Table of Contents

• Introduction
  
• Definitions
  
• Overview
  
• Duties and Responsibilities
  
• Registration of Biohazardous Materials
  
• Principles of Biosafety
  
• Laboratory Practice and Technique
  
• Safety Equipment (Primary Barriers)
  
• Laboratory Design and Construction (Secondary Barriers)
  
• Risk Assessment
  
• Biosafety Levels
  
• Animal Facilities
  
• Laboratory Biosafety Level Criteria
  
• Personal Protective Equipment
  
• Laboratory Safety Equipment
  
• Laboratory Equipment
  
• Signs and Posting
  
• Biohazardous/Biomedical Waste
  

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Introduction

As a leading research, medical, and educational institution, The Florida State University is morally and legally obligated to provide a safe working environment for all its employees, students, and guests.

This manual has been developed by the Biological Safety Office, Department of Environmental Health and Safety, The Florida State University, with the endorsement of the University's Biological Safety Committee. This manual comprises biological safety protocols for the laboratory.

With the great diversity of activities on campus, it is impossible for any one laboratory to have defined standard operating procedures (SOPs) for every possible activity related to biological safety. For this reason, the Biological Safety Manual was developed to provide information that can be used to protect workers and the surrounding environment from possible exposure to biohazardous agents.

This manual makes it possible to construct a general framework that can be used by individual departments or laboratories. It is designed to provide general information applicable to most laboratories, with specific information and procedures to be added as needed for each laboratory. Implementation of these procedures is the responsibility of the Principal Investigator and depends largely on the efforts of PI's, supervisors and employees. It is essential that staff and students seek additional advice and training when dealing with biohazardous agents to assure the safety of employees, students and the surrounding community. To assist in this, the services and resources of the Department of Environmental Health & Safety (EH&S) are available.

The Biological Safety Office has developed this manual based on guidelines developed by the Center for Disease Control (CDC) and the National Institute of Health (NIH), Biosafety in Microbiological and Biomedical Laboratories (4th edition); 1999, (BMBL), with the endorsement of the University's Biological Safety Committee. It is intended to provide information to protect workers and the surrounding environment from possible exposure to biohazardous agents. The information also serves to protect experiments and research by controlling the unwanted spread of contamination.

The premise is that no experiment should be considered so important that it jeopardizes the well-being of the worker or the environment. The planning and implementation of safety protocols to prevent laboratory-acquired infections and to eliminate the spread of contamination must be part of every laboratory's routine activities.

The handling of biological agents and recombinant DNA requires the use of precautionary measures dependent on the agents involved and the procedures being performed. It is the purpose of this manual to provide background information and guidelines to be used in conjunction with other resources for the evaluation, containment and control of biohazardous materials in the research laboratory.

The Biological Safety Office can be reached at 644-5374 or 644-9117.

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Definitions

Blood: human blood, human blood components that include plasma, platelets and wound exudates, and products made from human blood.

Bloodborne pathogens: pathogenic microorganisms present in human blood, which can cause disease in humans. Includes the hepatitis B virus (HBV), hepatitis C virus (HCV) and the human immunodeficiency virus (HIV).

Contaminated: presence or reasonably anticipated presence of blood or other potentially infectious materials (OPIM) on an item or surface.

Decontamination: use of physical or chemical means to remove, inactivate or destroy bloodborne pathogens on a surface or item to the point where they are no longer capable of transmitting infectious particles and the item or surface is rendered safe for handling, use, or disposal.

Engineering controls: controls such as sharp disposal containers or self-sheathing needle that isolate or remove the hazard from the workplace.

HIV: human immunodeficiency virus.

Other potentially infectious materials (OPIM): including the following human body fluids: semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva in dental procedures, any body fluid that is visibly contaminated with blood and all body fluids where it is difficult or impossible to differentiate between body fluids; any unfixed tissue from human and HIV/HBV containing culture medium.

Parenteral: entry into the body by other means than through the digestive tract such as by piercing mucous membranes or the skin by needle sticks, human bites, cuts and abrasions.

Personal protective equipment (PPE): special clothing worn by an employee to protect against a hazard. General work clothes (uniforms, pants, shirts, blouses) not intended to function as protection against a hazard are not considered to be personal protective equipment.

Regulated waste: defined in 64E-16.002(2) Florida Administrative Code, Biomedical Waste; any solid or liquid waste that may present a threat of infection to humans.

Examples include:

• Non-liquid or semi-liquid tissue and body parts from humans and other primates; laboratory and veterinary waste which contain disease-causing agents; discarded sharps; and blood, blood products and body parts from humans and other primates;
• Or, other potentially infectious materials; contaminated items that would release blood;
• Or other potentially infectious materials in a liquid or semi-liquid state if compressed;
• Items that are caked with dried blood or other potentially infectious materials and are capable of releasing these materials during handling; and
• Contaminated sharps and pathological and microbiological wastes containing blood or other potentially infectious materials.

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Overview

PI's working or proposing to work with recombinant DNA, human cells, pathogenic microorganisms, primate body fluids, certain animals, or biological toxins must seek approval by the FSU Biological Safety Committee. Included in this requirement is research undertaken by FSU appointees in facilities controlled by the University or supported by grants processed through the University. All proposals, regardless of funding source, are subject to review.

The Biological Safety Committee will determine if the research falls within the NIH Guidelines for Research Involving recombinant DNA. The Biological Safety Office will also review the research proposal and determine the appropriate level of biohazard containment.

Generally, the Biological Safety Committee receives research proposals from the grant processing office for the University, such as the Office for Sponsored Research or the Research Foundation. The Chairperson for the Biological Safety Committee will initially review and approve the research. If the Chairperson determines that the proposed research falls within the NIH Guidelines for Research Involving recombinant DNA or if the Chairperson determines that the research involves biological hazards, the Chairperson will notify the Biological Safety Officer to convene a full Biological Safety Committee review of the research proposal.

Once the review is complete, the Biological Safety Office will confirm that the facilities and safety equipment, including biological safety cabinets, are functioning properly and provide the required training for the PI, faculty, students, and staff conducting the research.

The PI will receive a memorandum from the Biological Safety Officer indicating whether the research is approved and stating the minimum level of containment necessary for the research. The research will not be approved if the appropriate safety equipment is not in place. The grant processing office at FSU will receive a copy of the memorandum as well.

The Biological Safety Office will routinely inspect the research facility to ensure that safety equipment; including biological safety cabinets and other safety equipment are functioning properly, and provide refresher training to the PI, faculty, students, and staff. Any deficiency will be reported to the Biological Safety Committee.

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Duties and Responsibilities

Principle Investigators

Principal Investigators (PI's) are responsible for implementing an appropriate biological safety program for their laboratories or departments. They should evaluate all their operations, perform risk assessments and develop plans for all activities accordingly. They are also responsible for establishing the appropriate biological safety containment levels in consultation with the University's Biological Safety Office and ensuring adherence to these levels. They must also assure strict adherence to biological safety practices and techniques for all work involving biohazardous materials. Individuals are responsible for their own safety and that of others potentially affected by biohazardous agents or substances, and for the protection of the environment.

Prior to the commencement of any activities involving the use of biohazardous materials as defined in the Centers for Disease Control and Prevention (CDC)/National Institute of Health (NIH) Biosafety in Microbiological and Biomedical Laboratories (BMBL) Manual and the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines), the PI must register all biohazardous agents with the Biological Safety Committee and the Biological Safety Office. It is also the responsibility of the PI to ensure that personnel receive the appropriate training on the potential hazards of biohazardous materials. This includes instruction in specific practices and techniques required for safely handling the agents. In addition, the PI should also ensure that personnel attend the Florida State University Biological Safety Laboratory Training.

Individuals

Individuals must adhere to biological safety practices and techniques. This includes working with biohazardous agents using the appropriate containment and personal protective equipment as directed by the supervisor and PI as outlined in the risk assessment.

The Biological Safety Committee

The Biological Safety Committee is responsible for reviewing practices, protocols and facilities for the handling of recombinant DNA and biohazardous materials at all facilities under the auspices of The Florida State University (FSU). The Committee is comprised of faculty members, the Biological Safety Officer, and representatives from the community. The Committee meets at least annually to review the overall Biological Safety Program and on an as needed basis for review of research proposals that involve recombinant DNA, including biohazardous materials, and to discuss and implement policies and procedures associated with an effective Biological Safety Program.

The University Biological Safety Committee can be reached by contacting the Biological Safety Officer, at 644-5374.

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Registration of Biohazardous Materials

All research proposals that involve biohazardous materials must notify the Biological Safety Committee and the Biological Safety Office by completing the Etio/Select Agents Registration Update Form (EHS 7-6), Recombinant DNA Registration Update Form (EHS 7-7) or the Select Agents Registration Form (EHS 7-1). These forms can be found online under the forms section.

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Principles of Biosafety

The term "containment" is used in describing safe methods for managing infectious materials in the laboratory environment. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents. There are two types of containment: primary and secondary.

Primary containment

Primary containment is the protection of personnel and the immediate laboratory environment from exposure to infectious agents; it is provided by both good microbiological technique and the use of appropriate safety equipment. The use of vaccines may provide an increased level of personal protection.

Secondary containment

Secondary containment is the protection of the environment external to the laboratory from exposure to infectious materials provided by a combination of good facility design and suitable operational practices. Therefore, the three elements of containment include laboratory practice and technique, safety equipment, and facility design. The risk assessment of the work to be done with a specific agent will determine the appropriate combination of these elements.

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Laboratory Practice and Technique

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

Each laboratory should develop or adopt a biological safety or operations manual that identifies the hazards that will or may be encountered and that specifies practices and procedures designed to minimize or eliminate exposures to these hazards. Personnel should be advised of special hazards and should be required to read and follow the required practices and procedures. A researcher trained and knowledgeable in appropriate laboratory techniques, safety procedures, and hazards associated with handling infectious agents must be responsible for the conduct of work with any infectious agents or material. This individual should consult with the Biological Safety Office or other health and safety professionals with regard to risk assessment.

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

Appropriate facility design and engineering features, safety equipment, and management practices must supplement laboratory personnel, safety practices, and techniques.

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Safety Equipment (Primary Barriers)

Safety equipment includes biological safety cabinets (BSCs), enclosed containers, and other engineering controls designed to remove or minimize exposures to hazardous biological materials. The biological safety cabinet (BSC) is the principal device used to provide containment of infectious splashes or aerosols generated by many microbiological procedures. Three types of biological safety cabinets (Class I, II, III) used in microbiological laboratories are described and illustrated in the BMBL 4th edition in Appendix A, Primary Containment: Biological Safety Cabinets. Open-front Class I and Class II biological safety cabinets are primary barriers that offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques. The Class II biological safety cabinet also provides protection from external contamination of the materials (e.g., cell cultures, microbiological stocks) being manipulated inside the cabinet. The gas-tight Class III biological safety cabinet provides the highest attainable level of protection to personnel and the environment.

An example of another primary barrier is the safety centrifuge bucket, an enclosed container designed to prevent aerosols from being released during centrifugation. To minimize aerosols, containment controls such as BSCs or centrifuge buckets must be used when handling infectious agents that can be transmitted through the aerosol route of exposure.

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

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Laboratory Design and Construction (Secondary Barriers)

The design and construction of the facility contributes to the laboratory workers' protection, provides a barrier for protection of individuals outside the laboratory, and protects persons or animals in the community from infectious agents which may be accidentally released from the laboratory. Laboratory management is responsible for providing facilities commensurate with the laboratory's function and the recommended biosafety level for the agents being manipulated.

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

When the risk of infection by exposure to an infectious aerosol is present, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Such design features include specialized ventilation systems to ensure directional airflow, air treatment systems to decontaminate or remove agents from exhaust air, controlled access zones, airlocks at laboratory entrances, or separate buildings or modules to isolate the laboratory. Design engineers for laboratories may refer to specific ventilation recommendations as found in the Applications Handbook for Heating, Ventilation, and Air-Conditioning (HVAC) published by the American Society of Heating, Refrigerating, and Air-Conditioning Engineers (ASHRAE).

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Risk Assessment

Bacteria, viruses, fungi or other infectious agents are studied because they cause disease. Since many of these agents are pathogenic to humans, animals, or other forms of life, their use poses risks, which vary with each agent and the way it is used. The CDC and the NIH have developed the Biosafety in Microbiological and Biomedical Laboratories (BMBL) Guidelines, which classify microorganisms into different Biosafety Levels. Pathogen risks are determined by weighing a number of factors associated with the microorganism. These factors include the severity of the disease that the microorganism causes, the routes of infection, its virulence, and infectivity. Other factors that are taken into account include the existence of effective therapies, possibilities for immunization, the presence of vectors, quantity of agent being used and whether the agent is indigenous to the United States.

Conditions under which an infectious agent is used in a laboratory is also of concern. Factors such as large volumes and high concentrations of an agent in growth media create a greater risk than working with smears of the same agent on microscope slides. Using agents in unusual or untried research settings can also alter the associated risks.

The BMBL uses the above factors to classify biohazards into four distinct Biosafety levels.

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Biosafety Levels

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

The recommended biosafety level(s) for the organism in Section VII (Agent Summary Statements) of the BMBL represent those conditions under which the agent ordinarily can be safely handled. The PI is specifically and primarily responsible for assessing the risks and appropriately applying the recommended biosafety levels. The Biological Safety Office can assist by verifying the recommended biosafety levels.

Generally, work with known agents should be conducted at the biosafety level recommended in the appropriate BMBL section. When specific information is available to suggest that virulence, pathogenicity, antibiotic resistance patterns, vaccine and treatment availability, or other factors are significantly altered, more (or less) stringent practices may be specified.

Biosafety Level 1

Practices, safety equipment, and facility design and construction are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other laboratories in which work is done with defined and characterized strains of viable microorganisms not known to consistently cause disease in healthy adult humans. Bacillus subtilis, Naegleria gruberi, infectious canine hepatitis virus, and exempt organisms under the NIH Recombinant DNA Guidelines are representative of microorganisms meeting these criteria. Many agents not ordinarily associated with disease processes in humans are, however, opportunistic pathogens and may cause infection in the young, the elderly, and immunodeficient or immunosuppressed individuals.

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

Biosafety Level 2

Practices, equipment, and facility design and construction are applicable to clinical, diagnostic, teaching, and other laboratories in which work is done with the broad spectrum of indigenous moderate-risk agents that are present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Hepatitis B virus, HIV, the salmonellae, and Toxoplasma spp. are representative of microorganisms assigned to this containment level. Biosafety Level 2 is appropriate when work is done with any human-derived blood, body fluids, tissues, or primary human cell lines where the presence of an infectious agent may be unknown. Laboratory personnel working with human-derived materials should refer to the OSHA Bloodborne Pathogen Standard for specific required precautions.

Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures, or ingestion of infectious materials. Extreme caution should be taken with contaminated needles or sharp instruments. Even though organisms routinely manipulated at Biosafety Level 2 are not known to be transmissible by the aerosol route, procedures with aerosol or high splash potential that may increase the risk of such personnel exposure must be conducted in primary containment equipment, or in devices such as a biological safety cabinet (BSC) or safety centrifuge buckets. Other primary barriers should be used as appropriate such as splash shields, face protection, gowns, and gloves.

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

Biosafety Level 3

Practices, safety equipment, and facility design and construction are applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with a potential for respiratory transmission, and which may cause serious and potentially lethal infection. Mycobacterium tuberculosis, St. Louis encephalitis virus, and Coxiella burnetii are representative of the microorganisms assigned to this level. Primary hazards to personnel working with these agents relate to ingestion, and exposure to infectious aerosols.

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

Biosafety Level 4

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

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

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

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

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Animal Facilities

Four biosafety levels are also described for activities involving infectious disease work with experimental animals. These four combinations of practices, safety equipment, and facilities are designated Animal Biosafety Levels 1, 2, 3, and 4, and provide increasing levels of protection to personnel and the environment. (For further information refer to BMBL, Section IV: Animals in the Laboratory) or FSU's Biological Safety Guidelines for Animal Use (OP-G-3.1.3) located on the web at www.safety.fsu.edu/animalbiosafety.html.

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Laboratory Biosafety Level Criteria

The essential elements of the four biosafety levels for activities involving infectious microorganisms and laboratory animals are summarized in Table 1 of this section. The levels are designated in ascending order, by degree of protection provided to personnel, the environment, and the community.

Biosafety Level 1 (BSL-1)

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

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

Standard Microbiological Practices

• Access to the laboratory is limited or restricted at the discretion of the PI when experiments or work with cultures and specimens are in progress.

• Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.

• Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.

• Mouth pipetting is prohibited; mechanical pipetting devices are used.

• Policies for the safe handling of sharps are instituted. (See Biomedical Waste Program (OP-G-3.1.4) at: www.safety.fsu.edu/biowaste.html).

• All procedures are performed carefully to minimize the creation of splashes or aerosols.

• Work surfaces are decontaminated at least once a day and after any spill of viable material.

• All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leak-proof container and closed for transport from the laboratory. Materials to be decontaminated outside of the immediate laboratory are packaged in accordance with applicable local, state, and federal regulations before removal from the facility

• A biohazard sign can be posted at the entrance to the laboratory whenever infectious agents are present. The sign may include the name of the agent(s) in use and the name and phone number of the investigator.

• An insect and rodent control program is in effect.

Special Practices

• None

Safety Equipment (Primary Barriers)

• Special containment devices or equipment such as a biological safety cabinet are generally not required for manipulations of agents assigned to Biosafety Level 1.

• It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.

• Gloves should be worn if the skin on the hands is broken or if a rash is present. Alternatives to powdered latex gloves should be available.

• Protective eyewear should be worn for procedures in which splashes of microorganisms or other hazardous materials is anticipated.

Laboratory Facilities (Secondary Barriers)

• Laboratories should have doors for access control.

• Each laboratory contains a sink for hand washing.

• The laboratory is designed so that it can be easily cleaned. Carpets and rugs in laboratories are not appropriate.

• Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surface and equipment.

• Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning.

• If the laboratory has windows that open to the exterior, they are fitted with fly screens.

Biosafety Level 2 (BSL-2)

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

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

Standard Microbiological Practices

• Access to the laboratory is limited or restricted at the discretion of the PI when experiments or work with cultures and specimens are in progress.

• Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.

• Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.

• Mouth pipetting is prohibited; mechanical pipetting devices are used.

• Policies for the safe handling of sharps are instituted. (See Biomedical Waste Program (OP-G-3.1.4) at: www.safety.fsu.edu/biowaste.html).

• All procedures are performed carefully to minimize the creation of splashes or aerosols.

• Work surfaces are decontaminated at least once a day and after any spill of viable material.

• All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leak-proof container and closed for transport from the laboratory. Materials to be decontaminated outside of the immediate laboratory are packaged in accordance with applicable local, state, and federal regulations before removal from the facility

• A biohazard sign can be posted at the entrance to the laboratory whenever infectious agents are present. The sign may include the name of the agent(s) in use and the name and phone number of the investigator.

• An insect and rodent control program is in effect.

Special Practices

• Access to the laboratory is limited or restricted by the PI when work with infectious agents is in progress. In general, persons who are at increased risk of acquiring infection, or for whom infection may have serious consequences, are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immunosuppressed may be at increased risk of acquiring infections. The PI has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory or animal room.

• The PI establishes policies and procedures whereby only persons who have been advised of the potential hazards and meet specific entry requirements (e.g., immunization) may enter the laboratory.

• A biohazard sign must be posted on the entrance to the laboratory when etiologic agents are in use. Appropriate information to be posted includes the agent(s) in use, the biosafety level, the required immunizations, the investigator's name and telephone number, any personal protective equipment that must be worn in the laboratory, and any procedures required for exiting the laboratory.

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

• Biosafety procedures are incorporated into standard operating procedures or in a biosafety manual adopted or prepared specifically for the laboratory by the laboratory director. Personnel are advised of special hazards and are required to read and follow instructions on practices and procedures.

• The PI ensures that laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates or additional training as necessary for procedural or policy changes.

• A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels.

  • Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

  • Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.

  • Syringes that re-sheathe the needle, needleless systems, and other safety devices are used when appropriate.

  • Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass are decontaminated before disposal, according to any local, state, or federal regulations.

• Cultures, tissues, specimens of body fluids, or other potentially infectious materials are placed in a container with a cover that prevents leakage during collection, handling, processing, storage, transport, or shipping.

• Laboratory equipment and work surfaces should be decontaminated with an effective disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated according to any local, state, or federal regulations before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, state, or federal regulations, before removal from the facility.

• Spills and accidents that result in overt exposures to infectious materials are immediately reported to the PI and the Biological Safety Office. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.

• Animals not involved in the work being performed are not permitted in the laboratory.

Safety Equipment (Primary Barriers)

• Properly maintained biological safety cabinets, preferably Class II, or other appropriate personal protective equipment or physical containment devices are used whenever:

  • Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals or embryonic eggs.

  • High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed rotor heads or centrifuge safety buckets are used, and if these rotors or safety buckets are opened only in a biological safety cabinet.

• Face protection (goggles, mask, face shield or other splatter guard) is used for anticipated splashes or sprays of infectious or other hazardous materials to the face when the microorganisms must be manipulated outside the BSC.

• Protective laboratory coats, gowns, aprons, or uniforms designated for laboratory use are worn while in the laboratory. This protective clothing is removed and left in the laboratory before leaving for non-laboratory areas (e.g., cafeteria, library, and administrative offices). All protective clothing is either disposed of in the laboratory or laundered by the institution; personnel should never take it home.

• Gloves must be worn when hands may contact potentially infectious materials, contaminated surfaces or equipment. Wearing two pairs of gloves may be appropriate. Gloves are disposed of when overtly contaminated, and removed when work with infectious materials is completed or when the integrity of the glove is compromised. Disposable gloves are not washed, reused, or used for touching "clean" surfaces (keyboards, telephones, etc.), and they should not be worn outside the laboratory. Alternatives to powdered latex gloves should be available. Hands are washed following removal of gloves.

Laboratory Facilities (Secondary Barriers)

• Provide lockable doors for facilities that house select agents (as defined in 42 CFR 72.6).

• Consider locating new laboratories away from public areas.

• Each laboratory contains a sink for hand washing.

• The laboratory is designed so that it can be easily cleaned. Carpets and rugs in laboratories are inappropriate.

• Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.

• Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning. Chairs and other furniture used in laboratory work should be covered with a non-fabric material that can be easily decontaminated.

• Install biological safety cabinets in such a manner that fluctuations of the room supply and exhaust air do not cause the biological safety cabinets to operate outside their parameters for containment. Locate biological safety cabinets away from doors, from windows that can be opened, from heavily traveled laboratory areas, and from other potentially disruptive equipment so as to maintain the biological safety cabinets' air flow parameters for containment.

• An eyewash station is readily available.

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

• There are no specific ventilation requirements. However, planning of new facilities should consider mechanical ventilation systems that provide an inward flow of air without re-circulation to spaces outside of the laboratory. If the laboratory has windows that open to the exterior, they are fitted with fly screens.

Biosafety Level 3 (BSL-3)

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

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

It is recognized, however, that some existing facilities may not have all the facility features recommended for Biosafety Level 3 (i.e., double-door access zone and sealed penetrations). In this circumstance, an acceptable level of safety for the conduct of routine procedures, (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, susceptibility testing, etc.), may be achieved in a Biosafety Level 2 facility, providing 1) the exhaust air from the laboratory room is discharged to the outdoors, 2) the ventilation to the laboratory is balanced to provide directional airflow into the room, 3) access to the laboratory is restricted when work is in progress, and 4) the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 are rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made by the PI and approved by the Biological Safety Committee and verified by the Biological Safety Office.

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

Standard Microbiological Practices

• Access to the laboratory is limited or restricted at the discretion of the PI when experiments or work with cultures and specimens are in progress.

• Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.

• Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.

• Mouth pipetting is prohibited; mechanical pipetting devices are used.

• Policies for the safe handling of sharps are instituted. (See Biomedical Waste Program (OP-G-3.1.4) at: www.safety.fsu.edu/biowaste.html).

• All procedures are performed carefully to minimize the creation of splashes or aerosols.

• Work surfaces are decontaminated at least once a day and after any spill of viable material.

• All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leak-proof container and closed for transport from the laboratory. Materials to be decontaminated outside of the immediate laboratory are packaged in accordance with applicable local, state, and federal regulations before removal from the facility

• A biohazard sign can be posted at the entrance to the laboratory whenever infectious agents are present. The sign may include the name of the agent(s) in use and the name and phone number of the investigator.

• An insect and rodent control program is in effect.

Special Practices

• Laboratory doors are kept closed when experiments are in progress.

• The PI controls access to the laboratory and restrict access to persons whose presence is required for program or support purposes. Persons who are at increased risk of acquiring infection or for whom infection may have serious consequences are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. The PI has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. No minors should be allowed in the laboratory.

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

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

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

• A biosafety manual specific to the laboratory is prepared or adopted by the PI and biosafety precautions are incorporated into standard operating procedures. Personnel are advised of special hazards and are required to read and follow instructions on practices and procedures.

• Laboratory and support personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates or additional training as necessary for procedural changes.

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

• A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels.

  • Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

  • Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.

  • Syringes that re-sheathe the needle, needleless systems, and other safe devices are used when appropriate.

  • Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, and disposed of according to any local, state, or federal regulations.

• All open manipulations involving infectious materials are conducted in biological safety cabinets or other physical containment devices within the containment module. No work in open vessels is conducted on the open bench. Clean up is facilitated by using plastic-backed paper toweling on non-perforated work surfaces within biological safety cabinets.

• Laboratory equipment and work surfaces should be decontaminated routinely with an effective disinfectant, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials.

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

  • Contaminated equipment must be decontaminated before removal from the facility for repair or maintenance or packaging for transport, in accordance with applicable local, state, or federal regulations.

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

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

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

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

Safety Equipment (Primary Barriers)

• Protective laboratory clothing, such as solid-front or wrap-around gowns, scrub suits, or coveralls are worn when in the laboratory. Protective clothing is not worn outside the laboratory. Reusable clothing is decontaminated before being laundered. Clothing is changed when overtly contaminated.

• Gloves must be worn when handling infectious materials, infected animals, and when handling contaminated equipment.

• Frequent changing of gloves accompanied by hand washing is recommended. Disposable gloves are not reused.

• All manipulations of infectious materials, necropsy of infected animals, harvesting of tissues or fluids from infected animals or embryonic eggs, etc., are conducted in a Class II or Class III biological safety cabinet.

• When a procedure or process cannot be conducted within a biological safety cabinet, then appropriate combinations of personal protective equipment (e.g., respirators, face shields) and physical containment devices (e.g., centrifuge safety buckets or sealed rotors) are used.

• Respiratory and face protections are used when in rooms containing infected animals.

Laboratory Facilities (Secondary Barriers)

• The laboratory is separated from areas that are open to unrestricted traffic flow within the building, and access to the laboratory is restricted. Passage through a series of two self-closing doors is the basic requirement for entry into the laboratory from access corridors. Doors are lockable. A clothes-changing room may be included in the passageway.

• Each laboratory room contains a sink for hand washing. The sink is hands-free or automatically operated and is located near the room exit door.

• The interior surfaces of walls, floors, and ceilings of areas where BSL-3 agents are handled are constructed for easy cleaning and decontamination. Seams, if present, must be sealed. Walls, ceilings, and floors should be smooth, impermeable to liquids and resistant to the chemicals and disinfectants normally used in the laboratory. Floors should be monolithic and slip-resistant. Consideration should be given to the use of coved floor coverings. Penetrations in floors, walls, and ceiling surfaces are sealed. Openings such as around ducts and the spaces between doors and frames are capable of being sealed to facilitate decontamination.

• Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and those chemicals used to decontaminate the work surfaces and equipment.

• Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning. Chairs and other furniture used in laboratory work should be covered with a non-fabric material that can be easily decontaminated.

• All windows in the laboratory are closed and sealed.

• A method for decontaminating all laboratory wastes is available in the facility and utilized, preferably within the laboratory (i.e., autoclave, chemical disinfectant, incineration, or other approved decontamination method). Consideration should be given to means of decontaminating equipment. If waste is transported out of the laboratory, it should be properly sealed and not transported in public corridors.

• Biological safety cabinets are required and are located away from doors, from room supply louvers, and from heavily traveled laboratory areas.

• A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air into the laboratory from "clean" areas and toward "contaminated" areas. The exhaust air is not re-circulated to any other area of the building. Filtration and other treatments of the exhaust air are not required, but may be considered based on site requirements, and specific agent manipulations and use conditions. The outside exhaust must be dispersed away from occupied areas and air intakes, or the exhaust must be HEPA-filtered. Laboratory personnel must verify that the direction of the airflow (into the laboratory) is proper. It is recommended that a visual monitoring device that indicates and confirms directional inward airflow be provided at the laboratory entry. Consideration should be given to installing an HVAC control system to prevent sustained positive pressurization of the laboratory. Audible alarms should be considered to notify personnel of HVAC system failure.

• HEPA-filtered exhaust air from a Class II biological safety cabinet can be re-circulated into the laboratory if the cabinet is tested and certified at least annually. When exhaust air from Class II safety cabinets is to be discharged to the outside through the building exhaust air system, the cabinets must be connected in a manner that avoids any interference with the air balance of the cabinets or the building exhaust system (e.g., an air gap between the cabinet exhaust and the exhaust duct). When Class III biological safety cabinets are used they should be directly connected to the exhaust system. If the Class III cabinets are connected to the supply system, it is done in a manner that prevents positive pressurization of the cabinets.

• Continuous flow centrifuges or other equipment that may produce aerosols are contained in devices that exhaust air through HEPA filters before discharge into the laboratory. These HEPA systems are tested at least annually. Alternatively, the exhaust from such equipment may be vented to the outside if it is dispersed away from occupied areas and air intakes.

• Vacuum lines are protected with liquid disinfectant traps and HEPA filters, or their equivalent. Filters must be replaced as needed. An alternative is to use portable vacuum pumps (also properly protected with traps and filters).

• An eyewash station is readily available inside the laboratory.

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

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

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

Biosafety Level 4 (BSL-4)

NOTE: A description of Biosafety Level 4 has being omitted due to the unlikelihood, at this time, of any research involving agents requiring such treatment. If, however, the need arises in the future to implement Biosafety Level 4 practices, an addendum to this manual will be issued at that time incorporating, as a minimum, the recommendations of the CDC and the NIH.

Table 1. Summary of Recommended Biosafety Levels for Infectious Agents NIH/CDC - BMBL 4th Edition

BSL	Agents	Practices	Safety Equipment (Primary Barriers)	Facilities (Secondary Barriers)
1	Not known to consistently cause disease in healthy adults	Standard Microbiological Practices	None required	Open bench top sink required
2	Associated with human disease, hazard = percutaneous injury, ingestion, mucus membrane exposure	BSL-1 practices plus: Limited access Biohazard warning signs "Sharps" precautions Biosafety manual defining any needed waste decontamination or medical surveillance policies	Primary barriers = Class I or II BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials; PPEs: laboratory coats, gloves, face protection as needed	BSL-1 plus: Autoclave available
3	Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences	BSL-2 practices plus: Controlled access Decontamination of all waste Decontamination of lab clothing before laundering Baseline serum	Primary barriers = Class I or II BSCs or other physical containment devices used for all open manipulations of agents; PPEs: protective lab clothing, gloves, respiratory protection as needed	BSL-2 plus: Physical separation from access corridors Self-closing, double door access Exhausted air not recirculated Negative airflow into laboratory
4	Dangerous/exotic agents which pose high risk of life-threatening disease; aerosol-transmitted lab infections; or related agents with unknown risk of transmission	BSL-3 practices plus: Clothing change before entering Shower on exit All material decontaminated on exit from facility	Primary barriers = all procedures conducted on Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure personnel suit	BSL-3 plus: Separate building or isolated zone Dedicated supply/exhaust, vacuum, and decon systems Other requirements outlined in the text

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Personal Protective Equipment

Personal Protective Equipment (PPE) is used to protect the individual from coming in direct contact with biohazardous materials. Furthermore, effective use of PPE minimizes the chances of contaminating the research materials. The following list outlines various standard PPE recommended for laboratory workers. For more information and guidance, please consult the Personal Protective Equipment Program (OP-G-2.4).

Eye Protection

Safety glasses with side shields that meet ANSI Standards must be worn when there is a potential splash of blood, body fluids or chemicals. If this potential for splashes of blood, body fluids, or chemicals is significant, splash goggles should be worn. Wearers of contact lenses should wear appropriate eye covering and face protection devices in a hazardous environment. At the discretion of the PI, chin-length face shields may substitute for safety glasses or splash goggles for some situations.

Face (Nose and Mouth) Protection

Surgical masks should be worn with protective eyewear if splashes, sprays or spatters are anticipated. This type of mask does not protect against aerosols. At the discretion of the PI, chin-length face shields may substitute for surgical masks.

Laboratory Clothing

Laboratory clothing consists of laboratory coats, aprons, scrub suits and gowns. A critical point to consider when selecting laboratory clothing is the length of the sleeve: a long sleeved garment is preferable to minimize contamination of the skin. Additionally, if splashes from liquids are anticipated, the appropriate water-resistant garments should be worn. If the garment is not disposable, it must be capable of withstanding sterilization in the event it becomes contaminated.

Laboratory clothing that has become contaminated with infectious material must be removed from the worker and kept in the laboratory area in such a way that contamination is contained. Additionally, all contaminated clothing shall be either discarded in the lab or laundered by the facility; personnel or students must never launder contaminated clothing at home.

Foot and leg wear

Closed-toe shoes and long pants should be worn at all times.

Gloves

Gloves must be worn when working with or around biohazardous materials. Appropriate glove selection should be based on the hazards involved. Latex gloves have fewer pores and lower in-use failure rates than vinyl gloves. However, latex allergies/sensitivities are real, thus non-latex, synthetic gloves such as nitrile or special washed latex gloves may be substituted.

Note, The National Institute for Occupational Safety & Health (NIOSH) warns workers and employers about dangers of latex exposure and possible allergic reactions such as skin rashes, hives, nasal symptoms, eye symptoms, sinus symptoms, asthma, and, rarely, shock. (If latex gloves are used, try powder-free low-protein latex gloves.)

Additionally, surgical latex gloves may be thicker and may have fewer holes than standard latex gloves.

Two pairs of gloves may be worn in higher risk areas such as HIV culture labs; one pair for general laboratory work, two pairs for work in a BSC. Some HIV research laboratories use three pairs: one pair for BSL-2 laboratory work, another pair for BSL-3 laboratory work, and a third pair for work with cultures in a BSC in the BSL-3 lab. When work in the BSC is completed, the outer pair of gloves is removed.

In order to maximize protection when working with hazardous materials, the glove shall overlap the lower sleeve and cuff of the lab coat; a long sleeved glove or disposable arm-shield may be worn. Similarly, temperature resistant gloves shall be worn when handling hot material and dry ice.

Finally, gloves should be disposed of immediately if they have been contaminated. Never wear gloves outside the laboratory, nor should anyone attempt to reuse or wash disposable gloves. Always consult with the PI or laboratory supervisor for assistance in selecting the appropriate type of glove.

Respirators

Respirators provide additional protection against infectious aerosols. Workers who are using respirators must be medically monitored and must be fitted to the individual and tested before being used in a potentially hazardous situation.

NOTE: Surgical masks are not respirators and are not certified as such; they do not protect the user adequately from most inhalation exposures. Please contact the Industrial Hygiene/Asbestos Control Office within EH&S for details and information prior to purchasing any respiratory protection equipment.

Various kinds of respirators fall within two major groups: Air-purifying respirators and Atmosphere-supplying respirators.

• Air Purifying Respirators

  Air-purifying respirators have filters, cartridges, or canisters that remove contaminants from the air by passing the ambient air through the air-purifying element before it reaches the user. Within this group exist three kinds of air-purifying respirators:

  • Particulate respirators:

    Particulate respirators capture particles in the air such as dusts, mists and fumes.

  • Gas and vapor respirators:

    Gas and vapor respirators are normally used when there are only gases and vapors in the air.

  • Combination respirators:

    Combination respirators are normally used in atmospheres that contain hazards of both particulates and gases or vapors.

• Atmosphere-supplying respirators

  Atmosphere-supplying respirators, on the other hand, supply air directly from a clean source rather than from the potentially contaminated air surrounding the user. Three kinds of atmosphere-supplying respirators have been developed to meet varying needs:

  • Air-supplied respirators:

    Air-supplied respirators that make use of a hose to deliver clean, safe air from a stationary source of compressed air.

  • Combination respirators:

    Combination respirators that have an auxiliary self-contained air supply that can be used if the primary supply fails.

  • Self-contained breathing respirators:

    Self-contained breathing respirators that consist of a wearable, clean-air supply pack.

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Laboratory Safety Equipment

The guidelines discussed in this section give rise to specific procedures that allow the laboratory workers to effectively use equipment for which the sole purpose is protection of the laboratory worker. In addition to these procedures, Material Safety Data Sheets (MSDS's) are available in the laboratory and should be reviewed by each individual to familiarize students and employees with the chemicals used in the laboratory. The Chemical Safety Office, within EH&S, maintains copies of most MSDS's for chemicals on FSU's campus. Additional links to other sources of MSDS's can be found on the Chemical Safety Office website at www.safety.fsu.edu/chem.html, under "Material Safety Data Sheets" link.

Biological Safety Cabinets (BSC's)

BSC's are designed to provide personnel, environmental, and product protection when appropriate practices and procedures are followed. They provide only one component of an overall biosafety program that requires consistent use of good microbiological practices. There are three kinds of biological safety cabinets: Class I, Class II, and Class III have been developed to meet varying research and clinical needs.

Class Descriptions

• Cabinet Types Class I

  This is an open fronted, negative pressure, ventilated cabinet with a minimum inward face velocity at the work opening of 75 feet per minute. A high efficiency particulate air (HEPA) filter filters the exhaust air from the cabinet. It is designed for general microbiological research with low and moderate risk agents, and is useful for containment of mixers, blenders, and other equipment in the laboratory. Personnel protection is provided by the inward flow of unfiltered air from the laboratory. Therefore, since this inward airflow can carry microbial contaminants into the cabinet, no product protection is achieved.

• Cabinet Types Class II

  Class II cabinets provide the microbe-free work environment necessary for cell culture propagation, and also may be used for the formulation of nonvolatile antineoplastic or chemotherapeutic drugs.

  This class is broken down even further into two more types: A and B. All Class II cabinets are designed for work involving microorganisms assigned to biosafety level 1, 2, and 3.

  • Type A

    This type of cabinet is usually unducted and thus suitable for work with microbiological research in the absence of volatile or toxic chemicals and radionuclides. An internal blower draws sufficient room air through the front grille to maintain a minimum calculated or measured average inflow velocity of at least 75 linear feet per minute. Approximately 30% of the air in the cabinet is exhausted into the lab and 70% is recirculated into the work zone. There are, however, "thimble", or canopy hood designs that allow for the exhausting of air out of the building. This design preserves the balance of the cabinet exhaust system, and does not disturb the internal airflow. An unducted cabinet is not to be used for work involving volatile or toxic chemicals. The buildup of chemical vapors in the cabinet (by the recirculated air) and in the laboratory (from exhaust air) could create health and safety hazards.

  • Type B

    This type is hard-ducted to allow for the exhausting of air out of the building and with a minimum calculated or measured inflow face velocity of 100 linear feet per minute. There are three types: B1, B2, and B3. These cabinets are differentiated by the amount of air that is discharged from the building: a B1 discharges 70% of the air while a B2 discharges 100%. The B3 features safety mechanisms to contain leakage of contaminated air within the cabinet.

Cabinet Types Class III

This cabinet type is a totally enclosed ventilated cabinet of gas-tight construction. Operations within the Class III cabinets are conducted through attached heavy-duty rubber gloves. Both supply and exhaust airs are HEPA filtered. Exhaust air must pass through two HEPA filters, or a HEPA filter and an air incinerator, before discharge to the outdoors. Airflow is maintained by a dedicated independent exhaust system exterior to the cabinet, which keeps the cabinet under negative pressure (usually about 0.5 inches of water pressure).

Proper Use of BSC's

Always follow the manufacturer's instructions when using a BSC to maximize the protection, the material on the work area, and the environment. Individuals using BSC's should be properly trained and safety instructions should be posted on or near the location of the unit. BSC'S SHOULD NOT BE USED AS STORAGE! The following are general guidelines for proper operation of a BSC.

• Startup
  • If necessary, turn off ultra violet sterilizer as soon as you enter the room.
  • Turn on all blowers and cabinet illumination lights.
  • Allow five minutes of operation to purge system; check flow alarm system and audiovisual alarm function (depending on system).
  • Decontaminate readily accessible interior surfaces with disinfectant appropriate for the agents or suspected agents present before use.
• Shut Down
  • Decontaminate and remove all unnecessary items from interior work area. Do not use cabinet as a storage area.
  • Decontaminate readily accessible interior surfaces with a disinfectant appropriate for the agents or suspected agents present after use.
  • Turn on ultraviolet sterilizer (if provided).
  • Allow five minutes of operation to purge the system.
  • Turn off the cabinet blower.

Moving and Installing BSC's

The operational integrity of a new BSC must be validated by certification before it is put into service or after a cabinet has been repaired or relocated. Relocating a BSC may break the HEPA filter seals or otherwise damage the filters or the cabinet. BSC's must be decontaminated prior to being moved. Certified professionals usually handle the decontamination of the BSC's motor and filters.

Certification

BSC's used on campus for handling of biohazards must be certified annually. As a service, the Biological Safety Office will pay for the annual certification of the BSC. For additional information, contact the Biological Safety Office at 644-5374 or 644-9117.

Fumehoods

Fumehoods are used to protect the individual from exposure to chemicals and gases. Fumehoods are not equipped with HEPA filters. Biohazardous materials must not be used in fumehoods.

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Laboratory Equipment

Centrifuges

Follow the manufacturer's instructions when using a centrifuge to minimize the risk of mechanical failure. Individuals using centrifuges shall be properly trained and safety instructions should be posted on or near the location of the unit.

Note that aerosols are created when filling centrifuge tubes, removing plugs, removing caps after use, and virtually anything related to the centrifugation.

Aerosol Creating Equipment

Blenders, ultrasonic disrupters, grinders, and pipettes can be a great source of aerosol production. These types of equipment should be used in a BSC when working with biohazardous materials. All devices should be decontaminated promptly after use.

Autoclaves

Autoclaving shall be used to render biohazardous waste noninfectious before disposal. Refer to the Biomedical Waste Program (OP-G-3.1.4) at: www.safety.fsu.edu/biowaste.html for the proper biohazardous/biomedical waste disposal guidelines. Note that a licensed biomedical waste broker is the only one who can remove biomedical waste and certain biohazardous wastes from campus. Call the Biosafety Office at 644-5374 or 644-9117 or consult the biomedical waste program website for information regarding disposal of biohazardous waste.

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Signs and Posting

For Biosafety Level 2 through 4 a completely filled out Biohazard sign must be posted prominently at entrances to laboratories working with biohazardous materials and at entrances to a tissue culture laboratory.

These signs display the international biohazard symbol and the legend "BIOHAZARD" or "BIOHAZARDOUS MATERIALS", specify the name of the biohazardous material in use, the location, the PI, and contact information.

These signs are available through the Biological Safety Office. The Biological Safety Office can be reached at 644-5374 or 644-9117.

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Biohazardous/Biomedical Waste

Waste disposal requires well-defined procedures to prevent exposure to pathogens. Improper disposal of biohazardous waste puts everyone at risk as well as jeopardizes the University's waste permits.

In order to prevent unwanted exposure, principal investigators, supervisors, technicians and students must be familiar with current waste disposal procedures for biohazardous materials as well as for chemical and radioactive materials.

For a complete detail of the biohazardous/biomedical waste consult the Biomedical Waste Program (OP-G-3.1.4) at www.safety.fsu.edu/biopic.html or contact the Biological Safety Office at 644-5374 or 644-9117.

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