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OCR for page 13
Safe Handling of Infectious
Agents
A. GUIDELINES FOR HANDLING
PATHOGENIC MICROORGANISMS
In 1984, the Centers for Disease Control (CDC)
and the National Institutes of Health ~ jointly
published a set of guidelines for the safe handling of
pathogenic microorganisms [1051. These guidelines,
developed over a period of several years in consulta-
tion with experts in the field, remain the best judg-
ments available; they are reproduced here in their
entirety, as Appendix A. The reader should consult
these guidelines in deciding on the appropriate level
of precaution to use in the handling of a particular
organism.
Guidelines for handling agents identified after the
CDC/NIH publication are published as Agent Sum-
mary Statements in Morbidity and Mortality Weekly
Report ~R), issued by the CDC. The Agent
Summary Statement for human immunodeficiency
virus (HIV) [36] is reprinted here as Appendix B.
and additional MMWR articles on HIV ("Recom-
mendations for Prevention of HIV Transmission in
Health-Care Settings" [34,381) are reprinted here as
Appendix C.
Throughout this and the following chapters, fre-
quent reference is made to Biosafety Levels 1 through
4. These levels are described in the CDC/NIH publi-
cation (Appendix A). Table A.1 of this appendix
summarizes the practices, techniques, and safety
equipment prescribed for each level.
B. ORGANISMS POSING SPECIAL RISKS
The risk of acquiring an infection in the labora-
tory is influenced by many variables. Among these
factors are the health and immune status of the labo
.
ratory worker, the suitability of the laboratory for
work with highly pathogenic agents, the characteris-
tics and the concentrations of the microbe being
handled, and the specific manipulations involved in
its handling.
Studies of infections acquired by personnel work-
ing in microbiological laboratories have been carried
out by several investigators over the past half-cen-
tury [42,84,101,105,120,121] and have identified a
number of potential human pathogens that are clearly
more frequent causes of laboratory-acquired illnesses
than are others (see Chapter 2, above). Organisms
falling in this category are to be found among vi-
ruses, bacterial rickettsiae, and fungi. Awareness of
those species with a high potential for invading nor-
mal humans should lead to the use of appropriate
precautions to minimize the risk of infection.
Among the agents that have been identified in
recent years as posing the greatest risk of infection to
laboratory and ancillary personnel of diagnostic labo-
ratories are the virus of hepatitis B. Mycobacterium
tuberculosis, and Shigella spp. [60,70,1211. A par-
tial list of other agents known to pose greater than
average risk to laboratory workers includes Brucella
spp. Salmonella spp., leptospires, Coxiella burnetii,
Rickettsia spp., and Coccidioides immitis. The re-
cently identified virus of AIDS (HIV), on the other
hand, poses a low risk of occupational infection to
laboratory workers, except to those working with
concentrated virus suspensions [37,1431. The sup-
plement to the CDC/NIH guidelines recommends,
therefore, that HIVs be handled according to the
standards and special practices of Biosafety Level 2
or 3, depending on the concentration or quantity of
virus or the type of laboratory procedure used (see
Appendix B).
13
OCR for page 14
14
No agent that is a component of the normal or
abnormal microbial flora of man should be regarded
as lacking totally in pathogenic potential, and all
microorganisms should be handled with appropriate
techniques. With the increase in research in virology
in the past half-century, laboratory infections with
viruses have increased relative to those caused by
bacteria and mycoplasmas.
An important defense against infection with some
viral agents is immunity induced by vaccination.
Whenever a vaccine is available (see Table 5.2), its
use should considered for those at risk of exposure
prior to their handling of the virus in question. Un-
der certain circumstances, when work with highly
virulent agents is contemplated, it may be necessary
to consider the administration of an experimental
vaccine. Because of the potential risk of injury to the
fetus from apparent or inapparent viral infection,
special precautions, including temporary reassign-
ment, may be considered for female personnel who
are pregnant or are contemplating pregnancy. (See
Chapter 5, Section D.)
All personnel working with infectious agents
should have documented evidence of immunization
with the vaccines required by most jurisdictions for
admission to elementary school, e.g., diphtheria, teta-
nus, pertussis, poliomyelitis, measles, mumps, and
rubella. In addition, vaccines for preventing infec-
tions with other agents to which they may be ex-
posed, if available, should be offered, and in certain
circumstances consideration should be given to mak-
ing such immunization mandatory.
Acceptance of immunization against, or demon-
stration of proven immunity to, hepatitis B virus
should be a precondition for the employment of all
workers who will be handling human blood or body
fluids. If the medical program of the hiring organiza-
tion includes a serum bank, a sample should be ob-
tained at the time of employment and stored in the
frozen state, to provide a baseline for subsequent
immunologic assays as required (see Chapter 5, Sec-
tion D).
C. HAZARDS FROM VERTEBRATE
ANIMALS AND INSECTS IN THE
LABORATORY
Personnel who work with experimental vertebrate
animals in the laboratory, or who receive and handle
BiOS~ETY IN THE ~O=TORY
specimens from vertebrate animals, should be cogni-
zant of the potential for exposure to zoonotic patho-
gens and to allergenic animal dancers, urine, and
saliva
A list of zoonotic pathogens and potential animal
sources of infection for humans is included in Ap-
pendix D; the information for this table was derived
from references 6, 17, 21, 26, 53, and 69. While it is
recognized that many of the agents listed are not
significant hazards under ordinary laboratory circum-
stances, laboratory staffs should recognize the dan-
gers of zoonotic pathogens and should realize, for
example, that protozoan cysts and larval stages of
certain helminths in fecal material can be infectious
[261. Application of the seven basic rules of bio-
safety cited in Section F of this chapter will greatly
reduce the risks of infection while handling verte-
brate animals or specimens obtained from them (see
also Section G of this chapter).
Strong consideration should be given to immu-
nizing employees with appropriate vaccines against
zoonotic agents, if available (see Table 5.2~.
Numerous agricultural, veterinary, and human
disease research laboratories are involved in the pro-
duction and maintenance of insects. Insects are also
produced for regulatory and control activities (e.g.,
screwworm control, which involves the release of
insects into the environment). The human health
hazards of insect production have been recognized
recently. In addition to the hazards associated with
insect bites, allergic reactions and respiratory dis-
eases may result from contact with, or aerosol expo-
sure to, various insect developmental stages, insect
waste products (e.g., body hairs and feces), ingredi-
ents used in insect diets, or mold spores and bacteria
that contaminate larval diets. Repeated exposure
over a period of months or years may produce respi-
ratory ailments or other manifestations of allergic
reactions in susceptible individuals.
During the preplacement medical evaluation at
the time of hiring or job assignment, a history of
allergies to vertebrate animals or insects that the
prospective employee is likely to encounter should
be elicited. After hiring, the periodic monitoring
medical examinations should include an evaluation
for the development of allergies (see Chapter 5, Sec-
tion D). The prevalence of allergies among person-
nel who work with or are exposed to vertebrate labs
ratory animals has been estimated to be 11 to 30
OCR for page 15
SAFE HANDLING OF INFECTIOUS AGENTS
percent [141. Some individuals may become very
sensitive to low concentrations of allergens [150,1511.
More than 300 cases of allergic reactions that proba-
bly resulted from the inhalation of insect-derived
materials have been reported [111. More than 40
species (among eight orders) of insects were associ-
ated with work-related allergic symptoms among U.S.
Department of Agriculture employees working with
insects [103. Insect allergy questionnaires and sur-
veys indicate that respiratory symptoms (e.g., sneez-
ing, coughing, and chest tightness) and eye and skin
irritation or skin rash are the mayor symptoms in
those with complaints of insect allergy r21,1461.
Inhalation of airborne material was reported as the
mechanism most frequently responsible for allergic
symptoms in persons working in insect-rearing fa-
cilities [21,1461.
Most insect-related health problems develop after
repeated exposure, and severity often increases with
continued exposure. Sensitivity and susceptibility
vary greatly among individuals. The allergic symp-
toms of conjunctivitis, rhinitis, sinusitis, asthma, or
pruritus and dermatitis can develop in from less than
one year to many years after initial exposure. Whether
or not people with allergies are more likely to de-
velop additional allergies to animal products is con-
troversial. Precluding allergic individuals from
employment does not eliminate the problem, since
nonallergic individuals also can become sensitized.
Reducing contamination levels and reducing ex-
posure are the best preventive measures. This may
be accomplished by engineering controls such as
fUtration and directional control of airflow, or by the
use of filter-top cages and directional airflow racks
to prevent the allergens from reaching the worker.
The selection, design, and utilization of such equip-
ment are the most important steps in controlling res-
piratory hazards. Respirators should be used only
for temporary or intermittent work, such as during
maintenance work on the ventilation equipment, and
should not be relied upon as a permanent solution
[151]. It may be appropriate for vertebrate animal
caretakers, insect production workers, laboratory
personnel, and others who work with animals or who
enter the animal holding areas to wear gloves, eye
protection, and a mask covering the nose and mouth.
It is good practice to change from street clothing to
laboratory garb. All persons who enter the animal
_
15
holding area should adhere to the protocols and the
regulations that apply to activities in the vivarium.
D. PRIMARY AND CONTINUOUS CELL
CULTURES
Cell cultures, in general, present few biohazards
in the laboratory, as evidenced by their extremely
wide usage and the rare cases of transmitted infec-
tions to laboratory personnel. Primary cell cultures
initiated with tissues from infected humans or ani-
mals are recognized hazards. Thus macaques, and
possibly other Old World monkeys, may have latent
Herpesvirus simiae (B-virus) infections and present
a hazard to personnel handling these animals and
their tissues. At least 24 documented cases of infec-
tions of laboratory workers handling primary cell
culture tissues (e.g., primary rhesus monkey kidney
cells) have occurred in the past 30 years [463. A
particularly noteworthy instance of the laboratory
infection of a number of workers by an adventitious
agent from monkeys occurred in 1967 in Marburg
and Frankfurt, Germany, and in Yugoslavia. Labo-
ratory workers handling tissues and cell cultures from
African green monkeys developed an acute febrile
illness. Seven deaths occurred among 31 documented
cases due to a previously unknown virus, subse-
quently named Marburg virus. It has not occurred in
laboratory workers since those incidents [1151. Tis-
sues from mice infected with lymphocytic chorio-
meningitis (LCM) virus or from chickens carrying
Newcastle Disease virus (NDV) also present poten-
tial hazards, but such laboratory infections have not
been reported. Clearly, primary cell cultures pre-
pared from humans infected with hazardous agents
(e.g., HIV) present danger of infection, and such
tissues must be handled with the precautions required
of the known or suspected infectious agent (see Ap-
pendix A).
Continuous cell cultures present no real docu-
mented risk in the laboratory unless they are care-
lessly contaminated with an infectious agent. All
continuous cell lines should be regularly monitored
for contamination with infectious agents, and it should
be emphasized that all nutrient media or other rea-
gents that may contain ingredients of biologic origin
must be treated as though they contain potentially
infectious agents.
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16
E. HANDLING OF NECROPSY AND
SURGICAL SPECIMENS
1. Introduction
Necropsy and surgical pathology expose health
care workers to various infectious agents that may be
in human tissues or associated body fluids. Proper
handling can minimize the risk of infection. Because
the consequences of infection are grave, agents of
principal concern are hepatitis B virus (KIEV), hu-
man immunodeficiency virus (HIV), Creutzfeldt-
Jakob agent (CJA), and M. tuberculosis, although a
number of other infectious agents, including viruses,
rickettsiae, bacteria, fungi, and parasites, pose poten-
tial risks. The principal means of acquiring infec-
tions when performing anatomic examinations are
through breaks in the skin caused by needle punc-
tures, cuts, or severe dermatitis, by contamination of
mucous membranes, and by inhalation. The risk of
infection is decreased by preventing breaks in the
body surfaces, preventing the formation of droplets
that might contaminate surface breaks or mucous
membranes, inserting barriers such as rubber gloves,
goggles, and masks between the infectious hazard
and the potential site of entry, and preventing the
generation of aerosols.
Fresh tissue may be infected with agents such as
HBV or HIV even if there is no history of such
infection. Those who perform autopsies or handle
fresh tissue or blood on a regular basis should have
immunity to HBV.
To date, in the United States there are few recom-
mendations for biosafety in necropsy and surgical
pathology [9,79,90], although such have been pro-
posed in Great Britain [471. Recommendations have
been developed for Creutzfeldt-Jakob disease (CJD)
[109], and a number of publications have helped to
define the risks associated with this agent [9,24,55,561.
The National Committee for Clinical Laboratory Stan-
dards DECALS) has published proposed guidelines,
entitled Protection of Laboratory Workers from In-
fectious Disease Transmitted by Blood and Tissue,
that include necropsy and tissue handling recom-
mendations [901. Independently, a committee of the
College of American Pathologists is developing rec-
ommendations for necropsy and surgical pathology.
In a given institution, there should be a clear defi-
nition of the responsibility for biosafety in the han-
dling of a body from the time of death until it is
BIOSAFETY IN THE LABORATORY
transferred to the mortician or incinerated, and for
surgically removed tissue. For an autopsy, the pro-
sector (generally a pathologist) is responsible for
biosafety. It is beyond the scope of this publication
to discuss autopsy biosafety in detail, but some ma-
jor points are considered below.
2. Necropsy
a. Routine Necropsies
Because of the high incidence of asymptomatic
carriers of BV and HIV in hospital or forensic
.
autopsies, all cases should be considered potentially
infectious and the necropsy performed carefully. Care
should be taken to minimize chances of needle sticks,
cuts, or abrasions. Risk of contamination of mucous
membranes should be decreased by wearing safety
goggles and a surgical mask, or a face shield.
b. Necropsies on Bodies Known to Be Infected
Bodies for necropsy should be appropriately la-
beled if they are known to be infected with such
agents as HBV, CJA, HIV, or M. t~erc~osis. The
medical record should also indicate the diagnosis.
Before beginning a dissection, it may be helpful to
discuss the case with the clinician to clarify the ex-
tent of examination required. Autopsy assistants
should be informed of the nature of the clinical diag-
nosis so that special disinfectants, such as sodium
hypochlorite solution (household breach diluted 1:100
in tap water), can be prepared prior to beginning the
dissection.
~ · .
In addition to the prosector and autopsy assistant,
it is helpful to have a "circulating" assistant who
remains "uncontaminated," thus preventing contami-
nation of telephones, cameras, drawer pulls, cultures,
papers, and other items by those doing the dissection,
and confining the contamination to the necropsy table
area
Protective clothing should include the following:
· a scrub suit covered with a long-sleeved gown
or a long-sleeved coverall suit plus an impervious
apron;
· impervious shoe covers;
· head covering;
· goggles or eyeglasses to prevent conjunctival
contamination;
OCR for page 17
SAFE HANDLING OF INFECTIOUS AGENTS
· face mask to decrease risk of droplet con-
tamination of mucous membranes, or inhalation
of aerosols; and
· double gloves (preferably including one pair
of heavy-duty gloves).
In performing the autopsy, it may be helpful to
cover rib ends with towels to decrease risk of cuts.
Dissection in the body should be limited to one pro-
sector at a time. Use of scissors when possible will
decrease the risks of cuts. Production of droplets and
aerosols should be minimized. Use of a Stryker saw
to open the skull or to cut bone is controversial
because of the potential for generation of droplets
and aerosols. Some authorities advocate using a
hand saw, whereas others recommend using the
Stryker saw with a ~PA-fUtered vacuum attach-
ment or covering the equipment with a wet towel.
The saw and aerosol control apparatus should be
adequately disinfected after use. In cases of CJD,
there should be special care not to cut the brain. A
new technique for the removal of the brain from
cases of AIDS at autopsy has been developed in
which the sawing is done inside a plastic bag [7X,791.
The British Committee on Dangerous Pathogens has
suggested performing limited postmortem examina-
tions with discrete tissue sampling for most AIDS
cases [il.
Any spills of blood or body fluid should be cleaned
immediately with a solution of household bleach di-
luted 1:100 in tap water.
Specimens for culture or other clinical laboratory
examinations should be handled in the same fashion
as in patient care areas, with care being taken not to
contaminate the outside of the container.
Disposable syringes and needles and knives should
be placed in a leak- and puncture-resistant container
for subsequent disposal.
If persons are cut or punctured while dissecting or
handling tissues or body fluids, the wound should be
encouraged to bleed, flushed with abundant water,
and treated with an antiseptic such as povidone-
iodine. The accident should be reported to the appro-
priate persons such as the safety officer, employee
health director, or laboratory supervisor, depending
on the institutional requirements.
At completion of the autopsy, the body should be
packed with absorbent material to prevent seepage of
liquids and should be washed with a 1:100 dilution
of household bleach or other appropriate disinfecting
17
agent. Tags on the body should note the infectious
hazard. The body should then be placed in a plastic
ban, which is also labeled with the appropriate haz-
ard warning (e.g., "Blood and Body Fluid Precau-
tions"~. In addition to labels on the body, the morti-
cian should be notified specifically of the infection
hazard. As discussed below in Section F of this
chapter, however, the use of special hazard warning
labels should not lead to the misconception that other
bodies are not potentially infectious.
When finished, prosectors and autopsy assistants
should remove protective clothing in the autopsy
room and place it in appropriate containers for incin-
eration or transport to the isolation laundry, and should
then shower. Soiled disposable items should tee placed
in biohazard bags for incineration. Soiled linens
should be double-bagged in durable, labeled isola-
tion bags and handled in the same manner as hospital
isolation linen.
Tissues that are to be saved should be placed in
formalin (1 part tissue to 10 parts formalin) and
should be cut thin enough (<2 cm thickness) to en-
sure penetration. Fixation in 10 percent formalin
will inactivate most infectious agents; mycobacteria
and CJA are exceptions (see below).
Instruments should be autoclaved or soaked in a
1:100 dilution of household bleach, or other appro-
priate disinfectant, for 30 minutes to 1 hour. Only
stainless steel can be placed in hypochlorite solution.
The table and the floor around the table should be
cleaned with a 1:100 dilution of household bleach, or
with a germicide approved (by FDA) for use as a
"hospital disinfectant" that is also tuberculocidal. If
a mop is used, it should be autoclaved.
Creutzfeldt-Jakob agent is particularly resistant
to killing, requiring autoclaving at 121°C for at least
30 minutes; it can survive in 10 percent formalin for
many months [9,561. Paraffin blocks may therefore
contain infectious CJA. CJA is usually inactivated
by household bleach at 0.5 to 5 percent concentra-
tions, with the higher concentration being more ef-
fective but also more corrosive [241. The agent is
most susceptible to IN NaOH. Contaminated mate-
rial should be autoclaved as above, inactivated with
one of the chemicals cited above, or incinerated. It
has recently been noted that formalin-fixed brain
tissue can be autoclaved to inactivate CJA and then
processed for histologic sections [801.
HIV and HBV are readily inactivated by a variety
of agents, including formalin, hypochlorite, and io
OCR for page 18
18
dine-based disinfectants. Special care should be ex-
ercised when performing autopsies on patients who
died of infections with these agents.
Mycobacterium tuberculosis is moderately resis-
tant to 10 percent formalin, requiring prolonged ex-
posure for complete killing [1101; formalin-fixed tis-
sue from recent cases may therefore be infective.
The usual route of infection is the inhalation of aero-
sols generated during necropsy, or the trimming of
tissue for histologic processing. Occasionally, the
organism is introduced into a cut ("prosector's wart').
3. Surgical Pathology
The hazards of surgical pathology are similar to
those of autopsy. Many tissues have been fixed in
formalin when received and are thus not infectious,
with the exceptions noted above. Such tissues are
best disposed of by incineration, more for aesthetic
reasons than those related to biohazard.
Cryostats used for frozen sections present a par-
ticular problem [1231. The operator should wear
gloves, gown, and mask when cutting the section,
whether or not the patient is known to have a disease
transmitted by blood or tissue. In addition, the cry-
ostat should be disinfected periodically (at least
weekly). If it is known that the patient has an infec-
tion that represents a hazard such as AIDS or tuber-
culosis, frozen sections should be prepared only when
absolutely necessary. The cryostat should be disin-
fected with an appropriate disinfectant as soon as
possible after the sections have been cut, to remove
contaminated tissue fragments and to decontaminate
surfaces.
All human anatomical waste and cadavers should
be disposed of by burial or incineration. The incin-
erator must be appropriately designed for handling
anatomical laboratory waste. Cadavers containing
radioactive isotopes or antineoplastic drugs require
special handling during autopsy and for disposal (see
Chapter 4~.
F. GOOD LABORATORY PRACTICES
1. Introduction
A number of reports and studies [S,15,40,67,
6S,77,84,96,101] attest to the potential for occupa-
tionally acquired infection by laboratory personnel
working directly with microbial agents. The signifi
BIOSAFETY IN THE LABORATORY
cant element to be derived from these reports is that
the exact source or cause of the infection could be
documented in fewer than 20 percent of the cases.
This finding provides strong evidence that exposures
and consequent infection occur not as the result of
overt accidents but during the performance of routine
procedures.
2. Routes of Exposure
The nature of infective contaminants dispersed
during the performance of any laboratory procedure
is a direct function of the amount of energy applied
during the procedure. Low-energy procedures (e.g.,
removal of screw caps and pouring of liquid me-
dium) principally yield droplets that are dispersed
onto body and work surfaces. Exposure of personnel
in these instances occurs usually through breaks in
the skin surface caused by cuts, scratches, and other
cutaneous lesions, or by ingestion of infectious mate-
rial transferred to the mouth by hands or objects. On
the other hand, procedures involving application of
large amounts of energy, such as homogenization
and centrifugation, have the potential for generating
respirable aerosols. It should be recognized that a
large number of procedures may result in the genera-
tion of a mixture of droplets and aerosols with the
result that exposure by more than one route is pos-
sible.
While it has been typical to focus on respirable
aerosols as the primary source of infection for labo-
ratory personnel, it is essential that other routes of
exposure be considered: contact, oral, ocular, and
inoculation.
a. Contact Route
The control of potential exposure by the contact
route requires that procedures be conducted in a
manner that avoids contamination of body or work
surfaces. This is accomplished through the use of
gloves and other personal protective clothing, pro-
tection of work surfaces with appropriate absorbent
disposable covering, use of care in the performance
of procedures, and cleaning and disinfecting work
surfaces. Procedures that can result in the generation
of droplets include decanting of liquids, pipetting,
removal of screw caps, vortex mixing of unsealed
containers, streaking inocula on agar surfaces, and
inoculation of animals.
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SAFE HANDLING OF INFECTIOUS AGENTS
It should be recognized also that dispersal of con-
taminants to other surfaces can occur by their trans-
fer on the gloves of the labo~auxy worker, by the
placement of contaminated equipment or laboratory
ware, and by the improper packaging of contami-
nated waste.
b. Oral Route
A number of procedures carried out in the labora-
tory and animal facility offer the potential for either
direct or indirect exposure by the mal route. The
procedure that offers the greatest potential for expo-
sure by ingestion is mouth pipetting. Clearly, such
exposures are completely avoidable through the use
of mechanical pipetting devices. Indirect oral expo-
sures can be avoided through the use of the personal
hygienic practice of regular hand washing, and by
not placing any objects, including fingers, into the
mouth. The wearing of a surgical mask or face
shield will serve to protect the worker against the
splashing of infectious material into the mouth.
c. Ocular Route
The wearing of a face shield, safety glasses, or
goggles will protect the worker against splashing
infectious material into the eyes.
d. Inoculation Route
The single procedure that presents the greatest
risk of exposure through inoculation is the use of a
needle and syringe. These are used principally for
the transfer of materials from diaphragm-stoppered
containers and for the inoculation of animals. Their
use in the transfer of materials from diaphragm-stop-
pered containers can, in addition, result in the disper-
sal of infectious material onto surfaces and into the
air. Depending upon the route of inoculation of
animals, the use of a needle and syringe may also
result in the contamination of their body surfaces.
Because of the imminent hazard of self-inoculation,
the use of the needle and syringe should be limited to
those procedures where there is no alternative, and
then the procedure should be conducted with the
greatest of care. Inoculation can also result from
animal bites and scratches.
19
e. Respiratory Route
Several procedures have the potential for generat-
ing respirable aerosols. Included are sonication,
homogenization, centrifugation, vigorous discharge
of fluids from pipettes, heating inoculating loops,
opening lyophilized preparations, and changing of
the litter in animal cages (see Chapter 3, Section I).
3. Prevention of Exposure
The time-honored approach for the safe handling
of infectious agents involves the use of a combina-
tion of strategies. This is accomplished by
· controlling the hazardous material at the
source to prevent release into the workplace,
· minimizing accidental release of the mate-
rial, and
· protecting the worker against contact with
the material.
However, He safe conduct of work with infec-
tious material is primarily dependent upon the appli-
cation of good laboratory practices by the laboratory
worker (see below).
4. The Seven Basic Rules of Biosafety
The most common means of exposure can be
essentially eliminated as occupational hazards by
following the seven basic rules of biosafety:
· Do not mouth pipette.
· Manipulate infectious fluids carefully to
avoid spills and the production of aerosols and
droplets.
· Restrict the use of needles and syringes to
those procedures for which there are no alterna-
tives; use needles, syringes, and other "sharps"
carefully to avoid self-inoculation; and dispose of
"sharps" in leak- and puncture-resistant contain-
ers.
· Use protective laboratory coats and gloves.
· Wash hands following all Laboratory activi-
ties, following the removal of gloves, and immedi-
ately following contact with infectious materials.
· Decontaminate work surfaces before and
after use, and immediately after spills.
· Do not eat, drink, store food, or smoke in the
laboratory.
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20
BIOSAPEIY IN THE LABORATORY
These simple and effective work practices can be tious agents. These practices are described in more
implemented readily by laboratory management at detail in subsequent sections of this chapter.
minimal cost and with no loss of employee effi
ciency or productivity. Even in the absence of more
sophisticated means for providing safety in the labo
ratory, these practices can achieve a major reduction
in the risk of accidental infection.
Laboratory activities that pose the risk of infec
tion via airborne aerosols or droplets demand the use
of special safeguards. For many "airborne patho
gens," the human infectious dose may be as low as
one viable microorganism, as demonstrated for tu
berculosis [107,1081. It is recommended that bio
logical safety cabinets or other primary containment
devices be used for all manipulations of materials,
including clinical specimens, known to contain or
suspected of containing microorganisms capable of
infecting by the respiratory route. In laboratories
where such materials are handled, the ventilation
system should provide directional airflow from
"clean" to "contaminated" areas, and the air should
not be recirculated.
The recommended procedures listed above, tar
geted at minimizing overt occupational exposures,
constitute the basic essentials of good laboratory prac
tice. Furthermore, these procedures are also effec
tive in reducing or eliminating overt exposure to the
variety of indigenous bacterial, viral, fungal, and
parasitic agents present in the community and com
monly found in clinical material submitted to the
laboratory for examination. The ultimate responsi
bility for assessing the risk of occupational infec
tions and for implementing appropriate practices, as
well as for providing adequate facilities and contain
ment equipment, rests with the laboratory director.
5. Summary
Virtually all laboratory procedures have the po
tential to disperse infectious material into the
workplace. Laboratory workers should be aware of
these potential hazards and exercise a high degree of
care during all manipulations of infectious materials.
As evidenced by the data accumulated in the review
of laboratory-acquired infections by Pike [1011, ex
posure of laboratory workers is not often associated
with overt accidents. More than 80 percent of labo
ratory-associated infections could not be ascribed to
any specific event. It is critical, therefore, that labo
ratory workers recognize that good microbiological
practices are required to prevent exposure to infec
G. TRANSPORTATION AND SHIPMENT
OF SPECIMENS
1. Introduction
Although it is obvious that biological specimens
should be properly packaged, labeled, shipped, and
received, concerned national and international or-
ganizations have found it necessary to develop rec-
ommendations and guidelines because of the fear of
accidents and spills involving such materials
[71,86,147,1481. Federal regulations govern the pack-
aging and shipping of hazardous materials. The im-
portation and subsequent transfer between laborato-
ries of etiologic agents and vectors of plant, animal,
and human diseases (including zoonotic agents) are
controlled through permit systems.
2. Packaging. Shipping, and Handling of
Biological Specimens
The shipment of diagnostic specimens, biological
products, and etiologic agents concerns everyone in-
volved in the process. Infectious materials that are
properly packaged and handled may pose considera-
bly lower risks of accidental exposure for nonlabora-
tory personnel who come in contact with the ship-
ment in transit. Proper packaging also may ensure
considerate and prompt handling of valuable speci
mens.
The shipping of unmarked and unidentified etio-
logic agents is prohibited. Requirements for the
proper method of containment in the packaging and
the use of the hazardous warning label are stipulated
in the U.S. Public Health Service Interstate Shipment
of Etiologic Agents Regulation [1291. Comparable
requirements of the International Civil Aviation Or-
ganization (ICAO) apply to the international ship-
ment of diagnostic specimens and infectious agents.
The containment packaging and hazard warning
labeling specified in the U.S. Public Health Service
Regulation [129] for the shipment of etiologic agents
is illustrated below in Figure 3.1. The package should
. ~
consist ot
· a securely closed, watertight primary con-
tainer (test tube, vial, or ampoule);
· a durable, watertight secondary container;
and
OCR for page 21
SAFE CANDLING OF INFECTIOUS AGENTS
r ~1', ~
ma
BIOMEDICAL
MATERIAL
STANDARD FORM 420 JUNE 1973
PRESCRIBED BY DEPT HEW (4.2 CFR)
420~lOt
1i
FIGURE 3.1 Containment packaging arid heard warning labeling specified
by die U.S. Public Heralds Service. Reprinted from US. Code of Federal
Regulations, Title 42, U.S. Public Heals Service, Part 72.
· a tertiary or outer shipping container.
The space between the primary and secondary
container must be filled with absorbent material suf-
f~cient to absorb the contents of the primary con-
tainer should there be leakage during transit. The
outside of the primary container should be examined
and cleaned to remove blood, feces, or other con-
taminants before it is packaged for shipment.
The exteriors of packages containing cultures of,
or suspensions of, etiologic agents should have af-
fixed to them the "Etiologic Agent Biomedical
Materials" hazard warning label illustrated in Figure
3.1. The packaging and the labeling requirements of
the regulation cited also apply to the local transport
of etiologic agents and diagnostic specimens by cou-
rier or by other delivery services. Similar require-
ments and restrictions applicable to the shipment of
etiologic agents, diagnostic specimens, and biologi-
cal products by all modes of transportation (i.e., air,
motor, rail, and water) are imposed by the Depart-
ment of Transportation [131] and the U.S. Postal
Senice (Postal Service Manua0, as well as by air-
line carriers and pilots' associations.
The importation of etiologic agents of human
diseases, as well as their subsequent transfer within
the United States, is regulated by the U.S. Public
Health Service (USPHS) [1281. The U.S. Depart-
ment of Agriculture (USDA) similarly regulates the
importation and transfer of etioloic agents of plant
and animal diseases [1251. In addition, the USDA
Animal and Plant Health Inspection Service (APHIS)/
Veterinary Services (VS) Memorandum 593.1 estab-
lishes procedures for the "Importation of Cell Cul
21
lures Including Hybridomas." Examples of the ap-
propriate USPHS and USDA application forms and
permits are included in Appendix E.
A summary of the requirements of the federal
agencies involved in the shipment of biological speci-
mens has been published recently by the American
Type Culture Collection (ATCC) (Rockville, MD
20852-1776~. This document also describes the pro-
cedures used for packaging and shipping the differ-
ent types of cultures of microorganisms and cells
maintained by the ATCC [31.
Procedures for receiving and unpacking etiologic
agents or other potentially infectious materials should
be established by laboratories receiving these items.
Often, such materials are received initially by ship-
ping, clerical, or other nonlaboratory personnel. These
employees should be given specific instructions to
notify laboratory staff promptly of the arrival of such
materials, and to deliver packages unopened directly
to a designated area or person. Shipments of etio-
logic agents or diagnostic specimens should never be
opened in offices or in shipping and other nonlabora-
tory areas.
In the laboratory, the designated specimen-receiv-
ing area should meet the facilities recommendation
for Biosafety Level 2 [86,1051. Microbiological prac-
tices, including the wearing of laboratory coats,
gloves, or other protective clothing, should be fol-
lowed as applicable. A Class I or Class II biological
safety cabinet provides the most suitable work sta-
tion for opening packages and for He initial handling
of incoming specimens [86] (see Chapter 3, Section
J). Specimens that show any evidence of damage or
leakage should be opened in a biological safety cabi
OCR for page 22
22
net only by trained personnel wearing appropriate
protective clothing.
Laboratories should have emergency contingency
plans [147,148] for handling damaged shipments.
Such plans are best prepared by the laboratory super-
visor in conjunction with the laboratory staff and the
safety officer. Emergency plans should be posted in
a conspicuous place in the laboratory for immediate
reference. Emergency plans should provide written
procedures for dealing with
· breakage or spillage of infectious materials,
· exposure of personnel to infectious materi-
als by accidental injection, cuts, or other injuries,
· accidental ingestion or contact of mucous
membranes with potentially hazardous material,
and
· aerosols.
Such emergency plans should include the following:
· decontamination procedures,
· emergency sernces (whom to contact), and
· emergency equipment and its location.
H. LABELING OF SPECIMENS WITHIN
THE LABORATORY
Some form of labeling is necessary to maintain
the identity of specimens in the laboratory and to
ensure that the analytical results obtained are prop-
erly recorded and reported. In addition, it is the
practice in many cases (and may be required as a
condition for accreditation) that special hazard warn-
ing labels be affixed to specimens that are known to
be hazardous (e.g., specimens obtained from patients
known to be infected with hepatitis B virus (REV) or
human immunodef~ciency virus (HIV), or from pa-
tients in high-risk groups for these infections, or
when previous tests of the specimen have shown it to
contain an etiologic agent).
The need for such special labeling is concerned
more with ethical or regulatory issues (e.g., workers'
right-to-know) than with laboratory safety. Unfortu-
nately, the use of special hazard warning labels can
inadvertently lead to the dangerous misconception
that other clinical specimens, not so labeled, can be
handled with less caution. Two levels of laboratory
practice may thus evolve: one for handling hazard-
labeled specimens and another for unlabeled samples.
This must be scrupulously avoided all clinical ma-
terial must be considered to be infectious, and must
BIOS~ETY IN THE STORY
be handled with exactly the same precautions as are
usedfor processing specimens with hazard warning
labels.
It is generally recognized that any clinical speci-
men may contain infectious agents (such as BV or
HIV) regardless of its source, the working clinical
diagnosis, or the testing requested. For example,
published reports indicate that from 1.0 to 1.5 per-
cent of the adults in the United States have serologi-
cal markers indicative of current or previous REV
infection [661. Thus, even though the percentage of
specimens containing an infectious agent may be
higher among samples collected from hepatitis pa-
tients, the total number will probably be greater among
routine specimens, which make up the vast majority
of the materials received in most laboratories. The
potential for worker exposure may, therefore, be ac-
tually greater from the more numerous routine speci-
mens, which would not be identified with a hazard
warning label. From the above considerations, it is
clear that the "Universal Precautions" described by
the CDC [34,38] must be followed when handling all
clinical specimens, whether labeled or unlabeled.
I. PREVENTION OF AEROSOL AND
DROPLET GENERATION
1. Introduction
Exposure to microorganisms dispersed or spread
in the form of infectious aerosols or droplets is an
important source of laboratory-acquired infection.
Infectious aerosols may be composed of dry or liquid
particles typically less than 5 microns in diameter,
which can be produced during the course of many
common laboratory processes. Such aerosols do not
settle quickly and can be dispersed widely through a
ventilation system or otherwise earned long distances
by air streams. If inhaled, the particles in an aerosol
are carried to the alveoli of the lungs. In contrast,
droplets (particles typically larger than 5 microns in
diameter) remain airborne only for a short period of
time and are nonrespirable. Because of their mass,
droplets tend to settle quickly on inanimate surfaces,
or may be deposited on skin or mucous membranes
of the upper respiratory tract. Accordingly, droplets
pose risks of infection associated with direct or indi-
rect contamination of the mucous membranes of the
eyes, nose, or mouth as well as of skin, clothing, and
laboratory equipment.
OCR for page 23
S~E HANDLING OF INFECTIOUS AGENTS
2. Control of Aerosols and Droplets
Almost any handling of liquids or of dry powders
is likely to generate aerosols and droplets; certain
operations such as pipetting, mixing, shaking, grind-
ing, filtering, sonicating, flaming, and centrifuging
have a high potential for aerosol production. Of
these, pipetting may be the most important. Various
reports indicate that pipettes are associated with many
laboratory-acquired infections [98,99,100,102,
104,1203. Hazards relating to pipetting include the
production of aerosols, aspiration of fluid into the
mouth, and contamination of the mouthpiece by the
operator's finger. The last two of these dangers can
be avoided if mouth pipetting is strictly prohibited,
as required by good laboratory practice. A wide
variety of mechanical pipetting devices are available
[63], and mouth pipetting under any circumstances is
absolutely unacceptable.
To minimize aerosol production, pipettes should
be drained gently with the tip against the inner wall
23
of the receiving tube or vessel. No infectious mate-
rial should be expelled forcibly from the pipette, and
air should never be bubbled through a suspension of
infectious agents in an open container. When han-
dling organisms for which Biosafety Level 3 precau-
tions are indicated (e.g., etiologic agents of tubercu-
losis, systemic mycoses, or Q fever), it is recom-
mended that pipetting procedures be carried out in a
biological safety cabinet. The equipment used in the
other operations mentioned above should be selected
for features designed to contain infectious liquids or
aerosols. For example, blenders should have
leakproof bearings and a tight-fitting casketed lid.
Blender bowls, tubes, and other devices likely to
contain aerosols should be opened, filled, and emp-
tied in a biological safety cabinet.
Centrifuges with sealed buckets, safety trunnion
cups, or sealed heads are effective in preventing es-
cape of liquids and aerosols Figure 3.2~. If fluid
should escape from a cup or rotor during high-speed
operation, the potential for extensive contamination
FIGURE 3.2 If a fluid containing an infectious agent were to escape from a centrifuge rotor or cup during high-speed op-
eration, the potential for extensive contamination and multiple infections would be great. The use of sealed buckets, safety
Caution caps, or sealed heads is an effective means of preventing the escape of liquids or aerosols. Courtesy, John H.
Richardson.
OCR for page 24
24
and multiple infections is great. For many speci-
mens, however, such as urine, the standard clinical
centrifuge is satisfactory. There have been compara-
tively few centrifuge accidents reported as the cause
of laboratory-acquired disease, but some of these
caused multiple infections because the accident cre-
ated a large volume of infectious aerosol [1411.
Instruments should be checked regularly to en-
sure that leakage does not occur during operational
procedures. For ultracentrifuges, a HEPA fluter should
be installed between the chamber and the vacuum
pump. If circumstances require such precautions,
centrifuges and other laboratory instruments that can
be enclosed and operated in specially designed safety
cabinets are available. Only those instruments and
cabinets intended for such a combined system should
be used together, otherwise the expected contain-
ment may not be achieved. For example, the air-
streams created by an ordinary benchtop centrifuge
operating in the work space of an ordinary Class II
BiOS~ETY IN THE FLORA TORY
biological safety cabinet can easily overwhelm the
protective air curtain.
Sputum and other clinical specimens submitted
for culture may contain unsuspected microorganisms,
such as mycobacteria, which are highly infectious by
the airborne route. Every effort should be made,
therefore, to minimize the risk of their aerosoliza-
tion. If generation of an aerosol is likely to occur
during the processing of these specimens, the use of
a biological safety cabinet is recommended strongly
for these procedures.
Improper technique in the flaming of inoculating
loops can result in the spread of infectious agents.
Spatter and release of droplets or aerosols can be
prevented by such methods as heating the shaft until
the sample has been heat-dried before flaming the
1Q°P itself (Figure 3.3~. Spatter can also be con-
trolled effectively by using a side-arm burner or elec-
tric microincinerator. Flaming itself can be avoided
by using sterile, disposable plastic loops.
i-! so
FIGURE 33 Proper technique in He flaming of inoculating loops is an important way to prevent the spread of infectious
agents. Courtesy, National Institutes of Health.
OCR for page 25
SAFE HANDING OF INFECTIOUS AGENTS
Early models of certain laboratory instruments,
such as cell sorters and other automated devices,
were not designed for containment and may be a
source of inadvertent contamination in the worl~lace.
Later models generally have overcome the problem,
but users are advised to test all equipment carefully
in order to identify any biological ha~is associated
with its operation.
Regardless of the type of equipment used or the
task performed, the objective is to prevent aerosol
release and to avoid exposure of personnel. These
ends can be accomplished by the laboratory practices
described above and by the use of appropriate equip-
ment, especially biological safety cabinets. Leaks or
escape of aerosols can be detected by using an indi-
cator such as fluorescein. It may be added to a sham
specimen or to water, and processed with the system
or procedure being tested. Its presence can then be
determined on surfaces, on material collected from
key locations, or in specimens from air samplers, by
using an ultraviolet lamp for excitation. Other suit-
able methods may be devised.
]. CONTAINMENT EQUIPMENT
1. Introduction
The risk of exposure of laboratory personnel can
be minimized by the use of carefully selected safety
equipment. A primary objective of containment is to
control aerosols, but in a broader sense safety equip-
ment should serve effectively to isolate the worker
from the toxic or infectious material being processed.
In many situations, however, the need is just the
reverse: i.e., to protect the product or the work from
contamination originating with the worker or the
environment. Finally, there is often the need to pro-
tect both the worker and the product, as in handling
cell cultures and some clinical specimens, or in sur-
gical procedures. The following examples are repre-
sentative of the types of equipment designed to avoid
the most common laboratory hazards, and these types
of equipment are, for that reason, among the most
important.
2. Biological Safety Cabinets
Most laboratory procedures generate aerosols that
may spread infectious material in the work area and
pose a risk of infection to the worker. Biological
25
safety cabinets are used extensively to prevent the
escape of aerosols or droplets and to protect materi-
als from airborne contamination Figure 3.4~. There
are three major types of this very useful safety de-
vice, referred to as Class I, Class II, and Class III.
These instruments are distinct from horizontal or
vertical laminar flow "clean benches," which should
never be used for handling infectious, toxic, or sensi-
tizing material. The Class I biological safety cabinet
is an enclosure with an inward airflow through the
front opening. It may be configured with a full-
width open front or with an installed front closure
panel to which arm-length rubber gloves may be
attached. The exhaust air from the biological safety
cabinet is passed through a HEPA filter so that the
equipment provides protection for the worker and
environment. The product in the cabinet, however,
FIGURE 3.4 Biological safety cabinets, combined with
protective gloves and laboratory coats, provide effective
isolation of die worker from the toxic or infectious mate-
rial being handled. Courtesy, John H. Richardson.
OCR for page 26
26
is subject to contamination by organisms that may be
present in the air supply.
Class II biological safety cabinets provide protec-
tion to the worker, the environment, and the product.
The airflow velocity at the face of the work opening
is at least 75 linear feet per minute Oxfam), and both
the supply and the exhaust air are ~PA-filtered.
Class I and Class II cabinets are partial containment
devices, which, if used in conjunction with good
laboratory practices, can dramatically reduce the risk
of exposure of operators to infectious aerosols and
droplets.
Figure 3.5 [73] shows the airflow patterns and
operating velocities for the five types of Class I and
Class II biological safety cabinets produced in the
United States. All of these biological safety cabinets
provide a comparable level of protection for the user
against exposure to infectious aerosols and droplets,
in that the velocity of the protective inward airflow
through the work opening is essentially the same.
The air quality within the Class I cabinets reflects
that of the laboratory room from which it is drawn,
since there is no filtration of the supply air. The
Class II types provide a very high quality, low-par-
ticulate or particulate-free atmosphere within the work
chamber. Class IIA cabinets are generally suitable
for procedures involving clinical specimens, and thus
are the most commonly used biological safety cabi-
net.
It is emphasized that biological safety cabinets
are not chemical fume hoods. Some of the air (30 to
70 percent) drawn in through the work opening of
Class IIA, IIB1, and IIB3 cabinets is recirculated
within the cabinet. Accordingly, users should be
aware of the possible buildup of hazardous concen-
trations within the cabinet if toxic, flammable, or
explosive materials are used. In addition, users of
Class IIA cabinets should know that nonparticulate
toxic, flammable, or explosive materials are not re-
moved by SPA filters, and are thus discharged
back into the laboratory room.
Class IIB3 units are functionally the same as those
of Class IIA except that the exhaust air from the
former is ducted to the outside directly or via a non-
recirculating exhaust system rather than back into the
laboratory room. Class IIB1 and IIB2 cabinets ex-
haust 70 percent and 100 percent, respectively, of the
intake air and provide containment of infectious aero-
sols. Those contemplating the purchase of Class IIB1
or IIB2 cabinets should be aware of their high air
BIOSAFETY IN THE LABORATORY
demand (700 to 1200 ft3/min), increased energy re-
quirements, and higher purchase and operating costs.
The Class III cabinet is a totally enclosed, gas-
tight work space equipped with protective gloves. It
is ventilated with HEPA-fUtered air and operated
with a negative air pressure of at least 0.5 inches of
water in the cabinet work space. The exhaust air is
passed through two HEPA filters, installed in series,
before being discharged to the outside of the build-
ing, usually through a dedicated exhaust system. Class
III cabinets provide the highest level of worker, prod-
uct, and environmental protection and are appropri-
ate for work with exotic high-risk biological agents,
including those in the Biosafety Level 4 category.
The operational efficiency of each biological safety
cabinet should be specifically tested and the system
certified before the instrument is placed in operation
after-installation, and subsequently on an annual ba-
sis. Recertification is also required if the unit is
relocated or if maintenance that may affect perform-
ance is done. Maintenance work on biological safety
cabinets should be performed by trained service per-
sonnel only (see Chapter 5, Section B). In addition,
cabinet users should understand the operation of the
equipment, its limitations, and the proper procedures
to be followed. Laboratory directors are responsible
for providing such training.
3. Pipetting Devices
Pipettes are among the most commonly used
pieces of equipment in the biomedical laboratory,
and their misuse has been related to a significant
number of laboratory-acquired infections [1001.
Regrettably, many laboratory workers were taught to
pipette by mouth, even after the associated hazards
were recognized. These individuals should be re-
quired to give up the old practice and learn to use the
pipetting aids that are now available for any applica-
tion [63] (Figure 3.6~. The importance of these aids
cannot be overemphasized, and any device requiring
mouth suction should be considered unsafe and inap-
propriate for use in the biological laboratory. Mouth
pipetiing of any material under any circumstances
should be explicitly prohibited.
4. Sonicators, Homogenizers, and Mixers
Operation of these or similar instruments may
create hazardous aerosols and lead to exposure of
OCR for page 27
27
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OCR for page 28
28
personnel unless extreme caution is exercised. If
indicated by the characteristics of the material being
processed or the agents involved, the instrument
should be operated in a biological safety cabinet.
Blenders should be designed to prevent leakage from
the rotor bearing or at the cover. Caps and gaskets
should be in good condition and the system checked
to ensure that leakage does not occur during op-
eration. When the risk of exposure to infectious
aerosols is present, blender bowls, tubes, and other
containers should be opened in a biological safety
cabinet.
5. Clothing, Masks, and Face Shields
Laboratory coats, gowns, safety glasses, face
shields, masks, and gloves offer some personal pro-
tection and are often used in combination with other
safety devices such as biological safety cabinets.
Special laboratory clothing protects street wear from
contamination. It should not be worn outside of the
laboratory. Each of these items has a particular use
in protecting the worker and should be used when
circumstances require. Gloves are especially impor-
tant when handling any potentially infectious mate-
rial such as blood or other biological specimens.
Safety glasses, face shields, and masks may protect
mucous membranes of the eye, nose, and mouth from
splash or droplet hazards during operations performed
outside of a biological safety cabinet.
K. BIOSAFETY IN LARGE-SCALE
PRODUCTION
1. Introduction
Microbial cultures of greater than 10 liters in
volume (defined as large-scale [1361) present bio-
safety concerns very similar to those described for
small-scale (~10 liters) experiments in the labora-
tory. All the recommendations described in the C DC/
NIH guidelines (Appendix A) for laboratory-scale
research should, therefore, be followed for large-
scale production, with the addition of the recommen-
dations described below. These recommendations
are equally applicable to cultures as small as 20 liters
and as large as 10,000 liters.
This section addresses only issues of biological
safety relative to infectious agents and does not deal
with other major areas of potential risk in large-scale
BIOSAFETY IN THE LABORATORY
FIGURE 3.6 The wide variety of available pipetting aids
make mouth pipetting an unnecessary and obsolete prac-
tice. Courtesy, John H. Richardson and National Institutes
of Health. (Figure continued on next page.)
production (e.g., end-products, by-products, media
components, and nonviable biological agents). These
subjects exceed the scope of this book, but other
sources [118], and references contained therein, pro-
vide pertinent information. Similarly, this section
does not address the biological safety issues that
pertain to the large-scale growth of mammalian cell
cultures. This topic is discussed briefly in Section D
of this chapter.
The physical containment typically implemented
for large-scale production serves to protect the worker.
The self-contained design used for large-scale pro-
duction should essentially eliminate the generation
of aerosols, one of the most common causes of labo-
ratory infections [1001. Standard operating proce-
dures (SOPs~including validation of equipment's
function, biological disposal, and specific methods
of operation should be written and closely followed
for all large-scale productions. Implementation of
these protocols should facilitate the maintenance of a
safer environment in which to work and produce the
highest quality of resultant product.
Special circumstances inherent in large-scale pro-
duction may actually reduce risks to levels lower
than those encountered in laboratory research. Po-
tential biological hazards associated with the specific
organisms used for large-scale production will typi-
cally have been defined and assessed in the research
OCR for page 29
SAFE HANDLING OF INFECTIOUS AGENTS
29
OCR for page 30
30
and developmental stages before large-scale produc-
tion is initiated. Because fewer biological unknowns
or variables are present, the biological hazards should
be reduced. This situation is especially true when
comparing large-scale culture of well-characterized
microorganisms with laboratory-scale culture of clini-
cal isolates or of soil isolates, where a tremendous
diversity of unknown organisms is present. When
the proper precautions are implemented and main-
tained, large quantities of potentially hazardous micro-
organisms may be grown safely [65,1351. Many of
the organisms or cell lines of interest for production
purposes (e.g., Bacillus and yeast) have had an ex-
tended industrial history of safety on the basis of
which risks can be accurately and confidently evalu-
ated.
Where large-scale production is initiated in the
process of commercialization of a desired product,
the constraints enforced to ensure product integrity
typically result in increased safety to personnel as
well. As numerous laboratories have developed large-
scale production facilities for the commercialization
of biological products, the need for carefully de-
signed and implemented procedures has become in-
creasingly important for the safety of laboratory
workers, the community, and the environment. The
following procedures are intended to allow handling
of suspected or known hazardous organisms at large-
scale levels with proper concern for health and safety.
The recommendations are not intended to apply rig-
idly to all situations. As experience is gained in
scale-up operations, more or less stringent guidelines
for individual operations may be indicated by the
safety department, biological safety officer, or bio-
safety committee. The procedures reflect the best
judgment of acceptable techniques and applicable
legal requirements [4,13,1S,51,82, 105,124,129,130,
132,133,134,1361. Therecommendationsrelyheav-
ily on the NIH "Guidelines for Research Involving
Recombinant DNA Molecules" [136], which have
proved dependable as a foundation for laboratory
safety programs whether or not recombinant DNA is
involved. These recommendations are broadly ap-
plicable because they are based on the potential pa-
thogenicity or infectivity for the host. These proce-
dures incorporate safety concepts and guidelines
published in documents of the National Institutes of
Health [133,134,1361, the Centers for Disease Con-
trol, in conjunction with the National Institutes of
Health [105], the U.S. Department of Agriculture
BIOSAFETY IN THE LABORATORY
[1241, the American Industrial Hygiene Association
[4], and the Medical Research Council of Canada
[821.
2. Organization and Responsibilities
Institutions planning to scale up the production of
biotechnology-related products should have, at least,
a safety department, a biological safety officer, and a
biosafety committee, with the responsibilities de-
scribed in Chapter 5. Scientists, technicians, equip-
ment workers, and maintenance and custodial per-
sonnel with access to the large-scale production area
should all be considered candidates for medical sur-
veillance, depending upon the organism being grown
and the product produced.
3. Containment
Containment levels for organisms such as fungi,
bacteria, and viruses are grouped into classes accord-
ing to their perceived or potential hazard to humans.
Selection of an appropriate biosafety level for work
with a particular agent depends upon such factors as
the virulence, pathogenicity, biological stability, mode
of transmission, endemic nature, and communicabil-
ity of the agent; the function of the laboratory; the
procedures and manipulations of the agent; the avail-
ability of effective vaccines or therapeutic measures;
and the quantity and concentration of the agent [1051.
The NIH "Guidelines for Research Involving Re-
combinantDNAMolecules" [136] considers cultures
greater than 10 liters in volume to be large-scale and,
for specific organisms, to require higher levels of
physical containment than cultures less than 10 liters
in volume. Increased containment for large-scale
culture is typically reflected in the requirement for
more extensive physical design, and not for operat-
ing at a higher biosafety level.
The risks associated with the use of microbial
cultures are controllable through two containment
approaches. For some work, an appropriate approach
is biological containment the use of species or strains
that pose a reduced risk to workers or to the environ-
ment. The more widely applicable approach, how-
ever, is physical containment a combination of labo-
ratory practices, design, and equipment. Physical
containment, through the implementation of good
laboratory practices and proper monitoring, cer~fi-
cation, and maintenance of the facility, leads to a
OCR for page 31
SAFE HANDLING OF INFECTIOUS AGENTS
significantly safer and more productive work envi-
ronment.
A National Institute for Occupational Safety and
Health (NIOSH) survey of six biotechnology compa-
nies [51] showed that companies with many years of
experience in fermentation technology tended to
emphasize more sophisticated, more effective, and
safer practices for handling infectious agents than
newly established companies.
All facilities and equipment used to provide con-
tainment should be tested before initiation of a pro-
gram and periodically thereafter by trained person-
nel. Such monitoring should include checks of room
air balance, biological safety cabinets, supply and
exhaust filters, sterilizers, and centrifuges. Labora-
tory personnel should ensure that biological safety
cabinets are appropriately certified prior to use. A
systematic, scheduled program of preventive mainte-
nance should be implemented for agitator seals, con-
trol valves, pressure relief valves, and equipment and
facility safeguards [511. Sterilization of fermenters,
feed lines, feed tanks, inoculating devices, exhaust
ports, sampling ports, and extraction devices should
be validated routinely.
Maintaining, testing, or cleaning of facilities or
equipment should not be allowed until all surfaces
needing servicing have been decontaminated. Spills
should be disinfected by laboratory personnel before
housekeepers are allowed to give assistance.
Typically, large-scale production facilities are
specifically engineered for maximal physical con-
tainment to ensure both personnel safety and product
integrity. The specific minimum requirements for
physical containment are described in the NIH
"Guidelines for Research Involving Recombinant
DNA Molecules" [1363. The large-scale contain-
ment classifications, BSL1-LS, BSL2-LS, and BSL3-
LS (Biosafety Levels 1, 2, and 3, large-scale, respec-
tively) are required for organisms for which the cor-
responding containment levels BSL1, BSL2, and
BSL3 (Biosafety Levels 1, 2, and 3, respectively) are
required for small-scale (<10 liters) research. No
provisions are made for large-scale growth of micro-
organisms that require the Biosafety Level 4 contain-
ment at the laboratory scale. (Consult Appendix A
for the small-scale containment levels for specific
microorganisms.) The appropriate implementation
of safeguards and of protective engineering controls
at the time of the design of the facility or laboratory
can reduce human exposure and avoid expensive
31
retrofitting. Specific details of engineering and de-
sign features that reduce the potential biological risks
associated with large-scale microbial production can
be found in the NIH guidelines [1361.
4. Inactivation
Vessels used for large-scale production are typi-
cally steam sterilized in place, in the absence or
presence of medium, prior to fermentation. Tem-
perature-sensitive tapes, crayons, or, preferably,
thermocouples, may be used to monitor sterilization
procedures. The growth medium is either sterilized
in the system or sterile medium is introduced via a
closed system designed to maintain physical contain-
ment. Connections should be sterilizable in place.
Metal containers, designed to preclude escape or en-
try of viable organisms during inoculation, should be
used. These inoculation vessels should be steril-
izable after the transfer process, and prior to their
removal from the production vessel, to minimize
release.
Following growth, cultures in the production ves-
sel should be inactivated either chemically or ther-
mally prior to initiating the product recovery pro
cesses. Inactivation is used in this context to refer to
the reduction in the total number of viable target
microorganisms in a large-scale culture to a number
comparable to or less than that obtained in a small-
scale laboratory culture (<10 liters). This reduction
enables a culture that is grown in a large-scale, closed
system to be handled according to the containment
level applicable for the corresponding laboratory-
scale culture. Inactivation, in this context, is there-
fore an interim action that is to be followed by
decontamination prior to the disposal of microbial
cultures. As an alternative to inactivating cultures in
the production vessel, cultures can be inactivated by
cell rupture or by further steps in subsequent stages
of the process, if these are designed into the same
closed system. When the viable biological agent
itself is the desired end-product (e.g., some vaccines),
the culture would not be inactivated but would be
maintained within a closed system to avoid human
exposure. The specific method of inactivation (see
Chapter 4) depends on the organism or cell culture
employed and upon the product to be isolated. Inac-
tivation efficiencies should be validated as described
in the SOPs. Physical containment during process-
ing steps following culture inactivation (i.e., steps in
OCR for page 32
32
valved in the recovery and purification of the defined
product) is not required but is recommended when
possible and feasible. All liquid and solid biological
waste should be decontaminated chemically or ther-
mally prior to disposal. In-line thermocouples are
especially effective in validating the thermal inacti-
vation of large quantities of liquid cultures prior to
disposal. If other primary equipment, such as centri-
fuges, is used in-line for harvesting cells prior to
inactivation, this equipment should also be decon-
taminated appropriately.
Water supplies or storage tanks provide ideal en-
try points for biological contaminants. Treatment of
water with ultraviolet light, ozone, or passage through
specifically selected filters significantly reduces the
potential for its contamination. The quality and type
of water processing should be specified in the SOPs.
5. Disposal
All biological waste generated through routine
procedures or as the result of accidents should be
decontaminated prior to its disposal, and should be
segregated from nonbiological waste and from radio-
active waste. Solid trash and small volumes of waste
should be disposed of by using the procedures de-
scribed for laboratory research. Contents of the pro-
duction vessel should be physically contained (e.g.,
with dikes or decontamination tanks) to confine spills
and leaks and to allow for their rapid and efficient
decontamination. All liquid collected by these pro-
cedures should be properly decontaminated by using
validated procedures to prevent the release of viable
organisms or cells into the environment. The con-
tents and all associated materials and equipment
should be inactivated either chemically or physically
prior to disposal. If decontamination tanks are used,
the method of inactivation should be validated for
each organism employed. Once inactivated, even
large quantities of liquid culture can be disposed of
by discharge to the sewer lines, provided that such
action is permitted by the relevant state and local
agencies. Air discharged from fermenters should be
faltered through a PUPA filter, incinerated, or other-
wise treated prior to release.
Personnel involved in the cleanup of accidentally
spilled waste should proceed as described in Chapter
5 for spills with laboratory-scale cultures. Particular
precautions should be taken to handle and decon
BIOSAFETY IN THE LABORATORY
laminate these large quantities of culture, as well as
the absorption materials used for cleanup.
6. Exposure
The self-contained design of the large-scale pro-
duction system, He inoculation method, and the in-
place inactivation of the vessel contents following
production essentially eliminate the release of aero-
sols, thereby reducing human exposure. Nonethe-
less, air and surfaces should be monitored to validate
the integrity of the systems during the fermentation
or processing procedures. Surfaces can be moni-
tored for microbial release by any of five basic meth-
ods: rinse, swab-rinse, agar contact, direct surface
agar plating, or vacuum probe surface method [41.
Other procedures are modifications of these basic
methods. In a survey of six genetic engineering
companies, agar contact was found to be the method
most frequently used [511. Organism detecting and
counting (RODAC) plates are commercially avail-
able with a variety of media and are used routinely.
The method works well and rapidly as a qualitative
procedure.
Numerous methods are available to sample air-
borne microorganisms [41. The more frequently used
methods include settling plates, air impingers, and
falters. Settling plates provide qualitative informa-
tion and consist simply of an open petri dish contain-
ing appropriate culture medium onto which particles
settle due to gravity. Air impingers are intrinsically
more quantitative because they pull air at a fixed rate
and volume onto the surface of the medium. Large
and defined quantities of airborne microorganisms
can be concentrated and analyzed. The most com-
mon method for sampling air is filtration. A fixed
volume of air can be passed through a filter of a
selected pore size. Particles and organisms can be
flushed from the filter, and the filtrate analyzed on an
appropriate medium, or the filter itself can be over-
laid directly with an appropriate medium. The use of
filters is limited, because of dehydrating effects, to
the detection of spores and of resistant vegetative
cells. Selection of an appropriate sampling method
depends upon the nature of the particles of interest,
their expected concentration, and the need for quan-
tif~cation. The reader can consult Table XVIII in
reference 6 for a more detailed description of these
and alternative sampling methods.
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SAFE HANDLING OF INFECTIOUS ACE=S
Surface and air sampling methods should be se-
lected to detect biological contaminants, as well as
the production organism itself. Adventitious agents
expected in large-scale production are similar to those
found in laboratory research (e.g., viruses, bacteria,
and fungi). Potential candidates depend upon the
specific organism under study and the medium em-
ployed.
7. Conclusion
Large-scale microbial culture operations can be
done safely, despite the risks associated with some
microorganisms. Personnel can function safely and
efficiently at any scale by using the proper facilities
and equipment.
L. BIOSAFETY IN PHYSICIANS' OFFICE
LABORATORIES AND OTHER SMALL
VOLUME CLINICAL LABORATORIES
Each small laboratory, including those in physi-
cians' offices, should have a safety program even if
the laboratory has only one employee (see Chapter
5~. For those employees without training in the
biological sciences, special effort should be made to
provide simple but effective instruction. The pro-
gram should be tailored to the laboratory function,
with proper emphasis on providing training in the
seven basic rules of good microbiological practices
as outlined in Section F of this chapter. Indeed, all of
the general principles outlined in this chapter for the
safe handling of infectious agents apply to small-
volume laboratories as well as large-volume ones.
Many of these small-volume laboratories may be
33
handling human blood or blood components that could
be infectious. If this is the case, the standard biologi-
cal practices as well as the special practices recom-
mended by the CDC/~IH guidelines for Biosafety
Level 2 should be used (see Appendix A), and the
Universal Precautions described in Appendix C should
be followed. Prudence should also be exercised to
minimize exposure to toxic chemicals and radionu-
clides.
Special precautions should be taken to ensure that
waste is managed in a safe, responsible manner.
Potentially infectious waste should preferably be
decontaminated on site. Liquid waste, which may be
contaminated with an infectious agent, can be steam
autoclaved or decontaminated by using a chemical
disinfectant that is effective for the intended use.
Decontaminated liquids can than be poured carefully
down a drain connected to the sanitary sewer. Solid
waste, including contaminated`'shaIps" (e.g., hypo-
dermic needles and broken glassware), should be
packaged in sealed, leak- and puncture-resistant con-
tainers for transport and disposal. Such properly
contained waste presents minimal risk to waste han-
dlers and can be safely managed as municipal solid
waste. If this practice is prohibited by local regula-
tions, then the prevailing practices should be fol-
lowed. Human excrete should be disposed of through
the sanitary sewer. The practices just described are
appropriate for the small laboratory that is involved
in testing patients' specimens. The safe disposal
methods discussed in Chapter 4 should be followed
in the small biomedical laboratory where infectious
agents are isolated and grown in cultures.
Further information on safety in the office labora-
tory may be found in references 12 and 54.
Representative terms from entire chapter:
biological safety
