Office of Environmental Health and Safety

Microbiology laboratories are special, often unique, work environments that may pose identifiable infectious disease risks to persons in or near them. Infections have been contracted in the laboratory throughout the history of microbiology. Published reports around the turn of the century described laboratory-associated cases of typhoid, cholera, glanders, brucellosis, and tetanus.¹⁹² In 1941 Meyer and Eddie¹²⁵ published a survey of 74 laboratory-associated brucellosis infections that had occurred in the United States, and concluded that the "handling of cultures or specimens or the inhalation of dust containing Brucella organisms is eminently dangerous to laboratory workers." A number of cases were attributed to carelessness or poor technique in the handling of infectious materials.

In 1949, Sulkin and Pike¹⁷⁹ published the first in a series of surveys of laboratory-associated infections summarizing 222 viral infections -- 21 of which were fatal. In at least a third of the cases the probable source of infection was considered to be associated with the handling of infected animals and tissues. Known accidents were recorded in 27 (12%) of the reported cases.

In 1951, Sulkin and Pike¹⁸⁰ published the second of a series of summaries of laboratory-associated infections based on a questionnaire sent to 5,000 laboratories. Only one-third of the 1,342 cases cited had been reported in the literature. Brucellosis outnumbered all other reported laboratory-acquired infections and, together with tuberculosis, tularemia, typhoid, and streptococcal infection, accounted for 72% of all bacterial infections and for 31% of infections caused by all agents. The overall case fatality rate was 3%. Only 16% of all infections reported were associated with a documented accident. The majority of these were related to mouth pipetting and the use of needle and syringe.

This survey was updated in 1965,¹⁵⁴ adding 641 new or previously unreported cases, and again in 1976,¹⁵¹ summarizing a cumulative total of 3,921 cases. Brucellosis, typhoid, tularemia, tuberculosis, hepatitis, and Venezuelan equine encephalitis were the most commonly reported. Fewer than 20% of all cases were associated with a known accident. Exposure to infectious aerosols was considered to be a plausible but unconfirmed source of infection for the more than 80% of the reported cases in which the infected person had "worked with the agent."

In 1967 Hanson et al.⁸⁶ reported 428 overt laboratory-associated infections with arboviruses. In some instances the ability of a given arbovirus to produce human disease was first confirmed as the result of unintentional infection of laboratory personnel. Exposure to infectious aerosols was considered the most common source of infection.

In 1974 Skinhoj¹⁷⁰ published the results of a survey which showed that personnel in Danish clinical chemistry laboratories had a reported incidence of hepatitis (2.3 cases per year per 1,000 employees), seven times higher than that of the general population. Similarly, a 1976 survey by Harrington and Shannon⁸⁸ indicated that medical laboratory workers in England had "a five times increased risk of acquiring tuberculosis compared with the general population". Hepatitis B and shigellosis were also shown to be continuing occupational risks and, along with tuberculosis, were the three most commonly reported occupation-associated infections in Britain.

Although these reports suggest that laboratory personnel were at increased risk of being infected by the agents they handle, actual rates of infection are typically not available. However, the studies of Harrington and Shannon⁸⁸ and of Skinhoj¹⁷⁰ indicate that laboratory personnel had higher rates of tuberculosis, shigellosis, and hepatitis B than does the general population.

In contrast to the documented occurrence of laboratory-acquired infections in laboratory personnel, laboratories working with infectious agents have not been shown to represent a threat to the community. For example, although 109 laboratory-associated infections were recorded at the Centers for Disease Control and Prevention from 1947-1973,¹⁵⁹ no secondary cases were reported in family members or community contacts. The National Animal Disease Center reported a similar experience,¹⁸¹ with no secondary cases occurring in laboratory and non-laboratory contacts of 18 laboratory-associated cases occurring from 1960-1975. A secondary case of Marburg disease in the wife of a primary case was presumed to have been transmitted sexually two months after dismissal from the hospital.¹¹⁷ Three secondary cases of smallpox were reported in two laboratory-associated outbreaks in England in 1973¹⁵⁷ and 1978.²⁰² There were earlier reports of six cases of Q fever in a commercial laundry cleaning linens and uniforms from a laboratory working with the agent,¹⁴⁰ one case of Q fever in a visitor to a laboratory,¹⁴⁰ and two cases of Q fever in household contacts of a rickettsiologist.¹⁰ One case of Monkey B virus transmission from an infected animal care giver to his wife has been reported, apparently due to contact of the virus with broken skin.⁹² These cases are representative of the sporadic nature and infrequency of community infections in laboratory personnel working with infectious agents.

In his 1979 review,¹⁵³ Pike concluded "the knowledge, the techniques, and the equipment to prevent most laboratory infections are available". In the United States, however, no single code of practice, standards, guidelines, or other publication provided detailed descriptions of techniques, equipment, and other considerations or recommendations for the broad scope of laboratory activities conducted with a variety of indigenous and exotic infectious agents. The booklet, Classification of Etiologic Agents on the Basis of Hazard,²³ served as a general reference for some laboratory activities utilizing infectious agents. This booklet, and the concept of categorizing infectious agents and laboratory activities into four classes or levels, served as a basic format for earlier editions of Biosafety in Microbiological and Biomedical Laboratories (BMBL). This third edition of the BMBL continues to specifically describe combinations of microbiological practices, laboratory facilities, and safety equipment, and recommend their use in four categories or biosafety levels of laboratory operation with selected agents infectious to humans.

The descriptions of Biosafety Levels 1-4 parallel those in the NIH Guidelines for Research Involving Recombinant DNA,^71,72,139 and are consistent with the general criteria originally used in assigning agents to Classes 1-4 in Classification of Etiologic Agents on the Basis of Hazards.²³ Four biosafety levels are also described for infectious disease activities utilizing small laboratory animals. Recommendations for biosafety levels for specific agents are made on the basis of the potential hazard of the agent and of the laboratory function or activity.

Since the early 1980's, laboratories have applied these fundamental guidelines in activities associated with manipulations involving the human immunodeficiency virus (HIV). Even before HIV was identified as the causative agent of acquired immunodeficiency syndrome (AIDS), the principles for manipulating a bloodborne pathogen were suitable for safe laboratory work. Guidelines were also promulgated for health care workers under the rubric of Universal Precautions.⁴³ Indeed, Universal Precautions and this publication have become the basis for safe handling of blood and body fluids, as described in the recent OSHA publication Bloodborne Pathogen Standard.¹⁸⁷

In the late 1980's, considerable public concern was expressed about medical wastes, which led to the promulgation of the Medical Waste Tracking Act of 1988.¹⁸⁶ The principles established in the earlier volumes of the BMBL for handling potentially infectious wastes as an occupational hazard were reinforced by the National Research Council's Biosafety in the Laboratory: Prudent Practices for the Handling and Disposal of Infectious Materials.¹²

As this edition goes to press, there is growing concern about safe practices, procedures and facilities integral to the issues associated with the re-emergence of tuberculosis and worker safety in laboratory and health care settings. The underlying principles of the BMBL are applicable in the control of this airborne pathogen, including multi-drug resistant strains of M. tuberculosis.^47,54

Experience has demonstrated the prudence of the Biosafety Level 1-4 practices, procedures and facilities described for manipulations of etiologic agents in laboratory settings and animal facilities. Although no national reporting system exists for reporting laboratory-associated infections, anecdotal information suggests that strict adherence to these guidelines does contribute to a healthier and safer work environment for laboratorians, their co-workers and the surrounding community. The guidelines presented here can be customized for each individual laboratory, and can be used in conjunction with other available scientific information on risk assessment, to further minimize the potential for laboratory-associated infections.

SECTION II

Principles of Biosafety

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

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

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

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

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

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

Safety Equipment (Primary 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 Appendix A. Open-fronted Class I and Class II biological safety cabinets are primary barriers which offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques. The Class II 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 cup, an enclosed container designed to prevent aerosols from being released during centrifugation. To minimize this hazard, containment controls such as BSCs or centrifuge cups must be used for handling infectious agents that can be transmitted through the aerosol route of exposure.

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

Facility Design (Secondary Barriers). The design of the facility is important in providing a barrier to protect persons working inside and outside of the laboratory within the facility, and to protect persons or animals in the community from infectious agents 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 be direct contact with the agents, or inadvertent contact exposures through contaminated work environments. Secondary barriers in these laboratories may include separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and handwashing facilities.

As the risk for aerosol transmission increases, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Such design features could include specialized ventilation systems to assure directional air flow, air treatment systems to decontaminate or remove agents from exhaust air, controlled access zones, airlocks as laboratory entrances, or separate buildings or modules for isolation of the laboratory. Design engineers for laboratories may refer to specific ventilation 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).²

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

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

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

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

Biosafety Level 2 practices, equipment, and facilities are applicable to clinical, diagnostic, teaching and other facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Hepatitis B virus, 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, or tissues where the presence of an infectious agent may be unknown. (Laboratory personnel working with human-derived materials should refer to the Bloodborne Pathogen Standard¹⁸⁷ 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 precaution with contaminated needles or sharp instruments must be emphasized. Even though organisms routinely manipulated at BSL2 are not known to be transmissible by the aerosol route, procedures with aerosol or high splash potential that may increase the risk of such personnel exposure must be conducted in primary containment equipment, or devices such as a BSC or safety centrifuge cups. Other primary barriers should be used as appropriate, such as splash shields, face protection, gowns, and gloves.

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

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

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

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

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

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

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

Animal 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 provide increasing levels of protection to personnel and the environment.

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

Except in extraordinary circumstances (e.g., suspected hemorrhagic fever), the initial processing of clinical specimens and identification of isolates can be done safely at Biosafety Level 2, the recommended level for work with bloodborne pathogens such as hepatitis B virus and HIV. The containment elements described in Biosafety Level 2 are consistent with the Occupational Exposure to Bloodborne Pathogens Standard¹⁸⁷ from the Occupational Safety and Health Administration (OSHA), that requires the use of specific precautions with all clinical specimens of blood or other potentially infectious material (Universal Precautions).⁴³ Additionally, other recommendations specific for clinical laboratories may be obtained from the National Committee for Clinical Laboratory Standards.¹³⁴

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

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

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

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

TABLE 1 & 2
Summary of Recommended Biosafety Levels for Infectious Agents

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

SECTION III

Laboratory Biosafety Level Criteria

The essential elements of the four biosafety levels for activities involving infectious microorganisms and laboratory animals are summarized in Tables 1 and 2 (see pages 14 and 15). The levels are designated in ascending order, by degree of protection provided to personnel, the environment, and the community.

Biosafety Level 1

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

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

A. Standard Microbiological Practices

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

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

3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas where there is reasonable likelihood of exposure to potentially infectious materials. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.

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

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

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

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

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

B. Special Practices: None

C. Safety Equipment (Primary Barriers)

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

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

3. Gloves should be worn if the skin on the hands is broken or if a rash exists.

4. Protective eyewear should be worn for anticipated

splashes of microorganisms or other hazardous materials to the face.

D. Laboratory Facilities (Secondary Barriers)

1. Each laboratory contains a sink for handwashing.

2. The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not appropriate, and should not be used because proper decontamination following a spill extremely difficult to achieve.

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

4. Laboratory furniture is sturdy. 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 that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists, (2) access to the laboratory is limited when work is being conducted, (3) extreme precautions are taken with contaminated sharp items, and (4) certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.

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

A. Standard Microbiological Practices

1. Access to the laboratory is limited or restricted at the discretion of the laboratory 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, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.

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

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

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

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

B. Special Practices

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

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

3. When the infectious agent(s) in use in the laboratory require special provisions for entry (e.g., immunization), a hazard warning sign incorporating the universal biohazard symbol is posted on the access door to the laboratory work area. The hazard warning sign identifies the infectious agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates the special requirement(s) for entering the laboratory.

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

5. When appropriate, considering the agent(s) handled, baseline serum samples for laboratory and other at-risk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the facility.

6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.

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

8. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

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

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

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

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

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

11. Spills and accidents which result in overt exposures to infectious materials are immediately reported to the laboratory director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.

12. Animals not involved in the work being performed are not permitted in the lab.

C. Safety Equipment (Primary Barriers)

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

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

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

2. Face protection (goggles, mask, faceshield or other splatter guards) 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.

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

4. Gloves are worn when handling infected animals and when hands may contact infectious materials, contaminated surfaces or equipment. Wearing two pairs of gloves may be appropriate; if a spill or splatter occurs, the hand will be protected after the contaminated glove is removed. Gloves are disposed of when contaminated, removed when work with infectious materials is completed, and are not worn outside the laboratory. Disposable gloves are not washed or reused.

D. Laboratory Facilities (Secondary Barriers)

1. Each laboratory contains a sink for handwashing.

2. The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not appropriate, and should not be used because proper decontamination following a spill is extremely difficult to achieve.

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

4. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.

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

6. A method for decontamination of infectious or regulated laboratory wastes is available (e.g., autoclave, chemical disinfection, incinerator, or other approved decontamination system).

7. An eyewash facility is readily available.

Biosafety Level 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 are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features.

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

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

A. Standard Microbiological Practices

1. Access to the laboratory is limited or restricted at the discretion of the laboratory 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, smoking, handling contact lenses, and applying cosmetics are not permitted in the laboratory. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.

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

5. All procedures are performed carefully to minimize the creation of aerosols.

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

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

B. Special Practices

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

2. The laboratory director controls access to the laboratory and restricts access to persons whose presence is required for program or support purposes. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. Persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. The director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.

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

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

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

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

7. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.

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

9. The laboratory director is responsible for insuring that, 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 laboratory director or other competent scientist proficient in safe microbiological practices and techniques.

10. 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.

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

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

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

12. Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials. Contaminated equipment should also be decontaminated before it is sent for repair or maintenance or package for transport in accordance with applicable local, state, or federal regulations, before removal from the facility. Plastic-backed paper toweling used on non-perforated work surfaces within biological safety cabinets facilitates clean-up.

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

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

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

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

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

C. Safety Equipment (Primary Barriers)

1. Properly maintained biological safety cabinets are used (Class II or III - see Appendix A) for all manipulation of infectious materials.

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

3. This equipment must be used for manipulations of cultures and of those clinical or environmental materials which may be a source of infectious aerosols; the aerosol challenge of experimental animals; harvesting of tissues or fluids from infected animals and embryonated eggs, and necropsy of infected animals.

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

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

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

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

D. Laboratory Facilities (Secondary Barriers)

1. The laboratory is separated from areas which are open to unrestricted traffic flow within the building. Passage through two sets of self-closing doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. A clothes change room (shower optional) may be included in the passage way.

2. Each laboratory contains a sink for handwashing. 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 so that they can be easily cleaned. Penetrations in these surfaces are sealed or capable of being sealed to facilitate decontamination.

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.

6. Windows in the laboratory are closed and sealed.

7. A method for decontaminating all laboratory wastes is available, preferably within the laboratory (i.e, autoclave, chemical disinfection, incineration, or other approved decontamination method).

8. A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air from "clean" areas into the laboratory toward "contaminated" areas. The exhaust air is not recirculated to any other area of the building, and is discharged to the outside with filtration and other treatment optional. The outside exhaust must be dispersed away from occupied areas and air intakes. Laboratory personnel must verify that the direction of the airflow (into the laboratory) is proper.

9. The High Efficiency Particulate Air (HEPA)-filtered exhaust air from Class II or Class III biological safety cabinets is discharged directly to the outside or through the building exhaust system. If the HEPA-filtered exhaust air from Class II or III biological safety cabinets is to be discharged to the outside through the building exhaust air system, it is connected to this system in a manner (e.g., thimble unit connection)¹³⁶ that avoids any interference with the air balance of the cabinets or building exhaust system. Exhaust air from Class II biological safety cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every twelve months.

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

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

12. An eyewash facility is readily available.

Biosafety Level 4

Biosafety Level 4 is required for work with dangerous and exotic agents which pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease. Agents with a close or identical antigenic relationship to Biosafety Level 4 agents are handled at this level until sufficient data are obtained either to confirm continued work at this level, or to work with them at a lower level. Members of the laboratory staff have specific and thorough training in handling extremely hazardous infectious agents; and they understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics. They are supervised by competent scientists who are trained and experienced in working with these agents. Access to the laboratory is strictly controlled by the laboratory director. The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. A specific facility operations manual is prepared or adopted.

Within work areas of the facility, all activities are confined to Class III biological safety cabinets, or Class II biological safety cabinets used with one-piece positive pressure personnel suits ventilated by a life support system. The Biosafety Level 4 laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment.

The following standard and special safety practices equipment, and facilities apply to agents assigned to Biosafety Level 4:

A. Standard Microbiological Practices

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

2. Persons wash their hands after handling infectious materials and animals; they take a decontaminating shower when they leave the laboratory.

4. Mouth pipetting is prohibited; only mechanical pipetting devices are used.

5. All procedures are performed carefully to minimize the creation of aerosols.

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

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

B. Special Practices

1. Only persons whose presence in the facility or individual laboratory rooms is required for program or support purposes are authorized to enter. Persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. Therefore, persons who may be at increased risk of acquiring infection or for whom infection may be unusually hazardous, such as children or pregnant women, are not allowed in the laboratory or animal rooms.

The supervisor has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. Access to the facility is limited by means of secure, locked doors; accessibility is managed by the laboratory director, biohazards control officer, or other person responsible for the physical security of the facility. Before entering, persons are advised of the potential biohazards and instructed as to appropriate safeguards for insuring their safety. Authorized persons comply with the instructions and all other applicable entry and exit procedures. A logbook, signed by all personnel, indicates the date and time of each entry and exit. Practical and effective protocols for emergency situations are established.

2. When infectious materials or infected animals are present in the laboratory or animal rooms, hazard warning signs, incorporating the universal biohazard symbol, are posted on all access doors. The sign identifies the agent, lists the name of the laboratory director or other responsible person(s), and indicates any special requirements for entering the area (e.g., the need for immunizations or respirators).

3. The laboratory director is responsible for insuring that, before working with organisms at Biosafety Level 4, all personnel demonstrate a high proficiency in standard microbiological practices and techniques, and in the special 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 laboratory director or other competent scientist proficient in these unique safe microbiological practices and techniques.

4. Laboratory personnel receive available immunizations for the agents handled or potentially present in the laboratory.

5. Baseline serum samples for all laboratory and other atrisk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory. The decision to establish a serologic surveillance program takes into account the availability of methods for the assessment of antibody to the agent(s) of concern. The program provides for the testing of serum samples at each collection interval and the communication of results to the participants.

6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.

8. Personnel enter and leave the facility only through the clothing change and shower rooms, and shower each time they leave the facility. Personnel use the airlocks to enter or leave the laboratory only in an emergency.

9. Personal clothing is removed in the outer clothing change room and kept there. Complete laboratory clothing, including under garments, pants and shirts or jumpsuits, shoes, and gloves, is provided and used by all personnel entering the facility. When leaving the laboratory and before proceeding into the shower area, personnel remove their laboratory clothing in the inner change room. Soiled clothing is autoclaved before laundering.

10. Supplies and materials needed in the facility are brought in by way of the double-doored autoclave, fumigation chamber, or airlock, which is appropriately decontaminated between each use. After securing the outer doors, personnel within the facility retrieve the materials by opening the interior doors of the autoclave, fumigation chamber, or airlock. These doors are secured after materials are brought into the facility.

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

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

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

12. Biological materials to be removed from the Class III cabinet or from the Biosafety Level 4 laboratory in a viable or intact state are transferred to a nonbreakable, sealed primary container and then enclosed in a nonbreakable, sealed secondary container. This is removed from the facility through a disinfectant dunk tank, fumigation chamber, or an airlock designed for this purpose.

13. No materials, except for biological materials that are to remain in a viable or intact state, are removed from the Biosafety Level 4 laboratory unless they have been autoclaved or decontaminated before they leave the facility. Equipment or material which might be damaged by high temperatures or steam may be decontaminated by gaseous or vapor methods in an airlock or chamber designed for this purpose.

14. Laboratory equipment is decontaminated routinely after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials. Contaminated equipment is also decontaminated before it is sent for repair or maintenance.

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

16. A system is set up for reporting laboratory accidents and exposures and employee absenteeism, and for the medical surveillance of potential laboratory-associated illnesses. Written records are prepared and maintained. An essential adjunct to such a reporting-surveillance system is the availability of a facility for the quarantine, isolation, and medical care of personnel with potential or known laboratory-associated illnesses.

17. Materials (e.g., plants, animals, and clothing) not related to the experiment being conducted are not permitted in the facility.

C. Safety Equipment (Primary Barriers)

1. All procedures within the facility with agents assigned to Biosafety Level 4 are conducted in the Class III biological safety cabinet or in Class II biological safety cabinets used in conjunction with one-piece positive pressure personnel suits ventilated by a life support system.

Activities with viral agents that require Biosafety Level 4 secondary containment capabilities can be conducted within Class II biological safety cabinets within the facility, without the one-piece positive pressure personnel suit being used if (a) the facility has been decontaminated, (b) no work is being conducted in the facility with other agents assigned to Biosafety Level 4, (c) all personnel are immunized against the specific agent being manipulated and demonstrate protective antibody levels, and (d) all other standard and special practices are followed.

2. All personnel entering the facility will don complete laboratory clothing, including undergarments, pants, and shirts or jumpsuits, shoes, and gloves. All such personal protective equipment is removed in the change room before showering and leaving the laboratory.

D. Laboratory Facility (Secondary Barriers)

1. The Biosafety Level 4 facility consists of either a separate building or a clearly demarcated and isolated zone within a building. Outer and inner change rooms separated by a shower are provided for personnel entering and leaving the facility. A double-doored autoclave, fumigation chamber, or ventilated airlock is provided for passage of those materials, supplies, or equipment which are not brought into the facility through the change room.

2. Walls, floors, and ceilings of the facility are constructed to form a sealed internal shell which facilitates fumigation and is animal and insect proof. The internal surfaces of this shell are resistant to liquids and chemicals, thus facilitating cleaning and decontamination of the area. All penetrations in these structures and surfaces are sealed. Any drains in the floors contain traps filled with a chemical disinfectant of demonstrated efficacy against the target agent, and they are connected directly to the liquid waste decontamination system. Sewer vents and other ventilation lines contain HEPA filters.

3. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize the horizontal surface area on which dust can settle.

4. Bench tops have seamless surfaces which are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

5. Laboratory furniture is of simple and sturdy construction, and spaces between benches, cabinets, and equipment are accessible for cleaning.

6. A foot, elbow, or automatically operated handwashing sink is provided near the door of each laboratory room in the facility.

7. If there is a central vacuum system, it does not serve areas outside the facility. In-line HEPA filters are placed as near as practicable to each use point or service cock. Filters are installed to permit in-place decontamination and replacement. Other liquid and gas services to the facility are protected by devices that prevent backflow.

8. If water fountains are provided, they are foot operated and are located in the facility corridors outside the laboratory. The water service to the fountain is not connected to the backflow-protected distribution system supplying water to the laboratory areas.

9. Access doors to the laboratory are self-closing and lockable.

10. Any windows are breakage resistant.

11. A double-doored autoclave is provided for decontaminating materials passing out of the facility. The autoclave door which opens to the area external to the facility is sealed to the outer wall, and automatically controlled so that the outside door can only be opened after the autoclave "sterilization" cycle has been completed.

12. A pass-through dunk tank, fumigation chamber, or an equivalent decontamination method is provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the facility.

13. Liquid effluents from laboratory sinks, biological safety cabinets, floor drains (if used), and autoclave chambers are decontaminated by heat treatment before being discharged to the sanitary sewer. Effluents from showers and toilets may be discharged to the sanitary sewer without treatment. The process used for decontamination of liquid wastes must be validated physically and biologically by use of a constant recording temperature sensor in conjunction with an indicator microorganism having a defined heat susceptibility profile.

14. A dedicated non-recirculating ventilation system is provided. The supply and exhaust components of the system are balanced to assure directional airflow from the area of least hazard to the area(s) of greatest potential hazard. The differential pressure/directional airflow between adjacent areas is monitored and alarmed to indicate malfunction of the system. The airflow in the supply and exhaust components is monitored and the components interlocked to assure inward (or zero) airflow is maintained.

15. The general room exhaust air from a facility in which the work is conducted in a Class III cabinet system is treated by a passage through a HEPA filter(s) prior to discharge to the outside. The air is discharged away from occupied spaces and air intakes. The HEPA filter(s) are located as near as practicable to the source in order to minimize the length of potentially contaminated ductwork. The HEPA filter housings are designed to allow for in situ decontamination of the filter prior to removal, or removal of the filter in a sealed gas-tight primary container for subsequent decontamination and/or destruction by incineration. The design of the HEPA filter housing should facilitate validation of the filter installation. The use of pre-certified HEPA filters can be an advantage. The service-life of the exhaust HEPA filters can be extended through adequate filtration of the supply air.

16. A specially designed suit area may be provided in the facility to provide personnel protection equivalent to that provided by Class III cabinets. Personnel who enter this area wear a one-piece positive pressure suit that is ventilated by a life support system. The life support system includes alarms and emergency backup breathing air tanks. Entry to this area is through an airlock fitted with airtight doors. A chemical shower is provided to decontaminate the surface of the suit before the worker leaves the area. The exhaust air from the suit area is filtered by two sets of HEPA filters installed in series. A duplicate filtration unit, exhaust fan, and an automatically starting emergency power source are provided. The air pressure within the suit area is lower than that of any adjacent area. Emergency lighting and communication systems are provided. All penetrations into the internal shell of the suit area are sealed. A double-doored autoclave is provided for decontaminating waste materials to be removed from the suit area.

17. The treated exhaust air from Class II biological safety cabinets, located in a facility in which workers wear a positive pressure suit, may be discharged into the animal room environment or to the outside through the facility air exhaust system. The biological safety cabinets are tested and certified at 12-month intervals. The air exhausted from Class III biological safety cabinets is passaged through two HEPA filter systems (in series) prior to discharge to the outside. If the treated exhaust is discharged to the outside through the facility exhaust system, it is connected to this system in a manner that avoids any interference with the air balance of the cabinets or the facility exhaust system.

TABLE 3
Comparison of Biological Safety Cabinets

SECTION IV

Vertebrate Animal Biosafety Level Criteria

If experimental animals are used, institutional management must provide facilities and staff and establish practices which reasonably assure appropriate levels of environmental quality, safety, and care. Laboratory animal facilities in many ways are extensions of the laboratory. As a general principle, the biosafety level (facilities, practices, and operational requirements) recommended for working with infectious agents in vivo and in vitro are comparable. It is well to remember, however, that the animal room is not the laboratory, and can present some unique problems. In the laboratory, hazardous conditions are caused by personnel or the equipment that is being used. In the animal room the activities of the animals themselves can introduce new hazards. Animals may produce aerosols, and they may also infect and traumatize animal handlers by biting and scratching.

These recommendations presuppose that laboratory animal facilities, operational practices, and quality of animal care meet applicable standards and regulations and that appropriate species have been selected for animal experiments (e.g., Guide for the Care and Use of Laboratory Animals, HEW Publication No. (NIH) 86-23, Rev. 1985, and Laboratory Animal Welfare Regulations - 9 CFR, Subchapter A, Parts 1, 2 and 3).

Ideally, facilities for laboratory animals used for studies of infectious or noninfectious disease should be physically separate from other activities such as animal production and quarantine, clinical laboratories, and especially from facilities that provide patient care. Animal facilities should be designed and constructed to facilitate cleaning and housekeeping. Traffic flow that will minimize the risk of cross contamination should be considered in the plans. A "clean/dirty hall" layout is useful in achieving this. Floor drains should be installed in animal facilities only on the basis of clearly defined needs. If floor drains are installed, the drain trap should always contain water or a suitable disinfectant.

These recommendations describe four combinations of practices, safety equipment, and facilities for experiments on animals infected with agents which produce, or may produce, human infection. These four combinations provide increasing levels of protection to personnel and to the environment, and are recommended as minimal standards for activities involving infected laboratory animals. These four combinations, designated Animal Biosafety Levels (ABSL) 1-4, describe animal facilities and practices applicable to work on animals infected with agents assigned to corresponding Biosafety Levels 1-4.

Facility standards and practices for invertebrate vectors and hosts are not specifically addressed in standards written for commonly used laboratory animals. "Laboratory Safety for Arboviruses and Certain other Viruses of Vertebrates,"¹⁷⁸ prepared by the Subcommittee on Arbovirus Laboratory Safety of the American Committee on Arthropod-Borne Viruses, serves as a useful reference in the design and operation of facilities using arthropods.

Animal Biosafety Level 1

A. Standard Practices

1. Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

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

3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in animal rooms. Persons who wear contact lenses in animal rooms should also wear goggles or a face shield.

4. All procedures are carefully performed to minimize the creation of aerosols.

5. Work surfaces are decontaminated after use or after any spill of viable materials.

6. Doors to animal rooms open inward, are self-closing and are kept closed when experimental animals are present.

7. All wastes from the animal room are appropriately decontaminated, preferably by autoclaving, before disposal. Infected animal carcasses are incinerated after being transported from the animal room in leakproof, covered containers.

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

B. Special Practices

1. The laboratory or animal facility director limits access to the animal room to personnel who have been advised of the potential hazard and who need to enter the room for program or service purposes when work is in progress. In general, persons who may be at increased risk of acquiring infection, or for whom infection might be unusually hazardous, are not allowed in the animal room.

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

3. Bedding materials from animal cages are removed in such a manner as to minimize the creation of aerosols, and are disposed of in compliance with applicable institutional or local requirements.

4. Cages are washed manually or in a cage washer. Temperature of final rinse water in a mechanical washer should be 180^oF.

5. The wearing of laboratory coats, gowns, or uniforms in the animal facility is recommended. It is further recommended that laboratory coats worn in the animal facility not be worn in other areas.

6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards, are required to read and to follow instructions on practices and procedures.

C. Safety Equipment (Primary Barriers)

Special containment equipment is not required for animals infected with agents assigned to Biosafety Level 1.

D. Animal Facilities (Secondary Barriers)

1. The animal facility is designed and constructed to facilitate cleaning and housekeeping.

2. A handwashing sink is available in the animal facility.

3. If the animal facility has windows that open, they are fitted with fly screens.

4. Exhaust air is discharged to the outside without being recirculated to other rooms, and it is recommended, but not required, that the direction of airflow in the animal facility is inward.

Animal Biosafety Level 2

A. Standard Practices

1. Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

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

4. All procedures are carefully performed to minimize the creation of aerosols.

5. Work surfaces are decontaminated after use or after any spill of viable materials.

6. Doors to animal rooms open inward, are self-closing and are kept closed when experimental animals are present.

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

B. Special Practices

3. When the infectious agent(s) in use in the animal room requires special entry provisions (e.g., the need for immunizations and respirators) a hazard warning sign, incorporating the universal biohazard symbol, is posted on the access door to the animal room. The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the animal facility supervisor or other responsible person(s), and indicates the special requirement(s) for entering the animal room.

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

5. When appropriate, considering the agents handled, baseline serum samples from animal care and other at-risk personnel are collected and stored. Additional serum samples may be collected periodically depending on the agents handled or the function of the facility. The decision to establish a serologic surveillance program must take into account the availability of methods for the assessment of antibody to the agent(s) of concern. The program should provide for the testing of serum samples at each collection interval and the communication of results to the participants.

6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and to follow instructions on practices and procedures.

8. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments are restricted in the animal facility for use only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

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

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

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

10. Cages are appropriately decontaminated, preferably by autoclaving, before they are cleaned and washed.

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

12. Animals not involved in the work being performed are not permitted in the lab.

C. Safety Equipment (Primary Barriers)

1. Biological safety cabinets, other physical containment devices, and/or personal protective equipment (e.g., respirators, face shields) are used whenever procedures with a high potential for creating aerosols are conducted.¹³⁹ These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, intranasal inoculation of animals, and manipulations of high concentrations or large volumes of infectious materials.

2. Appropriate face/eye and respiratory protection is worn by all personnel entering animal rooms housing nonhuman primates.

3. Laboratory coats, gowns, or uniforms are worn while in the animal room. This protective clothing is removed before leaving the animal facility.

4. Special care is taken to avoid skin contamination with infectious materials; gloves are worn when handling infected animals and when skin contact with infectious materials is unavoidable.

D. Animal Facilities (Secondary Barriers)

1. The animal facility is designed and constructed to facilitate cleaning and housekeeping.

2. A handwashing sink is available in the room where infected animals are housed.

3. If the animal facility has windows that open, they are fitted with fly screens.

4. If floor drains are provided, the drain traps are always filled with water or a suitable disinfectant.

5. Exhaust air is discharged to the outside without being recirculated to other rooms, and it is recommended, but not required, that the direction of airflow in the animal facility is inward.

6. An autoclave which can be used for decontaminating infectious laboratory waste is available in the building with the animal facility.

Animal Biosafety Level 3

A. Standard Practices

1. Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

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

4. All procedures are carefully performed to minimize the creation of aerosols.

5. W