Biological Safety Manual
Date of last revision: October 20097
Introduction______________________________________________________________ 94
Risk Assessment____________________________________________________________ 94
Biosecurity_________________________________________________________________ 97
Laboratory Biosafety_______________________________________________________ 97
Standard Microbiological Practices_________________________________________________________________________ 97
Biosafety Level 1______________________________________________________________________________________________ 98
Special Practices_____________________________________________________________________________________________ 99
Safety Equipment (Primary
Barriers and Personal Protective Equipment)_________________________________ 99
Laboratory Facilities (Secondary Barriers)__________________________________________________________________ 99
Biosafety Level 2______________________________________________________________________________________________ 99
Special Practices____________________________________________________________________________________________ 100
Safety Equipment (Primary
Barriers and Personal Protective Equipment)________________________________ 101
Laboratory Facilities (Secondary Barriers)_________________________________________________________________ 101
Biosafety Level 3_____________________________________________________________________________________________ 102
Special Practices____________________________________________________________________________________________ 102
Safety Equipment (Primary Barriers and Personal
Protective Equipment)_______________________________ 103
Laboratory Facilities (Secondary Barriers)_________________________________________________________________ 103
Biosafety Level 4_____________________________________________________________________________________________ 104
Experiments Using Human and non-Human Primate
Blood, Blood Products, Secretions or Other Potentially Infectious Materials 105
Recombinant DNA Experiments____________________________________________________________________________ 105
Cell Lines_____________________________________________________________________________________________________ 105
Viruses_______________________________________________________________________________________________________ 105
Bacteria______________________________________________________________________________________________________ 105
Parasites_____________________________________________________________________________________________________ 106
Fungi__________________________________________________________________________________________________________ 106
Animal Biosafety__________________________________________________________ 106
Zoonoses_____________________________________________________________________________________________________ 107
Standard Animal Biosafety Practices______________________________________________________________________ 108
Animal Biosafety Level 1____________________________________________________________________________________ 110
Special Practices____________________________________________________________________________________________ 110
Safety Equipment (Primary Barriers and Personal
Protective Equipment)_______________________________ 110
Laboratory Facilities (Secondary Barriers)_________________________________________________________________ 111
Animal Biosafety Level 2____________________________________________________________________________________ 112
Special Practices____________________________________________________________________________________________ 112
Safety Equipment (Primary Barriers and Personal
Protective Equipment)_______________________________ 113
Laboratory Facilities (Secondary Barriers)_________________________________________________________________ 113
Animal Biosafety Level 3____________________________________________________________________________________ 114
Special Practices____________________________________________________________________________________________ 115
Safety Equipment (Primary Barriers and Personal
Protective Equipment)_______________________________ 115
Laboratory Facilities (Secondary Barriers)_________________________________________________________________ 116
Animal Biosafety Level 4____________________________________________________________________________________ 117
Agriculture Pathogen Biosafety___________________________________________ 117
BSL3-AG for Work with Loose-Housed Animals__________________________________________________________ 117
Pathogens of Veterinary/Agricultural Significance_______________________________________________________ 120
Arthropod Containment___________________________________________________ 122
Arthropod Containment Levels (ACLs)____________________________________________________________________ 122
Arthropod Containment Level 1 (ACL-1)__________________________________________________________________ 122
Standard practices_________________________________________________________________________________________ 122
Special practices____________________________________________________________________________________________ 123
Safety equipment (primary barriers)______________________________________________________________________ 124
Facilities (secondary barriers)_____________________________________________________________________________ 124
Arthropod Containment Level 2 (ACL-2)__________________________________________________________________ 124
Standard practices_________________________________________________________________________________________ 124
Special practices____________________________________________________________________________________________ 126
Safety equipment (primary barriers)______________________________________________________________________ 127
Facilities (secondary barriers)_____________________________________________________________________________ 127
Arthropod Containment Level 3 (ACL-3)__________________________________________________________________ 128
Standard practices_________________________________________________________________________________________ 129
Special practices____________________________________________________________________________________________ 131
Safety equipment (primary barriers)______________________________________________________________________ 131
Facilities (secondary barriers)_____________________________________________________________________________ 132
Arthropod Containment Level 4 (ACL-4)__________________________________________________________________ 133
Biological Safety Cabinets_________________________________________________ 133
The Class I BSC_______________________________________________________________________________________________ 133
The Class II BSC______________________________________________________________________________________________ 134
The Class III BSC_____________________________________________________________________________________________ 134
Laminar
Flow ÒClean BenchesÓ_____________________________________________________________________________ 135
Chemicals in BSCS___________________________________________________________________________________________ 135
Radiological Hazards in the BSC____________________________________________________________________________ 135
BSC Use: Work Practices and Procedures_________________________________________________________________ 135
Preparing for Work within a Class II BSC__________________________________________________________________ 135
Operations within a Class II BSC___________________________________________________________________________ 137
BSC Decontamination_______________________________________________________________________________________ 138
Surface Decontamination__________________________________________________________________________________ 138
Gas Decontamination_______________________________________________________________________________________ 139
Certification of Biological Safety Cabinets_________________________________________________________________ 139
Containment Standards____________________________________________________________________________________ 139
General Biosafety Issues___________________________________________________ 139
Aerosol-Generating Processes_____________________________________________________________________________ 139
Aerosol Generating Activities_______________________________________________________________________________ 140
Measures to Decrease Hazards from Aerosols______________________________________________________________ 140
Biological Stains_____________________________________________________________________________________________ 141
Incubators___________________________________________________________________________________________________ 141
Freezer and Liquid Nitrogen Storage______________________________________________________________________ 141
Biological Spills______________________________________________________________________________________________ 142
Waste Decontamination and Disposal_____________________________________________________________________ 142
Bio-Hazardous Waste Management________________________________________________________________________ 143
Disinfectants_____________________________________________________________ 144
Alcohol_______________________________________________________________________________________________________ 144
Chlorine______________________________________________________________________________________________________ 144
Iodophor_____________________________________________________________________________________________________ 144
Ethylene Oxide_______________________________________________________________________________________________ 145
Transporting biological materials_________________________________________ 145
Regulations__________________________________________________________________________________________________ 146
Transfers_____________________________________________________________________________________________________ 146
Transfer Of USDA Plant Pests_______________________________________________________________________________ 148
General DOT Packaging Requirements For Transport Of
Infectious Substances By Aircraft__________ 148
Category A Infectious Substance (UN 2814 and UN
2900)________________________________________________ 149
Biological specimen, Category B (UN 3373)_______________________________________________________________ 149
Select Agent list___________________________________________________________ 149
HHS Select Agents____________________________________________________________________________________________ 149
USDA-HHS Overlap Agents__________________________________________________________________________________ 150
USDA High Consequence Livestock and Plant Pathogens
and Toxins____________________________________ 150
Integrated Pest Management (IPM)_________________________________________ 151
Controlling exposures (and the resulting
infections) requires an understanding of the factors involved in disease
transmission in the laboratory. The most common routes of exposure are
ingestion, inhalation, and self-inoculation. The development of an infection subsequent to an exposure to
an infectious agent depends upon individual susceptibility, the size of the
dose, and the pathogenicity of the organism. The only one of these three
factors within the control of the investigator is the size of the dose. If all exposures can be kept below
the infectious dose, the risk of infection is greatly minimized. This is the
basis for safety in the biological laboratory .(see Protective
Controls below).
This document was based in part on the CDC/NIH publications ÒBiosafety in the Microbiological and
Biomedical Laboratories, 54th EditionÓ and the ÒPrimary Containment for Biohazards:
Selection, Installation and Use of Biosafety CabinetsÓ. Also referenced were the , the American Public Health Association publication ÒControl of Communicable Diseases, 187th EditionÓ, the American Industrial Hygiene
Association publication ÒBiohazards Reference Manual, 2nd EditionÓ, and on the National Research Council publication ÒBiosafety in the Laboratory:
Prudent Practices for the handling and disposal of Infectious Materials.ÓÓ. The
biosafety cabinet graphics are from the CDC/NIH publication ÒPrimary
Containment...Ó and were scanned in by the ORCBS at Michigan State University.
Employees desiring more information than is presented here should refer to these references, or consult an Environmental Health and Safety (EHS) staff member.
In
keeping exposures at safe levels (below the Infectious Dose, or ID), OSHA recognizes three type of general protection;
in order of descending preference, they are: Engineering Controls,
Administrative Controls, and Personal Protective Equipment.
Engineering
Controls are called ÒPrimary BarriersÓ in the CDC/NIH ÒBiosafety in the
Microbiological and Biomedical Laboratories, 5th EditionÓ, and consists of such
items as biosafety cabinets and ventilation systems.
Administrative
controls are seldom
used in a biosafety environment since they involve limiting how
much time an individual spends
in a contaminated environment to keep exposures below the ID.
Personal
Protective Equipment (PPE) – equipment typically worn by the User, such
as gloves, surgical
masks, and respirators.
Primary
Barriers (Engineering Controls) are the preferred control mechanism, since they
depend less on the User than PPE. PPE is the least favored control mechanism
since it is dependent on proper fit use by the wearer. Whenever possible
Engineering Controls will be specified.
Institutions
that are NIH
grantees are required to provide safe and healthful working conditions
for their employees and foster work environments conducive to high-quality
research. Of
particular interest are the following laws and regulations.
P.L.
107-188 is designed to provide protection against misuse of Select Agents and
Toxins whether inadvertent or the result of terrorist actsÉimplemented, in
part, through regulations published by CDC at 42 CFR 73 Select Agents and Toxins.
Also,
animal and plant pathogen Select Agents are covered under the regulations
published by USDA-APHIS at 9 CFR 121 and 7 CFR 331.
Research
involving Select Agents and recombinant DNA molecules also is subject to the
NIH Guidelines for Research Involving DNA Molecules (NIH Guidelines) (see ÒNIH
Guidelines for Research Involving DNA Molecules and Human Gene Transfer
ResearchÓ)
P.L.
107-56 provides criminal penalties for possession of any biological agent,
toxin, or delivery system of a type or in a quantity that is not reasonably
justified by a prophylactic, protective, bona fide research, or other peaceful
purposeÉ [and] establishes restrictions on access to specified materials.
The NIH
Guidelines (April 2002 or latest revision) apply to all research projects that
involve recombinant DNA and are conducted at or sponsored by an organization
that receives NIH support for recombinant DNA research.
As
defined by the NIH Guidelines, recombinant DNA molecules are either
(1) molecules that are constructed outside of living
cells by joining natural or synthetic DNA segments to DNA molecules that can
replicate in a living cell or
(2) molecules that result from the replication of those
described in (1).
The NIH
Guidelines apply to both basic and clinical research studies. Recombinant DNA
research involving select agents also is subject to pertinent CDC and USDA
regulations.
Specific
guidance for the conduct of human gene transfer clinical trials appears in
Appendix M of the NIH Guidelines.
Failure
to comply with these requirements may result in suspension or termination of an
award for recombinant DNA research at the organization, or a requirement for NIH
prior approval of any or all recombinant DNA projects at the organization. In extreme cases all
Federal funding may be suspended until corrections are made.
Two
specific requirements of the NIH Guidelines are discussed below, but the
grantee should carefully review the NIH Guidelines in their entirety to ensure
compliance with all of the requirements for projects involving recombinant DNA
techniques.
1.
Institutional
Biosafety Committee
Each
organization that conducts research involving recombinant DNAÉmust have
policies and procedures to ensure compliance with the NIH Guidelines and must
establish a standing IBC.
The IBC
is required to review each proposed project for recombinant DNA experiments and
certify that the procedures, project, personnel, and facilities are adequate
and in compliance with the NIH Guidelines.
2.
Safety
and Annual Reporting for Human Gene Transfer Clinical Trials
Appendix
M-I-C-4 of the NIH Guidelines requires Serious Adverse Events (SAE) that are
unexpected and are possibly associated with human gene transfer intervention to
be reported to OBA and the IBC within 15 calendar days of investigator
notification of the sponsor, or within 7 days if life-threatening or fatal.
Annually,
investigators must submit to OBA certain information about protocols. Further
information about the content of these reports can be found in Appendix M-I-C-3
of the NIH Guidelines.
Although
still referred to as Òguidelines,Ó the
BMBL is in fact de facto regulations for NIH grantees and institutions receiving Federal funding. Failure to comply may result in the suspension or
loss of some or all Federal funding.
Grantees
are responsible for meeting Federal, State, and local health and safety
standards and for establishing and implementing necessary measures to minimize
their employeesÕ risk of injury or illness in activities related to NIH grants.
In
addition to applicable Federal, State, and local laws and regulations, the
following regulations must be followed when developing and implementing health
and safety operating procedures and practices for both personnel and
facilities:
á
29 CFR
1910.1030, Bloodborne pathogens;
á 29 CFR 1910.1450, Occupational exposure to
hazardous chemicals in laboratories; and,
á
Other
applicable occupational health and safety standards issued by the Occupational
Health and Safety Administration (OSHA) and included in 29 CFR Part 1910.
Risk assessment is a process used to identify the
hazardous characteristics of a known infectious or potentially infectious agent
or material, the activities that can result in a personÕs exposure to an agent,
the likelihood that such exposure will cause an infection, and the probable
consequences of such an infection. The information identified by risk
assessment will provide a guide for the selection of appropriate biosafety
levels and microbiological practices, safety equipment, and facility safeguards
that can prevent infection.
The primary factors to consider in risk assessment
and selection of precautions fall into two broad categories: agent hazards and procedure
hazards. In addition, the capability of the
laboratory staff to control hazards must be considered. This capability will
depend on the training, technical proficiency, and good habits of
all members of the laboratory, and the operational integrity of containment equipment and
facility safeguards.
The origin of the agent is also important in risk
assessment. Non-indigenous agents are of special concern because of their
potential to introduce risk of transmission, or spread of human and animal or
infectious disease from foreign countries into the United States.
The identification and assessment of hazardous
characteristics of genetically modified agents involve consideration of the
same factors used in risk assessment of the wild-type organism.
It is
important to address the possibility that the genetic modification could
increase an agentÕs pathogenicity or affect its susceptibility to antibiotics
or other effective treatments. It
is important to remain alert to the possibility that experimental alteration of
virulence genes may lead to increased risk. It also suggests that risk assessment is a continuing
process that requires updating as research progresses
Clemson EHS will perform a risk assessment when
requested; contact EHS (ehs@clemson.edu) for a
risk assessment.
Recent significant events have brought national and
international scrutiny to the area of laboratory security. These
events, including the anthrax attacks on U.S. citizens in October 2001 and the subsequent
expansion of the United States Select Agent regulations in December 2003, have led
scientists, laboratory managers, security specialists, biosafety professionals,
and other
scientific and institutional leaders to develop and improve the security of biological agents and
toxins within their facilities.
Section
VI of the BMBL 5th edition provides a good review of
issues to consider in developing a security plan for biological agents
and toxins capable of serious or fatal illness to humans or animals.
Contact EHS for a biosecurity review of your
facility, or if you desire to work with Select Agents and/or
Toxins.
To
determine the level of safety required in the biological/biomedical laboratory,
one must first determine the Risk Group (1, 2, 3, or 4) the agent and the
proposed activity. Some
agents have well defined Risk Grouping, while others do not. For agents with no
published Risk Group, a risk
assessment is necessary.
Note that the proposed activity can seriously
impact the Risk Group. For example, a Risk
Group 2 agent which is handled in high titers or large
volumes may have to be handled as a Risk Group 3 agent.
These practices are applicable for all biological/biomedical laboratories:
1. Laboratories must be locked whenever unoccupied.
2. Laboratories shall have a sink for hand washing.
3. Persons must wash their hands after working with
potentially hazardous materials and before leaving the laboratory.
4. Eating, drinking, smoking, handling contact lenses,
applying cosmetics, and storing food for human consumption is not
permitted in laboratory areas. Food must be stored outside the laboratory area
in cabinets or refrigerators designated and used for this purpose.
5. Mouth pipetting is prohibited; mechanical pipetting
devices must be used.
6. The following precautions must always be taken with
sharp items:
a. Careful management of needles and other sharps are
of primary importance. Needles must not be bent, sheared, broken, recapped,
removed from disposable syringes, or otherwise manipulated by hand before
disposal.
b. Used disposable needles and syringes must be
carefully placed in conveniently located puncture-resistant containers used for
sharps disposal.
c. Non-disposable sharps must be placed in a hard
walled container for transport to a processing area for decontamination,
preferably by autoclaving.
d. Broken glassware must not be handled directly.
Instead, it must be removed using a brush and dustpan, tongs, or forceps.
Plasticware should be substituted for glassware whenever possible.
e. Contact EHS for sharps containers.
7. Perform all procedures to minimize the creation of
splashes and/or aerosols.
8. Personnel using respirators must be enrolled in the
Clemson University respiratory protection program and be cleared medically by the Occupational
Health Nurse.
9. Decontaminate work surfaces after completion of
work and after any spill or splash of potentially infectious material with
appropriate disinfectant for the necessary contact time.
10. Decontaminate all cultures, stocks, and other
potentially infectious materials before disposal using an effective method.
Depending on where the decontamination will be performed, the following methods
should be used prior to transport:
a. Materials to be decontaminated outside of the
immediate laboratory must be placed in a durable, leak proof container and
secured for transport.
b. Materials to be removed from the facility for
decontamination must be packed in accordance with applicable local, state, and
federal regulations.
11. A sign incorporating the universal biohazard symbol
must be posted at the entrance to the laboratory when infectious agents are
present. The sign shall include the name of the agent(s) in use, and the name
and phone number of the laboratory supervisor or other responsible personnel.
12. An effective integrated pest management program is
required.
13. The laboratory supervisor must ensure that
laboratory personnel receive appropriate training regarding their duties, the
necessary precautions to prevent exposures, and exposure evaluation procedures.
Personnel must receive annual updates or additional training when procedural or
policy changes occur. All
training must be documented. OSHA mandates
annual training for work with Bloodborne Pathogens.
14. Personal health status may impact an individualÕs
susceptibility to infection, ability to receive immunizations or prophylactic
interventions. Therefore, all laboratory personnel, and
particularly women of child-bearing age, should be provided with information regarding
immune competence and conditions that may predispose them to infection.
Individuals having these conditions should be encouraged to self-identify to
the Occupational Health Nurse for appropriate counseling and guidance.
Biosafety
Level 1 is
suitable for work involving Risk
Group 1 (RG-1, RG1) agents – well
characterized agents not known to consistently cause disease in
immunocompetent adult humans, and present minimal potential hazard to laboratory personnel
and the environment. BSL-1 laboratories are not necessarily separated from the
general traffic patterns in the building.
Work is
typically conducted on open bench tops using standard microbiological
practices. Special containment equipment or facility design is
not required, but may be used as determined by appropriate risk assessment.
Laboratory personnel must have specific training in the procedures conducted in the
laboratory and must be supervised by a scientist with training in microbiology or a
related science.
The
following practices, safety equipment, and facility requirements apply to
BSL-1:
None
required.
1. Special containment devices or equipment, such as
BSCs, are not generally required, but
may be necessary if aerosol-generating procedures are used.
2. Protective laboratory coats, gowns, or uniforms must be worn to
prevent contamination
of personal clothing.
3. Wear protective eyewear while in the lab, especially when conducting procedures that have the potential to
create splashes of microorganisms or other hazardous materials. Persons who wear
contact lenses in laboratories must also wear eye protection.
4. Gloves must be worn to protect hands from exposure
to hazardous materials. Glove selection should be based on an appropriate
risk assessment. Alternatives to latex gloves (e.g. Nitrile gloves) are available. Wash
hands prior to leaving the laboratory. In addition, BSL-1 workers shall:
a. Change gloves when contaminated, integrity has been
compromised, or when otherwise necessary.
b. Remove gloves and wash hands when work with
hazardous materials has been completed and before leaving the
laboratory.
c. Do not wash or reuse disposable gloves. Dispose of
used gloves with other contaminated laboratory waste. Hand washing
protocols must be rigorously followed.
5. Gloves may
not be worn outside the laboratory.
1. The laboratory shall be routinely cleaned. Carpets and rugs in
laboratories are prohibited.
2. Laboratory furniture must be capable of supporting
anticipated loads and uses. Spaces between benches, cabinets, and equipment
should be accessible for cleaning.
3. Bench tops must be impervious to water and
resistant to heat, organic solvents, acids, alkalis, and other chemicals.
4. Chairs used in laboratory work must be covered with
a non-porous material that can be easily cleaned and decontaminated
with appropriate
disinfectant.
5. Laboratories windows that open to the exterior shall be
fitted with screens.
Biosafety Level 2 builds upon BSL-1. BSL-2 is
suitable for work involving Risk
Group 2 (RG-2, RG2) agents. RG2
agents are associated with human disease which is rarely
serious and for which preventive or therapeutic interventions are often
available (moderate individual risk; low community risk.) An RG2 agent
is a pathogen that can
cause human, animal, or
plant disease but is unlikely to be a serious hazard to laboratory
workers, the community, livestock,
plants, or the environment. Laboratory exposures may cause
serious infection, but effective treatment and preventive measures are
available and the risk of spread of infection is limited. BSL-2 differs from BSL-1 in that:
1. Laboratory
personnel have specific training in handling pathogenic agents and are supervised by scientists competent in handling
infectious agents and associated procedures;
2. Access to the laboratory is restricted when work is being
conducted; and
3. All procedures in which infectious aerosols or
splashes may be created are conducted in BSCs or other physical containment equipment.
The following special practices, safety equipment,
and facility requirements apply to BSL-2:
1. All persons entering the laboratory must be advised
of the potential hazards and meet specific entry/exit requirements. Requirements vary based on agent(s) in use.
2. Laboratory personnel must be provided medical
surveillance and offered appropriate immunizations for agents handled or
potentially present in the laboratory. Immunization costs will be borne by either the
Project or the Department; the employee shall not be charged.
3. In consultation with the Clemson Occupational Health
Nurse, each research
project may
choose to establish policies and procedures describing the collection and storage of serum samples from
at-risk personnel.
4. An agent-specific
biosafety manual/procedure must be prepared and adopted as policy. This document must be available and accessible.
5. The laboratory supervisor must ensure that
laboratory personnel demonstrate proficiency in standard and special microbiological
practices before working with BSL-2 agents. This shall be documented in writing and kept on
file in the laboratory.
6. Potentially infectious materials must be placed in
a durable, leak proof container during collection, handling, processing,
storage, or transport within a facility.
7. Laboratory equipment must be routinely decontaminated, as well as, after spills, splashes, or other potential contamination.
a. Spills involving infectious materials must be
contained, decontaminated, and cleaned up by staff properly
trained and equipped to work with infectious material.
b. Equipment must be decontaminated before repair,
maintenance, or removal from the laboratory.
8. Incidents that may result in exposure to infectious
materials must be immediately reported to Compendium, Risk Management, EHS, and the laboratory supervisor. If so directed by Compendium, the exposed
individual will report to Redfern (or other Facility as directed) for medical evaluation, surveillance, and treatment.
9. Animals and plants not associated with the work
being performed are not permitted in the laboratory.
10. All procedures involving the manipulation of
infectious materials that may generate an aerosol must be conducted within a BSC or other physical containment devices when indicated by risk assessment.
1. Properly maintained BSCs (preferably Class II; consult EHS for the appropriate BSC), other appropriate personal protective equipment, or other physical containment
devices must be used whenever:
a. Procedures with a potential for creating infectious
aerosols or splashes are conducted. These may include pipetting,
centrifuging, grinding, blending, shaking, mixing, sonicating, opening
containers of infectious materials, inoculating animals intranasally, and
harvesting infected tissues from animals or eggs.
b. High concentrations or large volumes of infectious
agents are used. Such materials may be centrifuged in the open
laboratory using sealed rotor heads or centrifuge safety cups.
2. Protective laboratory coats, gowns, smocks, or
uniforms designated for laboratory use must be worn while working with
hazardous materials. Remove protective clothing before leaving for
non-laboratory areas (e.g., cafeteria, library, administrative offices).
Dispose of protective clothing appropriately, or deposit it for laundering. EHS has several washing
machines on campus for laundering of lab clothing; laboratory
clothing may not be taken home.
3. Eye and face protection (goggles, mask, face shield
or other splatter guard) is used for anticipated splashes or sprays of
infectious or other hazardous materials when the microorganisms must be handled
outside the BSC or containment device. Eye and face protection must be
disposed of with other contaminated laboratory waste or decontaminated
before reuse. Persons who wear contact lenses in laboratories must also wear eye protection.
4. Gloves must be worn to protect hands from exposure
to hazardous materials. Glove selection should be based on an appropriate
risk assessment. Alternatives to latex gloves (e.g., Nitrile) are available. Gloves must not be worn outside the laboratory. In addition, BSL-2
laboratory workers should:
a. Change gloves when contaminated, integrity has been
compromised, or when otherwise necessary. Wear two pairs of
gloves when appropriate.
b. Remove gloves and wash hands when work with
hazardous materials has been completed and before leaving the
laboratory.
c. Do not wash or reuse disposable gloves. Dispose of
used gloves with other contaminated laboratory waste. Hand washing
protocols must be rigorously followed.
5. Eye, face and respiratory protection should be used
in rooms containing infected animals and as determined by the risk assessment.
1. All the requirements of BSL-1 plus:
2. Laboratory doors shall be
self-closing. Card key access is preferred, but not required,
for BSL-2.
3. Laboratory windows that open to the exterior are
not recommended. However, if a laboratory does have windows that open to the
exterior, they must be fitted with screens.
4. BSCs must be installed so that fluctuations of the
room air supply and exhaust do not interfere with proper operations. BSCs shall be located away from doors, windows that can be opened, heavily traveled
laboratory areas, and other possible airflow disruptions.
5. Vacuum lines must be protected with High Efficiency Particulate Air (HEPA)
filters, or their equivalent. Filters must be replaced as needed. Liquid disinfectant traps may be required.
6. An eyewash station must be readily available within the lab; South Carolina OSHA mandates than
an employee not have to pass through a doorway to access the eyewash/safety
shower.
7. Lab spaces must be negative to the corridor and
administrative spaces, and air may not be re-circulated from the lab to other areas of the
building.
8. BSCs must be tested and certified annually, and
whenever moved.
9. HEPA filtered exhaust air from a Class II BSC can
be safely re-circulated back into the laboratory environment if operated
according to manufacturerÕs recommendations. BSCs can also be connected to the laboratory
exhaust system by either a thimble (canopy) connection or a direct (hard)
connection. Provisions to assure proper safety cabinet performance and air
system operation must be verified.
10. A method for decontaminating all laboratory wastes
should be available in the facility (e.g., autoclave, chemical disinfection,
incineration, or other validated decontamination method).
Biosafety
Level 3 is suitable for Risk
Group 3 Agents (RG-3, RG3); RG3 agents are associated with serious or lethal
human disease for which preventive or therapeutic interventions may be
available (high individual risk but low community risk). Typically a
pathogen that usually causes serious human, animal, or plant disease
but does not ordinarily spread from one infected individual to another, and for
which effective
treatment and preventive measures are available.
Biosafety Level 3 is applicable to clinical,
diagnostic, teaching, research, or production
facilities where work is performed with indigenous or exotic agents that may cause serious or potentially lethal disease through
inhalation exposure. Laboratory personnel must receive specific training in
handling pathogenic and potentially lethal agents, and must be supervised by scientists
competent in handling infectious agents and associated procedures.
All procedures involving the manipulation of
infectious materials must be conducted within BSCs, other physical containment
devices, or by personnel wearing appropriate personal protective equipment.
A BSL-3 laboratory has special engineering and
design features.
The following standard and special safety
practices, equipment, and facility requirements apply to BSL-3:
1. All the requirements of BSL-2 plus:
2. Laboratory is locked at all times (even when
occupied).
3. Agent-specific biosafety procedures must be
prepared and followed.
4. The laboratory supervisor must ensure that
laboratory personnel demonstrate proficiency in standard and special
microbiological practices before working with BSL-3 agents. This shall be
documented in writing and kept on file in the laboratory.
5. All procedures involving the manipulation of
infectious materials that may generate an aerosol must be
conducted within a BSC or other physical containment devices.
6. No work with open vessels is conducted on the bench.
7. When a procedure cannot be performed within a BSC, a combination of personal
protective equipment and other containment devices, such as a centrifuge
safety cup or sealed rotor, must be used.
1. All the requirements of BSL-2 plus:
2. All procedures involving the manipulation of
infectious materials must be conducted within a BSC (preferably Class II or
Class III), or other physical containment devices. Contact EHS for assistance selection the
appropriate BSC.
3. Protective laboratory clothing with a solid-front
such as tie-back or wraparound gowns, scrub suits, or coveralls are worn by
workers when in the laboratory. Protective clothing may not be worn outside of the laboratory. Reusable clothing is decontaminated with appropriate
disinfectant before being laundered. Clothing must be changed when contaminated.
4. Eye, face, and respiratory protection must be used
in rooms containing infected animals.
1. All the requirements of BSL-2 plus:
2. Laboratory doors must be self-closing and be locked at all times. Key card
access is required.
3. The laboratory must be separated from areas that
are open to unrestricted traffic flow within the building.
4. Access to the laboratory is restricted to entry by
a series of two self-closing doors; the anteroom has facilities to change into
and out of laboratory
clothing.
5. No personal clothing (excluding undergarments) or
personal effects (jewelry, etc.) are worn into the lab.
6. Laboratories must have a sink for hand washing. The
sink must be hands-free or automatically operated. It should be located
near the exit door. If the laboratory is segregated into different
laboratories, a sink must also be available for hand washing in each zone. Additional sinks may be required as determined by
the risk assessment.
7. The laboratory must be designed so that it can be
easily cleaned and decontaminated. Carpets and rugs are not permitted.
Seams, floors, walls, and ceiling surfaces must be sealed. Spaces around doors and ventilation openings should be capable of being sealed to
facilitate space decontamination.
a. Floors must be slip resistant, impervious to
liquids, and resistant to chemicals. Floors shall be seamless, sealed, resilient or poured floors, with
integral cove bases.
b. Walls and ceilings shall be constructed to produce a sealed smooth finish
that can be easily cleaned and decontaminated.
8. The entire laboratory shall be decontaminated when there has been gross contamination of the space, significant
changes in laboratory usage, for major renovations, or maintenance shut
downs. Selection of the appropriate materials and methods used to
decontaminate the laboratory must be based on the risk assessment of the biological
agent(s) in use.
9. All windows in the laboratory must be sealed.
10. A ducted air ventilation system is required. This
system must provide sustained directional airflow by drawing air into
the laboratory from ÒcleanÓ areas toward Òpotentially contaminatedÓ areas. The
laboratory shall be designed such that under failure conditions the
airflow will not be reversed.
11. Laboratory personnel must be able to verify
directional airflow. A visual monitoring device which confirms directional
airflow must be provided at the laboratory entry. Audible alarms must be
installed to notify personnel of air flow disruption.
12. The laboratory exhaust air must not re-circulate to
any other area of the building.
13. The laboratory building exhaust air must be dispersed away from occupied areas and from building air intake
locations and must be HEPA filtered.
14. BSCs shall be certified at least annually to assure
correct performance.
15. HEPA filtered exhaust air from a Class II BSC can
be safely re-circulated into the laboratory environment if the cabinet is
operated according to manufacturerÕs recommendations. BSCs can also be connected to the laboratory exhaust
system by either a thimble (canopy) connection or a direct (hard) connection.
Provisions to assure proper safety cabinet performance and air system operation
must be verified. Class III BSCs must be directly (hard) connected up through
the second exhaust HEPA filter of the cabinet. Supply air must be provided
in such a manner that prevents positive pressurization of the cabinet.
16. A method for decontaminating all laboratory wastes shall be available
within the
laboratory (e.g., autoclave, chemical disinfection,
incineration, or other validated decontamination method).
17. Equipment that may produce infectious aerosols must
be contained in devices that exhaust air through HEPA filtration or other
equivalent technology before being discharged into the laboratory. These HEPA
filters must be tested and/or replaced at least annually.
18. All items, including large pieces of equipment, must be decontaminated before removal from the laboratory.
19. Enhanced environmental and personal protection may
be required by the agent summary statement, risk assessment, or applicable
local, state, or federal regulations. These laboratory enhancements may
include, for example, one or more of the following; an anteroom for clean
storage of equipment and supplies; gas tight dampers to facilitate laboratory isolation; final HEPA filtration of the
laboratory exhaust air; laboratory effluent decontamination; and advanced
access control devices such as biometrics. Consult EHS for information on what additional
measures may be required.
20. HEPA filter housings shall have gas-tight isolation dampers and bag-in/bag-out (with appropriate decontamination procedures) capability. The HEPA
filter housing shall allow
for leak testing of each filter and assembly. The filters and the housing will be certified at least annually.
21. The BSL-3 facility design, operational parameters,
and procedures must be verified and documented prior to operation.
Facilities must be re-verified and documented at least annually.
Biosafety
Level 4 is suitable for Risk
Group 4 Agents; these are agents that are likely to cause serious or lethal human disease for which preventive or therapeutic
interventions are not usually available
(high individual risk and high community risk). A pathogen that usually causes serious human,
animal, or plant disease
and that can be readily transmitted from one individual to another, directly
or indirectly. Effective treatment and preventive measures are not usually available.
There are
no Clemson University facilities
suitable for working with BSL4 agents; thus, work with such agents at Clemson
is prohibited.General
Precautions
Employees
who work with or around an agent for which there is a vaccine should consult
EHS or the Occupational Health Nurse for information about immunization for
that particular agent. Please inform EHS of the receipt of any material
classified as Biosafety Level 2 (BSL2) or above; include information about the
location, storage, use, precautions, and emergency procedures. Use a biohazard
warning symbol to designate labs or storage areas housing human blood, blood
products, or tissues, and any pathogenic agents. If a laboratory is conducting
work at BSL2 level or above, a warning sign identifying the agent, emergency
contact, and any special precautions required, must be posted on the laboratory
door as well. See the appendices for a summary of biosafety levels, and
listings of agents.
Note that receipt of any material deemed by the
CDC/USDA as a Select Agent requires prior approval; work with BSL2 Agents or
above require submitting a protocol for approval with the Institutional
Biosafety Committee.
Biological laboratories must be reviewed annually
by the Department Head, Primary Investigator or other responsible individual,
and copies of the reviews submitted to EHS (see Appendix D for a copy of a
Biological Laboratory checklist). EHS will conduct random inspections of
biological laboratories.
Please
observe the following basic precautions while working in a biological
laboratory:
Do not eat, drink, apply cosmetics or lip balm,
store food, or smoke in the laboratory.
Never
Do not
eat, drink, apply cosmetics or lip balm, store food, or smoke in the
laboratory.
Wear disposable, high cuff, latex or nitrile gloves
when working with biohazards (remember that latex gloves are permeable to
organic solvents, including ethanol).
Never
mouth pipette.
Thin
gloves offer little protection against cuts, bites, scratches, etc. Use the
thickest gloves allowed by your work (i.e. do not sacrifice the dexterity
required by your work.
Wearing
two pairs of thinner gloves permits the safe removal of the outer pair in case
of inadvertent contamination.
Wear the
lab coat while in the laboratory. The lab coat should be closed, and the
sleeves tucked into the gloves or otherwise restrained. Disposable Tyvek lab
coats are available and recommended for work conducted in biosafety cabinets.