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Biosafety in Microbiological and Biomedical Laboratories {3950}

Last modified on June 28, 2002

ABSL 1    ABSL2    ABSL3    ABSL4

UK Hazard Groups

Risk Assessment

Recommended Biosafety Levels for Infectious Agents and Infected Animals

    Bacteria    Prions

    Fungi        Rickettsia

    Parasites    Viruses

Laminar Flow Cabinets 

Biological Safety Cabinets  

Animal Biosafety Level 1

The pathogens carried by these animals are not known to cause disease in healthy adult humans. Standard practices should include:
  1. The director establishes policies, procedures and protocols for emergency situations. All projects must be pre-approved by the IACUC and the IBC.
  2. Access limited to those required for program or support, and they have been advised of the biohazards
  3. Medical surveillance program
  4. Safety manual advising people of special hazards. They must read and follow instructions.
  5. No eating, drinking, smoking, handling contact lenses, applying cosmetics, or human food storage except in designated areas.
  6. Perform all procedures carefully, to minimize creation of splatter or aerosol.
  7. Decontaminate work surfaces after use or any spill of viable materials.
  8. Wastes transported away in leak-proof, covered containers, preferably for incineration
  9. Policies for sharps are instituted.
  10. Wash hands after handling cultures or animals, removing gloves, and before leaving
  11. Biohazard sign posted on the entrance when infectious agents present, identifying the agent, listing the name and phone number of the responsible person, and indicating any special requirements for entering the room
  12. Insect and rodent control program in place

Primary barriers include:

  1. Lab coats and/or uniforms; lab coats left in the animal room; gowns and uniforms not worn outside the facility
  2. People working with NHPs wear eye and face protection

Facilities or secondary barriers should:

  1. Animal facility is separated from areas in the building that are open to unrestricted traffic
  2. Be protected by external doors that are self-closing and self-locking, with animal room doors that open inward, are self-closing and are kept closed
  3. Interior surfaces are water resistant, and the construction facilitates cleaning and housekeeping
  4. Horizontal surface area is minimized in appurtenances such as pipes, ducts and lights
  5. Windows not recommended; if present they must be break-resistant; also sealed or fitted with fly screens
  6. If there are floor drains, they always contain water and/or disinfectant in the traps
  7. Have ventilation in accordance with the Guide, no recirculation of exhaust air, and preferably always negative pressure to the hallways
  8. Facility has a hand-washing sink
  9. Wash cages manually or in a washer at 180° F (final rinse)
  10. Illumination adequate for all activities, avoiding reflections and glare

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Animal Biosafety Level 2

Agents in ABSL-2 are associated with human disease, and hazards from ingestion, percutaneous and mucous membrane exposure are addressed in the facilities, policies and procedures. With ABSL-2 work, the following should be standard practices in addition to those listed in ABSL-1:
  1. Special policies and procedures should be developed in addition to the SOPs
  2. Access to animal rooms limited to the fewest possible
  3. Biosafety manual is prepared or adopted, and people have to read instructions
  4. In the medical surveillance program, immunizations or tests for the agents should be provided, and a serum surveillance system implemented if appropriate
  5. Waste containers should be disinfected prior to disposal, and autoclaving before incineration is recommended
  6. Policies for sharps include: use only if there is no non-sharp alternative, use of safety syringes, and substitution of plastic for glassware

Special practices include:

  1. Training of personnel on potential hazards, how to prevent exposure, and exposure evaluation procedures, updated at least annually. Training records maintained.
  2. People who may be at increased risk of acquiring infection, or for whom infection might have serious consequences, are not allowed in unless special procedures can eliminate the extra risk. This assessment should be made by the occupational health physician.
  3. Only animals used for experiments allowed in the room
  4. Decontaminate equipment prior to removing it from the room
  5. Spills or accidents resulting in overt exposure immediately reported to the director, with medical evaluation provided and recorded

Primary barrier practices include:

  1. Gloves are worn in addition to lab coats, gowns or uniforms
  2. Personal protective equipment is worn after a risk assessment is completed
  3. Additional protection, including a biological safety cabinet and/or personal equipment such as face shields and respiratory protection is used when conducting procedures with a high potential for creating aerosols (necropsy, tissue or fluid harvest, or intranasal inoculation)
  4. If needed, animals are housed in containment equipment such as filter top cages, which must be handled in a biocontainment cabinet designed for rodents

Secondary barriers should meet the following criteria:

  1. External doors are securely locked
  2. If there are floor drains they are always filled with disinfectant
  3. There is no recirculation of exhaust air, and the direction of airflow in the facility is inward
  4. An autoclave is available in the facility
  5. Handwashing sinks are located in the animal rooms as well as elsewhere in the facility

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Animal Biosafety Level 3

These practices are suitable for work with indigenous or exotic agents that present the potential for aerosol transmission and of causing serious or potentially lethal disease.

Standard practices are much the same as those in effect for ABSL-1 and 2.

Additional special practices include:

  1. Cages are autoclaved or decontaminated before bedding is removed and before they are cleaned and washed. Equipment decontaminated before being packaged for removal
  2. Spill procedure is developed and posted. Only trained personnel are to deal with spills.
  3. All wastes must be autoclaved before incineration (or other appropriate terminal treatment).
  4. Materials not related to the experiment (plants, animals) must not be in the animal room

Primary barriers include:

  1. Personnel wear scrubs or uniforms, wrap-around or solid-front gowns (not front-button lab coats) which must be removed and left in the room; before leaving the facility clothes are removed, contained and decontaminated prior to laundering or disposal.
  2. PPE used for all activities involving handling; gloves aseptically removed and decontaminated with room wastes; face, eye and respiratory protection worn by all who enter animal rooms; boots, shoe covers or other protective footwear and disinfectant baths available.
  3. House animals in containment caging, such as in laminar flow hoods
  4. All work likely to generate aerosols done in a primary barrier, or else respirators worn by people in the room

Secondary barriers include:

  1. Access to the animal room is via a double door which includes a change room and shower, with another air-lock or double-door autoclave for movement of supplies and wastes
  2. Penetrations in walls, floors and ceilings are sealed; openings around ducts and spaces between doors and frames are capable of being sealed if needed for decontamination
  3. Hands-free washing sink near the animal room exit door, with trap filled with disinfectant after each use
  4. Ducted exhaust air ventilation system provided to create directional airflow; exhaust must be dispersed away from air intakes or be HEPA filtered; visual monitoring device to confirm inward air flow; audible alarms to notify if HVAC fails
  5. If class II biological safety cabinets used, air can be recirculated into the room if the cabinet is tested and certified annually. If air is exhausted to the outside from the cabinet, it must be connected so as to minimize interference with the air balance, for example by an air gap. If class III cabinets are used they should be directly connected to the exhaust system.
  6. Cages must be washed in a cage washer, not by hand.
  7. If there is a vacuum service (central or local), service connections are fitted with liquid disinfectant traps and in-line HEPA filters.
  8. The BSL-3 facility design and operation must be documented, tested for verification prior to operation, and re-verified annually.

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Animal Biosafety Level 4

These organisms are dangerous or exotic agents that pose high risk of life threatening disease, aerosol transmission or have unknown risk of transmission. Facilities can operate either with animals in class III cabinets, or personnel in suits.

Standard practices

  1. The medical surveillance plan must include the availability of post-exposure prophylaxis and counseling
  2. All wastes and trash, as well as clothing to be laundered, are sterilized in a double-door autoclave located in the secondary barrier wall.
  3. People should work in pairs when they work with animals.

Special precautions include:

  1. Additional measures for controlled access, such as a 24-hour guard and check in/out system. People enter and exit only through clothing change and shower rooms.
  2. Clothing is removed before entering and either complete laboratory clothing (including underwear and shoes) or a suit are worn.
  3. Accident reporting system for incidents, exposures and employee absenteeism is in place. A facility is available for quarantine, isolation and medical care.
  4. Serum samples are analyzed at intervals.

Primary barriers:

  1. Animals housed in a class III biological safety cabinet in a BSL-4 Cabinet Laboratory, or in partial containment systems in a BSL-4 Suit Laboratory (people wear one-piece positive-pressure ventilated suits).

Secondary barriers should conform to the facility requirements for BSL-4 laboratories. If it is to be a Cabinet Laboratory, the following are required:

  1. The area is to be a separate building or an isolated zone within a building. There are a minimum of two doors prior to entering the cabinet room, with outer and inner change rooms separated by a shower. For passage of materials that don’t go through the change room, a double-door autoclave, dunk tank, fumigation chamber or ventilated anteroom is provided.
  2. All containment parameters (directional airflow) and life support systems are inspected daily prior to starting work.
  3. Walls, floors, ceilings of the cabinet and inner change rooms are a sealed internal shell. Floors are integrally sealed and coved. Drains are directly to a liquid waste decontamination system. Sewer vents and other service lines have HEPA filters and vermin protection.
  4. Liquid effluents from the dirty side (inner change room, cabinet room, floor drains, autoclave chambers) are decontaminated by a proven method, preferably heat treatment. The process must be validated both physically and biologically.
  5. A dedicated non-recirculating ventilation system is provided.
  6. Supply air and exhaust air from the cabinet room, inner change room and anteroom pass through HEPA filters located as near as possible to the source. HEPA filters are tested and certified annually. Filter housings are designed for in situ decontamination of the filter prior to removal or are removed in a sealed primary container.
  7. Communication methods, such as voice, fax and computer are provided between the laboratory and the outside.

In a Suit Laboratory, the following are required in addition to or instead of the above:

  1. A specially-designed suit area is maintained. People wear a one-piece positive pressure ventilated suit protected by HEPA filtration. The life support system includes redundant breathing air compressors, alarms and emergency backup breathing air tanks. A chemical shower is used to decontaminate the suit surface. An automatically starting emergency power source is provided for the exhaust system, life support, alarms, lighting, entry and exit controls, and biological safety cabinets. All penetrations into the internal shell are sealed.
  2. All containment parameters inspected daily.
  3. Double-door autoclave for wastes.
  4. Doors to the chemical shower and airlocks are interlocked so that both doors can’t be opened simultaneously.
  5. In the ventilation system, redundant supply air fans are recommended and redundant exhaust fans are required. Exhaust air goes through two HEPA filters before release.

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United Kingdom Hazard Groups

These are established by the Advisory Committee on Dangerous Pathogens (ACDP), part of the Health and Safety Executive (HSE).
  1. Group 1: A biological agent unlikely to cause human disease
  2. Group 2: A biological agent that can cause human disease and may be hazardous to employees. It is unlikely to spread to the community and there is usually effective prophylaxis or effective treatment available.
  3. Group 3: A biological agent that can cause severe human disease and prsents a serious hazard to employees. It may present a risk of spreading to the community, but there is usually effective prophylaxis or treatment available.
  4. Group 4: A biological agent that causes severe human disease and is a serious hazard to employees; it is likely to spread to the community and there is usually no effective prophylaxis or treatment available.

Risk Assessment

Factors of interest in a risk assessment include:
  1. Pathogenicity, taking into account how the agent is transmitted, whether there is an effective vaccine, what the incidence of laboratory-acquired infection is
  2. Route of transmission; aerosols have caused the most laboratory-acquired infections
  3. Agent stability
  4. Infectious dose and lab workers’ immune status
  5. Concentration of organisms
  6. Origin of the infectious material, such as geographic location, host, whether the disease is zoonotic or has the potential to endanger American livestock
  7. Availability of data from animal studies if no human risk data are available
  8. Availability of effective prophylaxis or treatment
  9. Medical surveillance
  10. Evaluation of the experience and skill level of the people at risk

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Recommended Biosafety Levels for Infectious Agents and Infected Animals

Bacteria

Level 2:

Bacillus anthracis: vaccine available, no lab cases since 1950s, but a terrorist agent in 2001. Use BSL-3 for production quantities and activities with high risk of aerosol production. Lab registration with CDC required to send or receive this agent.

Bordetella pertussis

Burkholderia pseudomallei: causes melioidosis

Campylobacter: animal feces is known reservoir

Chlamydia: birds are major source of infection, no human vaccine

Clostridium botulinum: bioterrorism agent, exposure to toxin is the primary hazard, which can be absorbed or inhaled; toxoid available as investigational new drug (IND); bleach or NaOH inactivates the toxin

Clostridium tetani and Corynebacterium diphtheriae

Escherichia coli: cytotoxin-producing strains, a.k.a. enterohemorrhagic strains cause hemolytic uremic syndrome, usually in children; cattle are significant reservoirs

Francisella tularensis: has caused lab infections, and is a bioterrorism agent; infects via direct contact or aerosols; human ID25-50 is 10 organisms via respiratory route; use ABSL-3 for cultures and for animal studies; vaccine available

Helicobacter pylori: discovered in 1982, inhabits human gastric mucosa

Leptospira interrogans: well-documented laboratory hazard, shed in animal urine in enormous numbers for long periods

Listeria monocytogenes: usually from contaminated food such as soft cheeses, raw meat and unwashed vegetables; transplacental transmission may kill nearly 100% of human fetuses, so pregnant women must be fully informed

Legionella pneumophila: one case of lab-associated disease reported from animal studies with Pontiac Fever; guinea pigs infected experimentally, but rabbits and mice do not develop disease

Mycobacterium leprae: no cases reported in labs; humans are the only important reservoir although armadillos and NHPs can become infected

Mycobacterium spp. other than M. tuberculosis, M. bovis or M. leprae: these are ubiquitous and infectious, but not contagious; diseases are pulmonary diseases resembling TB (M. kansasii, M. avium), lymphadenitis, and skin ulcers or wound infections

Mycobacterium tuberculosis, M. bovis: are a proven laboratory hazard, including from NHPs; low infective dose (ID50<10 bacilli); all aerosol-generating activities must be conducted in a class I or II biological safety cabinet; infected NHPs and cultures should be ABSL-3; BCG is a modified live vaccine not used in the US

Salmonella typhi: Typhoid is a demonstrated laboratory hazard, but humans are the only reservoir; a vaccine is available; use BSL-3 for cultures; other Salmonella are BSL-2

Shigella: dozens of lab-acquired infections from feces; humans are the only significant reservoir but NHPs, guinea pigs and other rodents are proven sources

Treponema pallidum: humans are the only known reservoir, but the rabbit-adapted strains retain virulence for humans and there have been cases of transmission from a rabbit orchitis culture

Yersinia pestis: potential bioterrorism agent; vaccine available

Level 3

Brucella: most commonly-reported lab-acquired infection, mostly from organisms grown in large quantities or from sniffing bacterial cultures. Aerosols have been associated with large outbreaks. Use BSL-2 for clinical specimens; use ABSL-3 for cultures and infected animals; no human vaccine in the US.

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Fungi

Level 2

Blastomyces dermatitidis: infections have occurred from inoculation of yeast form and inhalation of conidia

Coccidioides immitis: several human cases; the arthroconidia are very tiny (2-5 millimicrons) and are readily inhaled deep into pulmonary spaces; the larger spherules are 30-60 millimicrons and usually cause local skin granulomas; use BSL-3 when handling cultures or soil known to be contaminated

Cryptococcus neoformans: has caused local granulomas after accidental inoculation

Histoplasma capsulatum: documented transmission usually in diagnostic labs; encapsulated spores are very resistant; infective conidia are <5 microns and prone to airborne dispersal

Sporothrix schenckii: skin infections have occurred in lab personnel, no pulmonary cases

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Parasites

Level 2

Blood and tissue protozoans: lab-associated infections with Plasmodium, Trypanosoma and Leishmania have occurred, particularly by droplet exposure; use either a biosafety cabinet or face shield when working with cultures or blood.

Intestinal protozoans: lab-associated infections with Toxoplasma, Isospora, Giardia, Entamoeba, Sarcocystis and Cryptosporidium have been reported, primarily by ingestion. Animals with Toxoplasma may infect lab animal workers who contact peritoneal fluid. Almost every lab working with calves as a source of Cryptosporidium has had infections.

Trematodes: infections with Schistosoma and Fasciola in lab workers contacting aquaria containing snails have occurred.

Cestodes: Echinococcus granulosus has not been reported to have been transmitted in a laboratory-associated infection, but people working with dog feces should be careful; cysts and fluids are not infectious but ingestion of eggs can occur. Ingestion of cysts (Cysticercus cellulosae) containing the larvae of Taenia solium can infect humans, which are the definitive hosts. Hymenolepis nana can be transmitted by rodents, and does not require an intermediate host.

Nematodes: lab-associated infections with Ascaris, Strongyloides, Enterobius, and hookworms have been reported, and allergic responses to aerosolized components can occur. Infective larvae of Strongyloides from NHPs are a potential risk.

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Prions

Prions are proteinaceous infectious particles that lack nucleic acids. They are composed of an abnormal isoform of a normal cell protein. All known mammalian infections result from the pathogenic isoform of the prion protein of scrapie, designated PrPSc. Scrapie and other neurodegenerative diseases caused by prions are also called transmissible spongiform encephalopathies.

Unlike viruses, the properties of prions change dramatically when passaged from one species to another. Prions passaged from human materials to transgenic mice probably are much less pathogenic.

Most animal prions are considered to be BSL-2; these include scrapie (sheep and goats), transmissible mink encephalopathy, chronic wasting disease (mule deer and elk), bovine spongiform encephalopathy, feline spongiform encephalopathy, and exotic ungulate encephalopathy. Human prions (kuru, Creutzfeldt-Jacob disease, Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia) and those propagated in NHPs are BSL-2 or 3 depending on the nature of the studies. BSE prions may also be handled as BSL-3 pathogens since they may have been transmitted to humans in Europe. An exception is mice which express either human genes or chimeric mouse-human genes, which should be handled at BSL-2 or 3.

Human prion diseases are not communicable or contagious, so precautions used for AIDS patients are adequate in affected people. Familial CJD, GSS and FFI are dominantly-inherited, and have been transmitted to apes, monkeys and mice carrying human PrP transgenes. Surgery should be minimized, and the operating room decontaminated as described if affected patients must undergo surgery.

Unfixed samples of human brain and other tissues (spleen, thymus, lymph nodes and lung) containing human prions are processed with extreme care under BSL-3 precautions. If accidental contamination of the skin occurs, the area should be swabbed with 1N NaOH for 5 minutes and then washed copiously with water. This may apply to handling of BSE samples as well.

Mice and hamsters are the experimental animals of choice for studies of prion disease. Transgenic mice highly susceptible to human prions are better choices than NHPs.

Prions are very small (55 kDa) and therefore pass through filters. They are extremely resistant to conventional inactivation procedures such as irradiation, boiling, dry heat, and chemicals. Sterilization of rodent brains requires autoclaving at 132° C for 4½ hours or use of 1N NaOH, 4M guanidinium chloride, or sodium hypochlorite with >2% free chlorine. Biosafety cabinets must be decontaminated with 1N NaOH followed by 1N HCl and rinsed with water. HEPA filters should be autoclaved and incinerated. Formaldehyde-fixed and embedded tissues remain infectious.

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Rickettsia

Coxiella burnetii is the second-most commonly reported infection in lab personnel, with a human ID50 of 10 organisms. Infectious aerosols or injection are the most likely routes of infection. BSL-3 practices are recommended for handling embryonated eggs (the only way to culture it), necropsy of infected animals and maintenance of guinea pigs or other rodents which are used in experiments. Pregnant sheep should be handled as described in Lab Anim 10(3):24-27, 1981 and Inf Control 3:461-465, 1982. There is an IND vaccine available from USAMRIID.

Rickettsia: Typhus and Rocky Mountain spotted fever have been transmitted in the laboratory, possibly from aerosols. The organisms are unstable in the environment. BSL-2 can be used for most work, but BSL-3 should be used for necropsy of experimentally-infected animals and handling of embryonated eggs. Animal studies with arthropods infected with human rickettsia should be conducted at ABSL-3. Antibiotic therapy is effective early in the disease.

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Viruses

Hantaviruses pose an extreme risk of aerosol infection, causing hantavirus pulmonary syndrome and/or hemorrhagic fever with renal syndrome. Materials assumed to be risky include excreta, fresh necropsy material, bedding, and animal bites. BSL-2 procedures are appropriate for human sera, but BSL-3 procedures in a BSL-2 facility should be used for cell culture of virus. Rodents known not to excrete the virus can be housed in ABSL-2 facilities, but all work with inoculation of virus into Peromyscus maniculatus or other permissive species should be conducted at ABSL-4.

Hepatitis A and hepatitis E are hazardous in people working with chimpanzees and other NHPs such as marmosets and owl monkeys. There is a European vaccine for hepatitis A that is available in the US.

Hepatitis B has been one of the most frequent lab-associated infections, with lab workers recognized as a high-risk group. Hepatitis D virus is defective and requires hepatitis B for replication. Hepatitis C infection can also occur in the lab, primarily from blood and serum.

Herpesvirus simiae (CHV-1) is usually acquired from an asymptomatic shedder, although the highest risk is from a bite of a monkey with active lesions. The agent may be present in thoracic and abdominal tissues as well as nervous tissues. Material known or suspected to contain CHV-1 should be handled at BSL-3, although cultures with high titers of virus should be handled in a BSL-4 facility. All macaque colonies should be presumed to be naturally infected.

Influenza may be transmissible from infected ferrets to man and vice versa. The cloaca of infected birds may also represent a hazard.

Lymphocytic choriomeningitis has been a hazard in hamsters, mice and guinea pigs used for research. Nude and SCID mice may harbor silent infections. Natural infections of NHPs include callitrichid hepatitis. Although most work with the virus can be conducted at BSL-2, infected hamsters should be handled at ABSL-3.

Poliovirus infection results in disease in only ~1% of cases, so it is impossible to estimate the numbers of lab-acquired infections. Animal-associated infections have not been reported. The WHO has issued guidance documents for people working with wild poliovirus. In 1999, BSL-2/polio laboratories were established, which had some extra restrictions imposed. One year after detection of the last wild poliovirus, labs will go to BSL-3/polio containment and will be listed on Agency/Institutional and National Inventories. When oral poliovirus immunization stops, all work with wild poliovirus will be restricted to BSL-4 labs.

Retroviruses such as HIV (16 cases) and SIV (2 cases) have infected lab personnel. In addition, humans working with NHPs have a 1.8% rate of infection with simian foamy viruses. People working with these viruses or other bloodborne pathogens should follow OSHA Bloodborne Pathogen Standards.

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Laminar Flow Cabinets

Laminar flow cabinets typically provide protection to the product inside, but not to the worker. There are two types: horizontal, in which the HEPA filter is at the back and the air flows into the front, across the surface and out the back; and vertical, in which air comes down from above through the HEPA filter.

Biological Safety Cabinets

Biological safety cabinets of all grades must be tested and certified in situ when they are installed, and re-certified at least annually and any time the cabinet is moved. They should be located away from traffic areas and doors in the room. Performance criteria for biological safety cabinets were established by the US National Sanitation Foundation.

Class I and IIA Cabinets

Class I BSCs are only manufactured on a limited basis, and are being replaced generally by Class II A BSCs. They are negative-pressure, open-front cabinets with a minimum face velocity at the work opening of 75 linear feet per minute (lfpm). All air is exhausted through a HEPA filter, either into the lab or outside. Since room air flows into the cabinet, there is no product protection in this cabinet.

Class IIA cabinets (and higher) utilize vertical laminar flow to protect the products inside from room air contaminants. Type A cabinets are useful for general microbiology work that does not generate volatile or toxic chemicals or radionuclides, since there is 70% air recirculation inside the cabinet. These may be exhausted either into the laboratory or outside via a thimble connection to the building exhaust.

 

Higher grade cabinets are all hard-ducted to the building exhaust and contain negative-pressure plena. In addition, all higher grade cabinets have a face velocity of at least 100 lfpm.

Class IIB Cabinets

Type B1 cabinets have 30% recirculation but they can be used for low-level radionuclides and toxic chemicals.

 

 

 

 

 

 

 

 

 

Type B2 are the same but air is not recirculated.

Type B3 cabinets have negative pressure compared to the room, and the exhaust is hard-ducted to the exterior of the building.

 

 

 

 

 

 

 

 

 

Class III Cabinets

In a Class III cabinet, gloves are used and the cabinet is completely enclosed and ventilated. They are used with organisms in BSL-3 or BSL-4. Since Class III cabinets must be connected to a double-door autoclave or dunk tank, several are generally set up as an interconnected system.

 

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©1999, Janet Becker Rodgers, DVM, MS, DipACLAM, MRCVS

All rights reserved.

Comments? Send an email to janet.rodgers@vet.ox.ac.uk