health Center during business hours, Monday-Friday. For more serious cases and during other hours, care is available that the Flagstaff Medical Center Emergency Room. In cases where individuals may be exposed to non-routine or exotic diseases, it is necessary to have emergency care sheets to provide basic information for medical personnel. Such information may include MSDS's, sheets copied from the "Control of Communicable Diseases Manual," or monographs written by experts in diseases such as Cercopithicine Herpes Virus. Such sheets should be readily available so affected individuals may easily take them when seeking emergency care. MSDS's for chemicals used in laboratories must also be readily available.

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NAU Bloodborne Pathogen ProgramIn December 1991, OSHA published the final rule covering occupational exposure to bloodborne pathogens. This was adopted by the state of Arizona and written into the Arizona Administrative Code in January 1993. NAU instituted its program in the spring of 1993.

The rule requires that those who handle human blood or other potentially infectious (human) materials as part of their job duties participate in an employer-generated program. This program shall include development and annual review of a site specific Exposure Control Plan, annual training regarding exposures, offer of hepatitis B vaccinations free-of-charge, and post-exposure health care services.

Northern Arizona University manages this program through the EH&S Biological Safety Office. The BBP program materials and information is available on our website at www4.nau.edu/regulatorycompliance

In addition to the program materials, the EH&S Biosafety Office provides training sessions annually.

Hepatitis B vaccinations are given by the Fronske Health Center. Official program medical records are kept by Fronske Health Center. There is a requirement that Flagstaff Medical Center staff or other providers send vaccination and post-exposure records to Fronske Health Center for record-keeping purposes. Declination statements from those that decline the vaccination series are kept by The EH&S Biosafety Office.

The EH&S Biosafety Office monitors for NAU compliance by requiring training and vaccination documentation and by confirming BBP participation during the annual laboratory safety survey which is conducted in each laboratory each year.

For further information, please call the EH&S Biological Safety Office.

BBP Exposure Information

For exposure incidents more than 1 hour away from Flagstaff, go to the nearest medical facility.

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4 -Medical Surveillance

HIV Research LaboratoryOccupational Medicine Policy

This policy is designed to protect employees who conduct research with HIV. Researchers who handle, manipulate, or assay live HIV cultures are covered under this policy.

Pre-employment Prior to beginning work in an HIV research lab, an employee shall be given, at no cost to the employee, the following:

1. A baseline serum sample shall be stored with the NAU Student Health Care Center. This is a requirement to work in the HIV lab.

2. A confidential HIV test shall be offered to the employee. The HIV test is available at the NAU Student Health Care Center. The test must be offered by the employer, but may be declined by the employee. This test shall include HIV counseling. Results of the test shall be given to the employee in person in a face-to-face meeting with a health care professional. No exceptions. All results of HIV testing shall remain completely confidential and shall be stored in a separate section in the employee’s medical record. At no time, will the employer or any administrator or official of NAU have access to the employee’s confidential record concerning HIV testing.

3. If the HIV researcher also works with human blood or other potentially infected material, all aspects of the NAU Bloodborne Pathogen Program under the OSHA Bloodborne Pathogen standard shall be implemented for that employee. The employer shall offer free HBV immunization, annual training, and have a written exposure control plan in the workplace.

Continuing employmentHIV researchers shall be given the following at no cost to the employee:1. Annual serum banking shall be offered to the employee, but may be declined.2. Annual HIV tests shall be offered to the employee, but may be declined.3. Annual training regarding HIV and post-exposure prophylaxis, including the risks of

chemoprophylaxis, shall be given to the employee. This is a required, not optional, component.

Post-exposure prophylaxisAn exposure to an HIV culture in a research laboratory is considered a “highest risk exposure” according to Public Health Service CDC guidelines. The following shall be implemented:1. If the exposure is to intact or broken skin, or in the event of a puncture wound, immediately

wash the affected area with water for five minutes. Soap may be used, if immediately available. If the exposure is to the eyes, they shall be rinsed for five minutes in an eyewash station. Other exposed mucous membranes (nose and mouth) shall be rinsed for five minutes with water.

2. The employee shall proceed immediately to the nearest health care facility. It is important for the employee to seek treatment within the first two hours of exposure.

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3. The employee may be seen at the Fronske Health Center between the hours of 8:00 a.m. and 4:00 p.m. Monday through Friday. After hours, employees should proceed to the Flagstaff Medical Center Emergency Department, where they will be seen immediately, free-of-charge.

4. The current Public Health Service (CDC) chemoprophylactic treatment/ post-exposure prophylaxis (PEP) shall be recommended and offered to an employee with the highest risk exposure, within the first two hours of exposure. However, medical use of chemoprophylaxis is a decision that shall be made by a health care professional in conjunction with the employee, based upon the most current CDC recommendations, the nature of the exposure event, and other medical factors. Flagstaff Medical Center pharmacy is well stocked with the appropriate medication.

5. Baseline tests and informed consent statements shall be required for PEP. 6. Counseling concerning the risks and benefits of the chemoprophylactic treatment shall be given

to the employee at the time of the exposure event. 7. The employee may contact an infectious disease specialist, if desired. 8. All HIV exposures that occur in the workplace are covered under Worker’s Compensation.

Flagstaff Medical Center and NAU personnel are familiar with the paperwork to process this type of Worker’s Compensation claim for NAU employees.

9. Post-exposure follow-up, including the offer of HIV testing and counseling, shall be given in accordance with the CDC recommendations and the OSHA Bloodborne Pathogen rule. The Flagstaff Medical Center protocol on HIV exposures will be followed.

ReferencesNAU Bloodborne Pathogen Program Compliance Materials; www4.nau.edu/regulatory complianceOSHA. 29 CFR Part 1910.1030. Occupational Exposure to Bloodborne Pathogens, Final Rule, Dec.

6, 1991CDC. Update: Provisional public health service recommendations for chemoprophylaxis after

occupational exposure to HIV; MMWR 1996; 45(22):468-472

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ImmunoprophylaxisThe Northern Arizona University follows the recommendations of the Centers for Disease Control and Prevention (CDC) and the Public Health Service Advisory Committee for Immunization Practices (ACIP) for vaccination of at-risk personnel.

Currently, NAU has written policies regarding the need for hepatitis B, anthrax, plague, tetanus, rabies, and vaccinia vaccination for certain at-risk personnel. Other vaccinations may be required or recommended, as needed. Particular attention is given to individuals who are or may become immunocompromised, as recommendations for vaccine administration may be different than for immunologically competent adults. Various other factors may be taken into account such as pregnancy, history of allergy, or HIV status.

When considering the need for immunization, a risk assessment will be conducted by the health care provider in conjunction with information regarding the experimental agent provided by the EH&S Biological Safety Office.

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Recommendations for Prophylactic Immunization of Laboratory Personnel Working with Infectious AgentsPlease note, the following is derived from the CDC publication, Biosafety in Microbiological and Biomedical Laboratories, 5th Edition, 2007 and the CDC publication, “Update on Adult Immunization: Recommendations of the Immunization Advisory Committee (ACIP),” 1991, MMWR vol. 40.

Bacterial agents 1. Bacillus anthracis

A licensed vaccine is available through the CDC. However, immunization of laboratory personnel is not recommended unless frequent work with clinical specimens or diagnostic cultures is anticipated and in settings in which production quantities, high concentrations, or procedures with a high potential for aerosol (all BSL-3 work) are encountered. In these settings, vaccination is recommended for all persons working with the agent, all persons working in the laboratory room where the cultures are handled, and all persons working with infected animals.

2. Clostridium botulinumA pentavalent (ABCDE) botulism toxoid is available through the CDC as an investigational new drug (IND). This toxoid is recommended for personnel working with cultures of C. botulinum or its toxins.

3. Clostridium tetaniThe adult diphtheria-tetanus toxoid is highly recommended for laboratory personnel who manipulate cultures or toxins and is required by the NAU Animal Contact Program for all NAU personnel who work with animals. This immunization should be given every 10 years.

4. Corynebacterium diphtheriaThe adult diphtheria-tetanus toxoid every 10 years is recommended for laboratory personnel who work with this agent.

5. Francisella tularensisVaccination for F. tularensis is available and should be considered for personnel working with infectious material or infected rodents. It is recommended for for persons working in or entering laboratories or animal rooms where cultures or animals are maintained.

6. Neisseria meningitidisVaccines for N. meningitidis are available and should be considered for personnel regularly working with infectious materials. The reader is advised to consult the current recommendations of the Advisory Committee on Immunization Practices (ACIP) published in the CDC Morbidity and Mortality Weekly Report (MMWR) for recommendations for vaccination against N. meningitidis.

7. Salmonella typhiVaccines for S. typhi are available and should be considered for personnel regularly working with potentially infectious materials. The reader is advised to consult the current recommendations of the Advisory Committee on Immunization Practices (ACIP) published in the CDC Morbidity and Mortality Weekly Report (MMWR) for recommendations for vaccination against S. typhi.

8. Yersinia pestisVaccination for Y. pestis is available and should be considered for personnel working with infectious materials or infected rodents. For example: laboratory personnel who routinely perform procedures that involve viable Y. pestis; and persons (e.g., mammalogists, ecologists, and other field workers) who have regular contact with wild rodents or their fleas in areas in which plague is enzootic or epizootic. The reader is advised to consult the current recommendations of the Advisory Committee on Immunization Practices (ACIP) published in the CDC Morbidity and Mortality Weekly Report (MMWR) for information on vaccination against Y. pestis.

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Rickettsial agents 1. Coxiella burnetii

An investigational Phase I, Q fever vaccine (IND) is available on a limited basis from the Special Immunizations Program (301-619-4653) of the USAMRIID, Fort Detrick, Maryland, for at-risk personnel under a cooperative agreement with the individual's requesting institution. The use of this vaccine should be restricted to those who are at high risk of exposure and who have no demonstrated sensitivity to Q fever antigen.

Viral agents 1. Hepatitis A virus

A vaccine is available and recommended for those who work with hepatitis A virus.2. Hepatitis B virus

A vaccine is available and highly recommended for those who work with hepatitis B virus. The hepatitis B vaccine is recommended for those who have occupational exposure to human blood or other potentially infectious human materials, according to the OSHA rule and the NAU Bloodborne Pathogen Program.

3. PoliovirusAll laboratory personnel who work directly with the agent are required to have documented polio vaccination or demonstrated serologic evidence of immunity to all three poliovirus types.

4. Poxviruses: vaccinia, cow pox, monkey poxVaccination is required for those who work with or around orthopox viruses.

5. Rabies virusImmunization is recommended for all individuals prior to work with rabies virus or infected animals, or engaging in diagnostic, production, or research activities with rabies virus. Immunization is also recommended for all individuals entering or working in the same room where rabies virus or infected animals are used. The NAU Animal Contact Program requires immunization for all employees (e.g. veterinarians and animal control personnel) who come into contact with unvaccinated carnivores.

6. Arboviruses For the following arthropod-borne viruses there is a vaccine available and it is recommended for those who work with the virus.

Eastern equine encephalitis JuninWestern equine encephalitis MucamboCabassue Rift Valley FeverChikungunya Venezualan equine encephalitisEverglades Yellow Fever

7. Measles, mumps, and German measles virus (Rubeola, paramyxovirus, and rubella virus)Immunizations are usually given in childhood. Vaccines are highly recommended for all unvaccinated or serologically negative adults.

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Vaccinia Immunization Policy

All personnel who work in an area where vaccinia, orthopoxviruses, or animals vaccinated with those agents are used, handled or housed must have received a vaccinia vaccination within the last ten years. Individuals must declare their vaccination status prior to engaging in such work. This may be accomplished by using the NAU Smallpox Vaccine Consent form.

To fulfill this requirement:

1. Request a Vaccinia Vaccination Information Packet from the EH&S Biosafety Office.

2. Following review of the material, questions should be directed as follows:

a. Medical questions – Fronske Health Centerb. Virology questions – BSO and/or principal investigator.

3. All projects involving “pox” viruses require vaccination.

4. Any medical condition precluding vaccination will also preclude participation in a project involving infectious orthopoxvirus.

It shall be the responsibility of each principal investigator to insure that:

1. Each project is evaluated to determine whether vaccination is required. These evaluations are conducted by the EH&S Biosafety Office and its various committees.

2. Each participant complies with the vaccine policy requirements prior to participation in any covered research project.

3. The EH&S Biosafety Office is notified of vaccination dates for all researchers working with vacccinia.

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Health Surveillance for Personnel Working with Infectious AgentsA health surveillance program is available for laboratory personnel who use agents that require it. Laboratory personnel should receive immunizations (see chapter entitled “Immunoprophylaxis”), such as hepatitis B vaccination, and medical tests, such as tuberculosis skin testing, when appropriate. The Animal Contact and Bloodborne Pathogen Programs (see appropriate chapters) provide for health assessments, risk assessments, medical tests, and immunizations for certain at-risk personnel.

Blood Serum SamplingFor those laboratory personnel working with certain agents under BSL-2 or BSL-3, baseline serum samples may be appropriate (see chapter entitled “Biological Safety Levels”). The collection and maintenance of baseline serum samples provide a tool for monitoring serological changes that may result from the employee’s work. For example, staff who work with non-human primates may be required to bank a blood sample.

The EH&S Biological Safety Office, with advice from the appropriate health care professional, will provide information regarding the need for, and the frequency of, blood serum sampling on a case-by-case basis. The need for serum sampling will be dependent upon the agent used and the type of research in which an individual is engaged. For example, researchers who work with large quantities of HIV are required to have serum sampling monitored annually and upon accidental exposure.

Health AssessmentsThe NAU Occupational Medicine Program provides for pre-placement and other health assessments depending upon the type of work in which an employee will be engaged.

Laboratory personnel who work with agents that are transmitted by aerosol or with certain chemicals or acute toxins may require a respirator at some stage of the research project. The EH&S Biosafety Office Occupational Medicine Respiratory Protection Program provides for health assessments that will determine an employee’s fitness for respirator use.

The EH&S Biosafety Office and Occupational Medicine Program also includes the Hearing Conservation Program, the Asbestos Monitoring Program, as well as the Animal Contact Program, discussed in a separate section. Occasionally, researchers who work with infectious agents will require a health assessment based on the requirements of one of these other programs.

Exposure to Mycobacterium Tuberculosis

All personnel who can be occupationally exposed to either TB research or patients with active TB should have a yearly TB test for health surveillance.

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5 – Appendices - Information for ResearchersBio-Agent / R-DNA –

NIH guidelines

Transgenic Animals

ACUTE TOXINPROJECT ADDENDUM

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