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OCR for page 83
Appendix A
Biosafety in Microbiological and
Biomedical Laboratories
C O N T E N TS
Section I. Introduction 85
Section II. Principles of Biosafety 86
Laboratory practice and technique 86
Safety equipment (primary barriers). 86
Facility design (secondary barriers) 87
Biosafety levels
Animal Biosafety levels
Importation and interstate shipment of certain
87
88
biomedical materials 89
Section III. Laboratory Biosafety
Level Criteria ................
Biosafety Level 1...............
Biosafety Level 2.......................
Biosafety Level 3............
Biosafety Level 4.............
. . 89
... . . ... 89
90
.92
94
-
Section IV. Vertebrate Animal Biosafety
Level Criteria 98
Animal Biosafety Level 1 98
Animal Biosafety Level 2 99
Animal Biosafety Level 3 .
Animal Biosafety Level 4 . .
.............. 100
..102
Section V. Recommended Biosafety
Levels for Infectious Agents and
Infected Animab 104
Riskassessment 106
· .... · ... ·· . ·· ... ···· ~
Agent Summary Statements
Parasitic Agents.........
Nematode parasites of humans .
Protozoal parasites of humans . .
Tremamde parasites of humans .
Cestode parasites of humans . . .
Fungal Agents ........................
Blastomyces dermatitidis ......
Coccidioides immitis .........
..107
..107
..107
..107
..107
..108
.108
..108
..108
.109
.109
109
Cryptococcus neoformans
Histoplasma capsulatum
Sporothrix schenc~i
Pathogenic members of the genera
Epidernmphyton, Microsporum, and
Trichophyton 109
.110
110
Bacmnal Agents ......................
Bacillus anthracis ....
Brucella (B. abortus, B. cants,
B. rnelitensis, B. suds) ..............
Chlamydia psittaci, C. trachomatis .....
Clostridium botulinum ...............
Clostridium tetani...................
Corynebacterium diphtheriae..........
Francisella tularensis................
Leptospira interrogans-fill strops ....
Legionella pneumophila; other
Legionella-like agents .............
Mycobacterium leprae ...............
0
0
111
111
2
2
2
3
3
Repnnted from J.H. Richardson and W.E. Baricley, Biosafety in Microbiological and Biomedical Laboratories, 1st ea., U.S. Public Health
Service, HHS Publication No. (CDC) 84-8395, U.S. Govemment Printing Office, Washington, D.C., March 1984. Figures, tables,
appendixes, and page numbers in this repented version have been renumbered to avoid confusion.
83
OCR for page 84
84
Mycobacterium spp. other than
M. tuberculosis, M. bovis, or M. Ieprae . . 113
Mycobacterium tuberculosis, M. bovis 114
Neisseria gonorrhoeae ................
Neisseria meningitidis .................
Pseudomonas pseudomallei ............
Salmonella cholera-suds, S. enteritidis"
aDserotypes 115
Salmonella typhi ~ 115
Shigellaspp 116
Treponemapallidum 116
Vibrionic enteritis (Campylobacterfetus
subsp. jejuni, Vibrio cholerae, V.
parahaemolyticus)
.114
.115
.115
. . .. .. . . .. .
Yersinia pestis ........................
Rickettsial Agents .
Coxiella burnetii
116
117
117
...................... 117
Rickettsia akari, Rochalimaea quintana,
end Rochalimaeavinsonii 117
Rickettsia prowaze~i, Rickettsia typhi
(R. mooseri), Rickettsia tsutsugamushi,
Rickettsia canada, and Spoued Fever
Group agents of human disease
other than Rickettsia rickettsii and
Rickettsia akari.....................
Rickettsm rickettsii ....................
Viral Agents .........
Hepatitis A virus ......................
Hepatitis B9 hepatitis non-A non-B
Herpesvirus sirruae (B-virus) ....
Herpesviruses ................
Influenza virus.............
Lymphocytic choriomeningitis
~CM) virus..................
Poliovirus e~ ee
Poxviruses
e-----~e~
APPENDIX A
Rabies virus . . . . . . . . . . . . . . . . . eeeeee122
Transmissible spongiform
encephalopathies (Creutzfeldt-Jakob
and kuru agen~. . . . . . . . . . . . ee..ee122
Vesicular stomatitis virus (VSV) e e 123
Arbonruses ~ eee123
Arboviruses Assigned to Biosafe~r
Level 2~ eeeeel23
Arboviruses and Arenaviruses Assigned to
Bio~ety Level 3e~
A1bov~u~e,AUenavOu~es, or
Filovinlses Assigned to Biosafety
Level 4 .
Appendix A.1. Biological Safety
.......... 126
128
Cabinets ~ eeeeeee129
Appendix A.2. Immunoprophylaxis eee~131
AppendLx A.3. Surveillance of Personnel
for Laboratory-Associated
RickettsialInfections 131
. 118 Appendix A.4. Importation andInterstate
. 118 Shipment of Human Pathogens and
119
.119
119
119
.. 120
120
Related Materials 132
Appendix A.5. Restricted Animal
Pathogens 132
Appendix A.6. Resources for
Information 133
. 121
121 Literature Cited
121
OCR for page 85
APPENDIX A
SECTIONI. INTRODUCTION
Microbiology laboratories are special, often
unique, work environments that may pose special
infectious disease risks to persons in or near them.
Personnel have contracted infections in the labora-
tory throughout the history of microbiology. Pub-
lished reports around the turn of the century described
laboratory-associated cases of typhoid, cholera,
"landers, brucellosis, and tetanus (123~. In 1941,
Meyer and Eddie (75) 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 Pike (113) 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 Pike (114) published the
second of a series of summaries of laboratory-associ-
ated 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 out-
numbered all other reported laboratory-acquired in-
fections and together with tuberculosis, tularemia,
typhoid, and streptococcal infection accounted for
72% of all bacteria infections and for 31% of infec-
tions 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 (93), adding
641 new or previously unreported cases, and again in
1976 (903, 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. Expo-
sure to infectious aerosols was considered to be a
plausible but unconfirmed source of infection for the
85
more than 80% of the reported cases in which the
infected person had "worked with the agent."
In 1967, Hanson et al. (53) 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 per-
sonnel. Exposure to infectious aerosols was consid-
ered the most common source of infection.
In 1974, Skinhoj (104) published the results of a
survey which showed that personnel in Danish clini-
cal 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 popula-
tion. Similarly, a 1976 survey by Hamngton and
Shannon (55) indicated that medical laboratory work-
ers in England had "a five times increased risk of
acquiring tuberculosis compared with the general
population." Hepatitis and shigellosis were also shown
to be continuing occupational risks and together with
tuberculosis were the three most commonly reported
occupation-associated infections in Britain.
Although these reports suggest that laboratory
personnel are at increased risk of being infected by
the agents they handle, actual rates of infection are
typically not available. However, the studies of Har-
rington and Shannon (55) and of Skinhoj (104) indi-
cate that laboratory personnel have higher rates of tu-
berculosis, shigellosis, and hepatitis than the general
population.
In contrast to the documented occurrence of labo-
ratory-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 in-
fections were recorded at the Center for Disease
Control in 1947-1973 (97), no secondary cases were
reported in family members or community contacts.
The National Animal Disease Center has reported a
similar experience (115), with no secondary cases
occurring in laboratory and nonlaboratory contacts
of 18 laboratory-associated cases occurring in 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 his dismissal
from the hospital (70~. Three secondary cases of
smallpox were reported in two laboratoqy-associated
outbreaks in England in 1973 (96) and 1978 (1303.
There were earlier reports of six cases of Q fever in
OCR for page 86
86
employees of a commercial laundry which handled
linens and uniforms from a laboratory where work
with the agent was conducted (84), and two cases of
Q fever in household contacts of a rickettsiologist
(5~. These cases are representative of the sporadic
nature and infrequent association of community in-
fections with laboratories working with infectious
agents.
In his 1979 review (92), Pike concluded, "the
knowledge, the techniques, and the equipment to
prevent most laboratory infections are available." No
single code of practice, standards, guidelines, or other
publication, however, provides detailed descriptions
of techniques, equipment, and other considerations
or recommendations for the broad scope of labora-
tory activities conducted in the United States with a
variety of indigenous and exotic infectious agents.
The booklet Classification of Etiologic Agents on the
Basis of Hazard (15) has, since 1969, served as a
general reference for some laboratory activities util-
izing infectious agents. That booklet and the concept
of categorizing infectious agents and laboratory ac-
tivities into four classes or levels served as a basic
format for Biosafety in Microbiological and Biomedi-
cal Laboratories. This publication will provide spe-
cific descriptions of combinations of microbiological
practices, laboratory facilities, and safety equipment
and recommendations for use in four categories or
biosafety levels of laboratory operation with selected
infectious agents of man.
The descriptions of biosafety levels 14 parallel
those of P14 in the NIH Guidelines for Research
Involving Recombinant DNA Molecules (43) and are
consistent with the general criteria used in assigning
agents to Classes 14 in Classification of Etiologic
Agents on the Basis of Hazard (15~. Four biosafety
levels are also described for infectious disease activi-
ties utilizing small laboratory animals. Recommen-
dations 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.
SECTION II. PRINCIPLES OF BIOSAFETY
The term 4'containment" is used in describing
safe methods for managing infectious agents in the
laboratory environment where they are being handled
or maintained. Primary containment, the protection
of personnel and the immediate laboratory environ-
ment from exposure to infectious agents, is provided
APPENDIX A
by good microbiological technique and the use of
appropriate safety equipment. The use of vaccines
may provide an increased level of personal protec-
tion. Secondary containment, the protection of the
environment external to the laboratory from expo-
sure to infectious materials, is provided by a combi-
nation of facility design and operational practices.
The purpose of containment is to reduce exposure of
laboratory workers and other persons to, and to pre-
vent escape into the outside environment of, poten-
tially hazardous agents. The three elements of con-
tainment include laboratory practice and technique,
safety equipment, and facility design.
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 infected
materials must be aware of potential hazards and
must be trained and proficient in the practices and
techniques required for safely handling such mate-
rial. The director or person in charge of the labora-
tory is responsible for providing or arranging for
appropriate training of personnel.
When standard laboratory practices are not suf-
f~cient to control the hazard associated with a par-
ticular 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.
Each laboratory should develop or adopt a bio-
safety or operations manual which identifies the haz-
ards that will or may be encountered and which speci-
f~es 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 sci-
entist trained and knowledgeable in appropriate labm
ratory techniques, safety procedures, and hazards
associated with handling infectious agents must di-
rect laboratory activities.
Laboratory personnel, safety practices, and tech-
niques 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 and a
variety of enclosed containers. The biological safety
OCR for page 87
APPENDIX A
cabinet is the principal device used to provide con-
tainment of infectious aerosols generated by many
microbiological procedures. Three types of biologi-
cal safety cabinets (Class I, II, III) used in microbio-
logical laboratories are described and illustrated in
Appendix A.1. Open-fronted Class I and Class II
biological safety cabinets are partial containment
cabinets which offer significant levels of protection
to laboratory personnel and to the environment when
used with good microbiological techniques. The gas-
tight Class III biological safety cabinet provides the
highest attainable level of protection to personnel
and the environment.
An example of an enclosed container is the safety
centrifuge cup, which is designed to prevent aerosols
from being released during centrifugation.
Safety equipment also includes items for per-
sonal protection such as gloves, coats, gowns, shoe
covers, boots, respirators, face shields, and safety
glasses. These personal protective devices are 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 devices may form the
primary barrier between personnel and the infectious
materials. Examples of such activities include certain
animal studies, animal necropsy, production activi-
ties, and activities relating to maintenance, service,
or support of the laboratory facility.
Facility design (secondary barriers). The de-
sign of the facility is important in providing a barrier
to protect persons working in the facility but outside
the laboratory and those in the community from in-
fectious agents which may be accidentally released
from the laboratory. Laboratory management is re-
sponsible for providing facilities commensurate with
the laboratory's function. Three facility designs are
described below, in ascending order by level of con-
tainment.
1. The basic laboratory. This laboratory pro-
vides general space in which work is done with vi-
able agents which are not associated with disease in
healthy adults. Basic laboratories include those fa-
cilities described in the following pages as Biosafety
Levels 1 and 2 facilities.
This laboratory is also appropriate for work with
87
infectious agents or potentially infectious materials
when the hazard levels are low and laboratory per-
sonnel can be adequately protected by standard labm
ratory practice. While work is commonly conducted
on the open bench, certain operations are confined to
biological safety cabinets. Conventional laboratory
designs are adequate. Areas known to be sources of
general contamination, such as animal rooms and
waste staging areas, should not be adjacent to patient
care activities. Public areas and general offices to
which nonlaboratory staff require frequent access
should be sepal from spaces which primarily
support laboratory functions.
2. The containment laboratory. This laboratory
has special engineering features which make it pos-
sible for laboratory workers to handle hazardous
materials without endangering themselves, the com-
munity, or the environment. The containment labora-
tory is described in the following pages as a Bio-
safety Level 3 facility. The unique features which
distinguish this laboratory from the basic laboratory
are the provisions for access control and a special-
ized ventilation system. The containment laboratory
may be an entire building or a single module or
complex of modules within a building. In all cases,
the laboratory is separated by a controlled access
zone from areas open to the public.
3. The maximum containment laboratory. This
laboratory has special engineering and containment
features that allow activities involving infectious
agents that are extremely hazardous to the laboratory
worker or that may cause serious epidemic disease to
be conducted safely. The maximum containment labs
ratory is described on the following pages as a Bio-
safety Level 4 facility. Although the maximum con-
tainment laboratory is generally a separate building,
it can be constructed as an isolated area within a
building. The laboratory's distinguishing character-
istic is that it has secondary barriers to prevent haz-
ardous materials from escaping into the environment.
Such barriers include sealed openings into the labo-
ratory, airlocks or liquid disinfectant barriers, a cloth-
ing-change and shower room contiguous to the labo-
ratory ventilation system, and a treatment system to
decontaminate exhaust air.
Biosafety levels. Four Biosafety levels are de-
scribed which consist of combinations of laboratory
practices and techniques, safety equipment, and labm
ratory facilities appropriate for the operations per
OCR for page 88
88
formed and the hazard posed by the infectious agents
and for the laboratory function or activity.
Biosafety Level 1. 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 rep-
resentative of those microorganisms meeting these
criteria. Many agents not ordinarily associated with
disease processes in humans are, however, opportun-
istic pathogens and may cause infection in the young,
the aged, and immunodef~cient or immunosuppressed
individuals. Vaccine strains which have undergone
multiple in viva passages should not be considered
av~rulent simply because they are vaccine strains.
Biosafety Level 2. 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
aerosols is low. Hepatitis B virus, the salmonellae,
and Toxoplasma spp. are representative of microor-
ganisms assigned to this containment level. Primary
hazards to personnel working with these agents may
include accidental autoinoculation, ingestion, and skin
or mucous membrane exposure to infectious materi-
als. Procedures with high aerosol potential that may
increase the risk of exposure of personnel must be
conducted in primary containment equipment or de-
vices.
Biosafety Level 3. Biosafety Level 3 practices,
safety equipment, and facilities are applicable to clini-
cal, diagnostic, teaching, research, or production fa-
cilities in which work is done with indigenous or
exotic agents where the potential for infection by
aerosols is real and the disease may have serious or
lethal consequences. Autoinoculation and ingestion
also represent primary hazards to personnel working
with these agents. Examples of such agents for which
Biosafety Level 3 safeguards are generally recom-
mended include Mycobacterium tuberculosis, St.
Louis encephalitis virus, and Coxiella burnetii.
APPENDIX A
Biosafety Level 4. Biosafety Level 4 practices,
safety equipment, and facilities are applicable to work
with dangerous and exotic agents which pose a high
individual risk of life-threatening disease. All ma-
nipulations of potentially infectious diagnostic
materials, isolates, and naturally or experimentally
infected animals pose a high risk of exposure and
infection to laboratory personnel. Lassa fever virus is
representative of the microorganisms assigned to
Level 4.
Animal Biosafety levels. Four Biosafety levels
are also described for activities involving infectious
disease activities 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 protec-
tion to personnel and the environment.
The laboratory director is directly and primarily
responsible for the safe operation of the laboratory.
Hisser knowledge and judgment are critical in as-
sessing risks and appropriately applying these rec-
ommendations. The recommended Biosafety level
represents those conditions under which the agent
can ordinarily be safely handled. Special characteris-
tics of the agents used, the training and experience of
personnel, and the nature or function of the labora-
tory may further influence the director in applying
these recommendations.
Work with known agents should be conducted at
the Biosafety level recommended in Section V unless
specific information is available to suggest that viru-
lence, pathogenicity, antibiotic resistance patterns,
and other factors are significantly altered to require
more stringent or allow less stringent practices to be
used.
Clinical laboratories, and especially those in
health care facilities, receive clinical specimens with
requests for a variety of diagnostic and clinical sup-
port services. Typically, clinical laboratories receive
specimens without pertinent information such as pa-
tient history or clinical findings which may be sug-
gestive of an infectious etiology. Furthermore, such
specimens are often submitted with a broad request
for microbiological examination for multiple agents
(e.g., sputum samples 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
OCR for page 89
APPENDIX A
hazard of clinical specimens. Except in extraordinary
circumstances (e.g., suspected hemorrhagic fever)
the initial processing of clinical specimens and iden-
tification of isolates can be and are safely conducted
using a combination of practices, facilities, and safety
equipment described as Biosafety Level 2. Biologi-
cal safety cabinets (Class I or II) should 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 in-
fectious aerosols is likely to be present. Class II
biological safety cabinets are also used to protect the
integrity of the specimens or cultures by preventing
contamination from the laboratory environment.
Segregating clinical laboratory functions and
limiting or restricting access to laboratory areas are
the responsibility of the laboratory director.
Importation' and interstate shipment of cer-
tain biomedical materials. The importation of etio-
logic agents and vectors of human diseases is subject
to the requirements of the Public Health Service For-
eign Quarantine regulations. Companion regulations
of the Public Health Service and the Department of
Transportation specify packaging, labeling, and ship-
ping requirements for etiologic agents and diagnostic
specimens shipped in interstate commerce (see Ap-
pendix A.4~.
The U.S. Department of Agriculture regulates
the importation and interstate shipment of animal
89
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 Add.
SECTION m. LABORATORY BIOSAFETY
LEVEL CRITERIA
The essential elements of the four Biosafety lev-
els for activities involving infectious microorgan-
isms and laboratory animals are summarized in Tables
A.1 and A.2. 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 agents of no known or minimal
potential hazard to laboratory personnel and the en-
vironment. The laboratory is not separated from the
general traffic patterns in the building. Work is gen-
erally conducted on open bench tops. Special con-
tainment equipment is not required or generally used.
Laboratory personnel have specific training in the
procedures conducted in the laboratory and are su-
pervised by a scientist with general training in micro-
biology or a related science.
The following standard and special practices,
safety equipment, and facilities apply to agents as-
signed to Biosafety Level 1.
TABLE A.1 Summary of Recommended Biosafety Levels for Infectious Agents
Biosafety level Practices and techniques
Safety equipment
None: primary containment provided
by adherence to standard laboratory
practices during open bench operations
Facilities
1 Standard microbiological practices
2
3
Basic
Level 1 practices plus: laboratory
coats; decontamination of all infectious
wastes; limited access; protective
gloves and biohazard warning signs as
indicated
Level 2 practices plus: special labora-
tory clothing; controlled access
Partial containment equipment (i.e., Basic
Class I or II Biological Safety
Cabinets) used to conduct mechanical
and manipulative procedures that have
high aerosol potential which may in
crease the risk of exposure to per
sonnel
Partial containment equipment used for Containment
all manipulations of infectious material
Level 3 practices plus: entrance Maximum containment equipment Maximum
through change room where street clo- (i.e., Class III biological safety cabinet containment
thing is removed and laboratory clo
thing is put on; shower on exit; all
wastes are decontaminated on exit
from the facility
~ , ~ , _ _ .
or partial containment equipment in
combination with full-body, air-
supplied, positive-pressure personnel
suit) used for all procedures and activi-
ties
OCR for page 90
Do
APPENDIX A
TABLE A.2 Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally
Infected Vertebrate Animals Are Used
Biosafety level Practices and techniques
Safety equipment
Facilities
Basic
1
Standard animal care and management None
practices
Laboratory coats; decontamination of
all infectious wastes and of animal
cages prior to washing; limited access;
protective gloves and hazard warning
signs as indicated
3 Level 2 practices plus: special labora
tory clothing; controlled access
Level 3 practices plus: entrance
through clothes change room where
street clothing is removed and labora
tory clothing is put on shower on exit;
all wastes are decontaminated before
removal from the facility
Partial containment equipment and/or
personal protective devices used for
activities and manipulations of agents
or infected animals that produce aero-
sols
Basic
Partial containment equipment and/or Containment
personal protective devices used for all
activities and manipulations of agents
or infected animals
Maximum containment equipment Maximum
(i.e., Class III biological safety cabinet containment
or partial containment equipment in
combination with full-body, air sup
plied, positive-pressure personnel suit)
used for all procedures and activities
A. Standard?rucrobiologicalpractices
1. Access to the laboratory is limited or re
stricted at the discretion of the laboratory direc
tor when experiments are in progress.
2. Work surfaces are decontaminated once
a day and after any spill of viable material.
3. All contaminated liquid or solid wastes
are decontaminated before disposal.
4. Mechanical pipetting devices are used;
mouth pipetting is prohibited.
5. Eating, drinking, smoking, and apply
ing cosmetics are not permitted in the work area.
Food may be stored in cabinets or refrigerators
designated and used for this purpose only. Food
storage cabinets or refrigerators should be lo
cated outside of the work area.
6. Persons wash their hands after they
handle viable materials and animals and before
leaving the laboratory.
7. All procedures are performed carefully
to minimize the creation of aerosols.
8. It is recommended that laboratory coats,
gowns, or uniforms be worn to prevent contami- washing.
nation or soiling of street clothes.
B. Specialpractices
1. Contaminated materials that are to be
decontaminated at a site away from the labora
tory are placed in a durable leakproof container
which is closed before being removed from the
laboratory.
2. An insect and rodent control program is
in effect.
C. Containment equipment
Special containment equipment is generally not re-
quired for manipulations of agents assigned to Bio-
safety Level 1.
D. Laboratoryfacilities
1. The laboratory is designed so that it can
be easily cleaned.
2. Bench tops are impervious to water and
resistant to acids, alkalis, organic solvents, and
moderate heat.
3. Laboratory furniture is sturdy. Spaces
between benches, cabinets, and equipment are
accessible for cleaning.
4. Each laboratory contains a sink for hand
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
OCR for page 91
APPENDIX A
of moderate potential hazard to personnel and the
environment. It differs in that (1) laboratory person-
nel 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, and (3) certain procedures in which
infectious aerosols are created are conducted in bio-
logical safety cabinets or other physical containment
equipment.
The following standard and special practices,
safety equipment, and facilities apply to agents as-
signed to Biosafety Level 2.
A. Standard~ucrobiologicalpractices
1. Access to the laboratory is limited or
restricted by the laboratory director when work
with infectious agents is in progress.
2. Work surfaces are decontaminated at
least once a day and after any spill of viable
material.
3. All infectious liquid or solid wastes are
decontaminated before disposal.
4. Mechanical pipetting devices are used;
mouth pipetting is prohibited.
5. Eating, drinking, smoking, and apply-
ing cosmetics are not permitted in the work area.
Food may be stored in cabinets or refrigerators
designated and used for this purpose only. Food
storage cabinets or refrigerators should be lo-
cated outside of the work area.
6. Persons wash their hands after handling
infectious materials and animals and when they
leave the laboratory.
7. All procedures are performed carefully
to minimize the creation of aerosols.
B. Specialpractices
1. Contaminated materials that are to be
decontaminated at a site away from the labora-
tory are placed in a durable leakproof container
which is closed before being removed from the
laboratory.
2. The laboratory director limits access to
the laboratory. 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. The
director has the final responsibility for assessing
91
each circumstance and determining who may
enter or work in the laboratory.
3. The laboratory director establishes poli-
cies and procedures whereby only persons who
have been advised of the potential hazard and
meet any specific entry requirements (e.g., im-
munization) enter the ldboramry or animal rooms.
4. When the infectious magentas) in use in
the laboratory require special provisions for en-
try (e.g., vaccination), 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 in-
fectious agent, lists the name and telephone
number of the laboratory director or other re-
sponsible personas), and indicates the special
requirementts) for entering the laboratory.
5. An insect and rodent control program is
in effect.
6. Laboratory coats, gowns, smocks, or
uniforms are worn while in the laboratory. Be-
fore leaving the laboratory for nonlaboratory
areas (e.g., cafeteria, library, administrative of-
f~ces), this protective clothing is removed and
left in the laboratory or covered with a clean
coat not used in the laboratory.
7. Animals not involved in the work being
performed are not permitted in the laboratory.
8. Special care is taken to avoid skin con-
tamination with infectious materials; gloves
should be worn when handling infected animals
and when skin contact with infectious materials
is unavoidable.
9. All wastes from laboratories and animal
rooms are appropriately decontaminated before
disposal.
10. Hypodermic needles and syringes are
used only for parenteral injection and aspiration
of fluids from laboratory animals and diaphragm
bottles. Only needle-locking syringes or dispos-
able syringe-needle units (i.e., needle is integral
to the syringe) are used for the injection or aspi-
ration of infectious fluids. Extreme caution should
be used when handling needles and syringes to
avoid autoinoculation and the generation of aero-
sols during use and disposal. Needles should not
be bent, sheared, replaced in the sheath or guard,
or removed from the syringe following use. The
needle and syringe should be promptly placed in
a puncture-resistant container and decontami
OCR for page 92
92
nated, preferably by autoclaving, before discard
or reuse.
11. Spills and accidents which result in ovens
exposures to infectious materials are immedi-
ately reported to the laboratory director. Medi-
cal evaluation, surveillance, and treatment are
provided as appropriate, and written records are
maintained.
12. When appropriate, considering the
agentts) handled, base-line serum samples for
laboratory and other at-risk personnel are col-
lected periodically, depending on the agents
handled or the function of the facility.
13. A Biosafety manual is prepared or
adopted. Personnel are advised of special haz-
ards and are required to read instructions on
practices and procedures and to follow them.
C. Containment equipment
Biological safety cabinets (Class I or II) (see
Appendix A.1) or other appropriate personal protec-
tive or physical containment devices are used when-
ever:
1. Procedures with a high potential for cre-
ating infectious aerosols are conducted (821.
These may include centrifuging, grinding, blend-
ing, vigorous shaking or mixing, sonic disrup-
tion, opening containers of infectious materials
whose internal pressures may be different from
ambient pressures, inoculating animals intrana-
sally, and harvesting infected tissues from ani-
mals or eggs.
2. High concentrations or large volumes of
infectious agents are used. Such materials may
be centrifuged in the open laboratory if sealed
heads or centrifuge safety cups are used and if
they are opened only in a biological safety cabi-
net.
D. Laboratoryfacilities
1. The laboratory is designed so that it can
be easily cleaned.
2. Bench tops are impervious to water and
resistant to acids, alkalis, organic solvents, and
moderate heat.
3. Laboratory furniture is sturdy, and spaces
between benches, cabinets, and equipment are
accessible for cleaning.
APPENDIX A
4. Each labo~auxy contains a sink for hand-
washing.
5. If the laboratory has windows that open,
they are fitted with fly screens.
6. An autoclave for decontaminating in-
fectious laboratory wastes is available.
Biosafety Level 3. Biosafety Level 3 is appli-
cable to clinical, diagnostic, teaching, research, or
production facilities in which work is done with in-
digenous or exotic agents which may cause serious
or potentially lethal disease as a result of exposure by
the inhalation route. Laboratory personnel have spe-
cif~c training in handling pathogenic and potentially
lethal agents and are supervised by competent scien-
tists who are experienced in working with these
agents. All procedures involving the manipulation of
infectious material are conducted within biological
safety cabinets or other physical containment de-
vices or by personnel wearing appropriate personal
protective clothing and devices. The laboratory has
special engineering and design features. It is recog-
nized, however, that many existing facilities may not
have all the facility safeguards recommended for
Biosafety Level 3 (e.g., access zone, sealed penetra-
tions, directional airflow, etc.~. In these circumstances,
acceptable safety may be achieved for routine or
repetitive operations (e.g., diagnostic procedures in-
volving the propagation of an agent for identifica-
tion, typing, and susceptibility testing) in laborato-
ries where facility features satisfy Biosafety Level 2
recommendations, provided the recommended "Stan-
dard Microbiological Practices," "Special Practices,"
and "Containment Equipment" for Biosafety Level 3
are rigorously followed. The decision to implement
this modification of Biosafety Level 3 recommenda-
tions should be made only by the laboratory director.
The following standard and special safety prac-
tices, equipment, and facilities apply to agents as-
signed to Biosafety Level 3.
A. Standard microbiologicalpractices
1. Work surfaces are decontaminated at
least once a day and after any spill of viable
material.
2. All infectious liquid or solid wastes are
decontaminated before disposal.
3. Mechanical pipetting devices are used;
mouth pipetting is prohibited.
OCR for page 93
APPENDIX A
4. Eating, drinking, smoking, storing food,
and applying cosmetics are not permitted in the
work area
5. Persons wash their hands after handling
infectious materials and animals and when they
leave the laboratory.
6. All procedures are performed carefully
to minimize the creation of aerosols.
B. Specialpractices
1. Laboratory doors are kept closed when
experiments are in progress.
2. Contaminated materials that are to be
decontaminated at a site away from the labora-
tory are placed in a durable leakproof container
which is closed before being removed from the
laboratory.
3. The laboratory director controls access
to the laboratory and restricts access to persons
whose presence is required for program or sup-
port purposes. 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 cir-
cumstance and determining who may enter or
work in the laboratory.
4. The laboratory director establishes poli-
cies 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 ani-
mal rooms.
5. When infectious materials or infected
animals are present in the laboratory or contain-
ment module, a hazard warning sign, incorporat-
ing 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 labo-
ratory director or other responsible personas),
and indicates any special requirements for enter-
ing the laboratory, such as the need for immuni-
zations, respirators, or other personal protective
measures.
6. All activities involving infectious mate-
rials are conducted in biological safety cabinets
or other physical containment devices within the
93
containment module. No work in open vessels is
conducted on the open bench.
7. The work surfaces of biological safety
cabinets and other containment equipment are
decontaminated when work with infectious ma-
terials is finished. Plastic-backed paper toweling
used on nonperforated work surfaces within bio-
logical safety cabinets facilitates cleanup.
8. An insect and rodent control program is
in effect.
9. Laboratory clothing that protects street
clothing (e.g., solid front or wrap-around gowns,
scrub suits, coveralls) is worn in the laboratory.
Laboratory clothing is not worn outside the labo-
ratory, and it is decontaminated before being
laundered.
10. Special care is taken to avoid skin con-
tamination with infectious materials; gloves
should be worn when handling infected animals
and when skin contact with infectious materials
is unavoidable.
11. Molded surgical masks or respirators are
worn in rooms containing infected animals.
12. Animals and plants not related to the
work being conducted are not permitted in the
laboratory.
13. All wastes from laboratories and animal
rooms are appropriately decontaminated before
disposal.
14. Vacuum lines are protected with high
efficiency particulate air (PAPA) filters and liq-
uid disinfectant traps.
15. Hypodermic needles and syringes are
used only for parenteral injection and aspiration
of fluids from laboratory animals and diaphragm
bottles. Only needle-locking syringes or dispos-
able syringe-needle units (i.e., needle is integral
to Be syringe) are used for the injection or aspi-
ration of infectious fluids. Extreme caution should
be used when handling needles and syringes to
avoid autoinoculation and the generation of aero-
sols during use and disposal. Needles should not
be bent, sheared, replaced in the sheath or guard,
or removed from the syringe following use. The
needle and syringe should be promptly placed in
a puncture-resistant container and decontami-
nated, preferably by autoclaving, before discard
or reuse.
16. Spills and accidents which result in overt
or potential exposures to infectious materials are
OCR for page 130
130
1~ ~-11
~4J~ ~ ~
FIGURE A.1 Class I biological safety cabinet
1~ 1
rim ~ ~ ~ ~
114~1
\ / ~
1':
Lit
~.~
Type A
1 1
~-~,1
At\
\N 11
...1- 1...
,: ~. ~'' ~
A. ~ I, ~ ~ ../..
,..../
.::....'.
.
NEW
li ~ ~ t ~
;~1
\ ~\
~ \\
IL I I I I
~ 11411
~ ,
W~
ll
Type B
FIGURE A.2 Class II biological safety cabinets
APPENDIX A
Class III. The Class III cabinet (Figure A.3) is a
totally enclosed ventilated cabinet of gas-tight con-
s~uction. Operations within the Class III cabinet are
conducted through attached nabber gloves. When in
use, the Class III cabinet is maintained under nega-
tive air pressure of at least 0.5 in. water gauge. Sup-
ply air is drawn into the cabinet through HEPA fil-
ters. The cabinet exhaust air is filtered by two HEPA
falters, installed in series, before discharge outside of
the facility. The exhaust fan for the Class III cabinet
is generally separate from the exhaust fans of the
facility's ventilation system.
Use of cabinets. Personnel protection provided
by Class I and Class II cabinets is dependent on the
inward airflow. Since the face velocities are similar,
they generally provide an equivalent level of person-
nel protection. The use of these cabinets alone,
however, is not appropriate for containment of
highest-risk infectious agents because aerosols may
accidentally escape through the open front.
The use of a Class II cabinet in the microbiologi-
cal laboratory offers the additional capability and
advantage of protecting materials contained within it
from extraneous airborne contaminants. This capa-
bility is provided by the HEPA-filtered, recirculated
mass airflow within the work space.
The Class III cabinet provides the highest level
of personnel and product protection. This protection
is provided by the physical isolation of the space in
which the infectious agent is maintained. When these
cabinets are required, all procedures involving infec
1
7W
5Z5Z
l
Was
11 ~
\
f TV
d, itt
O O O O
FIGURE AN Class m biological safety cabinet
OCR for page 131
APPENDIX A
tious agents are contained within them. Several Class
III cabinets are therefore typically set up as an inter-
connected system. All equipment required by the
laboratory activity, such as incubators, refrigerators,
and centrifuges, must be an integral part of the cabi-
net system. Double-doored autoclaves and chemical
dunk tanks are also attached to the cabinet system to
allow supplies and equipment to be safely introduced
and removed.
Personnel protection equivalent to that provided
by Class III cabinets can also be obtained with a
personnel suit area and Class I or Class II cabinets.
This area is one in which the laboratory worker is
protected from a potentially contaminated environ-
ment by a one-piece positive-pressure suit ventilated
by a life-support system. This area is entered through
an airlock fitted with airtight doors. A chemical
shower is provided to decontaminate the surfaces of
the suit as the worker leaves the area. The exhaust air
from the suit area is filtered by two HEPA units
installed in series.
APPENDIX A.2. IMMUNOPROPHYLAXIS
An additional level of protection for at-risk per-
sonnel may be achieved with appropriate prophylactic
vaccinations. A written organizational policy which
defines at-risk personnel, which specifies risks as
well as benefits of specific vaccines, and which dis-
tinguishes between required and recommended
vaccines is essential. In developing such an organiza-
tional policy, these recommendations and require-
ments should be specifically targeted at infectious
diseases known or likely to be encountered in a par-
ticular facility.
Vaccines for which the benefits (levels of
antibody considered to be protective) clearly exceed
the risks (local or systemic reactions) should be re-
quired for all clearly identified at-risk personnel.
Examples of such preparations include vaccines
against yellow fever, rabies, and poliomyelitis. Rec-
ommendations for giving less efficacious vaccines,
those associated with high rates of local or systemic
reactions, or those that produce increasingly severe
reactions with repeated use should be carefully con-
sidered. Products with these characteristics (e.g.,
cholera, tularemia, and typhoid vaccines) may be
recommended but should not ordinarily be required
131
for employment. A complete record of vaccines re-
ceived on the basis of occupational requirements or
recommendations should be maintained in the em-
ployee's permanent medical file.
Recommendations for the use of vaccines,
adapted from those of the Public Health Service
Advisory Committee on Immunization Practices, are
included in the agent summary statements in Section
V.
APPENDIX Am. SURVEILLANCE OF
PERSONNEL FOR LABORATORY
ASSOCIATED RICKETTSIAL INFECTIONS
Under natural circumstances, the severity of dis-
ease caused by rickettsial agents varies considerably.
In the laboratory, very large inocula which might
produce unusual and perhaps very serious responses
are possible. Surveillance of personnel for labora-
tory-associated infections with rickettsial agents can
dramatically reduce the risk of serious consequences
of disease.
Recent experience indicates that infections treated
adequately with specific anti-rickettsial chemother-
apy on the first day of disease do not generally pres-
ent serious problems. Delay in instituting appropriate
chemotherapy, however, may result in debilitating or
severe acute disease ranging from increased periods
of convalescence in typhus and scrub typhus to death
in R. rickettsii infections. The key to reducing the
severity of disease from laboratory-associated infec-
tions is a reliable surveillance system which includes
(1) round-the-clock availability of an experienced
medical officer, (2) indoctrination of all personnel
into the potential hazards of working with rickettsial
agents and advantages of early therapy, (3) a report-
ing system for all recognized overt exposures and
accidents, (4) the reporting of all febrile illnesses,
especially those associated with headache, malaise,
prostration, when no other certain cause exists, and
(5) a nonpunitive atmosphere that encourages report-
ing of any febrile illness.
Rickettsial agents can be handled in the labora-
tory with minimal real danger to life when an ade-
quate surveillance system complements a staff who
are knowledgeable about the hazards of rickettsial
infections and who put to use the safeguards recom-
mended in the agent summary statements.
OCR for page 132
132
APPENDIX A.4. IMPORTATION AND
INTERSTATE SHIPMENT OF HUMAN
PATHOGENS AND RELATED MATERIALS
The importation or subsequent receipt of etio-
logic agents and vectors of human disease is subject
to the Public Health Service Foreign Quarantine
Regulations (42 C:FR, Section 71.56~. Permits au-
thorizing the importation or receipt of regulated ma-
terials and specifying conditions under which the
agent or vector is shipped, handled, and used are
issued by the Centers for Disease Control.
The interstate shipment of indigenous etiologic
agents, diagnostic specimens, and biological prod-
ucts is subject to applicable packaging, labeling, and
shipping requirements of the Interstate Shipment of
Etiologic Agents (42 CFR, Part 72~. Packaging and
labeling requirements for interstate shipment of etio-
logic agents are summarized and illustrated in Figure
A.4.
Additional information on the importation and
interstate shipment of etiologic agents of human dis-
ease and other related materials may be obtained by
writing to:
Centers for Disease Control
Attention: Office of Biosafety
1600 Clifton Road, N.E.
Atlanta, GA 30333
Telephone: (404) 329-3883
FTS: 23~3883
APPENDIX A.5. RESTRICTED ANIMAL
PATHOGENS
Nonindigenous pathogens of domestic livestock
and poultry may require special laboratory design,
operation, and containment features not generally
addressed in this publication. The importation, pos-
session, or use of the following agents is prohibited
or restricted by law or by U.S. Department of Agri-
culture regulations or administrative policies:
African horse sickness virus
African swine fever virus
Besnoitia besnoia
Boma disease virus
APPENDIX A
Bovine ephemeral fever
Bovine infectious petechial
fever agent
Camelpax virus
Foot and mouth disease virus
Fowl plague virus
Histoplasma (Zymonema)
~ . .
larc~m~nosum
Hog cholera virus
Louping ill virus
Lumpy skin disease virus
Mycoplasma agalactiae
Mycoplasma mycoides
Nairobi sheep disease virus
(Ganjam virus)
Newcastle disease virus
(velogenic strains)
Pseudomonas mallet
Rickettsia ruminantium
Rift Valley fever virus
Rinde~pest virus
Swine vesicular disease virus
Teschen disease virus
Theileria annulata
Theileria bovis
Theileria hirci
Theileria lawrencei
~.
1 rypanosoma Evans
Trypanosoma vivax
Vesicular exanthema virus
Wesselsbron disease virus
The importation, possession, use, or interstate
shipment of animal pathogens other than those listed
above may also be subject to regulations of the U.S.
Deponent of Agriculture.
Additional information may be obtained by writ-
ing to:
Chief Staff Veterinarian
Organisms and Vectors
Veterinary Services
Animal and Plant Health Inspection Service
U.S. Department of Agriculture
Hyattsville, MO 20782
Telephone: (301) 436-8017
FTS 436-8017
OCR for page 133
APPENDIX A
APPENDIX A.6. RESOURCES FOR
INFORMATION
Resources for information, consultation, and
advice on biohazard control, decontamination proce-
dures, and other aspects of laboratory safety manage-
ment include:
Centers for Disease Control
Attention: Office of Biosafety
Atlanta, GA 30333
Telephone: (404) 329-3883
FTS 236-3883
133
National Institutes of Health
Attention: Division of Safety
Bethesda, MD 20892
Telephone: (301) 496-1357
FTS 496-1357
National Animal Disease Center
U.S. Department of Agriculture
Ames, IA 50010
Telephone: (515) 862-8258
FTS 862-8258
OCR for page 134
134
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Representative terms from entire chapter:
containment equipment
