Download Biosafety Level 2 - University of Guelph Physics

Department of Physics
Biosafety Level 2
Standard Operating Procedure
Contents
1.0 Purpose
2.0 Hazards
3.0 Responsibilities
4.0 Basic Biosafety Procedures (SOP)
5.0 Biohazards Handling Equipment
6.0 References
7.0 Appendix : Definitions, Risk Groups, Containment Laboratory, Containment
Levels, Organisms Lists, Agents Classification, Biosafety Level 2 Criteria, UoG Biosafety
Policy & Biosafety Procedures, Biological Safety Cabinets, Dept. of Microbiology Safety
Rules and Procedures, Decontamination and Disposal, Biosafety Training Checklist
Department of Physics
Standard Operating Procedure # SOP-BL2
Task/Process:
Prepared by:
Approved by:
Biosafety Level 2
David Atkinson
JHSC: Reggi Vallillee, Steve Wilson
Issue Date: Feb 6, 2007
Revision: draft only
Page:
2
1.0 Purpose
The purpose of this procedure is to ensure that persons working with biohazardous agents or
substances (bacterial, fungal, parasitic, viral, etc.) follow safe working practices in a
Biosafety Level 2 laboratory.
2.0 Hazards
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Contact with infectious substances may cause laboratory acquired infections (LAIs)
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Infectious substance may enter the body through ingestion, inhalation, contact with
mucous membranes, eyes, or non-intact skin (wounds, scrapes, bites, etc.)
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Risk of exposure may occur through infectious aerosols, spills, splashes, needlestick
injury, broken glass, pipet tips, centrifuge accidents, scalpel blades, sharp objects, etc.
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Secondary transmission of disease to the public may occur by workers carrying infectious
agents outside the laboratory
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Children, pregnant women, the elderly, immunocompromised or immunosuppressed
individuals are especially vulnerable to transmission of infectious substances
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Accidental release or transmission of pathogens into the environment may cause disease,
sickness, deleterious effect, or even mortality to human and nonhuman recipients
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Unlike chemical and physical agents, there is NO “safe” level of non-contained
pathogenic organisms
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Biosecurity of biohazardous materials is a growing concern for scientific institutions
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Department of Physics
Standard Operating Procedure # SOP-BL2
Task/Process:
Prepared by:
Approved by:
Biosafety Level 2
David Atkinson
JHSC: Reggi Vallillee, Steve Wilson
Issue Date: Feb 6, 2007
Revision: draft only
Page:
3
3.0 Responsibilities (prior to commencing work in a Level 2 laboratory)
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All workers must be WHMIS trained and attend the Department Safety course
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First time users must review University of Guelph Safety Policy 851.11.01 Biosafety
Policy - Section 9: Worker Responsibilities (see appendix)
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First time users must be trained by qualified personnel in biosafety procedures and sign
a Biosafety Training Checklist form
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A worker must report any illness, injury or medical condition that may be aggravated or
compromised by exposure to biohazardous materials
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A worker must understand that containment and safe handling of known and potentially
biohazardous materials requires strict adherence to prudent microbiological practices
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Eye and skin protection must be worn at all times during handling of any biohazardous
or potentially biohazardous substance
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A worker must be trained in the dissemination and containment of contaminants
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Users must be receive adequate instruction and training in appropriate aseptic technique
(see appendix for Decontamination and Disposal from Biohazards Reference Manual)
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Only approved containers may be used for disposal of biohazardous wastes
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Users must be trained and knowledgeable in spills cleanup and emergency procedures
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When in doubt always seek guidance from supervisory personnel
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All users must review Biosafety Level 2 Criteria (section 7.7 in appendix) including:
Standard Microbiological Practices, Special Practices, Safety Equipment and
Laboratory Facilities (Primary/Seconday barriers) Biological Safety Cabinets
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Department of Physics
Standard Operating Procedure # SOP-BL2
Task/Process:
Prepared by:
Approved by:
Biosafety Level 2
David Atkinson
JHSC: Reggi Vallillee, Steve Wilson
Issue Date: Feb 6, 2007
Revision: draft only
Page:
4
4.0 Basic Biosafety Procedures (for any laboratory dealing with infectious agents)
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KEEP THE LABORATORY NEAT, ORDERLY AND CLEAN
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KEEP LABORATORY DOORS CLOSED - ACCESS TO A LEVEL 2 LABORATORY IS LIMITED
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DO NOT EAT, DRINK OR STORE FOOD STUFF IN THE LABORATORY
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WASH HANDS WITH SOAP BEFORE LEAVING THE LABORATORY AND AFTER HANDLING ANY
CONTAMINATED MATERIALS
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PROTECT OPEN WOUNDS, CUTS, SCRATCHES WITH WATERPROOF DRESSINGS
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USE APPROPRIATE PERSONAL PROTECTIVE GEAR WHEN HANDLING CONTAMINATED,
INFECTIOUS OR POTENTIALLY INFECTIOUS MATERIALS
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WEAR A LAB COAT, DISPOSABLE GLOVES AND EYE PROTECTION IN THE LABORATORY
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DISINFECT WORK SURFACES BEFORE AND AFTER EACH WORK PERIOD
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AVOID PROCEDURES THAT CREATE AEROSOLS AND USE CONTAINMENT DEVICES
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DECONTAMINATE ALL CONTAMINATED OR INFECTIOUS MATERIALS PRIOR TO DISPOSAL
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PLACE BIO-WASTE IN ORANGE BIOHAZARD BAGS INSIDE A LEAK PROOF CONTAINER WITH
COVER FOR TRANSPORT TO AUTOCLAVE
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DISPOSE OF ALL SHARPS, SYRINGES, BROKEN GLASS IN PROPER CONTAINERS
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CLEAN-UP SPILLS IMMEDIATELY AND DISINFECT THE AREA
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REPORT ALL INCIDENTS, INJURIES OR SPILLS TO YOUR SUPERVISOR
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5.0 Biohazards Handling Equipment
WHMIS Class D – Division 3
Biohazardous Infectious signage
Post Biosafety Level 2
signage on laboratory door
Syringes, sharps, scalpels
dispose in SHARPS container
Sharps collector container
for biohazardous materials
Yellow waste bag used for
decontaminated glass only
Transport all biohazardous waste inside
a leak proof container with cover
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5.0 Biohazards Handling Equipment (cont)
Sharps and biohazard waste station
Standard autoclaving biohazard bag
Autoclave in New Science Complex
Biological safety cabinet
Containment devices
Personal protection
for eye, skin, lung
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6.0 References
Risk Group Classification for Infectious Agents
American Biological Safety Association
http://www.absa.org/XriskgroupsX/index.html
Biohazards Reference Manual
American Industrial Hygiene Association
1985 (Reprinted 1986), Prepared by AIHA Biohazards Committee
Laboratory Biosafety Level Criteria
CDC Department of Health and Human Services
Centers for Disease Control and Prevention
Office of Health and Safety
http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s3.htm
Human Pathogens and Organisms List
Public Health Agency of Canada
http://www.phac-aspc.gc.ca/ols-bsl/pathogen/organism_e.html
The Laboratory Biosafety Guidelines, 3rd Edition 2004
Minister of Health, Public Health Agency of Canada Office of Laboratory Security
Microbiology Teaching Laboratories Safety Rules and Procedures
University of Guelph, Dept. of Molecular and Cellular Biology
Biosafety Policy 851.11.01
Biosafety Procedures 851.11.02
University of Guelph Safety Policy Manual
http://www.uoguelph.ca/ehs/policies/Subject_Index.htm#BIOSAFETY
University of Guelph, EHS Department
Biosafety Videotape Catalogue
http://www.uoguelph.ca/ehs/videos.pdf
Protecting Yourself from Hantavirus Related Illness 17 min
Effective Use of the Laminar Flow Biological Safety Cabinet 16 min
Preventing Employee Exposure to Infectious Disease 19 min
Using the Gravity Displacement Steam Autoclave 29 min
Safe Use of Biological Safety Cabinets or The case of Contaminated Cultures 22 min
Biological Containment - Microbiological Safety Cabinets 25 min
Introduction to Biological Safety Cabinets - Part I 15 min
Introduction to Biological Safety Cabinets - Part 2 21 min
Controlling Your Risks: HIV in Research Laboratory 28 min
Mammalian Cell Culture Hazards 8:22 min
Bloodborne Pathogens 11 min
Biosafety 20 min
Germinology: An Introduction to Infection Control 20 min
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7.0 Appendix
7.1 Definitions
Biohazard
A biohazardous agent is one that is biological in nature, capable of self-replication and has the
capacity to produce deleterious effects upon other biological organisms, particularly humans.
A biological agent or substance present in or arising from the work environment which present
or may present a hazard to the health or well being of the worker or community. Biological
agents or substances which could be biohazards include: infectious or parasitic agents; non
infection microorganisms such as some fungi, yeasts and algae; plants and plant products, animal
and animal products which cause occupational disease.
Categories of Biohazardous Agents
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Bacteria
Fungi
Viruses
Rickettsiae
Chlamydiae
Parasites
Recombinant products
Allergens
Cultured animal cells and the potentially infectious agents these cells may contain
Infected clinical specimens (tissues, fluids, etc.)
Tissues from experimental animals (including animal dander)
Plant viruses, bacteria and fungi
Toxins (bacterial, plant, etc.)
7.2 Risk Group and Biosafety Level Definitions
Canadian Laboratory Safety Guidelines (2004; list not available)
Inherent risks of a pathogen made on basis of factors such as severity of disease caused, routes of
infection, virulence and infectivity takes into account existence of effective therapies,
possibilities for immunization, presence of vectors, quantity of agent and whether agent is
indigenous to Canada, possible effects on other species, including plants, or possible economic
environmental effects.
Risk Group 1 (low individual and community risk). Any biological agent that is unlikely to
cause disease in healthy workers or animals.
Risk Group 2 (moderate individual risk, limited community risk). Any pathogen that can cause
human disease, but under normal circumstances is unlikely to be a serious hazard to laboratory
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7.2 Risk Group and Biosafety Level Definitions (cont)
workers, the community, livestock or the environment. Laboratory exposures rarely cause
infection leading to serious disease, effective treatment and preventive measures are available
and the risk of spread is limited.
Risk Group 3 (high individual risk, low community risk). Any pathogen that usually causes
serious human disease, or can result in serious economic consequences but does not ordinarily
spread by casual contact from one individual to another, or that causes disease treatable by
antimicrobial or antiparasitic agents.
Risk Group 4 (high individual and community risk). Any pathogen that usually produces very
serious human disease, often untreatable, and may be readily transmitted from one individual to
another, or from animal to human or vice-versa, directly or indirectly, or by casual contact.
CDC/NIH Guidelines (1999)
"Biosafety in Microbiological and Biomedical Laboratories" 4th Edition, 1999. Section III gives
criteria for placing work into a biosafety containment level; not yet translated into Risk groups.
Biosafety Level 1 (BSL 1): well characterized agents not consistently known to cause disease in
healthy adult humans of minimal potential hazard to laboratory personnel and the environment
Biosafety Level 2 (BSL 2): agents of moderate potential hazard to personnel and the
environment
Biosafety Level 3 (BSL 3): indigenous or exotic agents which may cause serious or potentially
lethal disease as a result of exposure by the inhalation route (applicable to clinical, diagnostic,
teaching, research or production facilities)
Biosafety Level 4 (BSL 4): dangerous and exotic agents which pose a high individual risk of
aerosol-transmitted laboratory infections and life-threatening disease
7.3 The Containment Laboratory
This laboratory provides general space appropriate for work with infectious agents or potentially
infectious materials when the hazard levels are low and laboratory personnel can be adequately
protected by standard laboratory practice. Work is commonly conducted on the open bench with
certain operations confined to biological safety cabinets. Conventional laboratory designs are
adequate. Areas known to be sources of general contamination such as waste staging areas
should not be adjacent to media processing areas, tissue culture laboratories. Public areas and
general offices to which nonlaboratory staff require frequent access should be separated from
space which primarily supports laboratory functions.
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7.4 Containment Levels
The Public Health Agency of Canada is moving away from classifying organisms according to
risk group towards a classification system based on containment level. Containment levels are
more appropriate and give the end-user an indication of the containment required for handling
the organism safely in a laboratory setting. The containment level required for work with a
particular agent is based on the manipulations generally associated with laboratory-scale research
and clinical procedures.
Containment Level 1 (CL1)
This level applies to the basic laboratory handling agents requiring containment level 1. CL1
requires no special design features beyond those suitable for a well-designed and functional
laboratory. Biological safety cabinets are not required. Work may be done on an open bench top,
and containment is achieved through the use of practices normally employed in a basic
microbiology laboratory
Containment Level 2 (CL2)
This level applies to the laboratory handling agents requiring containment level 2. The primary
exposure hazards associated with organisms requiring CL2 are through the ingestion,
inoculation, and mucous membrane route. Agents requiring CL2 facilities are not generally
transmitted by the airborne route, but care must be taken to avoid the generation of aerosols
(aerosols can settle on bench tops and become an ingestion hazard by contamination of the
hands) or splashes. Primary containment devices such as biological safety cabinets and
centrifuges with sealed rotors or safety cups are to be used, as well as personal protective
equipment (gloves, laboratory coats, protective eyewear). Environmental contamination must
also be minimized by the use of hand washing sinks and decontamination facilities (autoclaves).
Containment Level 3 (CL3)
This level applies to diagnostic, research and clinical laboratories, production facilities, or
teaching laboratories handling agents requiring containment level 3. These agents may be
transmitted by the airborne route, often have a low infectious dose to produce effects, and can
cause serious or life-threatening disease. CL3 emphasizes additional primary and secondary
barriers to minimize the release of infectious organisms into the immediate laboratory and the
environment. Additional features to prevent transmission of CL3 organisms are appropriate
respiratory protection, HEPA filtration of exhausted laboratory air, and strictly controlled
laboratory access.
Containment Level 4 (CL4)
This is the maximum containment available and is suitable for facilities manipulating agents
requiring containment level 4. These agents have the potential for aerosol transmission, often
have a low infectious dose, and produce very serious and often fatal disease; there is generally no
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7.4 Containment Levels (cont)
treatment or vaccine available. This level of containment represents an isolated unit, functionally
and, when necessary, structurally independent of other areas. CL4 emphasizes maximum
containment of the infectious agent through complete sealing of the facility and testing to
pressure decay; isolating the researcher from the pathogen by containing the individual in a
positive pressure suit (most common) or containing the pathogen in a Class III biological safety
cabinet line (rare); and decontaminating air and other effluents produced in the facility.
http://www.phac-aspc.gc.ca/ols-bsl/pathogen/organism_e.html
7.5 Organism Lists
The Human Pathogens Importation Regulations (SOR/94-558) are the regulatory authority for
facilities wishing to import human pathogens into and transfer of specimens within Canada. Any
facility wishing to import human pathogens requiring containment levels 2, 3, or 4 must have a
valid Public Health Agency of Canada permit prior to importation. Pathogens requiring
containment level 1 facilities are not regulated by the HPIR, and therefore a permit is not
required for their importation.
The accompanying list of human pathogens (Bacteria, Viruses and Fungi) is a dynamic list
detailing required containment levels for manipulation. This list is regularly updated, and the
required containment levels are continually reassessed as new information becomes available. It
must also be understood that the accompanying list of human pathogens is not complete. The
containment level for unlisted organisms must be verified in consultation with the Office of
Laboratory Security, Public Health Agency of Canada.
Among the listed human pathogens, those classified as potentially harmful to animals have also
been indicated. Animal pathogens are regulated by the Canadian Food Inspection Agency
(CFIA). For importation of pathogens common to both humans and animals, an import permit is
required from the CFIA as well as the Public Health Agency of Canada.
Non-Pathogenic Organisms
35 KB Adobe Downloadable Document
The "Human Pathogens: Bacteria" list is currently being clarified.
The "Human Pathogens: Viruses" list is currently being clarified.
The "Human Pathogens: Fungi" lists is currently being clarified.
If you require assistance with classification of any of these agents into the
appropriate containment level please contact the Office of Laboratory Security
(613) 957-1779
[email protected]
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7.6 Classification of Agents (American Biological Safety Association)
Class 1
Agents of no or minimal hazard under ordinary conditions of handling
(biosafety level 1 standards of practice).
Class 2
Agents of moderate potential hazard to personnel and the environment This class includes
agents which may produce disease of varying degrees of severity from accidental
inoculation or injection or other means of cutaneous penetration but which are contained by
ordinary laboratory techniques (biosafety level 2 standards of practice and facility).
Class 3
Agents which may cause serious or potentially lethal disease as a result from exposure by
the inhalation route. Class 3 agents include those derived from outside the United States
which require a federal permit for importation unless they are specified for higher
classification. This class also includes pathogens which require special conditions for
containment (biosafety level 3 standards of practice and facility).
Class 4
Agents that require the most stringent conditions for their containment because they are
extremely hazardous to laboratory personnel or may cause serious epidemic disease.
Class 5
Foreign animal pathogens that are excluded from the United States by law or whose entry is
restricted by USDA administrative policy.
CLASS 1 AGENTS
(Class 1 includes all agents which have been assessed for risk and do not belong in higher classes. Call the
SHEA at 277-2753 to verify any unknown agent status).
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Bacillus cereus
Canine distemper virus (Snyder-Hill strain)
Influenza virus reference strains A/PR8/34, A/WS/33 or commonly used neurotropic variants Newcastle
virus strains licensed for vaccine use in US.
Lactobacillus acidophilus
Lactobacillus bulgaricus
Lactobacillus casei
Newcastle virus - strains licensed for vaccine use in US.
Parainfluenza virus 3, SF4 strain
Agents listed in Appendix C of "NIH Guidelines for Research Involving Recombinant DNA Molecules"
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7.6 Classification of Agents (cont)
CLASS 2 AGENTS
2B Bacterial Agents
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Acinetobacter baumannii (formerly A. calcoaceticus)
Actinobacillus (all species)
Actinomyces pyogenes (formerly Corynebacterium pyogenes)
Aeromonas hydrophila
Amycolata autotrophica
Arachnia propionica
Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum)
Arizona hinshawii (all serotypes)
Bacillus anthracis
Bacillus subtillis
Bacteroides (all species)
Bartonella henselae
Bartonella quintana
Bartonella vinsonii
Bordetella (all species)
Borrelia recurrentis
Borrelia vincenti
Borrelia burgdorferi
Campylobacter coli
Campylobacter fetus ssp. Fetus
Campylobacter jejuni
Chlamydia pneomoniae
Chlamydia psittaci
Chlamydia trachomatis
Clostridium botulinum
Clostridium chauvoei
Clostridium dificile
Clostridium haemolyticum
Clostridium histolyticum
Clostridium novyi
Clostridium perfringens
Clostridium septicum
Clostridium sordellii
Clostridium tetani
Corynebacterium bovis
Corynebacterium diptheriae
Corynebacterium equi
Corynebacterium ovis / pseudotuberculosis
Corynebacterium renale
Dermatophilus congolensis
Edwardsiella tarda
Enterbacter aerogenes
Erysipelothrix insidiosa
Erysipelothrix rhusiopathiae
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7.6 Classification of Agents (cont)
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Escherichia coli (all enteropathogenic, enterotoxigenic, enteroinvasive and strains bearing K-1 antigen,
including E. coli O157:H7)
Francisella novicida
Fusobacterium necrophorum
Haemophilus ducreyi
Haemophilus gallinarum
Haemophilus haemolyticus
Haemophilus influenzae
Haemophilus parahaemolyticus
Haemophilus parainfluenzae
Helicobacter pylori
Klebsiella (all species except oxytoca which is in class 1)
Legionella (including L. pneumophila)
Leptospira interrogans (all serotypes)
Listeria (all species)
Mixa polymorpha
Moraxella (all species)
Mycobacteria (all species except those listed in Class 3)
Mycobacterium africanum
Mycobacterium asiaticum
Mycobacterium avium
Mycobacterium bovis BCG vaccine strain
Mycobacterium chelonei
Mycobacterium fortuitum
Mycobacterium kansasii
Mycobacterium leprae
Mycobacterium malmoense
Mycobacterium marinum
Mycobacterium paratuberculosis
Mycobacterium scrofulaceum
Mycobacterium simiae
Mycobacterium szulgai
Mycobacterium ulcerans
Mycobacterium xenopi
Mycoplasma (all species except Mycoplasma mycoides and Mycoplasma agalctiae which are in Class 5)
Neisseria gonorrhoea
Neisseria meningitidis
Nocardia asteroides
Nocardia brasiliensis
Nocardia otitidiscaviarum
Nocardia transvalensis
Pasteurella (all species except those listed in class 3)
Plesiomonas shigelloides
Rhodococcus equi
Rochalimaea vinsonii
Salmonella (all species and all serotypes)
Serratia marcescens
Shigella (all species and all serotypes)
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7.6 Classification of Agents (cont)
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Sphaerophorus necrophorus
Staphylococcus aureus
Staphylococcus epidermidis
Streptobacillus moniliformis
Streptococcus agalactiae
Streptococcus pneumoniae
Streptococcus pyogenes
Treponema carateum
Treponema pallidum
Treponema pertenue
Vibrio cholerae (including biotype El Tor)
Vibrio parahemolyticus
Vibrio vulnificus
Yersinia enterocolitica
Yersinia pseudotuberculosis
2F Fungal Agents
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Absidia (all species)
Actinomyces (including Nocardia species and Actinomyces species and Arachnia propionica)
Aspergillus (all species)
Blastomyces dematitidis
Candida (all species)
Cladosporium bantianum
Cladosporium (xylohypha) trichoides
Crytococcus neoformans
Dactylaria galopava (Ochroconis gallopavum)
Epidermophyton (all species)
Exophiala (Wangiella) dermatitidis
Fonsecaea pedrosoi
Geotrichum (al species)
Loboa loboi
Madurella mycetomi
Microsporum (all species)
Mucor (all species)
Penicillium marneffei
Rhizopus (all species)
Sporothrix schenckii
Trichophyton (all species)
Trichosporon (all species)
2P Parasitic Agents
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Acanthocheilonema (all species)
Acanthamoeba (all species)
Ancylostoma (all species)
Angiostrongylus (all species)
Ascaris (all species)
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7.6 Classification of Agents (cont)
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Babesia (all species)
Balantidium (all species)
Brugia (all species)
Caprillaria (all species)
Clonorchis (all species)
Coccidia (all species)
Crytosporidium (all species)
Dicrocoelium (all species)
Dipetalonema (all species)
Diphyllobothrium (all species)
Dipylidium (all species)
Dracunculus (all species)
Echinococcus granulosus
Echinococcus multiocularis
Echinococcus vogeli
Entamoeba histolytica
• 2P Parasitic Agents (cont’d)
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Enterobius (all species)
Fasciola (all species)
Giardia (all species)
Heterophyes (all species)
Hymenolepis (all species)
Isospora (all species)
Leishmania (all species)
Linguatula (all species)
Loa (all species)
Macracanthrohynchus (all species)
Microsporidia
Naegleria fowleri
Naegleria gruberi
Necator (all species)
Onchocerca (all species)
Opisthorchis (all species)
Paragonimus (all species)
Plasmodium (all species)
Pneumocystis carinii
Sarcocystis
Schistosoma (all species)
Strongyloides (all species)
Taenia solium
Toxascaris (all species)
Toxocara (including T. canis)
Toxoplasma (all species)
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7.6 Classification of Agents (cont)
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Trichinella spiralis
Trichomonas vaginalis
Trichostrongylus (all species)
Trichuris trichiura
Trypanosoma (all species)
Wuchereria (all species)
2V Viral Agents (* denotes oncogenic viruses)
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Adv-Sv40, Ad2-SV40*
Adenoviruses (human - all types)*
Avian leukosis*
Avain Sarcoma virus
B-K virus
Bebaru virus
Bluetongue-indigenous
Bovine leukemia*
Bovine papilloma*
Buffalopox virus
Bunyamwera virus
Cache Valley virus
Calciviruses
California Encephalititis virus
Camelpox virus
CELO*
Chikkungunya vaccine strain 131/25
Coronaviruses
Cowpox virus
Coxpox virus
Coxsackie A and B viruses
Cytomegalovirus
Dengue virus serotypes 1,2,3,&4
Dog Sarcoma *
Eastern Equine Encephalomyelitis virus
Echoviruses (all types)
Encephalomyocarditis virus (EMC)
Epstein-Barr virus (EBV)*
FeLV/FeSV*
Fifth disease agent
Flanders viruses
Bibbon leukemia virus*
Guinea pig herpes*
Hamster leukemia*
Hart Park virus
Hepatitis viruses A,B,C,D,&E (associated antigen material)
Herpes Simplex virus*
other Herpes viruses (except Herpes virus simiae - Monkey B virus which is Class 4)
Human papilloma viruses
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7.6 Classification of Agents (cont)
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Human parvovirus (B19)
Influenza viruses (all types except A/PR8/34 which is in Class 1)
Junin, vaccine strain candidate #1
Kunjin
Langat virus
Lucke (frog)*
Lymphocytic choriomeningitis virus (LCM) (viscerotrophic strains)
Lymphogranuloma venereum agent
Marek’s Disease virus*
Mason-Pfizer monkey virus*
Measles virus
Milker’s Node virus
Molluscum contagiosum virus
Mopeia virus
Mouse mammary tumor*
Mumps virus
Murine leukemia*
Murine sarcoma*
Myxo-Paramyoviruses (all types except Canine distemper virus which is in class 1)
Newcastle disease virus
Norwalk virus
2V Viral Agents (cont)
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Orf virus
Papovaviruses
Parainfluenza viruses (all types except Parainfluenza virus 3, SF4 strain which is in Class 1)
Paravaccina virus
CLASS 3 AGENTS
3B Bacterial Agents
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•
•
•
•
•
•
•
•
•
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Bartonella (all species)
Brucella (all species)
Burkholderia mallei (formerlyPseudomonas mallei) (determined on a case by case basis)
Burkholderia pseudmallei (formerly Pseudomonas pseudomallei) (determined on a case by case basis)
Coxiella burnetii
Francisella tularensis
Mycobacterium bovis (except BCG strain which is in class 2)
Mycobacterium tuberculosis
Pasteurella multocida type B ("buffalo" and other foreign virulent strains)
Rickettsia akari
Rickettsia australias
Rickettsia canada
Rickettsia conorii
Rickettsia montana
Rickettsia prowazekii
Rickettsia rickettsii
18
7.6 Classification of Agents (cont)
•
•
•
•
•
•
Rickettsia sennetsu
Rickettsia siberica
Rickettsia tsutsugamushi
Rickettsia typhi (R. mooseri)
Rochalima quintana (determined on a case by case basis)
Yersinia pestis
3F Fungal Agents
•
•
•
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Coccidiodes immitis
Histoplasma capsulatum
Histoplasma capsulatum var. duboisii
Histoplasma farciminosum
3V Viral Agents
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Arboviruses (all strains except those in Class 2 and 4. Arboviruses indigenous to the United states are in
Class 3, except those listed in Class 2. West Nile and Semliki Forest viruses may be classified up or down,
depending on the conditions of use and geographic location of the laboratory.)
Creutzfeldt-Jakob disease agent (BSE agent, a prion)
Dengue virus, when used for transmission or animal inoculation experiments
Hantaviruses
HIV types 1&2, HTLV types 1&2, and SIV (all determined on a case by case basis)
Japanese encephalitis virus
Kuru disease agent
Lymphocytic choriomeningitis virus (LCM) (Neurotrophic strains)
Monkeypox virus, when used in vitro
Rabies street virus
Rift Valley fever virus
St. Louis encephalitis virus
Venezuelan equine encephalomyelitis virus (except strain TC-83 which is in class 2)
Vesicular stomatitis virus (VSV) (classification depends on strain and experimental design)
Vole rickettsia
Yellow fever virus (wild, when used in vitro)
Paracoccidioides brasilensis (determined on a case by case basis)
Bovine infectious petechial fever
Brucella melitensis
Camel pox virus
Cochliomyia hominvoraz (screw worm)
Ephemeral fever virus
Fowl plague virus
Goat pox virus
Histoplasma (Zymonema) farciminosum
Hog cholera virus
Louping ill virus
Lumpy skin disease virus
Mycoplasma agalactiae (contagious agalactia of sheep)
Mycoplasma mycoides (contagious bovine pleuropneumonia)
Nairobi sheep disease virus
Newcastle disease virus (Asiatic strains)
19
7.6 Classification of Agents (cont)
•
•
•
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•
•
•
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•
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Peste des petits ruminants (pest of small ruminants)
Pseudomonas ruinatium (heart water)
Rift valley fever virus
Rinderpest virus
Sheep pox virus
Swine vesicular disease virus
Teschen disease virus
Theileria parva (East Coast Fever)
Theileria Annulata
Theileria bovis
Theileria hirci
Theileria lawrencei
Trypanosoma vivax (Nagana)
Trypansoma evansi
Vesicular exanthema virus
Wesselsbron disease virus
Zyonema
3P Parasitic Agents
None
CLASS 4 AGENTS
4B Bacterial Agents
None
4F Fungal Agents
None
4P Parasitic Agents
None
4V Viral Agents
•
•
•
•
•
•
•
Ebola fever virus
Guanarito
Hemorrhagic fever agents, including Crimean hemorrhgic fever,Congo, Junin, and Machupo viruses, and
others as yet undefined
Herpesvirus simiae (Monkey B virus)
Lassa virus
Marburg virus
Monkeypox virus, when used for transmission or animal inoculation experiments
20
7.6 Classification of Agents (cont)
•
•
•
•
Tick-borne encephalitis virus complex, including Absettarov, Central European encephalitis viruses,
Hanzalova, Hypr, Kumlinge, Kyasanur forest disease, Omsk hemorrhagic fever, and Russian springsummer encephalitis
Venezuelan equine encephalitis virus, epidemic strains, when used for transmission or animal inoculation
experiments
Yellow fever virus (wild, when used for transmission or animal inoculation experiments)
Yellow fever virus (wild, when used for transmission or animal inoculation experiments)
CLASS 5 AGENTS
A. Animal Disease Organisms and Vectors which are Forbidden Entry into the United States by Law
•
Foot and Mouth disease virus
B. Animal Disease Organisms and Vectors which are Forbidden Entry into the United States by USDA
Policy
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African horse sickness virus
African Swine fever virus
Akabane virus
Besnoitia besnoiti
Borna disease virus
Poliomyelitis viruses (wild and attenuated)
Polioviruses (all types, wild and attenuated)
Polyoma*
Poxviruses (all types except Alastrim, Smallpox, and Whitepox which are in Class 5, and Monkeypox
which, depending on experiment, is in Class 3 or 4)
Pseudo-rabies virus
Rabbitpox virus
Rabies virus (all strains except Rabies "street" virus - fresh isolate which should be in Class 3)
Rat leukemia*
Rat mammary tumor*
Reoviruses (all types)
Respiratory syncytial virus
Rhinoviruses (all types)
Rift Valley Fever vaccine strain MP-12
Ross River virus
Rotaviruses
Rous sarcoma*
Rubella virus (Rubivirus)
Semliki Forest virus
Sendai virus
Shope fibroma*
Shope papilloma*
Simian viruses* (all types except Herpesvirus simiae - Monkey B virus, Class 4, and Marbug virus which is
a Class 4 hemorrhagic fever virus)
Sindbis virus
21
7.6 Classification of Agents (cont)
•
•
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Subsclerosing panencephalitis virus
SV 40 virus
Tacaribe complex
Tanapox complex
Tensaw virus
Transmissible Spongiform encephalopathies
Turlock virus
Vaccinia virus
Varicella virus
Venezuelan Equine Encephalomyelitis vaccine strain TC-83
Vescular stomatitus virus (laboratory adapted strains including VSV-Indiana, San Juan, and Glasgow)
Western Equine Encephalomyelitis virus
Yaba*
Yabapox virus
Yellow fever virus, 17D vaccine strain
C. Studied in the United States Organisms which may not be except at Specified Facilities
•
•
•
Alastrim
Small pox
White pox
22
7.7 Biosafety Level 2 Criteria
Biosafety Level 2 practices, equipment and facilities are those which are applicable to clinical,
diagnostic, teaching and other facilities working with the broad spectrum of indigenous
moderate-risk agents present in the community and associated with human disease of carrying
severity. Activities with low aerosol potential with these agents can be conducted on the open
bench using good microbiological techniques. Primary hazards to personnel working with these
agents relate to accidental auto-innoculation or ingestion of infectious materials. Procedures
with high aerosol potential may predictably and significantly increase the risk of exposure of
personnel to infectious aerosols and must be conducted in primary containment equipment or
devices.
Biosafety Level 2 (BSL-2) is suitable for work involving agents of moderate potential
hazard to personnel and the environment. Laboratory personnel have specific
training in handling pathogenic agents and are directed by competent scientists; access to the
laboratory is limited when work is being conducted; extreme precautions are taken with
contaminated sharp items; 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 apply to agents assigned to
Biosafety Level 2. (note: see appendix for list of Class 2 agents)
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. Doors are kept closed when experiments are in progress.
2. Persons wash their hands after they handle viable materials, 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. Food is stored outside the work area in cabinets or refrigerators designated for
this purpose only.
4. Mouth pipetting is prohibited; mechanical pipetting devices are used.
5. Policies for the safe handling of sharps are instituted.
6. All procedures are performed carefully to minimize the creation of splashes or aerosols.
7. Work surfaces are decontaminated on completion of work or at the end of the day and after
any spill or splash of viable material with disinfectants that are effective against the agents of
concern.
23
7.7 Biosafety Level 2 Criteria (cont)
8. 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 placed in a durable, leak proof container and closed for transport
from the laboratory. Materials to be decontaminated off-site from the facility are packaged in
accordance with applicable local, state, and federal regulations, before removal from the facility.
9. 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 have serious consequences, are not allowed in the
laboratory. For example, persons who are immunocompromised or immunosuppressed may be at
increased risk of acquiring infections, as well as children and pregnant women. 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 hazards and meet specific entry requirements (e.g., immunization)
may enter the laboratory.
3. A biohazard sign must be posted on the entrance to the laboratory to indicate the use or
presence of any infectious agents. Appropriate information to be posted includes the agent(s) in
use, the biosafety level, the required immunizations, the investigator's name and telephone
number, any personal protective equipment that must be worn in the laboratory, and any special
requirements for entering or exiting 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. Biosafety procedures are incorporated into standard operating procedures or in a biosafety
manual adopted or prepared specifically for the laboratory by the laboratory director. Personnel
are advised of special hazards and are required to read and follow instructions on practices and
procedures.
7. The laboratory director ensures that laboratory and support personnel receive appropriate
training on the potential hazards associated with the work involved, the necessary precautions to
prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates or
additional training as necessary for procedural or policy changes.
24
7.7 Biosafety Level 2 Criteria (cont)
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.
a. Needles and syringes or other sharp instruments should be restricted in the laboratory for use
only when there is no alternative. Plasticware should be substituted for glassware whenever
possible.
b. Only needle-locking syringes or disposable syringe needle units (i.e., needle is integral to the
syringe) are used for injection or aspiration of infectious materials.
Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable
syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed
in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable
sharps must be placed in a hard-walled container for transport to a processing area for
decontamination, preferably by autoclaving.
c. Syringes which re-sheathe the needle, needleless systems, and other safety devices are used
when appropriate.
d. 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, specimens of body fluids, or potentially infectious wastes are placed in a
leakproof container with cover that prevents leakage during collection, handling, processing,
storage, transport, or shipping.
Note: waste collected in biohazard bags must be transported in leakproof container to the
autoclave. The leakproof container must be periodically disinfected but need not be autoclaved.
10. Laboratory equipment and work surfaces should be decontaminated with an effective
disinfectant on a routine basis, after work with infectious materials is finished, and especially
after overt spills, splashes, or other contamination by infectious materials. Contaminated
equipment must be decontaminated according to any local, state, or federal regulations before it
is sent for repair or maintenance or packaged for transport in accordance with applicable local,
state, or federal regulations, before removal from the facility. (See UofG Biosafety Policy).
11. Spills and accidents that result in overt exposures to infectious materials are immediately
reported to the laboratory director. Medical evaluation, surveillance, and treatment are provided
as appropriate and written records are maintained.
12. Animals not involved in the work being performed are not permitted in the lab.
25
7.7 Biosafety Level 2 Criteria (cont)
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 embryonate 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, face shield or other splatter guard) is used for anticipated
splashes or sprays of infectious or other hazardous materials to the face when the
microorganisms must be manipulated outside the BSC.
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 hands may contact potentially infectious materials, contaminated
surfaces or equipment. Wearing two pairs of gloves may be appropriate. Gloves are disposed of
when overtly contaminated, and removed when work with infectious materials is completed or
when the integrity of the glove is compromised. Disposable gloves are not washed, reused, or
used for touching "clean" surfaces (keyboards, telephones, etc.), and they should not be worn
outside the lab. Alternatives to powdered latex gloves should be available. Hands are washed
following removal of gloves.
D. Laboratory Facilities (Secondary Barriers)
1. Provide lockable doors for facilities that house restricted agents.
2. Consider locating new laboratories away from public areas.
3. Each laboratory contains a sink for hand washing.
4. The laboratory is designed so that it can be easily cleaned. Carpets and rugs in laboratories are
inappropriate.
26
7.7 Biosafety Level 2 Criteria (cont)
5. Bench tops are impervious to water and are resistant to moderate heat and the organic
solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.
6. Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between
benches, cabinets, and equipment are accessible for cleaning. Chairs and other furniture used in
laboratory work should be covered with a non-fabric material that can be easily decontaminated.
7. Install biological safety cabinets in such a manner that fluctuations of the room supply and
exhaust air do not cause the biological safety cabinets to operate outside their parameters for
containment. Locate biological safety cabinets away from doors, from windows that can be
opened, from heavily traveled laboratory areas, and from other potentially disruptive equipment
so as to maintain the biological safety cabinets' air flow parameters for containment.
8. An eyewash station is readily available.
9. Illumination is adequate for all activities, avoiding reflections and glare that could impede
vision.
10. There are no specific ventilation requirements.
27
7.8 Biological Safety Cabinets
Biological safety cabinets (BSCs) are among the most effective, as well as the most commonly
used primary containment devices in laboratories working with infectious agents. The three
general types available (Class I, II, III) have various performance characteristics and applications
Class I Biological safety cabinet is designed for general microbiological research with low and
moderate risk agents, and is useful for containment of mixers, blenders, and other equipment.
These cabinets are not appropriate for handling research materials that are vulnerable to airborne
contamination, since the inward flow of unfiltered air from the laboratory can carry microbial
contaminants into the cabinet.
Class II Biological safety cabinets, (also called tissue culture hoods and biosafety cabinets),
provide personnel, environmental and product protection. As the diagram of the biosafety cabinet
shows, room air is drawn around the operator into the front grille of the cabinet. This provides
personnel protection by preventing contaminated air from spilling out.
In addition, the downward laminar flow of HEPA-filtered air provides a sterile environment for
tissue cultures by minimizing the chance of cross-contamination along the work surface of the
cabinet. Room air passes through the front grille and then flows upward through a plenum at
each side of the cabinet, and through the supply HEPA filter and then downward to the work area
through a back-pressure plate. Because air leaving the cabinet through the top of the unit has
passed through a HEPA filter, it is particle-free.
28
7.8 Biological Safety Cabinets (cont)
The Class II Biological safety cabinet is designed with inward air flow at a velocity to protect
personnel (75-100 lfpm), HEPA-filtered vertical laminar airflow for product protection, and
HEPA-filtered exhaust air for environmental protection.
Class II BSCs are classified into two types (A and B) based on construction, air flow velocities
and patterns, and exhaust systems. Basically, Type A cabinets are suitable for work with
microbiological research in the absence of volatile or toxic chemicals and radionuclides, since air
is recirculated within the work area. Type A cabinets may be exhausted through HEPA filters
into the laboratory, or to the outside via a 'thimble' connection to the exhaust ductwork.
Type B cabinets are further sub-typed into types B1 , B2, and B3. Type B cabinets are hardducted to the exhaust system, and contain negative pressure planes. These features, plus an
increased face velocity of 100 lfpm, allow work to be done with toxic chemical or radionuclides.
29
7.8 Biological Safety Cabinets (cont)
Class III Biological safety cabinet all operations in the work area of the cabinet are performed
through the attached rubber gloves. The Class III cabinet is operated under negative pressure. Air
supply is HEPA-filtered, and the cabinet exhaust air is filtered by two HEPA filters in series, or
HEPA filtration followed by incineration, before discharge outside the facility .
The Class III Biological safety cabinet is a totally enclosed, ventilated cabinet of gas-tight
construction and offers the highest degree of personnel and environmental protection from
infectious aerosols, as well as protection of research materials from microbiological
contaminants. Class III cabinets are most suitable for work with hazardous agents that require
Biosafety Level 3 or 4 containment.
Horizontal Laminar Flow "Clean Bench"
Horizontal Laminar flow clean air benches are not BSCs. They discharge HEPA-filtered air
across the work surface and toward the user. These devices only provide product protection.
They can be used for certain clean activities, such as the dust-free assembly of sterile equipment
or electronic devices. These benches should never be used when handling cell culture materials
or drug formulations, or when manipulating potentially infectious materials. The worker can be
exposed to materials (including proteinaceous antigens) being manipulated on the clean bench,
which may cause hypersensitivity. Horizontal clean air benches should never be used as a
substitute for a biological safety cabinet in research, biomedical or veterinary laboratories and/or
applications.
Vertical Laminar Flow "Clean Bench"
Vertical Laminar flow clean benches are also not BSCs. They may be useful, for example, in
hospital pharmacies when a clean area is needed for preparation of intravenous drugs. While
these units generally have a sash, the air is usually discharged into the room under the sash,
resulting in the same potential problems as the horizontal laminar flow clean benches.
30
7.8 Biological Safety Cabinets (cont)
Utility Services in biological safety cabinet
Utility services needed within a BSC must be planned carefully. The protection of vacuum
systems must be addressed. Electrical outlets inside the cabinet must be protected by ground
fault circuit interrupters and should be supplied by an independent circuit. When propane gas is
provided, a clearly marked emergency gas shut-off valve outside the cabinet must be installed for
fire safety. All non-electrical utility services should have exposed, accessible shut-off valves. As
a rule, propane gas burners should not be operated in a BSC. They disrupt the air flow pattern
and may pose fire hazards.
Ultraviolet Lamps in biological safety cabinet
Ultraviolet (UV) lamps are not required in BSCs. If installed, UV lamps must be cleaned
weekly to remove any dust and dirt that may block the germicidal effectiveness of the ultraviolet
light. The lamps should be checked periodically with a meter to ensure that the appropriate
intensity of UV light is being emitted. A sign must be placed on the entrance door when a UV
lamp is operating. UV lamps must be turned off when the room is occupied to protect eyes and
skin from UV exposure, which can burn the cornea and cause skin cancer.
Biosafety Cabinet Work Practices
1. Plan procedures carefully.
2. Start-up the biosafety cabinet and let it run at least 5-10 minutes before beginning
to work.
3. Do not overload cabinet. Separate clean from contaminated items (work from
clean to dirty).
4. Wear gloves and long sleeved lab coats.
5. Decontaminate the cabinet with disinfectant before and after work is completed.
6. Protect vacuum system by trap.
7. Avoid disruption of airflow (turbulence); extra items storage and movement in
and around the cabinet.
8. Perform work 10-15 cm. back from the front intake grille.
9. Equipment that causes turbulence should be placed in back 1/3 of work surface.
Do not block the back grille.
10. Avoid use of flame. Turbulence and filter damage may occur.
11. Avoid use of UV light.
12. Check the periodical test performance sticker affixed on the cabinet.
31