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BiologicalSafety

Manual

Finance and Administration

Division of

Environmental Health & Safety

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2007

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Biological

Safety

Manual

University of Florida

Finance and Administration

Environmental Health and Safety

Box 112190

Gainesville, Florida 32611

(352) 392-1591 phone

(352) 392-3647 fax

Email: [email protected]

Internet: http://www.ehs.ufl.edu/bio

July 2007

mailto:[email protected]

mailto:[email protected]

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Forward

By authority delegated from the University President, the Vice President for Finance andAdministration is responsible for the safety of all University facilities. Under thisauthority, policies are developed to provide a safe teaching, research, service, housingand recreational environment.

The division of Environmental Health and Safety was established in 1974 and given theresponsibility for the management of all safety practices and the administration of theprogram.

The mission of the division of Environmental Health and Safety (EH&S) is to minimizeinjury to faculty, staff, students and visitors and to minimize damage to Universityproperty. Inherent in this mission is the charge to provide a safe and healthy environmentin which the University’s activities can be pursued.

All applicable federal and state safety laws, rules and regulations are adopted by theUniversity. In order to carry out its duties and responsibilities, EH&S will adhere tostandards or codes related to safety which have been adopted and promulgated bynationally recognized standards-setting organizations. The interpretation of safety codesand standards is the responsibility of the Division of Environmental Health and Safety.

In order to assure an effective Environmental Health & Safety program for the Universityof Florida, it is imperative that all individuals associated with the University comply fullywith the policies and procedures set forth in the manual.

Cover photos are from Henry Wellcome Laboratory for Cell Imagery, University of East Anglia.

Dr. Michael Wormstone provided the one in the upper left corner. It’s a photo of FHL124(immortalized human lens) cells labeled with an antibody to the transcription factor SMAD4 (acomponent of the TGF-beta signaling pathway) and an Alexa-Fluor-488-conjugated secondaryantibody (green). The cells actin filaments were also labeled using Texas-Red-conjugatedphalloidin

Mr. David Moss contributed the one in the lower right corner of A UE-1 (immortalized mouseinner ear epithelial) cell labeled with an antibody to alpha-tubulin (microtubules) and stained withan Alexa-Fluor-488-conjugated secondary antibody (green). The cells nucleus was counterstainedwith DAPI (blue).

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Policy Statement

It is the policy of the University of Florida to provide a safe working environment. Theprimary responsibility for insuring safe conduct and conditions in the laboratory resideswith the principal investigator.

The UF Biological Safety Office is committed to providing up-to-date information,training, and monitoring to the research and biomedical community concerning the safeconduct 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, we provide this manual as a resource, to be used inconjunction with the CDC and NIH guidelines, the UF Select Agent Program, theLaboratory Safety Manual and other resource materials from Environmental Health &Safety.

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Introduction

The University of Florida Biological Safety Manual is intended for use as a guidancedocument for researchers and clinicians who work with biological materials. It should beused in conjunction with the UF Laboratory Safety Manual, which provides more general

safety information. These manuals describe policies and procedures that are required forthe safe conduct of research at the University of Florida.

Responsibilities

In the academic research/teaching setting, the principal investigator is responsible forensuring that all members of the laboratory are familiar with safe research practices. Inthe clinical laboratory setting, the faculty member who supervises the laboratory isresponsible 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 inlaboratories.

Employees who work with biological materials are responsible for reading this manual,carrying out the safety practices outlined here, and reporting any problems, accidents,and spills to the appropriate faculty member.

Environmental Health & Safety will provide guidance, information, review,monitoring, and training regarding biological safety programs, when appropriate. Thisincludes implementing registration activities for certain research projects, acting as aconsultant for departments regarding implementation and enforcement of biological

safety programs, evaluating work practices and personal protective equipment, providingeducational materials, tracking employee training, and medical monitoring.

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Emergency Phone Numbers

General

University Police Department 392-1111

Gainesville Fire Department 911

Gas leak 911

Spills/Accidents

Asbestos 392-3392

Biological or Recombinant Materials 392-1591

Chemicals (laboratory) 846-2550

Mercury 392-8400

Pesticides 392-1904

Radioactive Materials Campus 392-7359

HSC 392-1589

Select Agents 392-1591

392-6369

Medical Emergency

Student Health Care Center 392-1161

Shands Hospital Emergency Center 395-0050

Shands Occupational Health Services 265-0250(blood and body fluid exposures only)

Needle Stick Injury 1-866-477-6824 (OUCH)

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Environmental Health & Safety

Phone Numbers

Biological Safety 392-1591 Bldg. 179 Box 112190

Business Office 392-1591 Bldg. 179 Box 112190

Chemical Waste 392-8400 Surge Area Box 112725

Director’s Office 392-1591 Bldg. 179 Box 112190

Diving Science & Safety 392-1661 Bldg. 21 Box 118205

Facility & Fire Safety 392-1904 Bldg. 179 Box 112200

Fire Equipment Services 392-2365 Bldg. 179 Box 112200

Hazardous Materials Management 392-8400 Surge Area Box 112725

Industrial Hygiene 392-3393 Bldg. 104 Box 112195

Laboratory Safety 392-1591 Bldg. 179 Box 112190

Occupational Safety and Health 392-1591 Bldg. 104 Box 112195

Occupational Medicine 392-1591 Bldg. 179 Box 112195

Pest Control Services 392-1904 Bldg. 175 Box 112205

Program Support 392-1591 Bldg. 179 Box 112190

Radioactive Waste 392-8400 Surge Area Box 112725

Radiation Control & Rad. Services 392-7359 Bldg. 175 Box 112205

Radiation Control Delivery Services 392-8700 Bldg. 175 Box 100252

Radiation Control Health Center 392-1589 HSC D8-17 Box 112205

Risk Management 392-1591 Bldg. 104 Box 112195

Select Agents 392-1591 Bldg. 179 Box 112190

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Table of Contents

1 -- Biological Safety .......................................................................................................... 1 Principles of Biological Safety ..................................................................................... 2

Laboratory Practice and Technique ............................................................................ 2 Safety Equipment (Primary Barrier) ........................................................................... 3 Facility Design (Secondary Barrier) ........................................................................... 3 Biosafety Levels ......................................................................................................... 4 Animal Facilities ......................................................................................................... 6 Clinical Laboratories ................................................................................................... 6 Importation and Interstate Shipment of Certain Biomedical Materials ...................... 7

Biological Safety Levels ................................................................................................ 7 Biosafety Level 1 ........................................................................................................ 8 Biosafety Level 2 ........................................................................................................ 9 Biosafety Level 3 ...................................................................................................... 13 Animal Biosafety Level 1 (ABSL-1) ........................................................................ 18 Animal Biosafety Level 2 (ABSL-2) ........................................................................ 20 Animal Biosafety Level 3 (ABSL-3) ........................................................................ 23

Agents List ................................................................................................................... 30 Biological Safety Level 1 (BSL-1) ........................................................................... 30 Biological Safety Level 2 (BSL-2) ........................................................................... 30

BSL-2 - Bacterial Agents Including Chlamydia............................................... 31 BSL-2 - Fungal Agents ..................................................................................... 32 BSL-2 - Parasitic Agents .................................................................................. 32 BSL-2 - Viruses ................................................................................................ 33

Biological Safety Level 3 (BSL-3) ........................................................................... 35 BSL-3 - Bacterial Agents Including Rickettsia ................................................ 35 BSL-3 - Fungal Agents ..................................................................................... 35 BSL-3 - Parasitic Agents .................................................................................. 35 BSL-3 - Viruses and Prions .............................................................................. 35

Biological Safety Level 4 (BSL-4) ........................................................................... 36 BSL-4 - Bacterial Agents .................................................................................. 36 BSL-4 - Fungal Agents ..................................................................................... 36 BSL-4 - Parasitic Agents .................................................................................. 36 BSL-4 - Viral Agents ........................................................................................ 36

2 -- Information for Researchers .................................................................................... 38 Project Registration .................................................................................................... 39

Bio-Agent (BA) Registration .................................................................................... 39 Recombinant DNA (R-DNA) Registration ............................................................... 39 Acute Toxins (AT) Registration ............................................................................... 40 Regulated Biological Materials ................................................................................ 40 Table 3: Toxin Table ................................................................................................. 41 Select Agents ............................................................................................................ 43

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Select Agents List ............................................................................................. 44 Minors in Research Laboratories or Animal Facilities ............................................. 46

Biological Waste Disposal Policy ............................................................................... 47 Categories ................................................................................................................. 47

1) Infectious/potentially infectious/R-DNA ..................................................... 47 2) Non-infectious waste .................................................................................... 47 3) Mixed radioactive/biohazardous waste ........................................................ 47 4) Mixed chemical/biohazardous waste ............................................................ 48 5) Animal carcasses and materials .................................................................... 48 6) Human remains ............................................................................................. 48

Packaging .................................................................................................................. 48 1) Biohazard bags ............................................................................................. 48 2) Sharps ........................................................................................................... 48 3) Corrugated cardboard boxes ......................................................................... 48

Labeling .................................................................................................................... 48 1) Date ............................................................................................................... 49 2) Name/Location/Phone Number .................................................................... 49 3) Biohazard sign .............................................................................................. 49

Transport ................................................................................................................... 49 Training ..................................................................................................................... 49

Biological Waste Disposal Containers ....................................................................... 50 Biohazard Boxes ....................................................................................................... 50 Sharps Boxes ............................................................................................................. 50 Biohazard Bags ......................................................................................................... 51

Autoclave Use and Testing ......................................................................................... 51 Autoclave Testing ..................................................................................................... 51 Record-Keeping ........................................................................................................ 52 Autoclave Operating Procedures .............................................................................. 52 Autoclave Operation and Safety Training ................................................................ 53 Autoclave Guidelines ................................................................................................ 54

Disinfectants ................................................................................................................ 55 Liquids ...................................................................................................................... 55

Alcohols ............................................................................................................ 55 Chlorine compounds ......................................................................................... 56 Formaldehyde ................................................................................................... 56 Glutaraldehyde .................................................................................................. 56 Hydrogen peroxide ........................................................................................... 56

Iodine and Iodophors ........................................................................................ 57 Phenol and phenolic compounds ...................................................................... 57 Quaternary ammonium compounds .................................................................. 57

Gases ......................................................................................................................... 57 Ethylene Oxide ................................................................................................. 57 Vapor Phase Hydrogen Peroxide ...................................................................... 57 Chlorine Dioxide gas ........................................................................................ 57

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Ozone ................................................................................................................ 57 Formaldehyde Gas (from heating paraformaldehyde) ...................................... 58

Irradiation .................................................................................................................. 58 Ultraviolet, UV radiation .................................................................................. 58 Ionizing radiation .............................................................................................. 58 Electron Beam ................................................................................................... 58 Microwaves ....................................................................................................... 58

Disinfectants Bibliography ....................................................................................... 58 Shipment of Biological Materials .............................................................................. 63

Biological materials subject to shipping & transport regulations:............................ 64 Transporting biological material within and around UF: ......................................... 64 Permits: ..................................................................................................................... 65

Biological Safety Cabinets .......................................................................................... 67 Biological Safety Cabinets (BSCs) ........................................................................... 67

The Class I BSC ................................................................................................ 67 The Class II BSC .............................................................................................. 67

The Class II, Type A BSC ................................................................................ 67 The Class II, Type B1 BSC............................................................................... 68 The Class II, Type B2 BSC............................................................................... 68 The Class II, Type B3 BSC............................................................................... 69 The Class III BSC ............................................................................................. 69

Horizontal Laminar Flow “Clean Bench” ................................................................ 69 Vertical Laminar Flow “Clean Bench” ..................................................................... 69 Operations within a Class II BSC ............................................................................. 69

Laboratory Hazards ........................................................................................... 69 Decontamination ....................................................................................................... 71

Surface Decontamination .................................................................................. 71 Gas Decontamination ........................................................................................ 71 Engineering Requirements ........................................................................................ 72

Ultraviolet Lamps ............................................................................................. 72 BSC Placement ................................................................................................. 72 HEPA Filters ..................................................................................................... 72 Certification of BSCs ........................................................................................ 72

Emergency Procedures/Telephone Numbers ........................................................... 73 General Information .................................................................................................. 74 Medical Emergency .................................................................................................. 74 Accidental injection, cuts, skin exposures ................................................................ 75 Splashes to face and eyes .......................................................................................... 75 Accidental ingestion ................................................................................................. 76 Animal bites and scratches ....................................................................................... 76 Break in/Security Breach .......................................................................................... 77 Handling Biological Spills ........................................................................................ 77 Spill in the biosafety cabinet ..................................................................................... 78 Spill in the centrifuge ................................................................................................ 78 Spill inside the laboratory ......................................................................................... 79

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Spill outside the laboratory ....................................................................................... 80 Fire Safety ................................................................................................................. 81 Workplace Violence: ................................................................................................ 82 Hurricane: ................................................................................................................. 82 Tornadoes and other natural disasters:...................................................................... 83

3 -- Programs .................................................................................................................... 84 Animal Contact Medical Monitoring Program ........................................................ 85 UF Bloodborne Pathogen Program ........................................................................... 87

BBP Exposure Information ............................................................................... 87 4 -- Medical Surveillance ................................................................................................. 88

HIV Research Laboratory Occupational Medicine Policy ..................................... 89 Pre-employment ........................................................................................................ 89 Continuing employment............................................................................................ 89 Post-exposure prophylaxis ........................................................................................ 89 References ................................................................................................................. 90

Immunoprophylaxis .................................................................................................... 91 Recommendations for Prophylactic Immunization of Laboratory Personnel Workingwith Infectious Agents .............................................................................................. 92

Bacterial agents ................................................................................................. 92 Rickettsial agents .............................................................................................. 93 Viral agents ....................................................................................................... 93

Vaccinia Immunization Policy ................................................................................... 95 University of Florida ................................................................................................. 96

Health Surveillance for Personnel Working with Infectious Agents ..................... 97 Blood Serum Sampling ............................................................................................. 97 Health Assessments .................................................................................................. 97 Exposure to Mycobacterium Tuberculosis ............................................................... 98

5 -- Appendix A – Forms ................................................................................................. 99 R-DNA ...................................................................................................................... 99 Bio-Agent .................................................................................................................. 99 Transgenic Animals .................................................................................................. 99 Acute Toxin .............................................................................................................. 99 Project Addendum ................................................................................................... 99

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List of Tables

Table 1: Summary of Recommended Biosafety Levels for Infectious Agents ................ 28Table 2: Summary of Recommended Biosafety Levels for Activities in Which

Experimentally or Naturally Infected Vertebrate Animals Are Used ....................... 29Table 3: Toxin Table ......................................................................................................... 41Table 4: Dilutions of Household Bleach ........................................................................... 56Table 5: Summary and Comparison of Liquid Disinfectants (Page 1) ............................. 59Table 6: Summary of Practical Disinfectants ................................................................... 61Table 7: Reprocessing Methods for Equipment Used in the Health Care Setting ............ 62

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Principles of Biological SafetyThe following is from Biosafety in Microbiological and Biomedical Laboratories, 1999, HHS publication No. (CDC)93-8395, Centers for Disease Control & Prevention/National Institutes of Health

The term “containment” is used in describing safe methods formanaging infectious agents in the laboratory environment wherethey are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratoryworkers, other persons, and the outside environment topotentially hazardous agents.

Both good microbiological technique and the use of appropriatesafety equipment provide primary containment, the protection of personnel and the immediate laboratory environment fromexposure to infectious agents. The use of vaccines may provide

an increased level of personal protection. Secondarycontainment, the protection of the environment external to the laboratory from exposureto infectious materials, is provided by a combination of facility design and operationalpractices. Therefore, the three elements of containment include laboratory practice andtechnique, safety equipment, and facility design. The risk assessment of the work to bedone with a specific agent will determine the appropriate combination of these elements.

Laboratory Practice and Technique

The most important element of containment is strict adherence to standardmicrobiological practices and techniques. Persons working with infectious agents or

potentially infectious materials must be aware of potential hazards, and must be trainedand 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 orarranging for appropriate training of personnel.

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

activities.

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

Laboratory personnel, safety practices, and techniques must be supplemented by

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appropriate facility design and engineering features, safety equipment, and managementpractices.

Safety Equipment (Primary Barrier)

Safety equipment includes biological safety cabinets (BSCs), enclosed containers, andother engineering controls designed to remove or minimize exposures to hazardousbiological materials. The biological safety cabinet (BSC) is the principal device used toprovide containment of infectious splashes or aerosols generated by manymicrobiological procedures. Three types of biological safety cabinets (Class I, II, III)used in microbiological laboratories are described and illustrated in Appendix A. Open-fronted Class I and Class II biological safety cabinets are primary barriers which offersignificant levels of protection to laboratory personnel and to the environment when usedwith good microbiological techniques. The Class II biological safety cabinet alsoprovides protection from external contamination of the materials (e.g., cell cultures,microbiological stocks) being manipulated inside the cabinet. The gas-tight Class III

biological safety cabinet provides the highest attainable level of protection to personneland the environment.

An example of another primary barrier is the safety centrifuge cup, an enclosed containerdesigned to prevent aerosols from being released during centrifugation. To minimize thishazard, containment controls such as BSCs or centrifuge cups must be used for handlinginfectious 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. Personalprotective equipment is often used in combination with biological safety cabinets and

other devices that contain the agents, animals, or materials being worked with. In somesituations in which it is impractical to work in biological safety cabinets, personalprotective equipment may form the primary barrier between personnel and the infectiousmaterials. Examples include certain animal studies, animal necropsy, agent productionactivities, and activities relating to maintenance, service, or support of the laboratoryfacility.

Facility Design (Secondary Barrier)

The design of the facility is important in providing a barrier to protect persons workinginside 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 thelaboratory. Laboratory management is responsible for providing facilities commensuratewith the laboratory's function and the recommended biosafety level for the agents beingmanipulated.

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 BiosafetyLevel 1 and 2 facilities will be direct contact with the agents, or inadvertent contact

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exposures through contaminated work environments. Secondary barriers in theselaboratories 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 fromescaping into the environment. Such design features could include specializedventilation systems to assure directional air flow, air treatment systems to decontaminateor remove agents from exhaust air, controlled access zones, airlocks as laboratoryentrances, or separate buildings or modules for isolation of the laboratory. Designengineers for laboratories may refer to specific ventilation recommendations as found inthe Applications Handbook for Heating, Ventilation, and Air-conditioning (HVAC)published by the American Society of Heating, Refrigerating, and Air-conditioningEngineers (ASHRAE).

Biosafety Levels

Four biosafety levels (BSLs) are described which consist of combinations of laboratorypractices and techniques, safety equipment, and laboratory facilities used to controlhazardous biological materials. Each combination is specifically appropriate for theoperations performed, the documented or suspected routes of transmission of theinfectious agents, and for the laboratory function or activity.

The recommended biosafety level(s) for the organisms represent those conditions underwhich the agent can ordinarily be safely handled. The laboratory director is specificallyand primarily responsible for assessing risks and for appropriately applying therecommended biosafety levels. Generally, work with known agents should be conducted

at the biosafety level recommended. When specific information is available to suggestthat virulence, pathogenicity, antibiotic resistance patterns, vaccine and treatmentavailability, or other factors are significantly altered, more (or less) stringent practicesmay be specified.

Biosafety Level 1 practices, safety equipment, and facilities are appropriate forundergraduate and secondary educational training and teaching laboratories, and for otherfacilities in which work is done with defined and characterized strains of viablemicroorganisms not known to cause disease in healthy adult humans. Bacillus subtilis, Naegleria gruberi, and infectious canine hepatitis virus are representative of thosemicroorganisms meeting these criteria. Many agents not ordinarily associated with

disease processes in humans are, however, opportunistic pathogens and may causeinfection in the young, the aged, and immunodeficient or immunosuppressed individuals.Vaccine strains which have undergone multiple in vivo passages should not beconsidered avirulent simply because they are vaccine strains.

Biosafety Level 1 represents a basic level of containment that relies on standardmicrobiological practices with no special primary or secondary barriers recommended,other than a sink for hand washing.


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Biosafety Level 2 practices, equipment, and facilities are applicable to clinical,diagnostic, teaching, and other facilities in which work is done with the broad spectrumof indigenous moderate-risk agents present in the community and associated with humandisease of varying severity. With good microbiological techniques, these agents can be

used safely in activities conducted on the open bench, provided the potential forproducing splashes or aerosols is low. Hepatitis B virus, Salmonellae, and Toxoplasma spp. are representative of microorganisms assigned to this containment level. BiosafetyLevel 2 is appropriate when work is done with any human-derived blood, body fluids, ortissues where the presence of an infectious agent may be unknown. (Laboratorypersonnel working with human-derived materials should refer to the BloodbornePathogen Standard for specific, required precautions).

Primary hazards to personnel working with these agents relate toaccidental percutaneous or mucous membrane exposures, oringestion of infectious materials. Extreme precaution with

contaminated needles or sharp instruments must be emphasized.Even though organisms routinely manipulated at BSL-2 are notknown to be transmissible by the aerosol route, procedures with

aerosol or high splash potential that may increase the risk of such personnel exposuremust be conducted in primary containment equipment, or devices such as a BSC or safetycentrifuge cups. Other primary barriers should be used, as appropriate, such as splashshields, face protection, gowns, and gloves.

Secondary barriers such as hand washing and waste decontamination facilities must beavailable 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 withindigenous or exotic agents with a potential for respiratory transmission, and which maycause serious and potentially lethal infection. Mycobacterium tuberculosis, St. Louisencephalitis virus, and Coxiella burnetii are representative of microorganisms assigned tothis level. Primary hazards to personnel working with these agents relate toautoinoculation, ingestion, and exposure to infectious aerosols.

At Biosafety Level 3, more emphasis is placed on primary and secondary barriers toprotect personnel in contiguous areas, the community, and the environment fromexposure to potentially infectious aerosols. For example, all laboratory manipulations

should be performed in a BSC or other enclosed equipment, such as a gas-tight aerosolgeneration chamber. Secondary barriers for this level include controlled access to thelaboratory and a specialized ventilation system that minimizes the release of infectiousaerosols from the laboratory.

Biosafety Level 4 practices, safety equipment, and facilities are applicable for work withdangerous 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


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vaccine or therapy. Additionally, agents with a close or identical antigenic relationshipto Biosafety Level 4 agents should also be handled at this level. When sufficient data areobtained, work with these agents may continue at this level or at a lower level. Virusessuch as Marburg or Congo-Crimean hemorrhagic fever are manipulated at BiosafetyLevel 4.

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

The laboratory worker's complete isolation of aerosolized infectious materials isaccomplished primarily by working in a Class III BSC or a full-body, air-suppliedpositive-pressure personnel suit. The Biosafety Level 4 facility itself is generally aseparate building or completely isolated zone with complex, specialized ventilation and

waste management systems to prevent release of viable agents to the environment.

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 andappropriately applying these recommendations. The recommended biosafety levelrepresents those conditions under which the agent can ordinarily be safely handled.Special characteristics of the agents used, the training and experience of personnel, andthe nature or function of the laboratory may further influence the director in applyingthese recommendations.

Animal Facilities

Four 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 provideincreasing levels of protection to personnel and the environment.

Clinical Laboratories

Clinical laboratories, especially those in health care facilities, receive clinical specimenswith requests for a variety of diagnostic and clinical support services. Typically, theinfectious nature of clinical material is unknown, and specimens are often submitted with

a broad request for microbiological examination for multiple agents (e.g., sputa submittedfor “routine,” acid-fast, and fungal cultures). It is the responsibility of the laboratorydirector to establish standard procedures in the laboratory that realistically address theissue of the infectious hazard of clinical specimens.

Except in extraordinary circumstances (e.g., suspected hemorrhagic fever), the initialprocessing of clinical specimens and identification of isolates can be done safely atBiosafety Level 2, the recommended level for work with bloodborne pathogens such as


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hepatitis B virus and HIV. The containment elements described in Biosafety Level 2 areconsistent with the Occupational Exposure to Bloodborne Pathogens Standard from theOccupational Safety and Health Administration (OSHA), that requires the use of specificprecautions 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 LaboratoryStandards.

Biosafety Level 2 recommendations and OSHA requirements focus on the prevention of percutaneous and mucous membrane exposures to clinical material. Primary barrierssuch as biological safety cabinets (Class I or II) should be used when performingprocedures that might cause splashing, spraying, or splattering of droplets. Biologicalsafety cabinets should also be used for the initial processing of clinical specimens whenthe nature of the test requested or other information is suggestive that an agent readilytransmissible by infectious aerosols is likely to be present (e.g., M. tuberculosis), or whenthe 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 suchareas is the responsibility of the laboratory director. It is also the director's responsibilityto establish standard, written procedures that address the potential hazards and therequired precautions to be implemented.

Importation and Interstate Shipment of Certain Biomedical Materials

The importation of etiologic agents and vectors of human diseases is subject to therequirements of the Public Health Service Foreign Quarantine regulations. Companion

regulations of the Public Health Service and the Department of Transportation specifypackaging, labeling, and shipping requirements for etiologic agents and diagnosticspecimens 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 exoticanimal disease agents that pose a serious disease threat to domestic livestock and poultry.

Please see the section entitled “Shipment of Biological Materials” for more informationon permits.

Biological Safety LevelsThe following is from Biosafety in Microbiological and Biomedical Laboratories, 1999, HHS publication

No. (CDC) 93-8395, Centers for Disease Control & Prevention/National Institutes of Health


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Biosafety Level 1

Biosafety Level 1 is suitable for work involving well-characterized agents not known tocause disease in healthy adult humans, and of minimal potential hazard to laboratorypersonnel and the environment. The laboratory is not necessarily separated from thegeneral traffic patterns in the building. Work is generally conducted on open bench topsusing standard microbiological practices. Special containment equipment or facilitydesign is not required nor generally used. Laboratory personnel have specific training inthe procedures conducted in the laboratory and are supervised by a scientist with generaltraining in microbiology or a related science.

The following standard and special practices, safety equipment, and facilities apply to thehandling 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 specimensare in progress.

2. Persons wash their hands after they handle viablematerials and animals, after removing gloves, andbefore leaving the laboratory.

3. Eating, drinking, use of tobacco products, handlingcontact lenses, and applying cosmetics are notpermitted in the work areas where there isreasonable likelihood of exposure to potentially infectious materials.Persons who wear contact lenses in laboratories should also wear gogglesor a face shield. Food is stored outside the work area in cabinets orrefrigerators 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 tobe placed in a durable, leak-proof container and closed for transport fromthe laboratory. Materials to be decontaminated off-site from the

laboratory are packaged in accordance with applicable local, state, andfederal 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)


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1. Special containment devices or equipment such as a biological safetycabinet are generally not required for manipulations of agents assigned toBiosafety Level 1.

2. It is recommended that laboratory coats, gowns, or uniforms are worn toprevent contamination or soiling of street clothes.

3. Gloves should be worn if the skin on the hands is broken or if a rashexists.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 washing2. The laboratory is designed so that it can be easily cleaned. Walls and

floors must be constructed of water impervious materials that will stand upto 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 2

Biosafety 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.

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 containmentequipment.

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

A. Standard Microbiological Practices

1. Access to the laboratory is limited or restricted at the discretion of thelaboratory 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, andapplying cosmetics are not permitted in the work areas. Persons who wearcontact lenses in laboratories should also wear goggles or a face shield.


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Food is stored outside the work area in cabinets or refrigerators designatedfor 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 tobe placed in a durable, leak-proof container and closed for transport fromthe laboratory. Materials to be decontaminated off-site from thelaboratory are packaged in accordance with applicable local, state, andfederal regulations, before removal from the facility.

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

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 whoare at increased risk of acquiring infection or for whom infection may beunusually hazardous are not allowed in the laboratory or animal rooms.For example, persons who are immunocompromised orimmunosuppressed may be at risk of acquiring infections. The laboratorydirector has the final responsibility for assessing each circumstance anddetermining who may enter or work in the laboratory.

2. The laboratory director establishes policies and procedures whereby onlypersons who have been advised of the potential hazard and meet specific

entry requirements (e.g., immunization) enter the laboratory or animalrooms.

3. When the infectious agent(s) in use in the laboratory require specialprovisions or entry (e.g., immunization), a hazard warning signincorporating the universal biohazard symbol is posted on the access doorto the laboratory work area. The hazard warning sign identifies theinfectious agent, lists the name and telephone number of the laboratorydirector or other responsible person(s), and indicates the specialrequirement(s) for entering the laboratory.

4. Laboratory personnel receive appropriate immunizations or tests for theagents 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 serumspecimens may be collected periodically, depending on the agents handled or thefunction of the facility.

6. A Biosafety manual is prepared or adopted. Personnel are advised of specialhazards and are required to read and to follow instructions on practices andprocedures.


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7. Laboratory personnel receive appropriate training on the potential hazardsassociated with the work involved, the necessary precautions to preventexposures, and the exposure evaluation procedures. Personnel receive annualupdates, 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, andscalpels. Needles and syringes or other sharp instruments should be restricted inthe laboratory for use only when there is no alternative, such as parenteralinjection, phlebotomy, or aspiration of fluids from laboratory animals anddiaphragm bottles. Plasticware should be substituted for glassware wheneverpossible.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 otherwisemanipulated by hand before disposal; rather, they must be carefully placed

in conveniently located puncture-resistant containers used for sharpsdisposal. Non-disposable sharps must be placed in a hard-walledcontainer for transport to a processing area for decontamination,preferable by autoclaving.

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

c. Broken glassware must not be handled directly by hand, but must beremoved by mechanical means such as a brush and dustpan, tongs, orforceps. Containers of contaminated needles, sharp equipment, andbroken 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 thatprevents leakage during collection, handling, processing, storage, transport, orshipping.

10. Laboratory equipment and work surfaces should be decontaminated with anappropriate disinfectant on a routine basis, after work with infectious materials isfinished, and especially after overt spills, splashes, or other contamination byinfectious materials. Contaminated equipment must be decontaminated accordingto any local, state, or federal regulations before it is sent for repair ormaintenance. Equipment must also be decontaminated before removal from thefacility when it must be packaged for transport. Packaging and shipment shall bein accordance with applicable local, state, or federal regulations,.

11. Spills and accidents that result in overt exposures to infectious materials areimmediately 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)


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1. Properly maintained biological safety cabinets, preferably Class II, orother appropriate personal protective equipment or physical containmentdevices 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, vigorousshaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be differentfrom ambient pressures, inoculating animals intranasally, andharvesting infected tissues from animals or eggs.

b. Use Biological Safety Cabinets whenever high concentrations orlarge volumes of infectious agents are used. Such materials maybe centrifuged in the open laboratory if sealed rotor heads orcentrifuge safety cups are used, and if these rotors or safety cupsare 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 hazardousmaterials to the face, when the microorganisms must be manipulatedoutside the BSC.

3. Protective laboratory coats, gowns, smocks, or uniforms designated for labuse are worn while in the laboratory. This protective clothing is removedand left in the laboratory before leaving for non-laboratory areas (e.g.,cafeteria, library, and administrative offices). All protective clothing iseither 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 maycontact infectious materials, contaminated surfaces or equipment.

Wearing two pairs of gloves may be appropriate; if a spill or splatteroccurs, the hand will be protected after the contaminated glove isremoved. Gloves are disposed of when contaminated, removed whenwork with infectious materials is completed, and are never worn outsidethe 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 must be constructed of water impervious material that will stand upto harsh disinfectants. Carpeting is not allowed in laboratory facilities.

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

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

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


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6. A method for decontamination of infectious or regulated laboratory wastesis available (e.g., autoclave, chemical disinfection, incinerator, or otherapproved 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 tosurrounding areas to prevent accidental spread of potentially infectious orrecombinant agents.

9. Install biological safety cabinets in such a manner that fluctuations of theroom supply and exhaust air do not cause the biological safety cabinets tooperate outside their parameters for containment. Locate biological safetycabinets away from doors, from windows that can be opened, from heavilytraveled laboratory areas, and from other potentially disruptive equipmentso as to maintain the biological safety cabinets’ air flow parameters forcontainment.

Biosafety Level 3

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or productionfacilities in which work is done with indigenous or exotic agents which may causeserious or potentially lethal disease as a result of exposure by the inhalation route.Laboratory personnel have specific training in handling pathogenic and potentially lethalagents, and are supervised by competent scientists who are experienced in working withthese agents.

All procedures involving the manipulation of infectious materials are conducted withinbiological safety cabinets or other physical containment devices, or by personnel wearing

appropriate personal protective clothing and equipment. The laboratory has specialengineering and design features.

It is recognized, however, that many existing facilities may not have all the facilitysafeguards recommended for Biosafety Level 3 (e.g., access zone, sealed penetrations,directional airflow, etc.). In these circumstances, acceptable safety may be achieved forroutine or repetitive operations (e.g. diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in Biosafety Level 2facilities. However, the recommended Standard Microbiological Practices, SpecialPractices, and Safety Equipment for Biosafety Level 3 must be rigorously followed. Thedecision to implement this modification of Biosafety Level 3 recommendations should be

made only by the lab director. It is strongly recommended that persons have a minimumof 120 hour work experience in a Level 2 laboratory to qualify to work at Level 3.

The following standard and special safety practices, equipment, and facilities apply to theuse of agents assigned to Biosafety Level 3:

A. Standard Microbiological Practices


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1. Access to the laboratory is limited or restricted at the discretion of thelaboratory director when experiments are in progress.

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 gogglesor a face shield. Food is stored outside the work area in cabinets orrefrigerators 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 tobe placed in a durable, leak-proof container and closed for transport fromthe laboratory. Infectious wastes from BSL-3 laboratories must bedecontaminated 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 supportpurposes. For example, persons who are immunocompromised orimmunosuppressed, or for whom infection may be unusually hazardous,are not allowed in the laboratory or animal rooms. The director has thefinal responsibility for assessing each circumstance and determining whomay enter or work in the laboratory. No minors are allowed in level 3laboratories.

3. The laboratory director establishes policies and procedures whereby onlypersons who have been advised of the potential biohazard, who meet anyspecific entry requirements (e.g., immunizations), and who comply withall entry and exit procedures, enter the laboratory or animal rooms.

4. When infectious materials or infected animals are present in the laboratoryor containment module, a hazard warning sign incorporating the universalbiohazard symbol, is posted on all laboratory and animal room accessdoors. The hazard warning sign identifies the agent, lists the name andtelephone number of the laboratory director or other responsible person(s),and indicates any special requirements for entering the laboratory, such asthe need for immunizations, respirators, or other personal protectivemeasures.


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

6. Baseline serum samples are collected and stored for all laboratory andother at-risk personnel. Additional serum specimens may be collected

periodically, depending on the agents handled or the function of thelaboratory.7. A Biosafety Manual specific to the laboratory is prepared or adopted.

Personnel are advised of special hazards and are required to read and tofollow instructions on practices and procedures. An SOP manual isprepared to cover all procedures and activities.

8. Laboratory personnel receive appropriate training on the potential hazardsassociated with the work involved, the necessary precautions to preventexposures, and the exposure evaluation procedures. Personnel receiveannual 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 andtechniques, and in the practice and operations specific to the laboratoryfacility, before working with organisms at Biosafety Level 3. This mightinclude prior experience in handling human pathogens or cell cultures, ora specific training program provided by the laboratory director or othercompetent scientist proficient in safe microbiological practices andtechniques.

10. A high degree of precaution must always be taken with all contaminatedsharp items, including needles and syringes, slides, pipettes, capillarytubes, and scalpels. Needles and syringes or other sharp instrumentsshould 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. Plasticware shouldbe 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 oraspiration of infectious materials. Used disposable needles mustnot be bent, sheared, broken, recapped, removed from disposablesyringes, or otherwise manipulated by hand before disposal; rather,they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposablesharps must be placed in a hard-walled container for transport to a

processing area for decontamination, preferably 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, sharpequipment, and broken glass should be decontaminated beforedisposal, in accordance with any local, state, or federal regulations.


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

12. Laboratory equipment and work surfaces should be decontaminated with

an appropriate disinfectant on a routine basis, after work with infectiousmaterials is finished. It must be cleaned and decontaminated after overtspills, splashes, or other contamination with infectious materials.Contaminated equipment should also be decontaminated before it is sentfor repair or maintenance. In addition, it must be packaged for transport inaccordance with applicable local, state, or federal regulations, beforeremoval from the facility. Plastic-backed paper toweling can be used onnon-perforated work surfaces within biological safety cabinets to facilitateclean up.

13. Cultures, tissues, or specimens of body fluids are placed in a container thatprevents 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 andequipped to work with concentrated infectious material.

16. Spills and accidents that result in overt or potential exposures to infectiousmaterials are immediately reported to the laboratory director. Appropriatemedical evaluation, surveillance, and treatment are provided and writtenrecords 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 traveledareas. Biosafety Cabinets must be certified at least yearly.

2. Outside of a BSC, appropriate combinations of personal protectiveequipment are used (e.g., special protective clothing, masks, gloves, faceprotection, or respirators) in combination with physical containmentdevices (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 infectiousaerosols. The aerosol challenge of experimental animals; harvesting of tissues or fluids from infected animals, and embryonated eggs, andnecropsy of infected animals also require the use of BSCs.


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4. Face protection (goggles and mask, or face shield) is worn formanipulations of infectious materials outside of a biological safetycabinet.

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, thelaboratory. Reusable laboratory clothing is to be decontaminated beforebeing laundered. Change protective clothing immediately if contaminated.

7. Gloves must be worn when handling infected animals and when handsmay contact infectious materials and contaminated surfaces or equipment.Disposable gloves should be discarded when contaminated, and neverwashed 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 isthe basic requirement for entry into the laboratory from access corridors orother contiguous areas. A clothes change room (shower optional) may beincluded 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 water-resistant sothat they can be easily cleaned. Penetrations in these surfaces are sealedor capable of being sealed to facilitate decontamination. Floors should be

monolithic and coved to the walls.4. Bench tops are 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 createsdirectional airflow that draws air from “clean” areas into the laboratory

toward “contaminated” areas. The exhaust air is not recirculated to anyother area of the building, and is discharged to the outside through aHEPA filtration system. The outside exhaust must be dispersed awayfrom occupied areas and air intakes. Laboratory personnel must verifythat the direction of the airflow (into the laboratory) is proper. Visualmonitoring devices are recommended.

9. The High Efficiency Particulate Air (HEPA)-filtered exhaust air fromClass II or Class III biological safety cabinets is discharged directly to the


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outside or through the building exhaust system. If the HEPA-filteredexhaust air from Class II or III biological safety cabinets is to bedischarged to the outside through the building exhaust air system, it isconnected to this system in a manner (e.g., thimble unit connection) thatavoids any interference with the air balance of the cabinets or building

exhaust system. Exhaust air from Class II biological safety cabinets maybe recirculated within the laboratory if the cabinet is tested and certified atleast every twelve months.

10. Continuous flow centrifuges or other equipment that may produce aerosolsare contained in devices that exhaust air through HEPA filters beforedischarge into the laboratory.

11. Vacuum lines are protected with liquid disinfectant traps and HEPAfilters, or their equivalent, which are routinely maintained and replaced asneeded.

12. An eyewash/safety shower is readily available.13. A Biosafety Level 3 facility design and operational procedures must be

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

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

15. Level 3 laboratories and animal areas are audited by EH&S quarterly.16. Openings such as around ducts, spaces between doors and frames and

other penetrations must be capable of being sealed to facilitate

decontamination.

Animal Biosafety Level 1 (ABSL-1)

Animal Biosafety Level 1 (ABSL-1) is suitablefor work involving well characterized agentsthat are not known to cause disease in healthyadult humans, and that are of minimal potentialhazard to laboratory personnel and theenvironment.

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

for emergency situations. Each project is subject to pre-approval by theInstitutional Animal Care and Use Committee (IACUC) and the


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Institutional Biosafety Committee). Any special practices are approved atthis time.

2. Only those persons required for program or support purposes areauthorized to enter the facility. Before entering, persons are advised of thepotential 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 specialhazards, and are required to read and follow instructions on practices andprocedures.

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

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

7. Work surfaces are decontaminated after use or after any spill of viablematerials.

8. All wastes from the animal room (including animal tissues, carcasses, andcontaminated bedding) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance withapplicable institutional or local requirements. Incineration isrecommended.

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 identifiesthe infectious agent(s) in use, lists the name and telephone number of the

responsible person(s), and indicates the special requirements for enteringthe animal room (e.g., the need for immunizations and respirators).


B. Special Practices: None

C. 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 anduniforms 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


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experimental animals are present. Cubicle room inner doors may openoutward or be horizontal or vertical sliding.

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

4. Internal facility appurtenances, such as light fixtures, air ducts, and utilitypipes, 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 facilityhas windows that open, they are fitted with fly screens.

6. If floor drains are provided, the traps are always filled with water and/oran 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 airshould occur. It is recommended that animal rooms maintain negativepressure compared to adjoining hallways.

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 involves practices for work with those agents associated with human disease. It

addresses hazards from ingestion as well as frompercutaneous and mucous membrane exposure. ABSL-2builds upon the practices, procedures, containmentequipment, 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 policiesand procedures should be developed as needed and approved by the

Institutional Animal Care and Use Committee (IACUC) and theInstitutional Biosafety Committee (IBC).

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

3. An appropriate medical surveillance program is in place. All personnelreceive appropriate immunizations or tests for the agents handled or


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potentially present (e.g., hepatitis B vaccine, TB skin testing). Whenappropriate, 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 onpractices and procedures.

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


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

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

wastes from the animal room (including animal tissues, carcasses,contaminated bedding, unused feed, sharps, and other refuse) aretransported from the animal room in leak-proof, covered containers forappropriate disposal in compliance with applicable institutional or localrequirements. The outer surface of the containers is disinfected prior tomoving the material. Autoclaving of the contents prior to incineration isrecommended.

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 isno alternative, such as for parenteral injection, blood

collection, or aspiration of fluids from laboratory animalsand diaphragm bottles.

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

c. Plasticware should be substituted for glassware wheneverpossible.

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

11. A biohazard sign must be posted on the entrance to the animal roomwhenever infectious agents are present. The hazard warning sign identifiesthe infectious agent(s) in use, lists the name and telephone number of the

responsible person(s), and indicates the special requirements (e.g., theneed 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 necessaryprecautions to prevent exposures, and the exposure evaluation procedures.


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Personnel receive annual updates, or additional training as necessary forprocedural or policy changes. Records of all training provided aremaintained. In general, persons who may be at increased risk of acquiringinfection, or for whom infection might be unusually hazardous, are notallowed 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 recordsare 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 animalfacility. Gloves are worn when handling infected animals and when skincontact with infectious materials is unavoidable

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

3. Biological safety cabinets, other physical containment devices, and/orpersonal protective equipment (e.g., respirators, face shields) are usedwhenever conducting procedures with a high potential for creatingaerosols. 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 containmentequipment appropriate for the animal species. Filter top cages are alwayshandled in properly designed and operating animal bio-containmentcabinets recommended for rodents.

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

personnel 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, areself-closing, and are kept closed when experimental animals are present.Cubicle room inner doors may open outward or be horizontal or verticalsliding.

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


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

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

6. If floor drains are provided, the traps are always filled with an appropriatedisinfectant.7. Exhaust air is discharged to the outside without being recirculated to other

rooms. Ventilation should be provided in accordance with criteria fromGuide for Care and Use of Laboratory Animals, latest edition. Thedirection of airflow in the animal facility is inward; animal rooms shouldmaintain negative pressure compared to adjoining hallways.

8. Cages are washed manually or in an appropriate cage washer. Themechanical cage washer should have a final rinse temperature of at least180F.

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.


Animal Biosafety Level 3 involves practices suitable for work with animals infected withindigenous 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 Practices 1. Aside from the standard policies, procedures, and protocols for emergency

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

2. The laboratory or animal facility director limits access to the animal roomto the fewest number of individuals possible. Personnel who must enterthe 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 orpotentially present (e.g., hepatitis B vaccine, TB skin testing). Whenappropriate, a serum surveillance system should be implemented. Ingeneral, persons who may be at increased risk of acquiring infection, orfor whom infection might have serious consequences, are not allowed in


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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 onpractices and procedures.

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


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

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, coveredcontainers for appropriate disposal in compliance with applicableinstitutional or local requirements. Incineration is recommended. Theouter surface of the containers is disinfected prior to moving the material(see Special Practices #3 below).

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

the animal facility for use only when there is no alternative, suchas for parenteral injection, blood collection, or aspiration of fluidsfrom 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. Plasticware 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 identifiesthe infectious agent(s) in use, lists the name and telephone number of theresponsible person(s), and indicates the special requirements for enteringthe 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 necessaryprecautions to prevent exposures, and the exposure evaluation procedures.As necessary, personnel receive updates and/or additional training onprocedural or policy changes. Records of all training provided aremaintained.



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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 beforebeing packaged for transport or removal from the facility for repair ormaintenance.

2. A spill procedure is developed and posted. Only personnel properlytrained and equipped to work with infectious materials are to clean upspills. Spills and accidents that result in overt exposures to infectiousmaterials must be immediately reported to the facility director. Medicalevaluation, surveillance, and treatment are provided as appropriate andwritten records are maintained.

3. All wastes from the animal room must be autoclaved prior to incinerationor other appropriate terminal treatment.

4. Materials not related to the experiment (e.g., plants, animals) are notpermitted 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 gownsshould be worn over this clothing. Front-button laboratorycoats are unsuitable. The gown must be removed and leftin the animal room. Before leaving the animal facility,scrub suits and uniforms are removed and appropriatelycontained and decontaminated prior to laundering or

disposal.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 wastesbefore disposal.

c. Appropriate face/eye and respiratory protection (e.g., respiratorsand 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, suchas open cages placed in inward flow ventilated enclosures (e.g., laminarflow cabinets), solid wall and bottom cages covered with filter bonnets, orother equivalent primary containment systems.

4. Biological safety cabinets and other physical containment devices are usedwhenever conducting procedures with a potential for creating aerosols.


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These include necropsy of infected animals, harvesting of tissues or fluidsfrom infected animals or eggs, or intranasal inoculation of animals. AtBSL-3, all work should be done in a primary barrier; otherwise respiratorsshould be worn by personnel in the room.

D. Facilities (Secondary Barriers)1. The animal facility is separated from areas that are open to unrestrictedpersonnel 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, orproximity reader. Entry into the animal room is via a double-door entrywhich includes a change room and shower(s). An additional double-dooraccess (air-lock) or double-doored autoclave may be provided formovement of supplies and wastes into and out of the facility, respectively.Doors to animal rooms open inward and are self-closing. Doors tocubicles 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, andceilings) are water resistant. Penetrations in floors, walls and ceilingsurfaces are sealed and openings around ducts and the spaces betweendoors and frames are capable of being sealed to facilitate decontamination.

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

5. Internal facility appurtenances, such as light fixtures, air ducts, and utilitypipes, are arranged to minimize horizontal surface areas.

6. Windows are not recommended. Any windows must be resistant tobreakage and must be sealed.

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

8. Ventilation should be provided in accordance with criteria from the Guide

for Care and Use of Laboratory Animals, latest edition. A ducted exhaustair ventilation system is provided. This system creates directional airflowwhich draws air into the laboratory from "clean" areas and toward"contaminated" areas. The exhaust air is not recirculated to any other areaof the building. Filtration and other treatments of the exhaust air may notbe required, but should be considered based on site requirements, and

specific agent manipulations and use conditions. The exhaust must bedispersed away from occupied areas and air intakes, or the exhaust mustbe HEPA-filtered. Personnel must verify that the direction of the airflow(into the animal areas) is proper. It is recommended that a visualmonitoring device that indicates and confirms directional inward airflowbe provided at the animal room entry. Consideration should be given toinstalling an HVAC control system to prevent sustained positive


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pressurization of the animal spaces. Audible alarms should be consideredto notify personnel of HVAC system failure.

9. HEPA-filtered exhaust air from a Class II biological safety cabinet can berecirculated into the animal room if the cabinet is tested and certified atleast annually. When exhaust air from Class II safety cabinets is to be

discharged to the outside through the building exhaust air system, thecabinets must be connected in a manner that avoids any interference withthe air balance of the cabinets or the building exhaust system (e.g., an airgap between the cabinet exhaust and the exhaust duct). When Class IIIbiological safety cabinets are used, they should be directly connected tothe exhaust system. If the Class III cabinets are connected to the supplysystem, it is done in a manner that prevents positive cabinetpressurization.

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

11. An autoclave is available which is convenient to the animal rooms where

the biohazard is contained. The autoclave is utilized to decontaminateinfectious waste before moving it to other areas of the facility.

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

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

14. The completed Biosafety Level 3 facility design and operationalprocedures must be documented. The facility must be tested for

verification that the design and operational parameters have been metprior to operation. Facilities should be re-verified at least annually againstthese procedures as modified by operational experience.

15. Additional environmental protection (e.g., personnel showers, HEPAfiltration of exhaust air, containment of other piped services, and theprovision of effluent decontamination) should be considered if recommended by the agent summary statement, as determined by risk assessment of the site conditions, or other applicable federal, state, or localregulations.

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Table 1: Summary of Recommended Biosafety Levels for Infectious Agents

BSL Agents Practices Safety Equipment

(Primary Barriers)

1 Not known to consistently

cause disease in healthyadults

Standard Microbiological Practices None required

2 Associated with humandisease, hazard =percutaneous injury,ingestion, mucous membraneexposure

BSL-1 practice plus:

• Limited access

• Biohazard warning signs

• “Sharps” precautions

• Biosafety manual definingany needed wastedecontamination or medicalsurveillance policies

Primary barriers = Class I or IIBCSs or other physicalcontainment devices used for allmanipulations of agents that causesplashes or aerosols of infectiousmaterials; PPEs; laboratory coats;gloves; face protection as needed

3 Indigenous or exotic agents

with potential for aerosoltransmission; disease mayhave serious or lethalconsequences


• Controlled access• Decontamination of all

waste

• Decontamination of labclothing before laundering

• Baseline serum

• Decontamination of alleffluent

Primary barriers = Class I or II

BCSs or other physicalcontainment devices for all openmanipulations of agents; PPEs;protective lab clothing; gloves;respiratory protection as needed

4 Dangerous/exotic agents

which pose high risk of life-threatening disease, aerosol-transmitted lab infections; orrelated agents with unknownrisk of transmission


• Clothing change beforeentering

• Shower on exit

• All material decontaminatedon exit from facility

Primary barriers = All procedures

conducted in Class III BCSs orClass I or II BCSs in combinationwith full-body air-supplied,positive pressure personnel suit

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Agents List

The following agents have been listed according to the most appropriate BiologicalSafety Level to be used. The list presented below is based upon the risk groups given inAppendix B of the March 1996 Guidelines for Research Involving Recombinant DNA

Molecules (NIH Guidelines), the agent summary statements in the CDC/NIH publication, Biosafety in Microbiological and Biomedical Laboratories (BMBL), 4th edition (1999),guidance from state and local regulatory agencies, and recommendations of the CDC.

Please note that Biological Safety Levels are not inherent to an agent but are performancerecommendations and should be chosen after a risk assessment is completed.

A proper risk assessment takes into account the characteristics of the agent involved, theactivities to be performed, and the environment in which the work will be completed.Therefore, certain agents may be used at different Biological Safety Levels dependingupon 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 performedunder BSL-3 containment. Biological Safety Levels may be higher or lower than what isgiven below for a particular agent depending upon the circumstances of its use.

The Biological Safety Office (BSO) reviews all projects involving recombinant DNA,infectious disease agents, and agents of concern to livestock and agriculture and willassist you in the risk assessment process. Once the Institutional Biosafety Committee(IBC) and/or the Biological Safety Office assigns a Biological Safety Level, it must beadhered to unless new information to warrant a change, in most cases from peer-reviewedliterature, is provided. The IBC and/or BSO will review the literature and make anadjustment, if warranted.

Biological Safety Level 1 (BSL-1)

Agents that are not associated with disease in healthy adult humans, are of minimalpotential hazard to laboratory personnel, and of minimal potential hazard to theenvironment 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 4in low concentrations (<109 IP/ml)

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


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


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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

• Bacillus anthracis

• 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, C. trachomatis, C. pneumoniae

• Clostridium botulinum, Cl. chauvoei, Cl. 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 strainsbearing K1 antigen, including E. coli O157:H7

• Haemophilus ducreyi, H. influenzae

• Helicobacter pylori

• Klebsiella - all species except K. oxytoca (BSL-1)

• Legionella including L. pneumophila• 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.

leprae, M. malmoense, M. marinum, M. paratuberculosis, M. scrofulaceum, M.

simiae, M. szulgai, M. ulcerans, M. xenopi

• Mycoplasma, except M. mycoides and M. agalactiae which are restricted animalpathogens

• Neisseria gonorrhoeae, 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


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• Streptobacillus moniliformis

• Streptococcus including S. pneumoniae, S. pyogenes

• Treponema pallidum, T. carateum

• Vibrio cholerae, V. parahemolyticus, V. vulnificus

• Yersinia enterocolitica

BSL-2 - Fungal Agents

• Blastomyces dermatitidis• Cladosporium bantianum, C. (Xylohypha) trichoides

• Cryptococcus neoformans

• Dactylaria galopava (Ochroconis gallopavum)

• Epidermophyton

• Exophiala (Wangiella) dermatitidis

• Fonsecaea pedrosoi

• Microsporum

• Paracoccidioides braziliensis

• Penicillium marneffei

• Sporothrix schenckii

• Trichophyton

BSL-2 - Parasitic Agents

• Ancylostoma human hookworms including A. duodenal, A. ceylanicum

• Ascaris including Ascaris lumbricoides suum

• Babesia including B. divergens, B. microti

• Brugia filaria worms including B. malayi, B. timori

• Coccidia

• Cryptosporidium including C. parvum

• Cysticercus cellulosae (hydatid cyst, larva of T. solium)

• Echinococcus including E. granulosis, E. multilocularis, E. vogeli

• Entamoeba histolytica

• Enterobius

• Fasciola including F. gigantica, F. hepatica

• Giardia including G. lamblia

• Heterophyes

• Hymenolepis including H. diminuta, H. nana

• Isospora

• Leishmania including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana,

L. peruvania, L. tropica

• Loa loa filaria worms

• Microsporidium• Naegleria fowleri

• 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


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Human herpesvirus types 6 and 7Marek's disease virusMurine cytomegalovirusPseudorabies virus

OrthomyxovirusesInfluenza viruses types A, B, and C

Other tick-borne orthomyxoviruses as listed in the BMBL

Papovaviruses

• All human papilloma viruses

• Bovine papilloma virus

• Polyoma virus

• Shope papilloma virus

• Simian virus 40 (SV40)

ParamyxovirusesNewcastle disease virusMeasles virusMumps virusParainfluenza viruses types 1, 2, 3, and 4Respiratory syncytial virus

ParvovirusesHuman parvovirus (B19)

PicornavirusesCoxsackie viruses types A and B

Echoviruses - all typesPolioviruses - all types, wild and attenuatedRhinoviruses - all types

PoxvirusesVaccinia - all types except Monkeypox virus (BSL-3) and restricted poxviruses including

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

Reoviruses - all types including Coltivirus, human Rotavirus, and Orbivirus (Coloradotick fever virus)

RetrovirusesAvian leukosis virusAvian sarcoma virusBovine leukemia virusClinical samples from HIV-positive patientsFeline immunodeficiency virusFeline leukemia virusFeline sarcoma virus


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Gibbon leukemia virusMason-Pfizer monkey virusMouse mammary tumor virusMurine leukemia virusMurine sarcoma virusRat leukemia virus

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 beendemonstrated to be free of detectable replication competent retrovirus can be maintained,handled, and administered, under BL1 containment.

RhabdovirusesRabies virus - all strainsVesicular stomatitis virus - laboratory adapted strains ONLY including VSV-Indiana,

San Juan, and GlasgowTogaviruses (see Alphaviruses and Flaviviruses)

Rubivirus (rubella)


Agents to be used at BSL-3 are associated with serious or lethal human disease for whichpreventive or therapeutic interventions may be available.

BSL-3 - Bacterial Agents Including Rickettsia

• Bartonella

• Brucella including B. abortus, B. canis, B. suis

• Burkholderia (Pseudomonas) mallei, B. pseudomallei

• Coxiella burnetii• Francisella tularensis

• Mycobacterium bovis (except BCG strain, BSL-2), M. tuberculosis

• 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

• Coccidioides immitis (sporulating cultures; contaminated soil)

• Histoplasma capsulatum, H. capsulatum var.. duboisii

BSL-3 - Parasitic AgentsNone

BSL-3 - Viruses and Prions

Alphaviruses (Togaviruses) - Group A Arboviruses

• Semliki Forest virus

• St. Louis encephalitis virus


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• Venezuelan equine encephalomyelitis virus (except the vaccine strain TC-83 is BSL-2)

• Other viruses as listed in the BMBL

Arenaviruses

• Lymphocytic choriomeningitis virus (LCM) (neurotropic strains)

• Flexal

Bunyaviruses

• Hantaviruses including Hantaan virus

• Rift Valley fever virus

Flaviviruses (Togaviruses) - Group B Arboviruses

• Japanese encephalitis virus

• Yellow fever virus

• Other viruses as listed in the BMBL

Poxviruses• Monkeypox virus

Prions

• Transmissible spongiform encephalopathies (TME) agents, Creutzfeldt-Jacob diseaseand kuru agents (see BMBL for specific containment instruction)

Retroviruses

• Human immunodeficiency virus (HIV) types 1 and 2

• Human T cell lymphotropic virus (HTLV) types 1 and 2

• Simian immunodeficiency virus (SIV)

Rhabdoviruses

• Vesicular stomatitis virus


Agents to be used at BSL-4 are likely to cause serious or lethal human disease for whichpreventive or therapeutic interventions are not usually available.

BSL-4 - Bacterial Agents

None

BSL-4 - Fungal AgentsNone

BSL-4 - Parasitic Agents

None

BSL-4 - Viral Agents

Arenaviruses (Togaviruses) - Group A Arboviruses

• Guanarito virus


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• 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 Europeanencephalitis, 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)

Paramyxiviruses

• Equine morbillivirus

Hemorrhagic fever agents and viruses as yet undefined.

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2 -- Information for Researchers

38

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

Bio-Agent (BA) Registration

Use of the following materials requires that the principal investigator completes andsubmits the bio-agent registration document for approval by the Biological Safety Office.

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 containmentand handling (see previous section: “Agents List”) must be registered. Pleasenote that BSL-4 agents may not be used at UF.

2. Unknown human and animal pathogens must be registered. These are consideredBSL-2 until identified.

3. Cell lines or cultures that1) have been immortalized with a virus (such as EBV or a retrovirus),

2) are known to be tumorigenic in primates (including humans), or

3) are primary human tumor cells.These are considered BSL-2 (or higher in many cases).

4. Human blood or other tissues that are known to be HIV positive (or positive forany human disease-causing virus or other agent), when used in research, must be

registered.

Recombinant DNA (R-DNA) Registration

All R-DNA projects that involve a living recombinant organism (this excludes projectsthat involve DNA only, i.e. PCR) require registration with the Biological Safety Office.A subset of R-DNA projects requires review and approval from the InstitutionalBiosafety Committee (IBC). The UF IBC oversees all research projects and issuesinvolving R-DNA at UF. Use of the following requires that the principal investigatorcompletes and submits an R-DNA registration document.

1. All R-DNA projects, including the growth of recombinant bacteria for probeisolation (plasmid or phage preparations) require registration. Projects must beregistered regardless of where the material came from or who originallyconstructed it.

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

R-DNA projects are performed at BSL-1, BSL-2, BSL-3 or the corresponding levels forwhole plant (BSL-1P, BSL-2P, BSL-3P) or whole animal (BSL-1N, BSL-2N, BSL-3N)work. The Biological Safety Office, in conjunction with the IBC, will make the finaldetermination.

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Acute Toxins (AT) Registration

The use and storage of chemicals with a mammalian LD50 of < 100 μg/kg. For a partiallist, see the Toxins Table that follows.

Regulated Biological Materials

Agents, such as plant pathogens or exotic microorganisms, that are regulated by federalor state agencies (USDA/APHIS, EPA, FDA, DPI, etc.) shall be registered with theBiological Safety Office by submission of a biological agent registration form and aphotocopy of the permit and permit conditions that have been granted by that agency. Nospecial form is required unless the agent fits into one of the first three categories.

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Table 3: Toxin Table

Toxins with a mammalian LD50 of < 100 μg/kg must be registered with the BiologicalSafety Office. Therefore, use of the following toxins may require registration. If a toxinis not on the list, it still may require registration, depending upon the LD50. For moreinformation, please contact the Biological Safety Office at 392-1591.

Toxicity

LD50 (μg/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 0.0011

Botulinin toxin F 0.0025β-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 81

Clostridium perfringens beta toxin 400Clostridium perfringens delta toxin 5Clostridium perfringens epsilon toxin 0.1Conotoxin 12-30Crotoxin 82Diphtheria toxin 0.1Listeriolysin 3-12Leucocidin 50Modeccin 1-10Nematocyst toxins 33-70Notexin 25

Pertussis toxin 15

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Toxicity

LD50 (μg/kg)*

Pneumolysin 1.5Pseudomonas aeruginosa toxin A 3Ricin 2.7

Saxitoxin 8Shiga toxin 0.250Shigella dysenteriae neurotoxin 1.3Streptolysin O 8Staphylococcus enterotoxin B 25Staphylococcus enterotoxin F 2-10Streptolysin S 25Taipoxin 2Tetanus toxin 0.001Tetrodotoxin 8Viscumin 2.4-80

Volkensin 1.4Yersinia pestis murine toxin 10

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

Reference:

1. Gill, D. Michael; 1982; Bacterial toxins: a table of lethal amounts; Microbiological Reviews; 46: 86-94

2. Stirpe, F.; Luigi Barbieri; Maria Giulia Battelli, Marco Soria and Douglas A.

Lappi; 1992; Ribosome-inactivating proteins from plants: present status andfuture prospects; Biotechnology; 10: 405-412

3. Registry of toxic effects of chemical substances (RTECS): comprehensive guideto the RTECS. 1997. Doris V. Sweet, ed., U.S. Dept of Health and HumanServices, Public Health Service, Centers for Disease Control and Prevention,National Institute for Occupational Safety and Health; Cincinnati, Ohio

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Select Agents

The following lists of agents, toxins, and pathogens are classified by the Federalgovernment as Select Agents. Any possession, use, transfer or shipment of thesematerials is strictly controlled by regulation.

See the EH&S web site at www.ehs.ufl.edu/bio for additional information and the UF

Select Agent Policy currently in place.

Researchers considering work with any of these materials must first contact the UFResponsible Official at 392-1591 for the approvals, permits, clearances and othernecessary paperwork. Be aware that government clearance can take as much as 6 monthsto complete in advance of any project.

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

http://www.ehs.ufl.edu/bio

http://www.ehs.ufl.edu/bio

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Maximum Toxin Amounts Excluded from Regulation

HHS Toxins Amount

Abrin 100mg

Conotoxin 100mg

Diacetoxyscirpenol (DAS) 1000mg

Ricin 100mg

Saxitoxin 100mg

Shiga-like ribosome inactivating proteins 100mg

Tetrodotoxin 100mg

Overlap toxins Amount

Botulinum neurotoxins 0.5mg

Staphylococcal enterotoxins 5.0mg

Clostridium perfringens epsilon toxin 100mg

Shigatoxin 100mg

T-2 toxin 1000mg

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Minors in Research Laboratories or Animal Facilities

Unless enrolled as a University of Florida student, minors are not allowed to work or conduct

research in University of Florida research laboratories, greenhouses or animal facilitiesexcept as identified specifically below. In addition, minors are prohibited from operatingfarm 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 forrecombinant 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. See

ACS Policy # ACS-PY-012. (Note that this does not apply to individualPrincipal Investigator animal research laboratories).

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 above) if the following requirements are met in full:

a. The University of Florida EH&S Policy titled; Minors In Research

Laboratories Or Animal Facilities (www.ehs.ufl.edu/bio/minors.htm) hasbeen read and understood.

b. A minor’s research proposal registration form(www.ehs.ufl.edu/bio/MinorReg.pdf ) is submitted to and approved by theUniversity of Florida, Division of Environmental Health and Safety, or the

Institutional Biosafety Committee. Included in this form is The PotentialHazards information sheet which requires a parental/guardian signatureindicating he/she has read of potential risks

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

d. Personal protective equipment, specific to the hazard, is provided to the minorwith 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.

g. The laboratory is in full compliance with all applicable University of Florida safety programs and regulations.

http://www.ehs.ufl.edu/bio/minors.htm

http://www.ehs.ufl.edu/bio/MinorReg.pdf

http://www.ehs.ufl.edu/bio/MinorReg.pdf

http://www.ehs.ufl.edu/bio/minors.htm

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Biological Waste Disposal Policy

This policy is intended to provide guidance and insure compliance with the NIH/CDCguidelines, the State of Florida Administrative Code 64E-6, and restrictions of theAlachua County Landfill.

Categories

1) Infectious/potentially infectious/R-DNA

a) human pathogensb) animal pathogensc) plant pathogensd) recombinant DNA

e) 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.)

This waste must be inactivated prior to disposal. The preferred method is steamsterilization (autoclaving), although chemical inactivation or incineration may beappropriate in some cases. Storage of non-inactivated waste is restricted to within thegenerating laboratory. The material may not be stored longer than 24 hours prior toinactivation.

2) Non-infectious wasteThis category includes waste that is not contaminated with any of the biological wasteslisted in category 1. It includes solid waste and sharps generated in clinical or laboratorysettings. Sterile or unopened biomedical materials that require disposal are alsoconsidered biological waste.

IV packs test tubes petri dishesneedles* razor blades* tissue culture flaskssyringes culture dishesscalpels* flasksbroken glass and plasticware** 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 beinactivated prior to its release to Radiation Safety for disposal as radioactive waste.Steam-sterilization or chemical inactivation shall be employed as above. Although manyradioactive materials can be autoclaved safely, please check with the Radiation Safety

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Office (392-7359) 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 inactivatedprior to its release for chemical disposal. Precautions should be taken to prevent thegeneration 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 Biological Safety Office(392-1591) for guidance regarding particular chemicals.

5) Animal carcasses and materials

The disposal of animal carcasses and other animal materials shall bethrough the Animal Care Services incinerators or the Veterinary Medicinetissue digesters only. These incinerators and digesters are for animalmaterials only. Please contact Animal Care Services (392-2977) forfurther information. No animal bodies or material shall be disposed of asregular trash or through the biomedical waste receptacle.

6) Human remains

Please contact the State Anatomical Review Board (392-3588) for information regardingthe 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 (480grams), and heavy metal concentration (<100 PPM total of lead, mercury, chromium andcadmium) requirements. Written documentation (a test report) from the manufacturer

regarding these requirements must be on file. These bags must be placed in cardboardboxes (see #3, below) prior to disposal.

2) Sharps

Needles, scalpels and razor blades are required to be containerized inred plastic sharps containers. These are provided by Building Services(392-4414) at the Health Center. All other sharps (broken glass andplasticware, pipettes, etc.) shall be containerized in puncture-resistantcardboard boxes (see #3, below). These are also available from

Building Services.

3) Corrugated cardboard boxes

All biological waste must be containerized in rigid, leak proof, puncture resistant boxesas the terminal receptacle. The appropriate boxes are available from Building Services atthe Health Center, 392-4414.

Labeling

All 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 thatbiohazard bags must be labeled even though they will be placed inside a secondarycontainer for final disposal.

Sharps containers shall be labeled with the date the container is full.Corrugated boxes (biomedical waste boxes) shall be labeled with the date thebiohazardous waste was treated. Boxes used for non-biohazardous waste collection shallbe dated when the box is sealed.

2) Name/Location/Phone Number

Generator’s (principal investigator’s) name, lab location (room number) and phonenumber will be clearly printed on each container.

3) Biohazard sign

Only manufactured containers with the preprinted universal biohazard symbol and thewords “biomedical,” “biohazardous,” or “infectious” shall be used.

Transport

The transport of biohazardous waste outside of the laboratory (i.e., to an autoclave orincinerator) must be in a closed, leak-proof container that is labeled “biohazard.” Onlytrained personnel may transport biomedical waste. Labeling may be accomplished by useof a red biohazard bag or a biomedical waste box with the universal biohazard symbol.Only corrugated biomedical boxes and red plastic sharps containers may be used totransport biological waste to the biomedical waste receptacle. Waste receptacle personnelare instructed not to accept any other type of containers.

Transportation of red-bagged waste must be in closed, leak-proof containers, properlylabeled as “Biohazards.” Movement of regulated/biological waste through publiccorridors, along carpeted hallways, and on public elevators must be avoided. Anyleakage/spills from these containers must be immediately reported to the Biosafety Officeat 392-1591. Signs must be displayed to prevent tracking of the spills to other areas.

Training

All employees who handle biological waste shall be trained annually regarding its properhandling. All new employees shall be trained before they are allowed to handlebiological waste.

Training may be accomplished through the UF Bloodborne Pathogen Training Program,informally in the lab setting, or through formal training programs set up by individualdepartments or divisions. For assistance, please call the Biological Safety Office at(392-1591).

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

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Biological Waste Disposal ContainersThe following waste disposal containers for biohazardous or biomedical waste are to beused for teaching and research purposes at the University of Florida:

ALL BIOHAZARDOUS WASTE MUST BE TREATED BEFORE DISPOSAL

Biohazard Boxes

Biohazard boxes lined with red bags shall be used to dispose of treated (autoclaved orbleached) biomedical waste. These boxes are available from HSC Building Services(392-4414). Boxes shall be labeled with principal investigator’s name, phone number,date of treatment, and lab location (room number). The ¾ full boxes shall be labeled

with indelible ink markers (e.g. Sharpie®). The ¾ full boxes shall be sealed with a leak-resistant, clear, strapping tape in an H pattern. All edges where leakage could occur shallbe taped. Trained research or custodial staff only shall handle the full boxes.

H-Pattern Taping Sequence

1st 2nd 3rd

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

Sharps Boxes

Red plastic sharps boxes are used for disposal of needles, razor blades,scalpels, and small Pasteur pipettes. These are available from HSCBuilding Services (2-4414). Sharps boxes that contain infectious

materials must be inactivated by autoclaving the closed box. These boxesshall be labeled (with indelible ink marker as above) with the principalinvestigator’s name, phone number, date the sharps box is full, and lablocation (room number). Trained research or custodial staff only shallhandle the full sharps boxes.

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Biohazard Bags

Red plastic biohazard bags shall be used for collection of biohazardous tissue cultureitems, 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, thedate the first biohazard item is placed into the bag, and lab location (room number). Thebiohazardous waste items must be inactivated by autoclaving in biohazard autoclave bagsor treatment with bleach within 30 days of accumulation (Ch. 64E-16 F.A.C.). Aftertreatment, the bags shall be tightly sealed and placed in biohazard boxes. They may betransported to the biomedical waste receptacle by trained laboratory personnel or bytrained custodial staff. Biohazard bags should never be handled by non-research staff orplaced in the hallway. Double bagging may be required to prevent leakage, however,absorbent material must be added to prevent the presence of any liquid and fluid. Full,untreated biohazard bags shall be stored only in the lab where accumulation occurred.Full biohazard bags must be treated within 24 hours.


Fold box flaps down. Seal bag by twisting Double upper sectionInsert labeled liner and fold and taping. over and tape to

over edges. double seal the bag.

Autoclave Use and TestingRules governing the use and testing of autoclaves are based on Rule 64E-16 of theFlorida Administrative Code.

Autoclave Testing

Autoclaves shall be tested before being placed into service and then periodically foreffectiveness.

1) Periodicity a) Every 40 hours of use

Required for autoclaves that are used to inactivate human or non-humanprimate blood, tissues, clinical samples, or human pathogens.

b) Every 6 months

Required for autoclaves that are used to inactivate other material.2) Method

A commercially available test indicator kit that uses bacterial spores (Geobacillus

stearothermophilus) is the approved method of testing autoclave efficiency. Most

spore vial test kits require 56°C to 60°C incubation of the autoclaved test vial

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along with a non-autoclaved control vial. Incubation causes surviving spores togrow.

a) New autoclaves

Before placing an autoclave into service, a test load approximating theweight and density of the type of waste generated shall be autoclaved withtest spore vials. The spore vials should be placed at the bottom, top,

front, rear, and center of the autoclave chamber. This can be achieved byeither:

-placing vials at those positions within one large test load, OR -making several smaller test packs with 1 vial at the center of eachand placing the packs at those locations within the chamber.

The appropriate parameters for sterilization including temperature, pressure, andtreatment time shall be determined in this way.b) Autoclaves already in use

For periodic testing, place a spore vial in the very center of a test loadprior to autoclaving.

3) Storage Information

Please 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-Keeping

The following records regarding autoclave use must be kept:1) On-site maintenance records 2) 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 name

b) 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 sterilizationconditions because the tape will change color at lowertemperatures.

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 includethe following:1) Parameters

Appropriate parameters for sterilization shall be determined from the testing withspore vials. The time it takes to sterilize a load will change depending upon theload density and the sterilization cycle one chooses. Tests have been performedwhich imitate these various situations. Please follow the established guidelines.

2) Protocol

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Identification of standard treatment containers and proper load placement shall bemade.

3) Cleaning

The autoclave and work areas shall be cleaned after every use and the work areashall be disinfected, as needed.

Autoclave Operation and Safety Training

Autoclave training is available from the EH&S Biological Safety Office both quarterlyand upon request. The training is geared toward the research staff and goes over properuse of autoclaves and how they may be maintained and used properly. Safety training isalso given.


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Autoclave Guidelines

AUTOCLAVE GUIDELINES

STERILIZATION 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 MINUTES250mL --- 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 shouldbe autoclaved in order to achieve sterilization.

Questions? Please call Gary Smith at 392-1591

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Disinfectants“Disinfectant” refers to an agent that is applied to treat (usually) inanimate objects torender them free of pathogenic or infectious non-sporeforming microorganisms. Incontrast, the term “sterilant” refers to an agent that renders items free of all microorganisms, including spores. 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 itembefore 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 effectto the user. Therefore, Material Safety Data Sheets and other manufacturer’s productinformation should be available and thoroughly reviewed before using these products.

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

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

The EPA regulates moderate and low level disinfectants as “chemical germicides” undertheir pesticide regulations. They have published a list of 1) those products that areeffective against Mycobacterium spp. (tubercle bacilli), and 2) those products that areeffective 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 removegross 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, thesecompounds must be used in well-ventilated areas.

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

Liquids

Alcohols

The most commonly used alcohols, ethanol and isopropanol, are most effective atconcentrations of 70% (v/v) in water. Both higher and lower concentrations are lesseffective. Alcohols are active against vegetative bacteria, fungi, and lipid containingviruses but not against spores. Their action on lipid-containing viruses is variable.Alcohols are difficult to use as contact disinfectants because they evaporate rapidly anddo 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

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with other disinfectants such as formaldehyde (but see toxicity warning below) orchlorine (2000 ppm chlorine in alcohol). Alcohol is NOT a registered tuberculocidal orHIV 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 commonhousehold product (e.g. Clorox) is very effective as a general laboratory disinfectant anda 1:10 dilution supplying 5000 ppm available chlorine is effective against spills involvingblood or other organic material. Please note that the presence of high concentrations of protein can inactivate the action of chlorine products. Dilute hypochlorite solution mustbe prepared daily to be maximally effective. There are more concentrated sodiumhypochlorite solutions available for industrial use, so please read the product informationcarefully to determine the proper dilution.

Table 4: Dilutions of Household Bleach

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 bemelted to release the gas. Formaldehyde gas kills all microorganisms and spores but not

prions. It is used for space decontamination and to decontaminate biological safetycabinets, a dangerous process requiring specifically trained personnel. Formaldehydedissolved in water is active at 1-8% solutions and can be used to decontaminate hardsurfaces. However, because formaldehyde is an irritant at low concentrations (0.1 to 5ppm) and a probable carcinogen, its use as a hard surface disinfectant is limited tosituations 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 2% solution and requires activation by theaddition 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 allmicroorganisms, but is toxic, an irritant, and mutagenic and should be used only whennecessary. Please follow the manufacturer’s guidance when using glutaraldehyde-basedproducts because there are many different formulations that have been designed forspecific uses.

Hydrogen peroxide

Hydrogen peroxide is usually available as a 30% solution. It may be diluted 1:5 for useas a disinfectant to decontaminate work surfaces of laboratory benches and biosafetycabinets. It is active against a wide array of microorganisms. However, it is an oxidizing

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agent and should not be used on aluminum, copper, zinc, or brass. Hydrogen peroxide isunstable at high temperatures and in light.

Iodine and Iodophors

Iodine and iodophors, compounds in which the iodine is combined with a solubilizing orcarrier agent, are general, all-purpose disinfectants with an action similar to that of chlorine products. The appropriate concentration for iodine-containing products is 75ppm available iodine for disinfecting work surfaces. Concentrations may be much higherfor other purposes. Like chlorine compounds, the effectiveness of iodine compounds

may be diminished in the presence of protein/organic material. Iodophor compounds thatare used for antisepsis (germicide applied to tissue or skin) are not appropriate for use ashard surface disinfectants and vice versa. Please read the product material forappropriate dilutions and applications.

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 effectivenessagainst non-lipid viruses and, when properly formulated, show anti-mycobacterialactivity. There are many common disinfectants based on phenol and they should be usedaccording to the manufacturer’s recommendations.

Quaternary ammonium compoundsCompounds in this class are active at concentrations of 0.1 - 2%. They are active againstvegetative bacteria, lipid viruses, but not against bacterial spores, non-lipid viruses, ortubercle bacilli. These compounds should be used only when a low-level disinfectant isrequired.

Gases

Ethylene Oxide

Inactivates micro-organisms, cellular disruption; used only as surface sterilizerTemp 50°C-60°C, Humidity 30-40%Conc. 400-800 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°C-60°C,> temp results in > activityConc. 30%, less than 10 mg/literNon-toxic end products of water and oxygenLimited to surfaces, no penetration

Corrosive to some materialsDegrades natural rubber and nylon

Chlorine Dioxide gas

Dilute chlorine gas and sodium chlorite, less than 25 mg/literTemp 25°C-30°C, pre-humidification requiredLimited to surfaces, no penetrationCorrosive to some materialsMucous membrane irritant

Ozone

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Oxidizing properties, inhibits bacterial growth, reacts with amino acids,RNA/DNA

Temp 25°CConc. 2-5 mg/literSystems convert oxygen to ozoneLimited penetration

Formaldehyde Gas (from heating paraformaldehyde)Temp 20°C-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 resultantdamage to nucleic acids

Due to low energy, the power of penetration is poor.Dust and thin layers of proteins on surfaces reduce the virucidal activity

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, most common technique used for commercial sterilization of single-

use items, and Cesium 137

Electron Beam

Limited penetrationAccelerator generates high energy electrons

Microwaves

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

Disinfectants Bibliography

The following materials were consulted or used in this chapter: Disinfection, Sterilization, and Preservation. Fourth edition. 1991. Seymour S. Block ed., Lea & Fibiger,Philadelphia Laboratory Biosafety Manual. Third edition. 2004. World Health Organization, Geneva. Biological Safety: Principles and practices. Third edition. 2000. Diane O. Fleming et al. eds. ASM Press,Washington DC Manual of Clinical Microbiology. Eighth edition. 2003. Patrick Murray ed., ASM press, 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

70% Isopropylalcoholsolution

• Cleaning someinstruments

• Cleaning skin

• Changes proteinstructure of microorganism

• Presence of waterassists with killing action

• Fairly inexpensive • <50% solution not veryeffective

• Not active when organicmatter present

• Not active against certaintypes of viruses

• Evaporates quickly - contacttime not sufficient for killing

Chlorinecompounds

• Spills of human bodyfluids

• Bactericidal - Good

• Fungicidal - Good

• Sporicidal - Good at>1000 ppm SodiumHypochlorite

• Free available chlorinecombines with contentswithin microorganism -reaction byproducts cause

its death• Need 500 to 5000 ppm

• Produce chemicalcombination with cellsubstances

• 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 foodprep surfaces

• Corrode metals such asstainless, aluminum

• Organics may reduceactivity

• Increase in alkalinitydecreases bactericidal property

• Unpleasant taste and odor

• Tuberculocidal - withextended contact time

Gluteraldehyde • Bactericidal - Good

• Fungicidal - Good

• Tuberculocidal –slow acting

• Virucidal - Good

• Sporicidal - Good

• Coagulates cellularproteins

• Non-staining,relatively non-corrosive

• Useable as a sterilanton plastics, rubber,lenses, stainless steel,

and other items thatcan’t be autoclaved

• Not stable in solution (shelf life 14 days)

• Has to be in alkalinesolution

• Inactivated by organicmaterial


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Table 6: Summary of Practical Disinfectants

Quaternary AmmoniumCompounds

PhenolicCompounds

ChlorineCompounds Iodophor Ethyl Alcohol

IsopropylAlcohol

Inactivates

Vegetative Bacteria + + + + + +

Lipoviruses + +a + + +a +

Nonlipid Viruses - - + + - -

Bacterial Spores - - + + - -

Treatment Requirements

Use Dilution 0.1-2.0% 1.0-5.0% 500ppmb

25-1600ppmb 70-85% 70-85%

Contact Time-minutes

Lipovirus 10 10 10 10 10 10

Broad Spectrum NE NE 30 30 NE NE

Important Characteristics

Effective Shelf Life

> 1 week c

+ + - + + +

Corrosive - + + + - -

Flammable - - - - + +

Explosion Potential - - - - - - Inactivated by organicmatter

+ - + + - -

Skin Irritant + + + + - -

Eye Irritant + + + + + +

Respiratory Irritant - - + - - -

Toxicd + + + + + +

Applicability

Waste Liquids - - + - - -

Dirty Glassware + + + + + +

Equipment, SurfaceDecontamination

+ + + + + +

Proprietary Productse A-33, CDQ, End-Bac,

Hl-Tor, Mikro-QuatHll-Phene,

Metar, Mikro-

Bac, O-Syl

ChloramineT, Clorox,

Purex

Hy-Sine, Ioprep,Mikroklene,

WescodyneSource: Adapted from Laboratory Safety Monograph, A Supplement to the NIH Guidelines for Recombinant DNA Research. National Institu

Research Safety, National Cancer Institute, and the Special Committee of Safety and Health Experts, Bethesda, Maryland. January 1

+ Yesa Variable results depending on virus

dBy skin or mouth or both

- Nob Available Halogen

eSpace limitations preclude listing all products available.

NE Not Effectivec Protected from light and air do not imply endorsement (or rejection) of any product


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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 amendments

U.S. Public Health Service, 42 CFR Part 72, Interstate Shipment of Etiologic Agents

U.S. Department of Labor, Occupational Safety and Health Administration, 29 CFR Part

1910.1030, Bloodborne Pathogens

International Air Transport Association (IATA), Dangerous Goods Regulations

U.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, usethe IATA regulation (also referred to as the Dangerous Goods Regulation or DGR) astheir standard. Meeting the conditions of this standard will ensure meeting the provisionsof the other US regulations.

Many biological materials fall into the category of “dangerous goods” for shippingpurposes. All individuals involved in the transport of dangerous goods or the preparation of dangerous goods for transport must be trained to do so properly and safely. Inaddition, we require safe transport of items within facilities and around campus. Thesetopics are covered in the “Shipping and Transport of Biological Materials” training given

by the Biosafety Office.

You must have a training certificate from UF EH&S to ship biological materials. Call us or see our list of scheduled classes at http://www.ehs.ufl.edu/Bio/default.asp.

Training is valid for 2 years and is a federal requirement designed to protect yourself,your co-workers, the public - drivers, airline staff, crew, pilots, passengers, and packagerecipients.Training also ensures successful shipments; carriers or Federal regulators may open,delay, or reject your shipment if it’s not correct. Training also prevents penalties forviolations; civil penalties range from $250 to $27,500 per violation per day, and criminal

penalties for willful violations range up to $500K and 5 years in jail.

Classes cover the following:

1. Classifying the material – Is it regulated? Is it forbidden for transport?2. Identifying the material – select the proper shipping name3. Choosing the right packaging4. Packaging it correctly

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5. Marking & Labeling the shipment correctly6. Supplying additional required documentation – dangerous goods declaration

forms7. Making shipping arrangements – i.e. permits, customs documents for overseas

shipments

8. Transporting things safely around UF – hand carrying & vehicle transport

Biological materials subject to shipping & transport regulations:

In the context of shipping regulations, “Dangerous Goods” are “Articles or substanceswhich are capable of posing a risk to health, safety, property or the environment &

which are shown in the list of dangerous goods in the Regulations or which areclassified according to these Regulations.” (49 CFR Parts 100-185 & IATA 1.0).

Biological materials that fall under this definition include:

Biological toxinsInfectious substancesDiagnostic specimensBiomedical wasteCulturesGenetically Modified Organisms

Other biological material that may be regulated via state or federal permits:

Plants,Plant pestsInsectsCell culturesLive animals

Items that frequently accompany shipments of biological material are also regulated:

Dry iceEnvironmental pollutants (e.g. formalin)Alcohol

Fixative solutions

Transporting biological material within and around UF:

The following general guidelines apply:


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Double contain the items plastic leak-proof containers within sturdy outerpackaging. Include absorbent material within the containers as well as padding tominimize movement of the container(s) within the outer packaging. Wipe theouter container with an appropriate disinfectant before removing it from thelaboratory and apply a biohazard sticker if applicable. Put your name and contact

information on the package. Individuals transporting biohazardous agents shouldbe knowledgeable about handling spills.

UF policy states that dangerous goods are not to be transported in your personalvehicle. This is both a safety and liability issue. Use a state vehicle.

Shipping radioactives? Call 392-7359Shipping chemicals? Call 392-8400

Permits:

Federal or state permits may be required for some biological materials. See below orcontact us at 392-1591 for more information. Permits are issued in the name of the PIwho is required to keep them updated and current as necessary; the Biosafety Office doesnot hold any “centralized” permits.

1. CDC Import Permit http://www.cdc.gov/od/eaipp/ for import of etiological agentscausing disease in humans, non-sterilized human or animal tissues/fluids knownor suspected to contain disease agents, hosts/vectors known or suspected tocontain disease agents

2. USDA/APHIS Veterinary Permit http://www.aphis.usda.gov/vs/ncie/ needed forimport of materials derived from (livestock/poultry) animals or exposed to(livestock/poultry) animal-source materials, including: animal tissues, blood, cellsor cell lines of livestock or poultry origin, RNA/DNA extracts, hormones,enzymes, monoclonal antibodies for IN VIVO use in non-human species, certainpolyclonal antibodies and antisera, bulk shipments of test kit reagents, arthropodvectors of livestock diseases, and microorganisms infectious to livestock including bacteria, viruses, protozoa, and fungi.

3. Interstate movement of microorganisms infectious to livestock/poultry includingbacteria, viruses, protozoa, and fungi, arthropod vectors of livestock/poultry

diseases, and tissues, blood, serum, or cells from known infectedlivestock/poultry.

Note: A courtesy letter to the Florida Department of Agriculture andConsumer Services Division of Animal Industryhttp://www.doacs.state.fl.us/ai/contact.shtml is required for possession or useof any of the State of Florida reportable animal diseaseshttp://www.doacs.state.fl.us/ai/main/ani_diseases_main.shtml.


http://www.cdc.gov/od/eaipp/

http://www.aphis.usda.gov/vs/ncie/

http://www.doacs.state.fl.us/ai/contact.shtml

http://www.doacs.state.fl.us/ai/main/ani_diseases_main.shtml

http://www.doacs.state.fl.us/ai/main/ani_diseases_main.shtml

http://www.doacs.state.fl.us/ai/contact.shtml

http://www.aphis.usda.gov/vs/ncie/

http://www.cdc.gov/od/eaipp/

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4. USDA/APHIS Plant Protection & Quarantine Permithttp://www.aphis.usda.gov/ppq/permits/ is needed for import or interstatemovement of plant pests, plant pathogens, biological control agents, bees, plantpest diagnostic laboratories, soil microbe isolation laboratories, federal noxious

weeds and parasitic plants

5. USDA Biotechnology Regulatory Services Notification or Permithttp://www.aphis.usda.gov/brs/fieldtesting_importation.html for the import,interstate movement, or field release of genetically engineered plants, arthropods,and plant-associated microorganisms.

6. Florida Department of Agriculture and Consumer Services Division of PlantIndustry Permit http://www.doacs.state.fl.us/onestop/plt/entnempath.html for theimport into Florida of: arthropods, plant pathogens, nematodes, noxious weeds,genetically altered (insects, nematodes, plants, plant pests) organisms, biological

control agents.7. The transfer of Select Infectious Agents and Toxins is also regulated by the

USDA/CDC. Each shipment of listed agents must be registered with theUSDA/CDC through a responsible facility official at both the shipping andreceiving entities. Please contact EH&S Biosafety before sending orrequesting any Select Agents. The current list of Select Agents and Toxinscan be found on in this manual or on our website athttp://www.ehs.ufl.edu/Bio/select.htm.

8. Export of Etiologic Agents of Humans, Animals, Plants and Related Materialsis regulated by the US Department of Commerce, Dept. of State, and Dept. of

the Treasury. A wide variety of etiologic agents of human, plant and animaldiseases, including genetic material, and products which might be used forculture or production biological agents, will require an export license.Information may be obtained by calling the Biosafety Office at 352-392-1591.Export to certain countries is prohibited.


http://www.aphis.usda.gov/ppq/permits/

http://www.aphis.usda.gov/brs/fieldtesting_importation.html

http://www.doacs.state.fl.us/onestop/plt/entnempath.html

http://www.ehs.ufl.edu/Bio/select.htm

http://www.ehs.ufl.edu/Bio/select.htm

http://www.doacs.state.fl.us/onestop/plt/entnempath.html

http://www.aphis.usda.gov/brs/fieldtesting_importation.html

http://www.aphis.usda.gov/ppq/permits/

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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 theselection, function, and use of biologicalsafety cabinets (BSCs), which are theprimary means of containmentdeveloped for working safely withinfectious microorganisms. BSCs aredesigned to provide personnel,environment, and product protectionwhen appropriate practices andprocedures are followed.

Three kinds of biological safetycabinets, designated as Class I, II, andIII, have been developed to meetvarying research and clinical needs.

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

Biological Safety Cabinets (BSCs)

The similarities and differences in protection offered by the various classes of biosafetycabinets 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 airmovement to a chemical fume hood, but has a HEPA filter in the exhaust system toprotect 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 thefront grille of the cabinet, which provides personnel protection. In addition, thedownward laminar flow of HEPA-filtered air provides product protection by minimizingthe chance of cross-contamination along the work surface of the cabinet. Becausecabinet air has passed through the exhaust HEPA filter, it is contaminant-free(environmental protection), and may be recirculated back into the laboratory (Type ABSC) or ducted out of the building (Type B BSC).

The Class II, Type A BSC

An unducted Class II Type A BSC is not to be used for work involving volatile or toxicchemicals. The buildup of chemical vapors in the cabinet (by recirculated air) and in the


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such an interlock system should be verified; systems can be retrofitted if necessary. Apressure-independent device should monitor exhaust air movement.

The Class II, Type B3 BSC

This BSC is a ducted Type A cabinet. All positive pressure contaminated plenums withinthe 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 BSC was designed for work with biosafety level 4 microbiological agents,and provides maximum protection to the environment and the worker. It is a gas-tightenclosure with a non-opening view window.

Long, heavy-duty rubber gloves are attached in a gas-tight manner to ports in the cabinetand allow for manipulation of the materials isolated inside. Although these glovesrestrict 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 somewhatturbulent, airflow within the work environment.

Horizontal Laminar Flow “Clean Bench”

Horizontal laminar flow clean air benches are NOT BSCs. They discharge HEPA-filteredair across the work surface and toward the user. These devices only provide productprotection. They can be used for certain clean activities, such as the dust-free assemblyof sterile equipment or electronic devices. These benches should never be used whenhandling cell culture materials or drug formulations, or when manipulating potentiallyinfectious 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 safetycabinet 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, forexample, 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 dischargedinto the room under the sash, resulting in the same potential problems as the horizontallaminar flow clean benches.

Operations within a Class II BSC

Laboratory Hazards

Many common procedures conducted in BSCs may create splatter or aerosols. Goodmicrobiological techniques should always be used when working in a biological safetycabinet. For example, techniques to reduce splatter and aerosol generation will minimizethe potential for exposure to personnel from infectious materials manipulated within the


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cabinet. Class II cabinets are designed so that horizontally nebulized spores will becaptured 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 andsupplies 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 whenworking in a BSC. Work at least 6” back from the front edge and never cover the frontgrill. Opened tubes or bottles should not be held in a vertical position. Investigatorsworking with Petri dishes and tissue culture plates should hold the lid above the opensterile surface to minimize direct impact of downward air. Bottle or tube caps should notbe 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 biologicalsafety cabinet. On an open bench, flaming the neck of a culture vessel will create anupward air current that prevents microorganisms from falling into the tube or flask. Anopen flame in a BSC, however, creates turbulence that disrupts the pattern of air suppliedto the work surface. When deemed absolutely necessary, touch-plate microburnersequipped with a pilot light to provide a flame on demand may be used. Internal cabinetair disturbance and heat buildup will be minimized. The burner must be turned off whenwork is completed. Small electric “furnaces” are available for decontaminatingbacteriological 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. Thiscombination will provide protection to the central building vacuum system or vacuumpump, as well as to the personnel who service this equipment. Inactivation of aspiratedmaterials can be accomplished by placing sufficient chemical decontamination solutioninto 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 thatwill be removed from the BSC at the conclusion of the work. When chemical means areappropriate, suitable liquid disinfectant should be placed into the discard pan before work

begins. Items should be introduced into the pan with minimum splatter, and allowedappropriate contact time as per manufacturer's instructions. Alternatively, liquids can beautoclaved prior to disposal. Contaminated items should be placed into a biohazard bagor discard tray inside the BSC. Water should be added to the bag or tray prior toautoclaving.

When a steam autoclave is to be used, contaminated materials should be placed into abiohazard bag or discard pan containing enough water to ensure steam generation during


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the autoclave cycle. The bag should be taped shut or the discard pan should be coveredin the BSC prior to removal to the autoclave. The bag should be transported andautoclaved in a leak-proof tray or pan.

Decontamination

Surface Decontamination

All containers and equipment should be surface decontaminated and removed from thecabinet when work is completed. At the end of the workday, the final surfacedecontamination of the cabinet should include a wipe-down of the work surface, thecabinet's sides and back, and the interior of the glass. If necessary, the cabinet shouldalso be monitored for radioactivity and decontaminated when necessary. Investigatorsshould remove their gloves and gowns and wash their hands as the final step in safemicrobiological 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 itemswithin the cabinet, as well as the cabinet interior, should be immediately wiped with atowel 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 requiremore extensive decontamination. All items within the cabinet should be surfacedecontaminated 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'sdirections 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. Thedrain pan should be emptied into a collection vessel containing disinfectant. A flexibletube should be attached to the drain valve and be of sufficient length to allow the openend to be submerged in the disinfectant within the collection vessel. This procedureserves to minimize aerosol generation. The drain pan should be flushed with water andthe drain tube removed.

Should the spilled liquid contain radioactive material, a similar procedure can befollowed. Radiation safety personnel should be contacted for specific instructions (392-7359).

Gas Decontamination

BSCs that have been used for work involving infectious materials must bedecontaminated before HEPA filters are changed, internal repair work is done or before aBSC is relocated. The most common decontamination method uses formaldehyde gas,


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although more recently, hydrogen peroxide vapor has been used successfully. Thisenvironmentally benign vapor is useful in decontaminating HEPA filters, isolationchambers, and centrifuge enclosures. Call the Biosafety Office (392-1591) for the currentBSC 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 cleanedweekly to remove any dust and dirt that may block the germicidal effectiveness of theultraviolet light. The lamps should be checked periodically with a meter to ensure thatthe 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 burnthe cornea and cause skin cancer. Do not depend on UV lamps to disinfect the area.

BSC PlacementThe ideal location for the biological safety cabinet is remote from the entry (e.g., the rearof the laboratory away from traffic), since people walking parallel to the face of a BSCcan disrupt the air curtain. The air curtain created at the front of the cabinet is quitefragile, amounting to a nominal inward and downward velocity of 1 mph. Openwindows, 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 requirereplacement 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 UF policy. Please contact the EH&S Biological Safety Office forthe name and phone number of the current contractor performing this service. Prices andquality vary widely, so only BSO-approved contractors may be used.


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Emergency Procedures/Telephone Numbers

In the event of a major disaster affecting the campus, the UF Homepage http://www.ufl.edu/ is theofficial source of UF emergency related information.

Emergency Phone Numbers

Normal

Business

Hours

Evenings & Weekends

Fire/Police/Medical EmergencyNote: callers should be able to accurately statetheir location to emergency responders. Write thelocation/address of the lab here:

911Note that youmay need todial 9 first toget an outsideline.

911Note that you may need to dial 9 first to getan outside line.

Florida Poison Information Center 1-800-222-1222

1-800-222-1222

Suspected Gas Leak 392-1121 392-1121

Needlestick/Sharps Injury 1-866-477-6824

Chemical Spill http://www.ehs.ufl.edu/RiskMgmt/emergncy/chemspill.htm

392-8400 or1591

392-1111

Radiation Spill http://www.ehs.ufl.edu/Rad/RCGuide/rcg2.htm#2IX

392-7359 or1589

392-1111

Biological Spill http://www.ehs.ufl.edu/RiskMgmt/emergncy/biospill.htm

392-1591 392-1111

Physical Plant Trouble Desk* 392-1121 392-1121

HSC Physical Plant Trouble Desk 392-4411 392-4411IFAS Facilities Operations/Maintenance 392-1984 392-1121

* 24-hour building/maintenance repair hotline.

Other Contacts

PI or lab Supervisor home number and/or cell number(s):

_______________________________ (fill in)

Karen Gillis, UF BSO, 392-1591

Mark Yanchisin, UF lab safety Coordinator, 392-1591

Phil Collis, Assoc. Director of EH&S, 392-1591

Student Health Care Center 352-392-1161

Alachua County Emergency Management 352-264-6500

Red Cross, Alachua County Headquarters 352-376-4669


http://www.ufl.edu/

http://www.ufl.edu/

http://www.ehs.ufl.edu/RiskMgmt/emergncy/chemspill.htm



http://www.ehs.ufl.edu/Rad/RCGuide/rcg2.htm#2IX


http://www.ehs.ufl.edu/RiskMgmt/emergncy/biospill.htm








http://www.ufl.edu/

http://www.ufl.edu/

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General Information

It is important that persons handling biological agents have the proper training and

experience to work with these materials safely. This includes how to handle an accidental

release, exposure, injury, or evidence of agent theft or loss.

• Personnel should know what constitutes a potential exposure or release and

report this to the PI or lab supervisor immediately. The PI or lab supervisor

must then report this to the biosafety office and, as appropriate, to medical care

providers.

• All personnel must also be familiar with symptoms of disease or illness

associated with the materials they handle so that a previously undetected

exposure can be discovered and measures taken to prevent further exposures.

• Time and situation permitting, contain and secure all biological agents during

any and all emergencies. Agent storage areas shall remain locked.

• Know ahead of time the location and operation of emergency equipment such

as the eyewash and safety shower, first aid kit, chemical and biological spill kits,

emergency numbers, fire extinguishers, emergency exits.

• PIs or lab supervisors should periodically review this information with lab staff/

students and display this information in the laboratory.

• When possible, lab staff, PI/lab supervisor should meet and escort emergency

personnel on site.

Medical Emergency

1) Remain calm.2) Initiate lifesaving measures, as required.3) Call for EMERGENCY RESPONSE - MEDICAL EMERGENCY 9-1-1.4) Calling 911 automatically alerts the UF campus Police.5) Do not remove injured person unless there is a danger of further harm.6) Keep injured person warm.


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4) Forcibly hold eye open to ensure effective rinsing behind eyelids.5) Have injured worker move eye side-to-side and up-down during rinsing.6) Remove contact lenses.7) Always obtain medical attention for a hazardous material splash to the eye.8) Place contaminated clothing in a red biohazard bag for decontamination.

9) Watch for symptoms of exposure or delayed onset effects.10) Report incident to PI/supervisor and Biological Safety Office (392-1591).

Accidental ingestion

1) Seek medical treatment:a) During working hours go to the UF Student Health Center (SHCC):

i) Main Campus (Infirmary) location: Fletcher Drive next to the Florida Gymand the Racquet Club, 392-1161

ii) UF Health Science Center Satellite location: Second floor of the Dental

Towers of the J. Hillis Miller Health Science Center. Take a Dental Towerelevator to the second floor, turn left, and straight ahead is SHCC@Shands inRoom D2-49, phone 392-0627

b) After hours or on weekends, seek treatment at the nearest emergency room.

2) The PI or supervisor must notify the Biosafety office of the incident.

Animal bites and scratches

1) For small wounds – allow to bleed freely. If necessary, control bleeding by applyingdirect pressure with a sterile gauze or bandage.

2) Immediately wash with copious quantities of soap and water. Wash for at least 15minutes. A chlorhexidine soap such as Nolvason is recommended. Povidone – iodineor Betadine surgical soap may be used but is more likely to cause skin irritation andcellular damage. If eyes or mucous membranes are exposed, irrigate the area for atleast 15 minutes with water.

3) Seek medical treatment:a) During working hours go to the UF Student Health Center (SHCC):


ii) UF Health Science Center Satellite location: Second floor of the DentalTowers of the J. Hillis Miller Health Science Center. Take a Dental Towerelevator to the second floor, turn left, and straight ahead is SHCC@Shands inRoom D2-49, phone 392-0627

b) After hours or on weekends, seek treatment at the nearest emergency room.4) The PI or supervisor must notify the Biosafety office of the incident.


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5) If the bite or scratch is from a non-human primate, contact the followingphysician/specialists regarding Monkey B virus (Herpesvirus Simiae, CHV-1)exposure:a) Dr. Reuben Ramphal, Work phone: 352 392-4058, Pager: 352-413-7837b) Dr. Kenneth Rand, Work phone: 352 392-5621, Pager: 888-553-2503

c) The physicians will evaluate the injury and may decide to culture the wound forB-virus (Herpesvirus simiae) or collect blood for a baseline titer against B-virus,or use prescription drugs for preventative therapy. The physician directing thecare of the patient will contact the Director of Animal Care Services forinstructions regarding the need for cultures or serology from the monkeyinflicting the injury upon the patient.

d) Symptoms suggestive of B virus infection should be reported immediately to themedical consultant. When the possibility of B virus illness is seriouslyentertained, appropriate diagnostic studies should be performed and specificantiviral therapy should be instituted. The physician may wish to consult the ViralExanthems and Herpesvirus Branch, Division of Viral and Rickettsial Diseases,

CDC (Dr. Scott Schmitt, (404) 639-0066 or cell 404-725-5652 or Terri Hyde(404) 639-2696, for laboratory assistance, the National B Virus Resource Centerat GSU, (Dr. Julia Hilliard, (404) 651-0808).

e) The above procedures also apply to the employees of private contractors in Non-human Primate Facilities.

Break in/Security Breach

1. Call 352-392-1111 for University police department.

2. Notify PI/lab supervisor and the Biosafety Office of the break in.3. Escort University Police personnel at the scene.4. The PI and Biosafety Office will conduct an inventory check and report results to

appropriate authorities as needed.

Handling Biological Spills

Advance preparation for management of a spill is essential. A “bio spill kit” should beavailable and contain the following:• Forceps/scoop for broken glass/sharps

• Paper towels or absorbent material• Disinfectant• Respirators, if necessary• latex or nitrile gloves and safety glasses• Red bag


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Spill in the biosafety cabinet

1) Leave the cabinet on/running to prevent escape of contaminants from the cabinet.

2) Cover the area with paper towels or other absorbent material.

3) Pour appropriate disinfectant (e.g. a fresh 1:10 dilution of household bleach, 0.5%sodium hypochlorite) over the spill as a disinfectant solution to inactivatebiohazardous material. If necessary, sufficient disinfectant solution shall be used toensure that the drain pans and catch basins below the work surface containdisinfectant. Disinfect under the front exhaust grill if needed. Walls and equipment inthe biological safety cabinet that may have been splashed shall be wiped withdisinfectant.

4) Let disinfectant solution sit for 30 minutes.

5) Pick up absorbent materials and wipe up excess disinfectant solution.

6) Place material in biohazard bag.

7) Use tweezers, forceps, or tongs to pick up broken sharps and place in a sharpscontainer.

8) Rinse all disinfected areas with water and allow to dry.

9) Follow with 70% ethanol.

Spill in the centrifuge

1) Allow aerosols to settle for 30 minutes before attempting to clean up the spill. Keepthe centrifuge closed during this time. Post a sign on the centrifuge so others don’t tryto open it.

2) Gently open the centrifuge to prevent re-aerosolization.3) Place absorbent materials in the chamber and pour a fresh 1:10 dilution of household

bleach (0.5% sodium hypochlorite) over them. Let sit 30 minutes.4) Carefully remove carriers to a tub containing a fresh 1:10 dilution of household

bleach (0.5% sodium hypochlorite). Soak 30 minutes.5) Wipe the interior and lid of the centrifuge with a fresh 1:10 dilution of household

bleach (0.5% sodium hypochlorite).

6) Use tweezers, forceps, or tongs to pick up broken sharps – place in a sharps container.7) Wipe all areas with plenty of water to prevent corrosion. Dry and follow with 70%

ethanol.


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Spill inside the laboratory

1) Notify room occupants of the spill.2) All persons should leave the laboratory immediately.3) Close the door.4) If clothing is known (or suspected) to be contaminated, remove the clothing with

care, folding the contaminated area inward. Place the clothing into a red bag forautoclaving.

5) Wash all potentially contaminated body areas as well as the arms, face and hands.Shower if necessary.

6) Any exposed persons should seek medical advice or treatment:a) During working hours go to the UF Student Health Center (SHCC):

i) Main Campus (Infirmary) location: Fletcher Drive next to the Florida Gym

and the Racquet Club, 392-1161ii) UF Health Science Center Satellite location: Second floor of the Dental

Towers of the J. Hillis Miller Health Science Center. Take a Dental Towerelevator to the second floor, turn left, and straight ahead is SHCC@Shands inRoom D2-49, phone 392-0627

b) After hours or on weekends, seek treatment at the nearest emergency room.7) Post a sign on the door to keep people out.8) No one should enter for approximately 30 minutes so that aerosols can be cleared by

the ventilation system or allowed to settle.9) During this time, notify the PI or lab supervisor. Notify the biosafety office for large

spills, those you are uncomfortable handling, those involving BSL-3 agents, or thoseinvolving select agents.

10) Protective clothing should be worn when entering the laboratory to clean the spillarea. Latex or nitrile gloves, autoclavable, or disposable footwear, safety glasses, andan outer garment. If you have been issued an N95 respirator to work with this agent,put that on.

11) Take the “bio spill kit” into the laboratory room, and place paper towels, spillpillows, or other absorbent materials around and on the spill. If the spill was on thefloor, do not use a surgical gown that may trail on the floor when bending down.

12) Carefully pour a fresh 1:10 dilution of household bleach (0.5% sodium hypochlorite)over the spill as a disinfectant solution; avoid splashing and work from the outside

towards the center.


14) Use paper or cloth towels to wipe up the disinfectant and spill, working toward thecenter of the spill. Discard all towels and other clean up materials into a discardcontainer/red bag as they are used.

15) Wipe the outside of the discard containers, especially the bottom, with a towel soakedin a disinfectant.


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16) Place the discard container and other materials in an autoclave and sterilize.17) Remove shoes or shoe covers, outer clothing, respirator, and gloves and sterilize by

autoclaving.18) Wash hands, arms and face; shower if necessary.

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

Spill outside the laboratory

Safe transport of biohazardous material outside the laboratory is essential. Materialsshould be packaged securely (triple contained in unbreakable containers) to avoid suchspills. In addition, the person transporting the material should be knowledgeable aboutthe hazards of the material and how to respond to a spill. In the event of a spill outsidethe lab:

1) Clear area of all personnel and keep them out of the spill2) Have someone call the University of Florida Police Dept., 392-1111 and the

Biosafety Office 392-1591, for help.3) If clothing is known (or suspected) to be contaminated, remove the clothing with

care, folding the contaminated area inward. Place the clothing into a bag forautoclaving.

4) Wash all potentially contaminated body areas as well as the arms, face and hands.Shower if necessary.

5) Any exposed persons should seek medical advice or treatment:a) During working hours go to the UF Student Health Center (SHCC):


ii) UF Health Science Center Satellite location: Second floor of the DentalTowers of the J. Hillis Miller Health Science Center. Take a Dental Towerelevator to the second floor, turn left, and straight ahead is SHCC@Shands inRoom D2-49, phone 392-0627

b) After hours or on weekends, seek treatment at the nearest emergency room.6) Spill will be cleaned up by lab staff (if a spill kit is available) or by EH&S staff.7) Protective clothing should be worn to clean the spill area. Latex or nitrile gloves,

autoclavable, or disposable footwear, safety glasses, and an outer garment. If youhave been issued an N95 respirator to work with this agent, put that on.

8) Take the “bio spill kit” to the area, and place paper towels, spill pillows, or other

absorbent materials around and on the spill. If the spill was on the floor, do not use asurgical gown that may trail on the floor when bending down.

9) Carefully pour a fresh 1:10 dilution of household bleach (0.5% sodium hypochlorite)over the spill as a disinfectant solution; avoid splashing and work from the outsidetowards the center.



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11) Use paper or cloth towels to wipe up the disinfectant and spill, working toward thecenter of the spill. Discard all towels and other clean up materials into a discardcontainer/red bag as they are used.

12) Wipe the outside of the discard containers, especially the bottom, with a towel soakedin a disinfectant.

13) Place the discard container and other materials in an autoclave and sterilize.14) Remove shoes or shoe covers, outer clothing, respirator, and gloves and sterilize byautoclaving.

15) Wash hands, arms and face; shower if necessary.

Fire Safety

Predetermine two means of egress from your normal workplace.Learn the location of the nearest fire alarm pull station and portable fire extinguisher.

If you discover a fire in a University of Florida building, do the following:

1) Pull the fire alarm and call 911.2) Fire alarm pull stations are normally located near each exit. If the building is not

equipped with a fire alarm system, notify other occupants as you exit the building.3) Do not attempt to fight the fire with portable fire extinguishers or fire hoses unless

the fire is small and you have been trained in their proper use. DO NOT PUT YOURLIFE IN DANGER WHILE ATTEMPTING TO CONTROL A FIRE. When indoubt, evacuate.

4) Remain calm while talking to the operator. Be prepared to answer several questionsas to location, size of fire, your name, number of persons in building (if known) and

any injuries. Remain on the line until the operator is finished.5) Meet fire or police personnel when they arrive at the building. Stand by to answer any

questions they may have concerning the fire.6) Once out of the building DO NOT RE-ENTER THE BUILDING FOR ANY

REASON, unless emergency personnel have given the "ALL CLEAR" signal.

If the fire is INSIDE your room:

Leave your room and close the door.Pull the fire alarm and call 911.

Or, if the fire is small and you have been trained to use a fire extinguisher, attempt to put

it out. (Again, DO NOT PUT YOUR LIFE IN DANGER WHILE ATTEMPTING TOCONTROL A FIRE. When in doubt, evacuate.)Remember the acronym “PASS”

PASS =P Pull the pin.A Aim at the base of the fire.S Squeeze the trigger.S Sweep the nozzle from side to side.


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2) See the checklist to prepare lab areas for an impending hurricane:

http://www.ehs.ufl.edu/disasterplan/LabPrep.rtf

3) The UF homepage is the official source of UF Information. Official emergency

information for Alachua County is broadcast oni) Radio: WUFT-FM / WJUF-FM / WLUF-LP / WRUF / AM850 / Rock

104

ii) Television: WUFT-TV

iii) Additional information regarding safety and preparedness may befound at: www.ufl.edu/emerg.html

Tornadoes and other natural disasters:

Tornado Watch - conditions are favorable for the formation of tornados.Tornado Warning - indicates that a tornado has been sighted or is indicated on radar.There is generally little or no warning given at the approach of a tornado. In the event of a tornado:1) IMMEDIATELY seek shelter, preferably in the hallway of a main office building, an

interior bathroom or an interior closet.2) AVOID windows; flying debris can kill. Protect yourself by getting under a heavy

desk or table. Also, remember to protect your head.3) If time permits, open at least two windows to provide complete ventilation for each

building.




http://www.ufl.edu/emerg.html




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3 -- Programs

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Animal Contact Medical Monitoring ProgramThe University of Florida Animal Contact Medical Monitoring Programgrew out of the recommendations of the American Association for the

Accreditation of Laboratory Animal Care (AAALAC) reviewers.According to the US Public Health Service, an occupational healthprogram is required for institutions that employ personnel who have animal

contact.

The UF program is designed to protect our employees, students, and volunteers havinganimal contact from occupational exposure to conditions that may result in animal relatedillnesses. The requirements of the program are based upon those outlined in the PublicHealth Services document, Guide for the Care and Use of Laboratory Animals, publishedby the National Research Council. The current UF program requirements were revisedand approved, and put into place May 2006.

Individuals who will be working with animals or who will be working in proximity toanimals are required to participate in the medical monitoring program. They are providedwith animal contact medical monitoring information and immunizations relating to theiranimal contact as part of their pre-placement health assessment.

In addition, the Institutional Animal Care and Use Committee (IACUC) verifies that allpersonnel listed on new and continuing projects are registered with the UF AnimalContact Medical Monitoring Program. The IACUC committee notifies the PI of anypersonnel who are not thus cleared for animal contact. Principal Investigators areresponsible for ensuring that all personnel (including employees, students, colleagues,

collaborators, and volunteers) involved with their IACUC-approved project are givenprogram information. Investigators who do not respond to requests for registration mayhave their approval rescinded by the IACUC.

Individuals that have animal contact must participate in a risk assessment that includescontact information, health questionnaire and health assessment (physical examination,medical history, blood serum banking) based upon the type of animal contact, andimmunizations as needed. This risk assessment is provided at no cost to the employee.

Short-term visitors to UF from other institutions must provide documentation of participation in their home institution’s animal contact program or they must register inthe UF Animal Contact Program. Individuals involved in isolated one-time, non-recurrentexposures shall be informed of potential dangers and medical precautions, but are notrequired to participate in the program. The primary responsible party (principalinvestigator, research director, student research coordinator, etc.) shall be responsible forassuring compliance with the notification requirements for these individuals.


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Elements of the Program

1. Risk Assessment

• A Risk Assessment form is required to be completed by everyone working withanimals at UF or entering UF animal facilities. This form includes contact

information and a health questionnaire that will be evaluated by UF’sOccupational Medicine Physician or Licensed Health Care Professionals to assessthe risk of exposure and determine whether additional information and/orinteraction is necessary.

• A Renewal Risk Assessment form is required every three years or when any newspecies is contacted. This update will allow UF’s Occupational MedicinePhysician or Licensed Health Care Professionals to evaluate and, if necessary,address potential health risks to you resulting from a change to your health statusor changes to the type of animal exposure.

2. Specific requirements

• Tetanus Immunization within 10 years - All participants

• Rabies Immunization Series/Booster or Positive Titer within 2 years – Allindividuals handling unvaccinated carnivores or their tissue

• Respirator Clearance and Fit Test – All individuals required by the Q-Fever Policyor as medically necessary to prevent allergic reactions

• Serum Banking – All individuals who work with non-human-primates, all whohandle blood from alligators or birds housed outdoors, all who are required by the

Q-Fever Policy and all pre-menopausal females who handle cats or cat waste.

• TB Screening within 12 months – All individuals who enter any room with non-human primates.

• Medical consultation – As determined by the Occupational Medicine Physician.Examples are individuals with chronic disease, work-related injuries or illness,environmental or animal allergies.

3. Exemptions from the program

Individuals working on projects that involve observation of birds or other animals

outdoors/in their natural habitat are exempt from the program.

See http://www.ehs.ufl.edu/Bio/Animal/acweb.htm for more information.


http://www.ehs.ufl.edu/Bio/Animal/acweb.htm

http://www.ehs.ufl.edu/Bio/Animal/acweb.htm

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UF Bloodborne Pathogen ProgramIn December 1991, OSHA published the final rule coveringoccupational exposure to bloodborne pathogens. This wasadopted by the state of Florida and written into the Florida

Administrative Code in January 1993. UF instituted its programin 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-generatedprogram. This program shall include development and annual review of a site specificExposure Control Plan, annual training regarding exposures, offer of hepatitis Bvaccinations free-of-charge, and post-exposure health care services.

Environmental Health and Safety (EH&S) manages this program through the BiologicalSafety Office. Each January, EH&S sends program materials and instructions to

department chairs and directors of groups identified as having employees withoccupational exposure. These materials provide instructions and information necessary toachieve annual training compliance. The BBP program materials and information isavailable on our website at http://www.ehs.ufl.edu/bio/BBP/default.htm .

In addition to the program materials, EH&S provides “train-the-trainer” sessions annually.

Hepatitis B vaccinations are given by the Student Health Care Center (SHCC) or, in somecases, Shands Occupational Health Services, or other providers. Official program medicalrecords are kept by SHCC. There is a requirement that Shands or other providers sendvaccination and post-exposure records to SHCC for record-keeping purposes. Declination

statements from those that decline the vaccination series are kept by EH&S.

EH&S monitors for UF compliance by requiring training and vaccination documentationand by confirming BBP participation during the annual laboratory safety survey which isconducted in each laboratory each year.

For further information, please call the Biological Safety Office at 392-1591.

BBP Exposure Information

In Gainesville, call the Needlestick Hotline 24 hours aday following any exposure.

In Jacksonville, go to the Employee Health Office inSuite 505, Tower 1, 5th and Jefferson from 7 – 4:30.Go to the ER after hours. Phone: 904 244-9576

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


http://www.ehs.ufl.edu/bio/BBP/default.htm

http://www.ehs.ufl.edu/bio/BBP/default.htm

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4 -- Medical Surveillance

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HIV Research Laboratory

Occupational Medicine Policy

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

Pre-employment

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

1. A baseline serum sample shall be stored with the UF 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 isavailable at the UF Student Health Care Center. The test must be offered by theemployer, but may be declined by the employee. This test shall include HIVcounseling. 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 storedin a separate section in the employee’s medical record. At no time, will theemployer or any administrator or official of UF have access to the employee’sconfidential record concerning HIV testing.

3. If the HIV researcher also works with human blood or other potentially infectedmaterial, all aspects of the UF Bloodborne Pathogen Program under the OSHABloodborne Pathogen standard shall be implemented for that employee. Theemployer shall offer free HBV immunization, annual training, and have a writtenexposure control plan in the workplace.

Continuing employment

HIV 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 prophylaxis

An 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 beimplemented:


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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 beused, if immediately available. If the exposure is to the eyes, they shall be rinsedfor five minutes in an eyewash station. Other exposed mucous membranes (noseand mouth) shall be rinsed for five minutes with water.

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

exposure.3. The employee may be seen at the Student Health Care Center between the hours

of 8:00 a.m. and 4:00 p.m. After hours, or on weekends, employees shouldproceed to Shands 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 employeewith 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 CDCrecommendations, the nature of the exposure event, and other medical factors.Shands 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. Shands OHS at UF personnel are familiar with the paperwork toprocess this type of Worker’s Compensation claim for UF 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 OSHABloodborne Pathogen rule. The Shands protocol on HIV exposures will befollowed.

References

UF Bloodborne Pathogen Program Compliance Materials;http://www.ehs.ufl.edu/Bio/BBP/bio_bbp.htm

OSHA. 29 CFR Part 1910.1030. Occupational Exposure to Bloodborne Pathogens,Final Rule, Dec. 6, 1991

CDC. Update: Provisional public health service recommendations for chemoprophylaxis

after occupational exposure to HIV; MMWR 1996; 45(22):468-472Human immunodeficiency virus (HIV) and acquired immune deficiency syndrome

(AIDS) exposure (includes chemoprophylaxis treatment protocol); Shands OHSat UF, rev. July 17, 1996.


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ImmunoprophylaxisThe University of Florida follows the recommendations of the Centers for DiseaseControl and Prevention (CDC) and the Public Health Service Advisory Committee forImmunization Practices (ACIP) for vaccination of at-risk personnel.

Currently, UF has written policies regarding the need for hepatitis B, tetanus, rabies, andvaccinia vaccination for certain at-risk personnel. Other vaccinations may be required orrecommended, as needed. Particular attention is given to individuals who are or maybecome immunocompromised, as recommendations for vaccine administration may bedifferent than for immunologically competent adults. Various other factors may be takeninto account such as pregnancy, history of allergy, or HIV status.

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


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Rickettsial agents

1. Coxiella burnetii

An investigational new Phase I Q fever vaccine (IND) is available from theSpecial Immunizations Program, U.S. Army Medical Research Institute forInfectious Diseases (USAMRIID), Fort Detrick, MD. The use of this vaccine

should be limited to those at high risk of exposure (e.g., individuals engaged inresearch with pregnant sheep, or live C. burnetii organisms) and who have nodemonstrated sensitivity to Q fever antigen.

Viral agents

1. Hepatitis A virusA vaccine is available and recommended for those who work with hepatitis Avirus.

2. Hepatitis B virusHepatitis B virus infection is a major infectious hazard for health care personnel.A vaccine is available and highly recommended for workers performing tasksinvolving exposure to blood or blood contaminated body fluids, according to the

OSHA rule and the UF Bloodborne Pathogen Program.3. Influenza:

Recommended during influenza season for health care workers involved inpatient care, those with chronic medical conditions, to prevent staff illness andreduce spread of the disease.

4. PoliovirusAll laboratory personnel who work directly with the agent are required to havedocumented polio vaccination or demonstrated serologic evidence of immunity toall three poliovirus types.

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

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

7. ArbovirusesFor the following arthropod-borne viruses there is a vaccine available and it isrecommended for those who work with the virus.Eastern equine encephalitis* Junin CabassueWestern equine encephalitis* Mucambo Rift Valley FeverVenezuelan equine encephalitis* Chikungunya EvergladesYellow Fever(*No commercial vaccine available for human infections)


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8. Measles, Mumps, and German measles virus (Rubeola, paramyxovirus, andrubellavirus)Immunizations are usually given in childhood. Vaccines are highly recommendedfor all unvaccinated or serologically negative adults.


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Vaccinia Immunization Policy

All personnel who work in an area where vaccinia, orthopoxviruses, or animalsvaccinated with those agents are used, handled or housed must have received a vacciniavaccination within the last ten years. Individuals must declare their vaccination statusprior to engaging in such work. This may be accomplished by using the UF Pox VirusVaccine Form.

To fulfill this requirement:

1. Request a Vaccinia Vaccination Information Packet from Environmental Health& Safety (EH&S) at 392-1591.

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

a. Medical questions – Student Health Care Center’s Occupational MedicineProgram (391-1161 X4259)

b. Virology questions – EH&S and/or principal investigator.3. All projects involving “pox” viruses require vaccination.

4. Any medical condition precluding vaccination will also preclude participation in aproject 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. Theseevaluations are conducted by EH&S and its various committees.

2. Each participant complies with the vaccine policy requirements prior to

participation in any covered research project.

3. EH&S is notified of vaccination dates for all researchers working with vaccinia.


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University of Florida

Pox Virus Vaccine Form

Name______________________________ Department _________________________UF ID _____________________________ Title ______________________________Date ______________________________ Position # __________ Phone __________

I have read the informational package concerning biosafety considerations for workingwith vaccinia virus and related orthopoxviruses and I have had an opportunity to ask questions about this information of both a subject expert and a health care professional. Iunderstand that I may obtain a smallpox vaccination administered by university medicalpersonnel at no cost to me.

I understand that failure to consent to vaccine will prohibit me from working with any“pox” viruses.

Previously Vaccinated

Date: ___________________________Location: ___________________________Administered by: ___________________________Employee Signature: ___________________________________

Consent to Vaccination

I authorize and request the University of Florida and its designated employees toadminister the vaccine to me.

Employee Signature: _____________________________________Date Given: ______________________Lot Number: ____________Administered By: ________________________________________


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Health Surveillance for Personnel Working with

Infectious Agents

A health surveillance program is available for laboratory personnel who use agents thatrequire it. Laboratory personnel should receive immunizations (see chapter entitled“Immunoprophylaxis”), such as hepatitis B vaccination, and medical tests, such astuberculosis skin testing, when appropriate. The Animal Contact and Bloodborne PathogenPrograms (see appropriate chapters) provide for health assessments, risk assesments,medical tests, and immunizations for certain at-risk personnel.

Blood Serum Sampling

For those laboratory personnel working with certain BSL-2 or BSL-3 agents, baselineserum samples may be appropriate (see chapter entitled “Biological Safety Levels”). The

collection and maintenance of baseline serum samples provide a tool for monitoringserological changes that may result from the employee’s work. For example, staff whowork with non-human primates may be required to bank a blood sample.

The Biological Safety Office, with advice from the appropriate health care professional,will provide information regarding the need for, and the frequency of, blood serumsampling on a case-by-case basis. The need for serum sampling will be dependent uponthe 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 samplingmonitored annually and upon accidental exposure.

Health Assessments

The UF Occupational Medicine Program provides for pre-placement and other healthassessments 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 withcertain chemicals or acute toxins may require a respirator at some stage of the researchproject. The EH&S Occupational Medicine Respiratory Protection Program provides forhealth assessments that will determine an employee’s fitness for respirator use.

The EH&S Occupational Medicine Program also includes the Pesticide Medical

Monitoring Program, the Hearing Conservation Program, the Diving Science and SafetyProgram, and the Asbestos Monitoring Program, as well as the Animal Contact Program,discussed in a separate section. Occasionally, researchers who work with infectiousagents will require a health assessment based on the requirements of one of these otherprograms.


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Exposure to Mycobacterium Tuberculosis

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


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5 -- Appendix A – Forms

R-DNA

http://www.ehs.ufl.edu/bio/Files/rdna.doc

Bio-Agent

http://www.ehs.ufl.edu/bio/Files/bio.doc

Transgenic Animals

http://www.ehs.ufl.edu/bio/Files/rdnata.doc

Acute Toxin

http://www.ehs.ufl.edu/bio/Files/toxin.doc

Project Addendum

http://www.ehs.ufl.edu/bio/Files/addendum.doc
















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