UW-Stout
Biological Safety Program
Policy Manual
Table of Contents
1.
Introduction ................................................................................................................................1
2.
Terminology ................................................................................................................................1
3.
Biosafety Subcommittee of the Research Safety Committee ...........................................................4
4.
Review and approval of biohazardous materials studies and instruction ..........................................5
5.
Roles and responsibilities .............................................................................................................8
6.
Practices and procedures ........................................................................................................... 10
7.
Biohazardous waste disposal ...................................................................................................... 14
8.
Biohazardous spill cleanup .......................................................................................................... 14
9.
Transportation and shipping of infectious agents ......................................................................... 16
10.
Select agents (Health and Human Services and US Department of Agriculture) .......................... 17
11.
Controlled substances (Drug Enforcement Agency) .................................................................. 18
12.
Forbidden agents ................................................................................................................... 18
APPENDIX A KEY CONTACTS ............................................................................................................. 19
APPENDIX B PROJECT REGISTRATION AND APPROVAL ...................................................................... 20
APPENDIX C PROJECT REGISTRATION FORMS AND SIGNS ................................................................. 21
APPENDIX D STANDARD PRACTICES AND PROCEDURES..................................................................... 32
Autoclave procedures and safety .................................................................................................... 32
Biological safety cabinets (BSCs) .................................................................................................... 33
Blenders, ultrasonic disrupters, grinders and lyophilizers .................................................................. 35
Bloodborne pathogens ................................................................................................................... 36
Centrifuge equipment .................................................................................................................... 36
Cryostats ...................................................................................................................................... 37
Decontamination ........................................................................................................................... 38
Housekeeping ............................................................................................................................... 40
Labeling ........................................................................................................................................ 40
Loop sterilizers and Bunsen burners ............................................................................................... 41
Personal protective equipment (PPE) .............................................................................................. 41
Pipettes and pipetting aids ............................................................................................................. 42
Sharps safety ................................................................................................................................ 43
APPENDIX E TRAINING ..................................................................................................................... 46
APPENDIX E-1 BIOSAFETY LEVEL 1 (BSL-1) LABORATORY WORKER TRAINING ................................ 47
APPENDIX E-2 BIOSAFETY LEVEL 2 (BSL-2) LABORATORY WORKER TRAINING ................................ 50
APPENDIX E-3 ............................................................................................................................... 54
APPENDIX F TREATMENT, HANDLING AND DISPOSAL OF BIOLOGICAL MATERIALS ............................. 55
APPENDIX G SELECT AGENTS AND TOXINS........................................................................................ 59
APPENDIX H ACRONYMS ................................................................................................................... 60
1. Introduction
1.1. This manual, in conjunction with the latest editions of the U.S. Department of Health and
Human Services Publication Number (CDC) 21-1112, Biosafety in Microbiological and Biomedical
Laboratories (the BMBL) (http://www.cdc.gov/biosafety/publications/index.htm) and the
National Institutes of Health (NIH) Guidelines for Research Involving Recombinant or Synthetic
Nucleic Acid Molecules (NIH Guidelines) (http://oba.od.nih.gov/rdna/nih_guidelines_oba.html),
outlines the requirements for safely working with biohazardous materials at the University of
Wisconsin - Stout. The Research Safety Committee (RSC) is responsible for the regulation of
biohazardous materials on the UW-Stout campus. Registration with the RSC is required for all
instructional, research, and outreach projects involving potentially pathogenic microorganisms,
etiologic agents of Risk Group 2/biosafety level 2 or higher, infectious agents, oncogenic viruses,
human tissue and bloodborne pathogens, and in-vitro construction or propagation of
recombinant nucleic acid molecules and synthetic nucleic acid molecules. The RSC may be
contacted through Safety and Risk Management at 232-1793.
1.2. The concern with all biohazardous material is the safety of University employees and students
working with the material and the prevention of release of that material.
1.3. An integral component of the UW-Stout Biosafety Program is a group of generally accepted
standard practices and procedures, included in Appendix D. Anyone working with a biohazardous
agent or biohazardous material at any facility at UW-Stout is expected to follow these practices
and procedures.
1.4. Investigators may work with vertebrate animals in conjunction with their work with some
biohazardous materials. All vertebrate animal work conducted at UW-Stout must be approved by
the Institutional Animal Care and Use Committee (IACUC), which can be contacted through
Research Services at 232-1126.
1.5. Any work with biohazardous materials that involves human subjects must also be approved by
the Institutional Review Board (IRB), which can be contacted through Research Services at 2321126.
1.6. Inevitably there are special situations that the existing program is not designed to
accommodate. In those situations, consult with the RSC before initiation of the work to prevent
misunderstandings after work begins, whether the work is research, teaching or outreach.
1.7. The principal investigator (PI) or instructor involved in teaching, research, and/or outreach
activities involving biohazardous materials has the primary ethical and legal responsibility to
ensure the safety of students, faculty, staff, visitors and the environment. For that reason PIs
are urged to carefully read this manual and understand its contents. If there are specific
questions concerning this manual or the UW-Stout Biological Safety Program please contact
Safety and Risk Management at 232-1793.
2. Terminology
2.1. Traditionally, microorganisms have been classified according to the biological containment they
require. Four levels of containment have been defined and termed biosafety levels (BSLs 1-4);
thus an organism requiring, for example, BSL-2 containment is termed a BSL-2 or a Class 2
1
agent or organism. More recently, NIH has introduced classification of agents into four risk
groups based on their relative pathogenicity for healthy adult humans. The following table lists
the four groups and the basis for classification.
Risk Group 1 (RG1)
Risk Group 2 (RG2)
Risk Group 3 (RG3)
Risk Group 4 (RG4)
Agents that are not associated with disease in
healthy adult humans
Agents that are associated with human disease
that is rarely serious and for which preventive
or therapeutic interventions are often available
Agents that are associated with serious or
lethal human disease for which preventive or
therapeutic interventions may be available
(high individual risk but low community
risk)
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)
2.1.1.
Risk group classifications correlate with but do not equate to biosafety levels. A risk
assessment performed by the PI will determine the degree of correlation between an
agent’s risk group classification and the required biosafety level. Under some
circumstances, the containment level required may be raised or lowered as a result of a
comprehensive risk assessment as detailed in Section II-A-3 of the NIH Guidelines. If you
have any concerns regarding the risk group or appropriate level of containment for an
organism, consult the NIH Guidelines.
2.1.2.
There are several references for assistance with classification of organisms by risk group:
2.1.2.1. Appendix B of the NIH Guidelines lists biological agents by risk group and includes
those agents known to infect humans as well as selected animal agents that may
pose theoretical risks if inoculated into humans. Included are lists of representative
genera and species known to be pathogenic. Mutated, recombined, and nonpathogenic species and strains are not considered. Non-infectious life cycle stages
of parasites are excluded. The list includes the more commonly encountered agents
and is not meant to be all-inclusive.
2.1.2.2. The American Biological Safety Association maintains a searchable database
(http://www.absa.org/riskgroups/index.html) of international risk group
classifications for bacteria, viruses, fungi, and parasites that provides information to
be used as a starting point for the risk assessment and the determination of the
biosafety level to be used when working in the laboratory.
2.1.2.3. The Public Health Agency of Canada has produced Pathogen Safety Data Sheets for
common human pathogens (http://www.phac-aspc.gc.ca/lab-bio/res/psdsftss/index-eng.php) that include a section on recommended exposure
controls/precautions, including risk group/biosafety level.
2
2.1.3.
All PIs using human organ or cell cultures (primary cultures, cell strains, cell lines) must
handle all such cultures under BSL-2 conditions and in accordance with the Bloodborne
Pathogen Standard (29 CFR 1910.1030) unless the RSC has specifically approved a lower
standard of containment.
2.2. For the purposes of this policy, a biohazardous material is any biological material capable of
causing harm to humans, animals or plants, including both biohazardous agents and nonreplicating materials such as toxins. In addition, this term may also be used to refer to material
that harbors a biohazardous agent. A biohazardous agent is a pathogen capable of replication
and is a disease-causing microorganism capable of causing diseases in humans, animals, or
plants. Toxic, mutagenic, and teratogenic chemicals are not considered biohazards for the
purpose of this policy.
2.2.1.
There are several categories of biohazards or potentially infectious materials, including:
2.2.1.1. Human, animal and plant pathogens: bacteria (including those with drug resistance
plasmids), rickettsiae, fungi, viruses (including oncogenic viruses and viroids),
parasites, and prions.
2.2.1.2. All human and/or non-human primate blood, blood products, tissues and certain
body fluids. Researchers or instructors contemplating use of human blood, blood
products, tissues and body fluids should also contact the IRB through Research
Services at 232-1126; more information is available at
http://www.uwstout.edu/rs/humansubjects.cfm.
2.2.1.3. Cultured cells (all human and certain animal species) and potentially infectious
agents these cells may contain.
2.2.1.4. Biological toxins (bacterial, fungal, botanical, etc.).
2.2.1.5. Certain recombinant nucleic acid molecules and synthetic nucleic acid molecules,
and cells, organisms and viruses containing such molecules.
2.2.1.6. Infected animals and animal tissues.
2.3. Research involving experimentation and manipulation of various types of potentially infectious
systems can be divided into four general categories. They are those that expose the worker or
the environment to (1) blood, bodily fluids, and organs/tissues of human origin; (2) infectious
agents and their potentially infectious products; (3) recombinant or synthetic nucleic acid
molecules and their products; and (4) miscellaneous biohazardous materials.
2.3.1.
Experimentation or manipulations of human blood or other materials of human origin,
including, but not limited to excreta, secreta, blood and its components, unfixed tissue,
and tissue fluids, all of which may or may not contain an infectious agent, may place the
worker at risk of exposure to bloodborne pathogens. Examples that could result in
exposure would be clinical sciences laboratories performing tests or analyses on human
blood or other potentially infectious materials or research labs performing experiments
and/or manipulations with human blood or unfixed tissues or organs.
3
2.3.2.
Infectious agents, pathogens or substances are defined as those substances containing
viable microorganisms or their toxins that are known or suspected to cause disease in
animals, plants or humans. Pathogens are classified as bacteria, fungi, rickettsiae,
viruses, parasites, oncogenic viruses, and prions. Any materials that come in contact with
infectious agents or their byproducts must be handled and disposed of in the same
manner appropriate for disposing of the infectious agents themselves.
2.3.3.
In the context of the NIH Guidelines, recombinant and synthetic nucleic acid molecules
are defined as:
2.3.3.1. Molecules that a) are constructed by joining nucleic acid molecules and b) that can
replicate in a living cell (i.e., recombinant nucleic acids);
2.3.3.2. Nucleic acid molecules that are chemically or by other means synthesized or
amplified, including those that are chemically or otherwise modified but can base
pair with naturally occurring nucleic acid molecules (i.e., synthetic nucleic acids); or
2.3.3.3. Molecules that result from the replication of those described in 2.3.3.1 and 2.3.3.2.
2.3.4.
Miscellaneous biohazardous materials include materials not directly covered by the above
definitions, such as allergens, cultured animal cells and their potentially infectious agents,
tissues from experimental animals (including animal dander), plant viruses, bacteria and
fungi, toxins (bacterial, plant, etc.), and those as yet unnamed elements or agents that
may produce disease. In regard to allergens, it is not the intent of the RSC to require the
registration of research projects involving only allergens (i.e. projects involving allergens
not listed as biohazardous agents or materials). Allergens are almost ubiquitous in the
environment. However, it is important that PIs consider the hazard associated with
allergens because manipulations of these allergens or materials containing allergens may
result in human exposures to allergens at concentrations orders of magnitude greater
than would normally occur. Because severe allergenic reactions may be life-threatening,
the control of allergens in any teaching, research or outreach project must be an
important consideration in experimental/procedural design.
3. Biosafety Subcommittee of the Research Safety Committee
3.1. The RSC is charged by the University Chancellor to formulate policy and procedures related to
biosafety, chemical hygiene and radiation safety. The Biosafety Subcommittee of the RSC
(referred to as the Biosafety Subcommittee for the remainder of this document) functions to
approve experiments involving human gene therapy, formation of transgenic animals or plants,
and the generation and/or use of recombinant or synthetic nucleic acid molecules in accordance
with the requirements of the NIH Guidelines. UW-Stout also requires Biosafety Subcommittee
registration and approval for use of RG2 biohazardous agents and toxins.
3.2. Structure and responsibilities
3.2.1. Members of the Biosafety Subcommittee will be selected from the general RSC
membership based on their experience with and expertise in biohazardous materials and
their capability to assess the safety of work with these materials and to identify any
potential risk to public health or the environment.
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3.2.2. The Biosafety Subcommittee has the responsibility of assessing risks and potential
environmental impacts associated with investigations involving biological agents and
making recommendations for safe conduct of such studies. It also functions on behalf of
the institution to ensure that the experimental work is performed in compliance with
current policies and guidelines promulgated by government granting and regulatory
agencies.
3.2.3. Registrations approved by the Biosafety Subcommittee will be active for three years from
the date of approval. Written notification that the registration will expire will be sent out
at least 30 days prior to the expiration date. The PI will then be required to submit a new
registration application for review and approval by the Biosafety Subcommittee.
3.2.4. The Biosafety Subcommittee will meet as necessary to conduct business.
4. Review and approval of biohazardous materials studies and instruction
4.1. The Biosafety Subcommittee and RSC can effectively carry out their designated functions only if
they have adequate prior knowledge of potentially hazardous research projects and instruction.
Therefore, all instructional, research, and outreach projects involving potentially pathogenic
microorganisms, etiologic agents of RG1 or higher, infectious agents, oncogenic viruses, human
tissue and bloodborne pathogens, and work with recombinant or synthetic nucleic acid
molecules must be registered with and approved in writing by the Biosafety Subcommittee.
4.2. It is not the purpose of the Biosafety Subcommittee to pass judgment on scientific merits or
even to consider "risk" versus "expected benefits" of potentially hazardous research projects
involving an organism of RG1 or RG2 to be undertaken on the University’s premises. Rather, it is
the concern of this Committee to know whether or not the safety precautions proposed for the
experimental work are adequate for protection of personnel and the environment. In general,
the review process will focus on (1) qualifications of the investigator, (2) agents to be employed,
(3) risks presented by experimental procedures, (4) adequacy of containment equipment and
facilities, (5) training level of persons directly associated with the work, and (6) other factors
relevant to safe conduct of the study.
4.3. Due to a lack of facilities designed to contain them, UW-Stout does not allow RG3 or RG4
organisms or biohazardous materials requiring BSL-3 or BSL-4 containment or facilities on the
campus or at any of its outlying units or off-campus locations. There are a limited number of
approved and certified BSL-4 facilities within the U.S., such as those at the Centers for Disease
Control and Prevention (CDC) in Atlanta, GA and the U.S. Army Medical Research Institute for
Infectious Diseases in Fort Detrick, MD.
4.4. The PI and the Biosafety Subcommittee must concur on all matters relating to containment
requirements, safe practices, and handling and disposal procedures for biohazardous materials.
In the event of non-concurrence, the recommendations of the Biosafety Subcommittee shall
prevail until such time as they are modified or rescinded by appellate decision of an
administrative review that may include outside reviewers. Questions relating to recombinant or
synthetic nucleic acid molecule studies that are not covered by the NIH Guidelines will be
referred to the NIH Office of Biotechnology Activities (OBA) for resolution.
5
4.5. Some laboratories maintain cultures and/or archival samples of biohazardous materials. In these
instances, a “generic” registration for maintenance and storage of reference or other
samples/cultures shall be submitted to the Biosafety Subcommittee. An inventory of agents
should be attached and updated whenever significant changes are made, either additions or
deletions. Significant changes include additional species or additional strains of inventoried
agents that demonstrate a need for more stringent containment.
4.6. All projects involving recombinant or synthetic nucleic acid molecules must comply with the NIH
Guidelines, regardless of the source of funds supporting the research. It is the definitive
reference for this research in the United States.
4.6.1.
UW-Stout requires that all experiments involving the generation and/or use of
recombinant or synthetic nucleic acid molecules be registered with the Biosafety
Subcommittee, EVEN IF THEY ARE EXEMPT from the NIH Guidelines. There may be
recombinant or synthetic nucleic acid molecule experiments that are not covered by the
NIH Guidelines that require review and approval by outside agencies before initiation or
funding. These experiments are most common in the environmental sciences. If you
have specific questions about a particular host-vector system not covered by the
guidelines, please call the OBA, National Institutes of Health at (301) 496-9838 or FAX
(301) 496-9839.
4.6.2.
Experiments involving recombinant or synthetic nucleic acid molecules are divided into
categories denoted as classes. Presently there are six classes (III-A, III-B, III-C, III-D,
III-E and III-F). It is the responsibility of the PI to read the NIH Guidelines in order to
determine which class is applicable to the work conducted.
4.6.3.
Exempt experiments are listed in the NIH Guidelines. To register exempt recombinant or
synthetic nucleic acid molecule research complete the “Registration Cover Sheet,” noting
the reason the research is exempt, and a “Registration Form for Exempt Research” and
send both to the Biosafety Subcommittee. Forms are found in Appendix C. The Biosafety
Subcommittee review will certify the project as exempt. All such research must be
conducted using BSL-1 practices. Exempt experiments include:
4.6.3.1. Those that are not in organisms or viruses.
4.6.3.2. Those that consist entirely of DNA segments from a single nonchromosomal or viral
DNA source, though one or more of the segments may be a synthetic equivalent.
4.6.3.3. Those that consist entirely of DNA from a prokaryotic host including its indigenous
plasmids or viruses when propagated only in that host (or a closely related strain of
the same species), or when transferred to another host by well-established
physiological means.
4.6.3.4. Those that consist entirely of DNA from a eukaryotic host including its chloroplasts,
mitochondria, or plasmids (but excluding viruses) when propagated only in that host
(or a closely related strain of the same species).
4.6.3.5. Those that consist entirely of DNA segments from different species that exchange
DNA by known physiological processes, though one or more of the segments may
6
be a synthetic equivalent. A list of such exchangers will be prepared and periodically
revised by the NIH Director with advice of the Recombinant DNA Advisory
Committee (RAC) after appropriate notice and opportunity for public comment. See
the NIH Guidelines, Appendices A-I through A-VI, Exemptions Under Section III-F-5-Sublists of Natural Exchangers, for a list of natural exchangers that are exempt.
4.6.3.6. Those that do not present a significant risk to health or the environment as
determined by the NIH Director, with the advice of the RAC, and following
appropriate notice and opportunity for public comment. See the NIH Guidelines,
Appendix C, Exemptions under Section III-F-6 for other classes of experiments that
are exempt.
4.6.4.
The following experiments require Biosafety Subcommittee review and approval
simultaneous with initiation (prior approval is not required); see the NIH Guidelines,
Section III-E. To register non-exempt recombinant or synthetic nucleic acid molecule
research, complete “Registration Cover Sheet” and a “Recombinant DNA Registration
Form for Non-Exempt Research” and send to the RSC.
4.6.4.1. Experiments involving the formation of recombinant or synthetic nucleic acid
molecules containing no more than two-thirds of the genome of any eukaryotic
virus.
4.6.4.2. Experiments involving nucleic acid modified-whole plants, and/or experiments
involving recombinant or synthetic nucleic acid molecule-modified organisms
associated with whole plants, except those that fall under Section III-A, III-B, III-D,
or III-F of the NIH Guidelines (as described in the NIH Guidelines, Section III-E-2).
4.6.4.3. Experiments involving transgenic rodents in which the animal’s genome has been
altered by stable introduction of recombinant or synthetic nucleic acid molecule, or
nucleic acids derived therefrom, into the germ line (transgenic rodents).
4.6.5.
The following experiments require approval from the Biosafety Subcommittee prior to
initiation; see the NIH Guidelines, Section III-D. To register these projects, complete
“Registration Cover Sheet” and a “Registration Form for Non-Exempt Research” and send
to the Biosafety Subcommittee with the applicable supporting documentation.
4.6.5.1. Experiments using RG2 (or higher) or restricted agents as host-vector systems.
4.6.5.2. Experiments in which DNA from RG2 (or higher) or restricted agents is cloned into
nonpathogenic prokaryotic or lower eukaryotic host-vector systems.
4.6.5.3. Experiments involving the use of infectious DNA or RNA viruses or defective DNA or
RNA viruses in the presence of a helper virus in tissue culture systems.
4.6.5.4. Experiments involving whole animals.
4.6.5.5. Experiments involving whole plants.
4.6.5.6. Experiments involving more than 10 liters of culture.
7
4.6.5.7. Experiments involving influenza viruses.
4.6.6.
The use of any campus greenhouse facility for research with transgenic plants must be
approved by the Biosafety Subcommittee and greenhouse management personnel.
4.7. Research and instruction involving infectious biological agents (RG2 organisms or above) and
biologically-derived toxins must be registered with and approved by the Biosafety Subcommittee
prior to the start of work. Additional information and forms are found in Appendices B and C.
4.8. All animal protocols involving the use of recombinant or synthetic nucleic acid molecules;
infectious or transmissible agents; human blood, body fluids or tissues; or toxins must be
submitted to the Biosafety Subcommittee for registration and approval prior to final approval by
the IACUC. The PI must notify the IACUC in writing prior to initiation of experimentation. IACUC
guidelines are available online (http://www.uwstout.edu/rs/iacuc.cfm). Investigators who are
uncertain how to categorize agents should contact the Biosafety Subcommittee.
5. Roles and responsibilities
5.1. The Biosafety Officer (BSO) is primarily responsible for implementation and the day-to-day
management of the biological safety program. Major duties or activities of the BSO include the
following:
5.1.1.
Monitor compliance with University safety practices and procedures regarding potentially
infectious and biohazardous materials.
5.1.2.
Assist in the preparation and periodic updating of a biosafety manual that is in
accordance with University policy and consistent with government regulatory guidelines.
5.1.3.
Provide consultation to investigators and instructors on matters relating to laboratory
safety, appropriate handling and containment of biohazardous materials,
decontamination, and disposal of biohazardous wastes.
5.1.4.
Aid investigators and instructors in the development of appropriate emergency measures
for dealing with accidental spills and contamination.
5.1.5.
Conduct surveillance of laboratories in which biohazardous materials are employed to
ensure compliance with the approved protocol, prescribed safety guidelines, prescribed
safety training and rectification of any deficiencies.
5.1.6.
Monitor all intra-campus transport to ensure compliance with the rigorous containment
procedures described herein. Provide information for off-campus shipment of
biohazardous materials.
5.1.7.
Develop and conduct training programs for laboratory personnel using biohazardous
materials, specifically to promote techniques for safe handling and disposal.
5.1.8.
Serve as liaison between the University and outside regulatory agencies concerned with
the use of biohazardous materials.
8
5.1.9.
The BSO, with concurrence from the Co-chairs of the RSC, or with concurrence of three
members of the RSC if the Chairs are unavailable, may stop any work with microbial
agents or any hazardous research project that creates an unreasonable hazard to
personnel or involves experiments prohibited by the University. The entire RSC then will
review the problem and will complete the review within a working week. Written
recommendation(s) will be forwarded from the RSC to the Vice Chancellor for Academic
and Student Affairs for final action.
5.1.10. In the event of a mechanical malfunction, systems breakdown or shutdown of any
nature, or preventive maintenance of primary containment equipment or components,
the BSO must be notified immediately. In the case of an unplanned event and if Physical
Plant mechanical staff is not already on the scene, the BSO will notify appropriate
Physical Plant staff. Proper precautions must be taken immediately. All experiments must
be halted and the biological agents secured (e.g. containers sealed or placed in freezer
or refrigerator). The area must be cordoned off during the entire time of the shutdown.
No further activities will be allowed until the BSO certifies that the facility is safe to use.
5.2. Principal investigators (PIs) and Laboratory Instructors:
5.2.1.
The PI is expected to know and understand all relevant safety guidelines described in this
manual, to personally monitor day-to-day operation of the laboratory, and to take all
necessary steps for protection of staff, students, and the general public against
undesirable consequences of experimental work conducted within UW-Stout facilities.
Ultimate responsibility for the safe conduct of research involving biohazardous materials
rests with the PI. The PI is aware of the complexities of the research and its associated
hazards and must adequately inform all who are working in the research laboratory or
otherwise involved in the research of the hazards involved. Because working with
biohazardous materials may pose special risks, the PI must assure compliance with the
biohazardous materials standard policies and procedures.
5.2.2.
File a registration form with and obtain written approval of the Biosafety Subcommittee
before performing work involving the use of oncogenic viruses, infectious agents,
recombinant or synthetic nucleic acid molecules or experiments involving the use of
human blood, bloodborne pathogens or other potentially infectious materials such as
unfixed human tissues, primary human cell lines, and certain bodily fluids. See Section 4
and Appendix B of this manual for the registration and approval process.
5.2.3.
Thoroughly inform and train all persons (including research personnel such as graduate
students, undergraduate students, technicians and laboratory researchers) directly
involved in potentially hazardous experiments on the biohazardous materials and harmful
chemicals, the potential health risk presented and the safety procedures necessary to
minimize exposure. Training requirements are outlined in Appendix E. Each laboratory
must maintain a current inventory book of biohazardous material, maintain a current
record of personnel training, post safety standards for the required biosafety level and
maintain safety information for each human pathogen in the inventory.
5.2.4.
Establish emergency procedures to be followed in the event of an overt spill or
contamination with potentially hazardous biological material. These procedures should be
9
posted in a prominent place in the laboratory. Moreover, it is recommended that a
responsible member of the laboratory staff be designated to handle emergency situations
whenever the PI is absent from the premises. Section 9 of this manual provides
guidelines for spill cleanup.
5.2.5.
Immediately report any unusual incident, such as spill, break in containment, or overt
contamination to the BSO or Safety and Risk Management.
5.2.6.
Post working areas and facilities with biohazard warning signs. Standardized signs will be
provided by the BSO, and are included in Appendix C. The PI should consult the BSO if
assistance is required in placement of signs.
5.3. Laboratory personnel and students
5.3.1.
All research personnel (researchers, graduate students and undergraduate students) and
teaching lab students under the direction of the PI are expected to participate in the
safety training and to use safety procedures established by the PI.
5.3.2.
Perform all tasks using established safety practices and shall comply with the safety
guidelines for the work being performed.
5.3.3.
Keep themselves informed of the risks in the laboratory and ask for and participate in
training that increases their knowledge and ability to deal safely with the risks.
5.3.4.
Report all unsafe practices, accidents and injuries to the PI/instructor (or the laboratory
manager or BSO if the PI/instructor is unavailable).
6. Practices and procedures
6.1. Standard microbiological practices will be followed when working with all biohazardous
materials, regardless of BSL or risk group.
6.1.1.
The laboratory supervisor must enforce UW-Stout policies that control access to the
laboratory (e.g. granting key card access only to authorized students and staff, keeping
doors closed).
6.1.2.
Persons must wash their hands after working with potentially hazardous materials and
before leaving the laboratory.
6.1.3.
Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food
for human consumption must not be permitted in laboratory areas. Food must be stored
outside the laboratory area in cabinets or refrigerators designated and used for this
purpose.
6.1.4.
Mouth pipetting is prohibited; mechanical pipetting devices must be used.
6.1.5.
Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken
glassware must be developed and implemented; see Appendix D for recommended
practices. Whenever practical, laboratory supervisors should adopt improved engineering
10
and work practice controls that reduce risk of sharps injuries. Precautions, including
those listed below, must always be taken with sharp items. These include:
6.1.5.1. 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.
6.1.5.2. Used disposable needles and syringes must be carefully placed in conveniently
located puncture-resistant containers used for sharps disposal.
6.1.5.3. Non-disposable sharps must be placed in a hard walled container for transport to a
processing area for decontamination, preferably by autoclaving.
6.1.5.4. Broken glassware must not be handled directly. Instead, it must be removed using a
brush and dustpan, tongs, or forceps. Plastic ware should be substituted for
glassware whenever possible.
6.1.6.
Perform all procedures to minimize the creation of splashes and/or aerosols.
6.1.7.
Decontaminate work surfaces after completion of work and after any spill or splash of
potentially infectious material with appropriate disinfectant.
6.1.8.
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.
6.1.8.1. Materials to be decontaminated outside of the immediate laboratory must be placed
in a durable, leak proof container and secured for transport.
6.1.8.2. Materials to be removed from the facility for decontamination must be packed in
accordance with applicable local, state, and federal regulations.
6.1.9.
A sign incorporating the universal biohazard symbol must be posted at the entrance to
the laboratory or classroom when infectious agents are present. The sign should include
the name of the agent(s) in use, and the name and phone number of the laboratory
supervisor or other responsible personnel. For BSL-2 agents, the sign should also include
the procedure required for entry and exit (e.g. PPE) and special practices (e.g. use of
BSC, immunizations). Signs are available in Appendix C, or from the BSO.
6.1.10. An effective integrated pest management program is required (see Appendix G of the
BMBL).
6.1.11. 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. Personal health status may impact an
individual’s susceptibility to infection, ability to receive immunizations or prophylactic
interventions. Therefore, all laboratory personnel and particularly women of childbearing
age should be provided with information regarding immune competence and conditions
that may predispose them to infection. Individuals having these conditions should be
11
encouraged to self-identify to their healthcare provider for appropriate counseling and
guidance.
6.2. Work with BSL-2 agents requires special practices:
6.2.1.
Access to the laboratory must be restricted when work is being conducted.
6.2.2.
All persons entering the laboratory must be advised of the potential hazards. This can be
accomplished by posting the BSL-2 Point Paper (Appendix E-3) along with a sign-in sheet
to record the names of people who have reviewed the paper. The paper can also be
reviewed with classes or groups as a whole.
6.2.3.
Laboratory personnel must be provided medical surveillance, as appropriate, and offered
available immunizations for agents handled or potentially present in the laboratory.
6.2.4.
A laboratory-specific biosafety manual must be prepared and adopted as policy. The
biosafety manual must be available and accessible.
6.2.5.
The laboratory supervisor must ensure that laboratory personnel demonstrate proficiency
in standard and special microbiological practices before working with BSL-2 agents.
6.2.6.
Potentially infectious materials must be placed in a durable, leak proof container during
collection, handling, processing, storage, or transport within a facility.
6.2.7.
Laboratory equipment should be routinely decontaminated, as well as after spills,
splashes, or other potential contamination.
6.2.8.
Spills involving infectious materials must be contained, decontaminated, and cleaned up
by staff properly trained and equipped to work with infectious material.
6.2.9.
Equipment must be decontaminated before repair, maintenance, or removal from the
laboratory.
6.2.10. Incidents that may result in exposure to infectious materials must be immediately
evaluated and treated according to procedures described in the laboratory biosafety
manual. All such incidents must be reported to the laboratory supervisor. Medical
evaluation, surveillance, and treatment should be provided and appropriate records
maintained.
6.2.11. Animal and plants not associated with the work being performed must not be permitted
in the laboratory.
6.2.12. All procedures involving the manipulation of infectious materials that may generate an
aerosol should be conducted within a biological safety cabinet (BSC) or other physical
containment devices.
12
6.3. The following safety equipment should be used when working with biohazardous materials:
6.3.1.
For BSL-1 agents, special containment devices or equipment, such as BSCs, are not
generally required. For BSL-2 agents, properly maintained BSCs, other appropriate
personal protective equipment, or other physical containment devices must be used
whenever:
6.3.1.1. 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.
6.3.1.2. 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.
6.3.2.
Protective laboratory coats, gowns, smocks, or uniforms designated for laboratory use
are recommended for work with BSL-1 agents and required for BSL-2 agents. Remove
protective clothing before leaving the laboratory. Dispose of protective clothing
appropriately, or deposit it for laundering. BSL-2 laboratory clothing should not be taken
home.
6.3.3.
Safety glasses are required to be worn by everyone in the laboratory if anyone is working
with a liquid chemical or biohazardous material. Splash goggles (and mask, face shield or
other splatter guard as needed) should be 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 should also wear eye protection.
6.3.4.
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
should be available. Gloves must not be worn outside the laboratory. In addition,
laboratory workers should:
6.3.4.1. Change gloves when contaminated, glove integrity is compromised, or when
otherwise necessary.
6.3.4.2. Remove gloves and wash hands when work with hazardous materials has been
completed and before leaving the laboratory.
6.3.4.3. Do not wash or reuse disposable gloves. Dispose of used gloves with other
contaminated laboratory waste. Hand washing protocols must be rigorously followed.
6.3.5.
For work with BSL-2 agents and animals, a risk assessment should be used to determine
if eye, face and/or respiratory protection is needed in rooms containing infected animals.
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6.4. Emergency procedures:
6.4.1.
All injuries and accidental autoinoculation, ingestion or inhalation of infectious agents
must be reported immediately to the lab director or supervisor and the BSO. Affected
employees should be sent to their primary care physician for evaluation, possible
treatment and/or possible referral. Dial 911 immediately for any medical emergency.
6.4.2.
In case of natural disasters, fires, or power failure the following precautions must be
taken:
6.4.2.1. In power failures, immediately discontinue all work until power is restored. If a BSC is
being used, all open containers and the hood sash must be closed.
6.4.2.2. In case of fire, personnel must immediately follow standard emergency procedures
(evacuate area, call 911). Temperatures sufficient to ignite materials will inactivate
infectious agents used in the laboratory. However, emergency personnel should don
PPE before entering the lab and follow disinfecting procedures described in Section 8
for decontaminating any released infectious materials not involved in the fire.
6.4.2.3. In natural disasters, personnel must immediately follow standard emergency
procedures (take shelter, wait for further instructions). Upon return to the facility, PPE
must be used when entering a lab to decontaminate any disaster-related release of
infectious material. Contain released material and decontaminate using spill
procedures outlined in Section 8. Emergency personnel should don PPE before
entering lab and/or areas housing infected animals.
7. Biohazardous waste disposal
7.1. All disposal of infectious waste, autoclave bags, pipettes, sharps and biological waste must be
performed in accordance with Wisconsin Department of Natural Resources medical waste
regulations. UW-Stout has developed procedures for the proper disposal of biological waste
materials. Specific information on the disposal of biological materials and biological waste
management is found in Appendix F.
8. Biohazardous spill cleanup
8.1.
If a small or moderate spill (<100 mL) occurs inside the BSC:
8.1.1.
Wear lab coat, safety goggles and gloves during cleanup.
8.1.2.
Allow BSC to run during spill cleanup.
8.1.3.
Apply disinfectant and allow a minimum of 30 minutes contact time.
8.1.4.
Wipe up spillage with disposable disinfectant-soaked cloth.
8.1.5.
Wipe the walls, work surface and any equipment in the cabinet with a disinfectantsoaked cloth.
8.1.6.
Discard contaminated disposable materials in an appropriate autoclavable waste
container(s) and autoclave before discarding.
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8.1.7.
Place contaminated reusable items in autoclave bags, autoclavable pans with lids or wrap
in newspaper before autoclaving and cleanup.
8.1.8.
Expose non-autoclavable materials to disinfectant for a minimum of 20 minutes of
contact time before removal from the BSC.
8.1.9.
Remove protective clothing used during cleanup and place in an autoclave bag for
autoclaving. Wash hands whenever gloves are removed.
8.1.10. Allow the BSC to run for 10 minutes after cleanup before resuming work or turning
cabinet off.
8.2.
If a small or moderate spill (<100 mL) occurs in the lab, outside of the BSC:
8.2.1.
Clear the area of all personnel. Wait for aerosol to settle before entering the spill area.
The time required will depend on ventilation within the area but a general rule of thumb
is to wait approximately 30 minutes.
8.2.2.
Remove any contaminated clothing and place in an autoclave bag to be autoclaved.
8.2.3.
Don a lab coat, safety goggles and gloves.
8.2.4.
Soak paper towels in disinfectant and place over the spill.
8.2.5.
Encircle the spill with additional disinfectant, being careful to minimize aerosolization
while assuring adequate contact.
8.2.6.
Decontaminate all items within the spill area.
8.2.7.
Allow a minimum of 30 minutes of contact time to ensure germicidal action of
disinfectant.
8.2.8.
Wipe equipment with a 1:10 bleach solution, followed by water and 70% alcohol.
8.2.9.
Place disposable contaminated spill materials in appropriate autoclavable container(s) for
autoclaving.
8.2.10. Place contaminated reusable items in autoclave bags, autoclavable pans with lids or wrap
in newspaper before autoclaving and cleanup.
8.2.11. Remove protective clothing used during cleanup and place in an autoclave bag for
autoclaving. Wash hands whenever gloves are removed.
8.3.
If a large spill (>100 mL) occurs inside or out of the BSC:
8.3.1.
Evacuate the room, close the doors, prevent others from entering, and wait 30 minutes
for aerosols to settle.
8.3.2.
Follow procedures for small and moderate spills. If it is not safe for staff to perform the
cleanup, call 911.
15
8.3.3.
8.4.
8.5.
For large spills in the BSC, contact the BSO for assistance in determining if professional
decontamination of the BSC is indicated.
If a spill occurs outside of the lab, during transport:
8.4.1.
Should a spill occur in a public area, do not attempt to clean it up without appropriate
PPE (gloves, safety goggles, lab coat).
8.4.2.
As an interim measure, wear gloves and place paper towels, preferably soaked in
disinfectant, directly on spilled materials to prevent spread of contamination. To assure
adequate contact, surround the spill with disinfectant, if available, taking care to
minimize aerosols.
8.4.3.
Call 911 to report the spill and request outside assistance. Also call the BSO and/or
Safety and Risk Management for assistance in managing the situation.
If a centrifuge tube breaks while the centrifuge is running:
8.5.1.
Turn off the motor. Allow the machine to be at rest for 30 minutes before opening. Or, if
breakage is discovered after the machine has stopped, re-close the lid immediately and
allow the unit to be at rest for 30 minutes.
8.5.2.
Unplug the centrifuge before initiating clean-up.
8.5.3.
Don strong, thick rubber gloves, safety goggles and a lab coat.
8.5.4.
Flood the centrifuge bowl with a germicidal disinfectant. Place paper towels soaked in a
disinfectant over the entire spill area. Allow a minimum of 30 minutes of contact time.
8.5.5.
Use mechanical means (such as forceps) to remove broken tubes and glass fragments.
Place them in a sharps container for autoclaving and disposal as infectious waste.
8.5.6.
Remove buckets, trunnions and the rotor and place in disinfectant for 24 hours or
autoclave. Place paper towels soaked in a disinfectant over the equipment if transport is
necessary.
8.5.7.
Unbroken, capped tubes may be placed in disinfectant and recovered after 30 minute
contact time or autoclaved.
8.5.8.
Use mechanical means to remove remaining disinfectant soaked materials from
centrifuge bowl and discard as infectious waste.
8.5.9.
Place paper towels soaked in a disinfectant in the centrifuge bowl and allow it to soak
overnight, wipe down again with disinfectant, wash with water and dry. Discard
disinfectant soaked materials as infectious waste.
8.5.10. Remove protective clothing used during cleanup and place in a biohazard bag for
autoclaving. Wash hands whenever gloves are removed.
9. Transportation and shipping of infectious agents
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9.1.
All shippers of hazardous materials (biological, chemical, radioisotopes) must have
documented, U.S. Department of Transportation-mandated training. There are significant fines
associated with improper packaging and shipping of hazardous materials. Please contact Safety
and Risk Management for further information.
9.2.
Regulations on the transportation of biological agents are aimed at ensuring that the public and
the workers in the transportation chain are protected from exposure to any agent that might be
in the package. Protection is achieved through (a) the requirements for rigorous packaging that
will withstand rough handling and contain all liquid material within the package without leakage
to the outside, (b) appropriate labeling of the package with the biohazard symbol and other
labels to alert the workers in the transportation chain to the hazardous contents of the
package, (c) documentation of the hazardous contents of the package should such information
be necessary in an emergency situation, and d) training of workers in the transportation chain
to familiarize them with the hazardous contents so as to be able to respond to emergency
situations.
9.3.
For transportation within and between buildings on campus biohazardous materials need to be
packaged in a sealed, leak-proof primary container that is securely positioned in a secondary
leak-proof and closable container (e.g. cooler, ice chest) containing a clearly visible biohazard
symbol on the outside. Please contact Safety and Risk Management regarding the transport of
biohazardous materials over public highways utilizing a state of Wisconsin vehicle. The use of
private vehicles for the transportation of such materials on or off campus is prohibited.
9.4.
Receiving and sending animals, animal-derived materials, infectious or biohazardous agents,
biological toxins, and genetically modified organisms require the approval of federal agencies
such as the CDC, the U.S. Department of Agriculture (USDA), or the U.S. Fish and Wildlife
Service. Regulations that govern the transfer of biological materials help to minimize or
eliminate the possible threats to public health and agriculture. Current resources regarding the
transportation of infectious substances are found in Appendix C of the BMBL.
10. Select agents (Health and Human Services and US Department of Agriculture)
10.1. The U.S. Department of Health and Human Services (HHS) and the USDA have developed a list
of select biological agents and toxins that have the potential to pose a severe biosecurity threat
to public health, animals, and agricultural crops. A list of those materials currently classified as
a select agent is on a joint USDA Animal and Plant Health Inspection Service (APHIS)/CDC
website (http://www.selectagents.gov) and in Appendix G. HHS and USDA have adopted strict
regulations for the obtaining, possession, use, or transfer of any of these selected agents.
Failure to comply with the established regulations can result in significant civil and criminal
penalties. Therefore, any plans for obtaining such materials must approved by the Biosafety
Subcommittee well in advance of any planned use. Anyone contemplating select agent use
should understand that a decision to do so means accepting a significant level of personal
responsibility for meeting all aspects of the requirements mandated by the federal rules. At the
time this manual was developed there were no select agents on campus.
10.2. HHS regulations in 42 CFR Part 73 and the companion USDA regulations in 7 CFR Part 331 and
9 CFR Part 121 require federal registration and a security risk assessment; safe handling;
provisions for securing the agents against unauthorized access, theft, loss or release; and
17
restricted laboratory access.
11. Controlled substances (Drug Enforcement Agency)
11.1. A controlled substance is a drug that is regulated by state and federal laws that aim to control
the danger of addiction, abuse, harm and illegal trafficking. They may be illegal for sale or use,
but can be permitted for legitimate use in research.
11.2. When planning for research that involves drugs, the PI shall identify whether the drug is a
controlled substance and act accordingly. The U.S. Drug Enforcement Agency (DEA) publishes
schedules of controlled substances: http://www.deadiversion.usdoj.gov/schedules/.
11.3. The use of controlled substances for research requires obtaining both state (Wisconsin
Controlled Substances Board Special Use Authorization) and federal (DEA) registration. The
state registration must be obtained first. Penalties for using controlled substances without
proper registration can be severe. The regulations strictly limit who can handle or administer
the drugs and imposes physical security and inventory requirements.
11.4. The permitting process is between and individual researcher and the state and DEA.
Registrants cannot share controlled substances with non-registered users who are not under
their supervision.
11.5. Possession of expired substances can bring additional scrutiny from the U.S. Department of
Agriculture since administration of expired substances is not allowed.
11.6. Disposal is strictly regulated by the DEA.
11.7. Neither the BSO nor Safety and Risk Management play a role in the permitting process,
although the BSO can provide limited guidance.
11.8. The UW-Madison Research Animal Resources Center has very good guidance on the use of
controlled substances: https://www.rarc.wisc.edu/documents/printable_lab_notebook.pdf.
12. Forbidden agents
12.1. UW-Stout policy prohibits the use or possession of RG3 or RG4 organisms on campus or at any
off-campus locations. The only exclusion to this policy is reference material necessary for
identification purposes. Reference material must be labeled as such and stored in an inactive
state, either frozen or lyophilized. All freezers and storage cabinets must be clearly labeled with
signs that include the biohazard symbol, the name of the organism, and emergency contact
information. A list of all such viable reference material must be provided to the Biosafety
Subcommittee.
18
APPENDIX A
KEY CONTACTS
Position
Research Safety Committee Co-Chair
Chemical Hygiene Officer, Research Safety
Committee Co-Chair
Biological Safety Officer
Executive Director, Health and Safety
Research Administrator
IACUC Administrative Assistant
Institutional Review Board (Human Subjects)
University Police
Name
John Kirk
Rebecca Hoeft
Phone number
232-2209
232-2151
Jim Burritt
Jim Uhlir
Elizabeth Buchanan
Landon Kafka
232-5048
232-2188
232-2477
232-1126
232-1126
232-2222
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APPENDIX B
PROJECT REGISTRATION AND APPROVAL
University Researchers are required to obtain Biosafety Subcommittee of the Research Safety Committee
(RSC) review and approval for work involving:

Recombinant or synthetic nucleic acid molecules. All work must be registered with the
Biosafety Subcommittee.
Submit: “Registration Cover Sheet for All Recombinant or Synthetic Nucleic Acid
Molecule Research” and “Recombinant or Synthetic Nucleic Acid Molecule
Registration Form” (either exempt or non-exempt) (see Appendix C).

Infectious Biological Agents (organisms in Risk Group 2 or above). This includes a broad
spectrum of organisms able to cause human disease.
Submit: “Biological Agent Registration Form” (see Appendix C).

Biologically Derived Toxins. Examples include endotoxins, staphlococcus, entertoxins,
saxitoxin, cholera toxin, botulinum toxin, etc.
Submit: “Biological Toxin Registration Form” (see Appendix C).
To initiate the review process, University researchers must submit a Biosafety Subcommittee registration
form. Forms are in Appendix C. Project protocols that include the following items must accompany the
completed form:

Identification of potential hazards during sample preparation and experimental manipulations
(e.g. aerosol generation, use of sharps, excretion of animals).

Staff training and investigator experience.

Safety procedures that will be employed to minimize risks and prevent release (e.g. protective
clothing, use of biological safety cabinets, sharps disposal).

Accidental spill and/or exposure procedures specific to the project.
Send the documents to the BSO, who will process the forms for Biosafety Subcommittee review. The
principal investigator will be invited to attend the review to summarize the project and answer questions.
The Biosafety Subcommittee and/or RSC may request modification of the procedures or request that the
BSO inspect labs or other facilities before granting approval. A copy of the approved form will be sent to
the principal investigator.
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APPENDIX C
PROJECT REGISTRATION FORMS AND SIGNS
21
UNIVERSITY OF WISCONSIN – STOUT
RESEARCH SAFETY COMMITTEE
REGISTRATION COVER SHEET
FOR ALL RECOMBINANT OR SYNTHETIC NUCLEIC ACID
MOLECULE RESEARCH
Principal Investigator:
Project Title:
Click here to enter text.
Click here to enter text.
PROTOCOL #
Click here to enter text.
APPROVAL –
SUBCOMMITTEE
APPROVAL – FULL
COMMITTEE
Click here to enter text.
WITH MODIFICATION
Click here to enter text.
Department:
Click here to enter text.
Click here to enter text.
UW-Stout adheres to the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules
(http://oba.od.nih.gov/rdna/nih_guidelines_oba.html) with regard to all uses of recombinant or synthetic nucleic acid
molecules. Experiments involving recombinant or synthetic nucleic acid molecules are divided into six classes listed below;
it is the responsibility of the PI to read the NIH Guidelines in order to determine which class is applicable to the work.
UW-Stout requires that all recombinant or synthetic nucleic acid molecule work done at the University be registered with
the Research Safety Committee EVEN IF IT IS EXEMPT from the NIH Guidelines. Registration is accomplished by
completion of this cover sheet and the appropriate registration form for either exempt or non-exempt research.
III-F. Experiments that are exempt from the NIH Guidelines.
☐ Those synthetic nucleic acids that (1) can neither replicate nor generate nucleic acids that can replicate in any living cell, and (2)
are not designed to integrate into DNA, and (3) do not produce a toxin that is lethal for vertebrates at an LD 50 of less than 100
nanograms/kilogram body weight.
☐ Those that are not in organisms, cells or viruses and that have not been modified or manipulated to render them capable of
penetrating cellular membranes.
☐ Those that consist solely of the exact recombinant or synthetic nucleic acid sequence from a single source that exists
contemporaneously in nature.
☐ Those that consist entirely of nucleic acids from a prokaryotic host, including its indigenous plasmids or viruses when propagated
only in that host (or a closely related strain of the same species), or when transferred to another host by well-established
physiological means.
☐ Those that consist entirely of nucleic acids from a eukaryotic host including its chloroplasts, mitochondria or plasmids (but
excluding viruses) when propagated only in that host (or a closely related strain of the same species).
☐ Those that consist entirely of DNA segments from different species that exchange DNA by known physiological processes, though
one or more of the segments may be a synthetic equivalent.
☐ Those genomic DNA molecules that have acquired a transposable element, provided the transposable element does not contain
any recombinant and/or synthetic DNA.
☐ Those that do not present a significant risk to health or the environment, as determined by the NIH Director.
III-E. Experiments that require RSC registration simultaneous with initiation.
☐ Those involving the formation of recombinant or synthetic nucleic acid molecules containing no more than two-thirds of the
genome of any eukaryotic virus.
☐ Those involving recombinant or synthetic nucleic acid molecule-modified whole plants, and/or those involving recombinant or
synthetic nucleic acid molecule-modified organisms associated with whole plants.
☐ Those involving transgenic rodents.
III-D. Experiments that require RSC approval before initiation.
☐ Those using RG2 (or higher) or restricted agents as host-vector systems.
☐ Those in which DNA from RG2 (or higher) or restricted agents is cloned into nonpathogenic prokaryotic or lower eukaryotic hostvector systems.
☐ Those involving the use of infectious/defective DNA/RNA viruses in the presence of helper virus tissue culture systems.
☐ Those involving whole animals.
☐ Those to genetically engineer plants by recombinant or synthetic nucleic acid molecule methods, to use such plants for other
experimental purposes, to propagate such plants, or to use plants together with microorganisms or insects containing recombinant
or synthetic nucleic acid molecules.
☐ Those involving more than 10 liters of culture.
☐ Those involving influenza viruses.
III-C. Experiments that require RSC and IRB approvals and NIH RAC review before participant enrollment.
☐ Those involving the deliberate transfer of recombinant or synthetic nucleic acid molecules, or DNA/RNA derived from recombinant
or synthetic nucleic acid molecules, into one or more human research participants.
III-B. Experiments that require NIH/OBA and RSC approval before initiation.
☐ Those involving the cloning of toxin molecules with LD50 of less than 100 nanograms/kilogram body weight.
III-A. Experiments that require RSC approval, NIH RAC review and NIH Director approval before initiation.
☐ Those considered as Major Actions under the NIH Guidelines.
Oct 13
UNIVERSITY OF WISCONSIN – STOUT
RESEARCH SAFETY COMMITTEE
PROTOCOL #
Click here to enter text.
REVIEW –
SUBCOMMITTEE
Click here to enter text.
WITH MODIFICATION
Click here to enter text.
REGISTRATION FORM FOR EXEMPT RESEARCH
RECOMBINANT OR SYNTHETIC NUCLEIC ACID MOLECULES
Principal Investigator:
Campus Mailing Address:
☐New Submission
Project Title:
Funding Agency:
Phone No.:
Click here to enter text.
E-mail:
Click here to enter text.
☐Grant Renewal of RSC# Click here to
☐Revision/Resubmission of RSC#Click here to
Click here to enter text.
Click here to enter text.
enter text.
Click here to enter text.
Click here to enter text.
enter text.
Project Dates:
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enter text.
to
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enter text.
1. Attach a project summary describing your research in such a way that a scientist from another field will understand.
2. Location. Provide information regarding the research facilities and type of use.
Building
Room No.
Type of Use
(e.g. Procedure room, animal housing, cold room)
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
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3. Assurance by Principal Investigator. Electronic submission of this form from the principal investigator’s UW-Stout
email address confirms his/her agreement that all work on this project will be conducted according to the National
Institutes of Health (NIH) "Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules" and
using Biosafety Level 1 practices as described in the most current version of the Centers for Disease Control and
Prevention/NIH publication entitled "Biosafety in Microbiological and Biomedical Laboratories." Additional stipulations
required by the Research Safety Committee on behalf of UW-Stout will also be followed.
Date of submission: Click here to enter a date.
Save an electronic copy of this completed form and submit it to burrittj@uwstout.edu along with required attachments
(including the Registration Cover Sheet). The email must come from the principal investigator’s/instructor’s UW-Stout
email address. You will receive an email within 2-3 days acknowledging receipt of your submission.
Oct 13
UNIVERSITY OF WISCONSIN – STOUT
RESEARCH SAFETY COMMITTEE
REGISTRATION FORM FOR NON-EXEMPT RESEARCH
RECOMBINANT OR SYNTHETIC NUCLEIC ACID MOLECULES
Principal Investigator:
Campus Mailing Address:
☐New Submission
Click here to enter text.
APPROVAL –
SUBCOMMITTEE
APPROVAL – FULL
COMMITTEE
Click here to enter text.
WITH MODIFICATION
Click here to enter text.
Click here to enter text.
Phone No.:
Click here to enter text.
E-mail:
Click here to enter text.
☐Grant Renewal of RSC# Click here to
☐Revision/Resubmission of RSC#Click here to
Click here to enter text.
Click here to enter text.
enter text.
Click here to enter text.
Click here to enter text.
Project Title:
Funding Agency:
PROTOCOL #
enter text.
Project Dates:
Click here to
enter text.
to
Click here to
enter text.
1. Attach a project summary describing your research in such a way that a scientist from another field will understand.
2. Recombinant or Synthetic Nucleic Acid Molecules
Source(s)
(Gene, species, strain)
Name
(Spell out acronyms)
Name of Insert or
Protein Expressed
Use on Construct
(e.g. toxin, marker train, virulence
factor, DNA repair gene, oncogene,
transcription factor)
(Cloning for sequencing; PCR
expression in a microbe;
expression in Organ, Tissue or
Cell Culture; expression assoc. w/
organism)
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
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3. Vector Description(s). Attach a construct map if available. Please indicate any regions that increase the safety of
this construct.
Gene Transfer Method
(e.g. conjugation, liposome,
viral, infection, electroporation,
CaPO4, polyplexes, naked DNA
uptake)
Vector Backbone
Source
(e.g. bacterial plasmid, cosmid,
phage, virus synthetic,
transposition, YAC, BAC)
Risk Attenuation
Vector Technical Name
(Include commercial vendor if
applicable)
(Replication defective?
Helper dependent?
Disarmed? K-12 derivative?
Restricted to prokaryotic
expression?)
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4. Toxic Molecules. Do the DNA clones contain genes for the biosynthesis of toxic molecules lethal for vertebrates?
☐Yes
Oct 13
☐No
If yes, provide LD50 information: Click here to enter text.
UW-Stout Registration Form for Non-Exempt Research
5. Scale. Does an individual experiment involve more than 10 liters of culture?
☐Yes
☐No
6. Location. Please provide information regarding the research facilities, type of use and the biosafety level (BSL-1 or
BSL-2) that will be used during this project.
Building
Room No.
Type of Use
(e.g. procedure room, animal housing,
cold room)
Biosafety Level
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text.
Click here to enter
text.
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Click here to enter text.
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text.
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text.
Click here to enter text.
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text.
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text.
Click here to enter text.
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7. Provide laboratory protocols specific to this research. Be sure to include the following:
a. Identification of potential exposure hazards during sample preparation and experimental manipulations (e.g.
aerosol generation when transferring, mixing or centrifuging; use of sharps; excretion by animals).
b. Staff training.
c. Safety procedures that will be employed to minimize risk and prevent release of the agent (e.g. protective
clothing, use of biological safety cabinet, sharps handling and disposal)
d. Accidental spill/exposure procedures.
8. Research Personnel. Identify personnel conducting the experiments (including students). Specify degree, applicable
training and experience including duration, and project responsibilities.
Name
Degree
Training/Experience
Project Responsibilities
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text.
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text.
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text.
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text.
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text.
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text.
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9. Assurance by Principal Investigator. Electronic submission of this form from the principal investigator’s UW-Stout
email address confirms his/her agreement that all work on this project will be conducted according to the National
Institutes of Health (NIH) "Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules" and
using appropriate biosafety practices as described in the most current version of the Centers for Disease Control and
Prevention/NIH publication entitled "Biosafety in Microbiological and Biomedical Laboratories." Additional stipulations
required by the Research Safety Committee on behalf of UW-Stout will also be followed.
Date of submission: Click here to enter a date.
Save an electronic copy of this completed form and submit it to glenztr@uwstout.edu and burrittj@uwstout.edu along
with required attachments (including the Registration Cover Sheet). The email must come from the principal
investigator’s/instructor’s UW-Stout email address. You will receive an email within 2-3 days acknowledging receipt of
your submission.
Oct 13
UW-Stout Registration Form for Non-Exempt Research
UNIVERSITY OF WISCONSIN – STOUT
RESEARCH SAFETY COMMITTEE
BIOLOGICAL AGENT REGISTRATION FORM
PROTOCOL #
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APPROVAL –
SUBCOMMITTEE
APPROVAL – FULL
COMMITTEE
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WITH MODIFICATION
Click here to enter text.
Click here to enter text.
Principal Investigator/Instructor:
Phone No.:
Click here to enter text.
Click here to enter text.
Campus Mailing Address: Click here to enter text.
E-mail:
Click here to enter text.
☐New Submission
☐Grant Renewal of RSC# Click here to
☐Revision/Resubmission of RSC#Click here to
enter text.
enter text.
Project Title/Course Number: Click here to enter text.
Funding Agency:
Click here to enter text.
Project/Course Dates:
Click here to
enter text.
to
Click here to
enter text.
1. Attach a project summary describing your research or instruction in such a way that a scientist from another field
will understand. Include in detail all methods that will be used that are directly involved with the biological agent,
including the experimental/instructional design and goals, the source of the agent, assessment of the biohazardous
potential and containment methods.
2. Biological Agents. Identify the agent(s), biosafety level and check the applicable categories*.
Microorganism
Biosafety Level
(Gene, species, strain)
Human
Pathogen*
Animal
Pathogen*
Plant
Pathogen*
Click here to enter text.
Choose an item.
☐
☐
☐
Click here to enter text.
Choose an item.
☐
☐
☐
Click here to enter text.
Choose an item.
☐
☐
☐
3. Source. How will the agent be acquired?
☐Laboratory stock
☐Off-campus collection
☐Directly from animal/plant tissues
☐Off-campus researcher
☐Other (describe): Click here to enter text.
4. Storage. How will the agent be maintained?
☐Lypholized
☐Chemical-fixation
☐Frozen
☐Refrigerated
☐Incubator
☐Room Temperature
☐Other (describe): Click here to enter text.
5. Location. Where will experiments be performed?
Building and room number: Click here to enter text.
6. Animals. Does the experiment involve infection of animals?
All research involving animals must also be submitted to the IACUC.
If yes, can the organism be released from the animal to the environment?
7. Scale. Does an individual experiment involve more than one liter of culture?
Oct 13
☐Yes
☐No
☐Yes
☐No
☐Yes
☐No
UW-Stout Biological Agent Registration Form
8. Safety Equipment. Identify equipment to be used.
Biosafety cabinet?
☐Yes
Autoclave available?
☐No
☐Yes
☐No
Model: Click here to enter text.
Location: Click here to enter text.
Location: Click here to enter text.
Monitored?
☐Yes
☐No
Certification date: Click here to enter text.
9. Disposal. How are the compound and/or other materials containing biological agents (e.g. animal excrement, blood,
bedding, carcasses) inactivated or disposed of? If a chemical disinfectant is used, state the type and concentration.
Material to be Disposed Of
Method/Procedure for Inactivation/Disposal
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
10. Provide laboratory protocols specific to this research/instruction. Be sure to include the following:
a. Identification of potential exposure hazards during sample preparation and experimental manipulations
(e.g. aerosol generation when transferring, mixing or centrifuging; use of sharps; excretion by animals).
b. Staff and/or student training.
c. Safety procedures that will be employed to minimize risk and prevent release of the agent (e.g.
protective clothing, use of biological safety cabinet, sharps handling and disposal).
d. Accidental spill/exposure procedures. Include information on vaccines available and their recommended
use for persons handling the agent.
11. Research/Instruction Personnel. Identify personnel conducting the experiments or assisting with the instruction
(including students). Specify degree, applicable training and experience including duration, and project
responsibilities.
Name
Click here to enter
text.
Click here to enter
text.
Click here to enter
text.
Degree
Click here to enter
text.
Click here to enter
text.
Click here to enter
text.
Training/Experience
Project Responsibilities
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
12. Assurance by Principal Investigator/Instructor. Electronic submission of this form from the principal
investigator’s/instructor’s UW-Stout email address confirms his/her agreement to perform all work using appropriate
biosafety practices as described in the most current version of the Centers for Disease Control and Prevention/NIH
publication entitled "Biosafety in Microbiological and Biomedical Laboratories." Additional stipulations required by the
Research Safety Committee on behalf of UW-Stout will also be followed.
Date of submission: Click here to enter a date.
Save an electronic copy of this completed form and submit it to glenztr@uwstout.edu and burrittj@uwstout.edu along
with required attachments. The email must come from the principal investigator’s/instructor’s UW-Stout email address.
You will receive an email within 2-3 days acknowledging receipt of your submission.
Oct 13
UW-Stout Biological Agent Registration Form
UNIVERSITY OF WISCONSIN – STOUT
RESEARCH SAFETY COMMITTEE
BIOLOGICAL TOXIN REGISTRATION FORM
PROTOCOL #
Click here to enter text.
APPROVAL –
SUBCOMMITTEE
APPROVAL – FULL
COMMITTEE
Click here to enter text.
WITH MODIFICATION
Click here to enter text.
Click here to enter text.
Principal Investigator/Instructor:
Phone No.:
Click here to enter text.
Click here to enter text.
Campus Mailing Address: Click here to enter text.
E-mail:
Click here to enter text.
☐New Submission
☐Grant Renewal of RSC# Click here to
☐Revision/Resubmission of RSC#Click here to
enter text.
Project Title/Course Number: Click here to enter text.
Funding Agency:
Click here to enter text.
enter text.
Project/Course Dates:
Click here to
enter text.
to
Click here to
enter text.
1. Attach a project summary describing your research or instruction in such a way that a scientist from another field
will understand. Include in detail all methods that will be used in the project that are directly involved with the
biological toxin, including the experimental/instructional design and goals, the source of the agent, assessment of the
biohazardous potential and containment methods.
2. Biological Toxins. Identify the toxin(s), biosafety level, LD50 and source of the LD50 data (e.g. mouse, rat).
Biological Toxin
Biosafety Level
LD50
LD50 Source
Click here to enter text.
Choose an item.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Choose an item.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Choose an item.
Click here to enter text.
Click here to enter text.
3. Quantity. What quantity of the biotoxin will be kept on hand? Please note method used to inventory or account for
quantity.
Describe: Click here to enter text.
4. Security. How will the security of the toxin be maintained?
☐Locked cabinet
☐Locked refrigerator/freezer
☐Other (describe): Click here to enter text.
5. Location. Provide information regarding the facilities where the biotoxin will be produced, stored and/or used in
experiments.
Building
Room No.
Produced, Stored, and/or Used for Experiments?
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
6. Animals. Does the experiment involve infection of animals?
If yes, can the organism be released from the animal to the environment?
☐Yes
☐Yes
☐No
☐No
All research involving animals must also be submitted to the IACUC.
Oct 13
UW-Stout Biological Toxin Registration Form
7. Safety Equipment. Identify equipment to be used.
Biosafety cabinet?
☐Yes
Autoclave available?
☐No
☐Yes
☐No
Model: Click here to enter text.
Location: Click here to enter text.
Location: Click here to enter text.
Monitored?
☐Yes
☐No
Certification date: Click here to enter text.
8. Disposal. How are the compound and/or other materials containing biotoxins (e.g. animal excrement, blood,
bedding, carcasses) inactivated or disposed of? If a chemical disinfectant is used, state the type and concentration.
Material to be Disposed Of
Method/Procedure for Inactivation/Disposal
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
9. Provide laboratory protocols specific to this research. Be sure to include the following:
a. Identification of potential exposure hazards during sample preparation and experimental manipulations
(e.g. aerosol generation when transferring, mixing or centrifuging; use of sharps; excretion by animals).
b. Staff training.
c. Safety procedures that will be employed to minimize risk and prevent release of the toxin (e.g. protective
clothing, use of biological safety cabinet, sharps handling and disposal)
d. Accidental spill/exposure procedures. Include information on the availability of antidotes for persons
exposed to the toxin.
10. Research Personnel. Identify personnel conducting the experiments (including students). Specify degree,
applicable training and experience including duration, and project responsibilities.
Name
Degree
Training/Experience
Project Responsibilities
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
11. Assurance by Principal Investigator/Instructor. Electronic submission of this form from the principal
investigator’s/instructor’s UW-Stout email address confirms his/her agreement to perform all work using appropriate
biosafety practices as described in the most current version of the Centers for Disease Control and Prevention/NIH
publication entitled "Biosafety in Microbiological and Biomedical Laboratories." Additional stipulations required by the
Research Safety Committee on behalf of UW-Stout will also be followed.
Date of submission: Click here to enter a date.
Save an electronic copy of this completed form and submit it to glenztr@uwstout.edu and burrittj@uwstout.edu along
with required attachments. The email must come from the principal investigator’s/instructor’s UW-Stout email address.
You will receive an email within 2-3 days acknowledging receipt of your submission.
Oct 13
UW-Stout Biological Toxin Registration Form
BIOSAFETY
1
Standard Microbiological Practices Apply
LEVEL
Biohazardous material(s): Click here to enter text.
Notice
Call or See
Building
Room
Entry or
Information
Click here to enter
text.
Click here to enter
text.
Click here to enter
text.
Click here to enter
text.
Click here to enter
text.
Click here to enter
text.
Click here to
enter text.
Click here to
enter text.
Click here to
enter text.
Emergency
Emergency
Building: Click here to enter text.
Campus Phone
Emergency Phone
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Room: Click here to enter text. Date posted: Click here to enter a date.
Additional copies of this sign available from the UW-Stout Laboratory Safety intranet website:
https://www.uwstout.edu/healthandsafety/safety/intranet/laboratory-safety.cfm
BIOSAFETY
Limited
Access
LEVEL
For work with:
Biohazardous material(s)
Procedure required for entry/exit (e.g.
PPE)
Special practices (e.g. use of BSC,
immunizations)
Click here to enter text.
Click here to enter text.
Click here to enter text.
Notice
Call or See
Building
Room
Entry or
Information
Click here to enter
text.
Click here to enter
text.
Click here to enter
text.
Click here to enter
text.
Click here to enter
text.
Click here to enter
text.
Click here to
enter text.
Click here to
enter text.
Click here to
enter text.
Emergency
Emergency
Building: Click here to enter text.
2
Room: Click here to enter text.
Campus Phone
Emergency Phone
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Click here to enter text.
Date posted: Click here to enter a date.
Additional copies of this sign available from the UW-Stout Laboratory Safety intranet website:
https://www.uwstout.edu/healthandsafety/safety/intranet/laboratory-safety.cfm
APPENDIX D
STANDARD PRACTICES AND PROCEDURES
Autoclave procedures and safety
1. All users must undergo and document training for operation of the autoclave.
2. Studies have shown that the processing time necessary to achieve decontamination of biological
material depends on load size, type of container, and moisture content. A 60 minute autoclave
cycle (excluding exhaust time) is recommended to allow enough time for the center of the load
to come up to temperature and have sufficient steam penetration. This will insure that the
temperature in ALL parts of the load has reached 121°C (250°F) for at least 20 minutes.
3. In order to standardize the autoclaving of wastes and to assure that all loads, regardless of size
or content, are properly decontaminated, the following procedures must be followed.
a. Place materials in an autoclavable container. Plastics must be labeled as autoclavable or
they may melt.
i. Autoclave bags should be loosely packed and not more than 3/4 full. Add water
to a bag if the load is dry. Do not seal the bag shut; steam must be able to
penetrate the bag to decontaminate the contents. If using the supplied rubber
bands place on loosely, do not twist.
ii. The caps or lids on containers of liquids should be loosened to avoid dangerous
pressure build-up and possible explosion.
iii. Do not put sharp or pointed contaminated objects directly into an autoclave bag.
Place them in an appropriate rigid disposal container (such as a sharps
container) first.
b. Use autoclave indicator tape as a check that the correct temperature has been reached.
To validate adequate steam penetration to the center of a load, tape can be buried in the
load. Tape does not prove decontamination effectiveness; see “Autoclave testing” below.
c.
Load the autoclave according to the manufacturer’s instructions.
i. Place containers in a tray and load the tray into the autoclave; this is easier to
load and unload and will catch spills. Do not place items directly on the autoclave
chamber floor.
ii. Do not overload the autoclave; an over-packed autoclave chamber does not
allow sufficient steam distribution. Be sure there is ample room between bags so
steam circulation is not impaired.
d. Autoclave at 121°C for 45-60 minutes.
e. After autoclaving, any bag displaying the biohazard symbol should be placed in a nontransparent plastic bag or other secondary non-transparent container (e.g. closed
cardboard box, dark colored trash bag) prior to disposal into the normal trash. Bags with
32
the biohazard symbol, regardless of use, must not be placed into the regular waste
stream without defacing the symbol or over-bagging.
f.
Autoclaved liquid culture waste can be sewered unless hazardous chemical waste is
present, in which case it must be disposed of in accordance with hazardous waste
regulations. Do not sewer melted agar as it will congeal and clog plumbing.
4. Autoclave testing: All autoclaves used for waste decontamination should be regularly tested for
effectiveness. Autoclave indicator tape is used to verify that an adequate temperature was
reached during the autoclaving cycle, but it does not prove decontamination effectiveness.
a. Integrator strips (e.g. B. stearothermophilus spore strips) provide immediate test results.
The spores, which can survive 121ºC for five minutes but are killed at 121ºC in 13
minutes, are more resistant to heat than most, thereby providing an adequate safety
margin when validating decontamination procedures. Each type of container employed
should be individually tested with these spores because efficacy varies with the load, fluid
volume, etc.
i. Attach the indicator strip to a stick or string and put in the center of the load and
place strips at locations throughout the autoclave.
ii. Retrieve after autoclaving for confirmation that the entire load has been exposed
to the conditions necessary for decontamination.
iii. If the indicator reads unsafe, autoclave again.
b. An autoclave testing log must be kept for two years.
5. Personal safety precautions:
a. Do not autoclave materials containing solvents, volatile or corrosive chemicals (such as
phenol, chloroform, bleach, etc.) or radioactive materials.
b. When unloading an autoclave, wear heat resistant gloves, eye protection, and lab coat.
c.
To prevent steam burns, make sure that the autoclave pressure is near zero before
opening the door.
d. Allow steam to escape gradually by slowly cracking open the autoclave door. Allow load
to cool for 10 minutes before removing.
e. Do not autoclave sealed containers or full bottles with narrow necks as they may
explode.
Biological safety cabinets (BSCs)
1. BSCs are often referred to as “tissue culture hoods” or “laminar flow hoods.” “Biological safety
cabinet” is the correct term. All BSCs use high efficiency particulate air (HEPA) filters to treat
exhaust air.
a. Class I and II cabinets are designed for work involving low to moderate risk biological
33
agents (BSL-1, BSL-2 and BSL-3). Class I cabinets provide worker and environmental
protection, but no product protection, while Class II cabinets provide worker,
environmental and product protection. Class III cabinets are fully enclosed and are used
for work with high risk biological agents (BSL-4).
b. All BSCs in Jarvis Science Addition are Class II, Type A2; they recirculate 70% of the air
and exhaust 30% and provide intake air velocity of 100 feet per minute. Both room air
and interior cabinet air are drawn into a front grille creating an air barrier that provides
personnel protection. In addition, downward laminar flow of HEPA-filtered air provides
product protection. Air exhaust passes through a certified exhaust HEPA filter and may
be recirculated back into the laboratory or exhausted out of the building, providing
environmental protection. All UW-Stout BSCs exhaust back into the laboratory
and therefore cannot be used for applications involving volatile chemicals,
cytotoxic drugs or radionuclides.
2. Laminar flow clean benches are not BSCs and they offer no worker or environmental protection.
Clean benches must never be substituted for BSCs. Air is discharged across the work surface to
protect product on the bench from contamination, but the worker is exposed to whatever is being
used on the bench. Clean benches should not be used for work involving cell cultures, toxins,
volatile chemicals, infectious materials or materials that may cause hypersensitivity to the worker
(e.g. animal dander).
3. To ensure safe use:
a. Make sure the BSC is certified when it is installed or after it is moved, and annually
thereafter (there is a sticker on the front of the BSC indicating when it was last certified).
Do not use a BSC that is marked or signed as “Failed.” Check the blower gauge or LCD
screen regularly for an indication of a problem.
b. Do not disrupt the protective airflow pattern of the BSC. Rapidly moving your arms in
and out of the cabinet, people walking rapidly behind you, and open lab doors may
disrupt the airflow pattern and reduce the effectiveness of the BSC.
c.
Only one person at a time should work in the BSC to minimize air disturbance.
d. Minimize the storage of materials in and around the BSC.
e. It is not necessary to flame items in the near microbe-free environment of a BSC. This
creates turbulence in airflow and will compromise sterility; heat buildup may damage the
filters. See Appendix A of the BMBL; the CDC instead recommends, when deemed
necessary, touch-plate micro burners equipped with a pilot light to provide a flame on
demand, small electric furnaces for decontamination of bacteriological loops and needles,
or disposable or recyclable sterile loops. Alcohol lamps should not be used in BSCs.
4. Operational directions:
a. Remove all unnecessary equipment and supplies from the cabinet; clutter alters air flow.
Do not place objects over the front air intake grille or block the rear exhaust grille.
34
b. Plan your experiment. Gather all required materials, think through the steps and lay out
materials in a logical manner that will prevent excessive movements while the BSC is in
use.
c.
Turn on the blower at least 15 minutes before starting work to remove particulates in the
cabinet.
d. Before using, wipe work surfaces with an appropriate disinfectant. Wipe off each item
you need for your procedures with the disinfectant and place in cabinet.
e. Place a pan with disinfectant and/or a sharps container inside the BSC for pipette
discard. Collect waste materials inside the cabinet to avoid excessive movement inside
and outside the BSC. Do not use vertical pipette discard canisters on the floor outside
cabinet. Be sure to seal bags and cover open containers before removing them from
them from the BSC.
f.
Delay work for approximately one minute after placing hands/arms inside the cabinet. Do
not rest arms on the front grille; raising arms slightly will lessen disruption of air flow.
g. Work as far back in the cabinet as practical (at least four inches inside the front grille
edge).
h. Segregate contaminated and clean items; as a general rule of thumb, keep clean
materials at least one foot away from aerosol-generating activities to minimize crosscontamination. Work from “clean” (left) to “dirty” (right).
i.
If you use a piece of equipment that creates air turbulence in the BSC (such as a
centrifuge, blender) place equipment in the back third of the cabinet; stop other work
while equipment is operating.
j.
Protect the building vacuum system from biohazards by placing a HEPA cartridge filter or
its equivalent between the vacuum trap and the source valve in the cabinet.
k.
Clean up all spills in the cabinet immediately. Wait 10 minutes before resuming work.
l.
When work is finished, wipe down the surfaces of containers and equipment with an
appropriate disinfectant and remove from the BSC.
m. Leave the blower on for 15 minutes with no activity so that airborne contaminants are
purged from the work area.
n. Wipe all interior surfaces with disinfectant.
o. Remove lab coat and gloves and wash hands thoroughly before leaving laboratory.
Blenders, ultrasonic disrupters, grinders and lyophilizers
1. The use of any of these devices results in considerable aerosol production. Blenders, grinders and
cell-disruption equipment should be used in a BSC when working with biohazardous materials.
2. Safety blenders, although expensive, are designed to prevent leakage from the bottom of the
35
blender jar, provide a cooling jacket to avoid biological inactivation and withstand sterilization by
autoclaving. If blender rotors are not leak-proof, they should be tested with sterile saline or dye
solution prior to use with biohazardous material. The use of glass blender jars is not
recommended because of the breakage potential. If they must be used, glass jars should be
covered with a polypropylene jar to prevent spraying of glass and contents in the event the
blender jar breaks. A towel moistened with disinfectant should be placed over the top of the
blender during use. Before opening the blender jar allow the unit to rest for at least one minute
to allow the aerosol to settle. The device should be decontaminated promptly after use.
3. Depending on the lyophilizer design, aerosol production may occur when material is loaded or
removed from the lyophilizer unit. If possible, sample material should be loaded in a BSC. The
vacuum pump exhaust should be filtered to remove any hazardous agents or, alternatively, the
pump can be vented into a BSC. After lyophilization is completed, all surfaces of the unit that
have been exposed to the agent should be disinfected. If the lyophilizer is equipped with a
removable chamber, it should be closed off and moved to a BSC for unloading and
decontamination. Handling of cultures should be minimized and vapor traps should be used
wherever possible.
4. Opening an ampoule containing liquid or lyophilized culture material should be performed in a
BSC to control the aerosol produced. Gloves must be worn.
a. To open, nick the neck of the ampoule with a file, wrap it in disinfectant soaked towel,
hold the ampoule upright and snap it open at the nick.
b. Reconstitute the contents of the ampoule by slowly adding liquid to avoid aerosolization
of the dried material.
c.
Mix the contents without bubbling and withdraw it into a fresh container.
d. Discard the towel and ampoule top and bottom as infectious waste.
5. Ampoules used to store biohazardous material in liquid nitrogen have exploded causing eye
injuries. The use of polypropylene tubes eliminates this hazard. These tubes are available dustfree or pre-sterilized and are fitted with polyethylene caps with silicone washers. Heat sealable
polypropylene tubes are also available.
Bloodborne pathogens
1. All principal investigators using human or non-human primate blood or blood products, unfixed
tissue, body fluids, or organ or cell cultures of human or non-human primate origin must follow
the procedures outlined in the UW-Stout Bloodborne Pathogens Exposure Control Plan.
2. Bloodborne pathogens training is required for employees and students working with the abovelisted products and can be provided by Safety and Risk Management or numerous other online
providers.
Centrifuge equipment
1. Hazards associated with centrifuging include mechanical failure and the creation of aerosols. To
minimize the risk of mechanical failure, centrifuges must be maintained and used according to
36
the manufacturer’s instructions. Users should be properly trained, and operating instructions that
include safety precautions should be prominently posted on the unit.
2. Aerosols are created by practices such as filling centrifuge tubes, removing plugs or caps from
tubes after centrifugation, removing supernatant, and re-suspending sedimented pellets. The
greatest aerosol hazard is created if a tube breaks during centrifugation. To minimize the
generation of aerosols when centrifuging biohazardous material, the following procedures should
be followed:
a. Use sealed tubes and safety buckets that seal with O-rings. Before use, inspect tubes, Orings and buckets for cracks, chips, erosions, bits of broken glass, etc. Do not use
aluminum foil to cap centrifuge tubes because it may detach or rupture during
centrifugation.
b. Fill and open centrifuge tubes, rotors and accessories in a biological safety cabinet. Avoid
overfilling of centrifuge tubes so that closures do not become wet. After tubes are filled
and sealed, wipe them down with disinfectant.
c.
Add disinfectant to the space between the tube and the bucket to disinfect material in
the event of breakage during centrifugation.
d. Always balance buckets, tubes and rotors properly before centrifugation.
e. Do not decant or pour off supernatant. Use a vacuum system with appropriate in-line
reservoirs and filters.
f.
Work in a BSC when re-suspending sedimented material. Use a swirling rotary motion as
necessary, wait a few minutes to permit the aerosol to settle before opening the tube.
3. Small low-speed centrifuges may be placed in a BSC during use to reduce the aerosol escape.
High-speed centrifuges pose additional hazards. Precautions should be taken to filter the exhaust
air from vacuum lines, to avoid metal fatigue resulting in disintegration of rotors and to use
proper cleaning techniques and centrifuge components. Manufacturers’ recommendations must
be meticulously followed to avoid metal fatigue, distortion and corrosion.
4. Avoid the use of celluloid (cellulose nitrate) tubes with biohazardous materials. Celluloid
centrifuge tubes are highly flammable and prone to shrinkage with age. They distort on boiling
and can be highly explosive in an autoclave. If celluloid tubes must be used, an appropriate
chemical disinfectant must be used to disinfect them.
Cryostats
1. Frozen sections of unfixed tissue infected with an etiologic agent pose a risk because accidents
can occur. Freezing tissue does not necessarily inactivate infectious agents. Freezing propellants
under pressure should not be used for frozen sections as they may cause spattering of droplets
of infectious materials. Gloves should be worn during preparation of frozen sections. When
working with biohazardous material in a cryostat, the following is recommended:
a. Consider the contents of the cryostat to be contaminated and decontaminate it
frequently with disinfectant.
37
b. Consider trimmings and sections of tissue that accumulate in the cryostat to be
potentially infectious and remove them during decontamination.
c.
Defrost and decontaminate the cryostat with a tuberculocidal hospital disinfectant once a
week and immediately after tissue known to contain bloodborne pathogens, M.
tuberculosis or other infectious agents is cut.
d. Handle microtome knives with extreme care. Stainless steel mesh gloves should be worn
when changing knife blades.
2. Consider solutions for staining potentially infected frozen sections to be contaminated.
Decontamination
1. Decontamination is a term used to describe a process or treatment that renders a medical device,
instrument, or environmental surface safe to handle. A decontamination procedure can range
from sterilization to simple cleaning with soap and water. Sterilization, disinfection and antisepsis
are all forms of decontamination.
a. Sterilization is the use of a physical or chemical procedure to destroy all microbial life,
including highly resistant bacterial endospores.
b. Disinfection eliminates virtually all pathogenic non-spore forming microorganisms but not
necessarily all microbial forms on inanimate objects (e.g. work surfaces, equipment).
Effectiveness is influenced by the kinds and numbers of organisms, the amount of
organic matter, the object to be disinfected and chemical exposure time, temperature
and concentration.
c.
Antisepsis is the application of a liquid antimicrobial chemical to skin or living tissue to
inhibit or destroy microorganisms. It includes swabbing an injection site on a person or
animal and hand washing with germicidal solutions. Although some chemicals may be
utilized as either a disinfectant or an antiseptic, adequacy for one application does not
guarantee adequacy for the other. Manufacturers’ recommendations for appropriate use
of germicides should always be followed.
2. There are four main categories of physical and chemical means of decontamination. They are
heat (wet, dry and incineration), liquid disinfection, vapors and gases, and radiation. Each
category is discussed briefly below.
a. Wet heat is the most dependable method of sterilization. Autoclaving (saturated steam
under pressure of approximately 15 psi to achieve a chamber temperature of at least
121ºC for a prescribed time) is the most convenient method of rapidly achieving
destruction of all forms of microbial life. In addition to proper temperature and time,
prevention of entrapment of air is critical to achieving sterility. See “Autoclave procedures
and safety” in this Appendix.
b. Dry Heat is less efficient than wet heat and requires longer times and/or higher
temperatures to achieve sterilization. It is suitable for the destruction of viable organisms
on impermeable non-organic surfaces such as glass, but it is not reliable in the presence
38
of shallow layers of organic or inorganic materials that may act as insulation. Sterilization
of glassware by dry heat can usually be accomplished at 160 - 170º C for periods of two
to four hours. Dry heat sterilizers should be monitored on a regular basis using
appropriate biological indicators (e.g. B. subtilis [globigii] spore strips).
c.
Incineration is another effective means of decontamination by heat. As a disposal
method incineration has the advantage of reducing the volume of the material prior to its
final disposal.
d. Liquid disinfection is most practical for surface decontamination and, when used in
sufficient concentration, as a decontaminate for liquid wastes prior to final disposal in the
sanitary sewer. If liquid disinfectants are used, they must have been shown to be
effective against the organism(s) present. Liquid disinfectants are available under a wide
variety of trade names. In general, these can be classified as halogens, acids, alkalis,
heavy metal salts, quaternary ammonium compounds, phenolic compounds, aldehydes,
ketones, alcohols and amines. The more active a compound is, the more likely it is to
have undesirable characteristics such as corrosivity. No liquid disinfectant is equally
useful or effective under all conditions and for all viable agents.

A 0.5% sodium hypochlorite solution (1:10 dilution of household bleach) is
effective for decontamination of equipment and work surfaces. In locations
where bleach would cause corrosion, an iodophor (e.g. Wescodyne) should be
used.
e. Vapors and gases are primarily used to decontaminate BSCs and associated systems,
bulky or stationary equipment not suited to liquid disinfectants, instruments or optics that
might be damaged by other decontamination methods, and rooms, buildings and
associated air-handling systems. Agents included in this category are glutaraldehyde and
formaldehyde vapor, ethylene oxide gas, peracetic acid and hydrogen peroxide vapor.
When used in closed systems and under controlled conditions of temperature and
humidity, excellent disinfection can be obtained. Great care must be taken during use
because of the hazardous nature of many of these compounds. Contact Safety and Risk
Management for monitoring requirements if these compounds are to be used.
f.
Although ionizing radiation will destroy microorganisms, it is not a practical tool for
laboratory use. Non-ionizing radiation in the form of ultraviolet (UV) radiation is used for
inactivating viruses, bacteria and fungi. It will destroy airborne microorganisms and
inactivate microorganisms on exposed surfaces or in the presence of products of
unstable composition that cannot be treated by conventional means. UV lamps are not
recommended for decontamination unless they are properly maintained. Because UV
lamp intensity or destructive power decreases with time, it should be checked with a UV
meter yearly. Frequent lamp cleaning (at least every few weeks) is necessary to prevent
accumulation of dust and dirt that drastically reduces its effectiveness. If UV must be
used, it should be used when areas are not occupied.
3. All infectious materials and all contaminated equipment or apparatus should be decontaminated
before being washed, stored or discarded. Autoclaving is the preferred method. Each individual
working with biohazardous material should be responsible for its proper handling.
39
Housekeeping
1. Good housekeeping in laboratories is essential to reduce risks and protect the integrity of
biological experiments. Routine housekeeping must be relied upon to provide work areas free of
significant sources of contamination. Housekeeping procedures should be based on the highest
degree of risk to which personnel and experimental integrity may be subjected.
2. Laboratory personnel are responsible for cleaning laboratory benches, equipment and areas that
require specialized technical knowledge. Additional laboratory housekeeping concerns include:
a. Keeping the laboratory neat and free of clutter; surfaces should be clean and free of
infrequently used chemicals, glassware and equipment.
b. Maintaining access to sinks, eyewashes, emergency showers and fire extinguishers; they
should not be blocked.
c.
Proper disposal of chemicals and waste; old and unused chemicals should be disposed of
promptly and properly. Contact Safety and Risk Management for instructions.
d. Providing a workplace that is free of physical hazards; aisles and corridors should be free
of tripping hazards. Attention should be paid to electrical safety, especially as it relates to
the use of extension cords, proper grounding of equipment, avoidance of overloaded
electrical circuits and avoidance of the creation of electrical hazards in wet areas.
e. Removing unnecessary items on floors, under benches or in corners.
f.
Properly securing all compressed gas cylinders.
g. Never using BSCs or chemical fume hoods for storage of chemicals or other materials.
3. Practical custodial concerns include:
a. Dry sweeping and dusting that may lead to the formation of aerosols is not permitted.
b. The usual wet or dry industrial type vacuum cleaner is a potent aerosol generator and,
unless equipped with a HEPA filter, must not be used in the biological research
laboratory.
Labeling
1. All containers of biohazardous material should be labeled with the scientific name of the
contents, the date stored and the name of the individual who stored them. Unlabeled and
obsolete material should be autoclaved and disposed of.
2. Bottles with alcohol-containing solutions should be clearly labeled to avoid autoclaving.
3. Refrigerators and freezers that are used to store biohazardous materials should be labeled with
“Biohazard: No Food Or Drink” or similar wording and the universal biohazard symbol on the
exterior of the door.
4. A sign incorporating the universal biohazard symbol must be posted at the entrance to the
40
laboratory or classroom when infectious agents are present. The sign should include the name of
the agent(s) in use, and the name and phone number of the laboratory supervisor or other
responsible personnel. For BSL-2 agents, the sign should also include the procedure required for
entry and exit (e.g. PPE) and special practices (e.g. use of BSC, immunizations). Signs are
available in Appendix C, online
(http://www.uwstout.edu/healthandsafety/safety/intranet/Biosafety.cfm), or from the BSO.
Loop sterilizers and Bunsen burners
1. Sterilization of inoculation loops or needles in an open flame generates small-particle aerosols
that may contain viable microorganisms. The use of a shielded electric incinerator minimizes
aerosol production during loop sterilization. Alternatively, disposable plastic loops and needles
may be used for culture work where electric incinerators or gas flames are not available. The
loops are semi-quantitative and can be used for counting bacteria.
2. Continuous flame gas burners should not be used in BSCs. These burners can produce turbulence
that disturbs the protective airflow patterns of the cabinet. Additionally, the heat produced by the
continuous flame may damage the HEPA filter. If a gas burner must be used, one with a pilot
light should be selected.
Personal protective equipment (PPE)
1. PPE is used to protect personnel from contact with hazardous materials and infectious agents.
Appropriate clothing may also protect the experiment from contamination. The following PPE is
recommended for regular use:
a. Eye and face protection: Goggles, in combination with masks or chin length face shields
or other splatter guards, are required whenever there is the possibility of splashes,
sprays or splatters of infectious or other hazardous materials to the face.
b. Laboratory clothing: This category includes laboratory coats, smocks, scrub suits, and
gowns.

Long sleeved garments should be used to minimize the contamination of skin or
street clothes and to reduce shedding of microorganisms from the arms.

In circumstances where it is anticipated that splashes may occur, the garment
must be resistant to liquid penetration (in order to protect clothing from
contamination).

If the garment is not disposable, it must be capable of withstanding sterilization
in the event it becomes contaminated.

Additional criteria for selecting clothing are comfort, appearance, closure types
and location, anti-static properties and durability.

Protective clothing must be removed before leaving the laboratory.

Disposables should be available for visitors, maintenance and service workers in
the event it is required.
41

c.
All protective clothing should be either discarded in the laboratory or laundered
by the facility. Employees and students must not launder laboratory clothing at
home.
Gloves:

Gloves must be selected based on the hazards involved and the activity to be
conducted.

Gloves must be worn when working with biohazards, toxic substances and other
physically hazardous agents.

Temperature-resistant gloves must be worn when handling hot material or dry
ice.

Delicate work requiring a high degree of precision dictates the use of thin walled
gloves. Protection from contact with toxic or corrosive chemicals may also be
required. More information on glove selection when working with chemicals is
available in the UW-Stout Chemical Hygiene Plan.

When working with hazardous materials, the lower sleeve and the cuff of the
laboratory garment should be overlapped by the glove. A long sleeved glove or
disposable arm-shield may be worn for further protection of the garment.

In some instances “double gloving” may be appropriate. If a spill occurs, hands
will be protected after the contaminated outer gloves are removed. Gloves must
be disposed of when contaminated, removed when work with infectious material
is completed, and not worn outside the laboratory. Disposable gloves must not
be washed or reused.
d. Respirators: In certain instances BSCs or other controls may not be sufficient to control
exposure to airborne hazards and a respirator is required. Respirator selection is based
on the hazard and the protection factor required. Employees and/or students who require
respiratory protection must contact Safety and Risk Management for inclusion in the UWStout Respiratory Protection Program. The program provides a medical examination to
ensure no health conditions exist that would be exacerbated by respirator usage, annual
fit testing to ensure proper respirator size and type and training to ensure proper
respirator use and maintenance. Under no circumstances shall anyone wear a respirator
unless he/she is a participant in the program.
2. Contact the BSO or Safety and Risk Management for assistance in selection of other PPE.
Pipettes and pipetting aids
1. Pipettes are used for volumetric measurements and transfer of fluids that may contain infectious,
toxic, corrosive or radioactive agents. Laboratory-associated infections have occurred from oral
aspiration of infectious materials, mouth transfer via a contaminated finger and inhalation of
aerosols.
2. Exposures to aerosols may occur when liquid from a pipette is dropped onto the work surface,
42
when cultures are mixed by pipetting or when the last drop of an inoculum is blown out. A
pipette may become a hazardous piece of equipment if improperly used. The safe pipetting
techniques outlined below are required to minimize the potential for exposure to hazardous
materials:
a. Never mouth pipette. Always use a pipetting aid.
b. If working with biohazardous or toxic fluid, confine pipetting operations to a BSC.
c.
Always use cotton plugged pipettes when pipetting biohazardous or toxic materials, even
when safety pipetting aids are used.
d. Do not prepare biohazardous materials by bubbling expiratory air through a liquid with a
pipette.
e. Do not forcibly expel biohazardous material out of a pipette.
f.
Never mix biohazardous or toxic material by suction and expulsion through a pipette.
g. When pipetting, avoid accidental release of infectious droplets. Place a disinfectant
soaked towel on the work surface and autoclave the towel after use.
h. Use "to deliver" pipettes rather than those requiring "blowout."
i.
Do not discharge material from a pipette at a height. Whenever possible allow the
discharge to run down the container wall.
j.
Place contaminated, reusable pipettes horizontally in a pan containing enough liquid
disinfectant to completely cover them. Do not place pipettes vertically into a cylinder.
k.
Discard contaminated disposable pipettes in an appropriate sharps container. See
Appendix F for more information on the disposal of pipettes or sharps.
l.
Pans or sharps containers for contaminated pipettes should be placed inside the BSC, if
possible.
Sharps safety
1. A high degree of precaution must always be taken with any sharp items used in the laboratory,
including needles, glass slides and cover slips, Pasteur pipettes, capillary tubes, and scalpels or
other blades. Two of the most common causes of needle sticks are re-capping needles and
improper disposal of needles. All needle sticks, and other sharps injuries, carry the risk of
secondary infections as well as exposure to the needle's content and/or contamination on the
outside of the needle or other sharp instrument.
a. Plastic ware should be substituted for glassware whenever possible.
b. Safety engineered sharps should always be considered first. The University of Virginia
has a website devoted to safety-engineered sharp devices at
http://www.healthsystem.virginia.edu/pub/epinet/new/safetydevice.html.
43
c.
Do not pick up broken glass with hands. Use mechanical means such as a brush and
dustpan, tongs or forceps.
2. Syringes and hypodermic needles are dangerous instruments. The use of needles and syringes
should be restricted to procedures for which there is no alternative. Blunt cannulas should be
used as alternatives to needles wherever possible (i.e. for procedures such as oral or intranasal
animal inoculations). Needles and syringes should never be used as a substitute for pipettes.
When needles and syringes must be used with biohazardous or potentially infectious agents, the
following procedures are recommended:
a. Use syringes that re-sheath the needle, needleless systems, or other safety engineered
syringes when possible.
b. Use needle-locking syringes to prevent exposure to infectious agents or other hazardous
materials via sprays or aerosols (and to prevent the loss of valuable samples).
c.
Work in a BSC whenever possible.
d. Wear gloves.
e. Fill the syringe carefully to minimize air bubbles.
f.
Expel air, liquid and bubbles from the syringe vertically into a cotton pledget moistened
with disinfectant.
g. Do not use a syringe to mix infectious fluid forcefully.
h. Do not contaminate the needle hub when filling the syringe in order to avoid transfer of
infectious material to fingers.
i.
Wrap the needle and stopper in a cotton pledget moistened with disinfectant when
removing a needle from a rubber-stoppered bottle.
j.
Bending, recapping, clipping or removal of needles from syringes is prohibited. If it is
essential that a contaminated needle be recapped or removed from a syringe, a resheathing needle, mechanical device or the one handed scoop method (in order of
preference) must be used. The use of needle nipping devices is prohibited. NOTE: the
BMBL does not permit recapping of needles used for BSL-2 agents for any reason.
k.
Use a separate pan of disinfectant for reusable syringes and needles. Do not place them
in pans containing pipettes or other glassware to eliminate sorting later.
3. Only “contaminated” broken glass, plastic vials, laboratory slides, etc. are considered infectious
waste. However, all discarded sharps (contaminated or not) such as hypodermic needles, scalpel
blades, lancets and syringes with needles attached are considered infectious waste. See Appendix
F and/or contact the BSO or Safety and Risk Management for more information on the disposal of
sharps.
4. Sharps usage should be reviewed annually. Can a procedure be modified so that a sharp is not
needed? Is there a safety engineered sharp available?
44
45
APPENDIX E
TRAINING
The principal investigator (PI) is responsible for the training of everyone working in his/her laboratory.
Training is possibly the single most important action a PI can take to promote a safe and healthy
laboratory.
Laboratory-specific biosafety training
1. Laboratory specific biosafety training shall be provided by the PI and will include generalized
training for the biosafety level at which the laboratory operates and specialized training for
specific hazards present in that laboratory. For workers in laboratories operating at Biosafety
Level 1 (BSL-1), it is suggested that information included as Appendix E-1 be reviewed with each
worker. The document should be signed by both the laboratory worker and the PI. This training
shall be reviewed annually. If the suggested training form is not used, an equivalent document
shall be developed to document that training in all standard and special practices, safety
equipment and facilities related to work at BSL-1 has been provided.
2. For workers in laboratories operating at BSL-2, it is suggested that information included as
Appendix E-2 be reviewed with each worker. The document should be signed by both the
laboratory worker and the PI. Note that if CDC select agents are used in the laboratory their
presence should be noted on training documentation. This training shall be reviewed annually. If
the suggested training form is not used, an equivalent document shall be developed to document
that training in all standard and special practices, safety equipment and facilities related to work
at BSL-2 has been provided.
3. The signed training document should be retained by the PI as record of appropriate laboratory
worker training.
Bloodborne pathogens training
1. UW-Stout has developed a bloodborne pathogens exposure control plan that should be reviewed
by all laboratory workers.
2. Bloodborne pathogens training is necessary for any laboratory worker who works with human
blood, human blood components, products made from human blood, human organs or human
body fluids. Training can be provided by Safety and Risk Management or other providers.
Teaching laboratories: Instructors in teaching laboratories should provide specific training for the
hazards expected to be encountered in the laboratory procedures utilized. Because of the brevity of
courses and the large number of students involved it is recommended that each instructor develop a
streamlined training form emphasizing the particular elements required for their course. Both the
instructor and student should sign the training form.
General BSL-1 and -2 entry training: The point paper in Appendix E-3 is intended to provide general
awareness training to anyone who needs access to the BSL-2 labs. It is posted on the doors to all BSL-2
labs along with a sign-in sheet, and can be used by instructors to inform students enrolled in courses in
the BSL-2 labs that are not doing BSL-2 work. It is not intended to be complete training for employees
and students who will be working with agents that require BSL-2 containment.
46
APPENDIX E-1
BIOSAFETY LEVEL 1 (BSL-1) LABORATORY WORKER TRAINING
BSL-1 is suitable for work involving well-characterized agents not known to consistently cause disease in
healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. The
laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally
conducted on open bench tops using standard microbiological practices. Special containment equipment
or facility design is neither required nor generally used. Laboratory personnel have specific training in the
procedures conducted in the laboratory and are supervised by a scientist with general training in
microbiology or a related science.
The following standard and special practices, safety equipment and facilities apply to agents assigned to
BSL-1:
Standard microbiological practices
1. The laboratory supervisor must enforce UW-Stout policies that control access to the laboratory
(e.g. granting key card access only to authorized students and staff, keeping doors closed).
2. Persons must wash their hands after working with potentially hazardous materials and before
leaving the laboratory.
3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for
human consumption must not be permitted in laboratory areas. Food must be stored outside the
laboratory area in cabinets or refrigerators designated and used for this purpose.
4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.
5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware
must be developed and implemented. Whenever practical, laboratory supervisors should adopt
improved engineering and work practice controls that reduce risk of sharps injuries. Precautions,
including those listed below, must always be taken with sharp items. These include:
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. Plastic ware should be substituted for glassware
whenever possible.
6. Perform all procedures to minimize the creation of splashes and/or aerosols.
47
7. Decontaminate work surfaces after completion of work and after any spill or splash of potentially
infectious material with appropriate disinfectant.
8. 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.
9. A sign incorporating the universal biohazard symbol must be posted at the entrance to the
laboratory or classroom when infectious agents are present. The sign should include the name of
the agent(s) in use, and the name and phone number of the laboratory supervisor or other
responsible personnel.
10. An effective integrated pest management program is required (see Appendix G of the BMBL).
11. 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. Personal health status may impact an individual’s susceptibility to infection,
ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel
and particularly women of childbearing 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 their healthcare provider for appropriate
counseling and guidance.
Special practices: None
Safety equipment (primary barriers and personal protective equipment)
1. For BSL-1 agents, special containment devices or equipment, such as BSCs, are not generally
required.
2. Protective laboratory coats, gowns, smocks, or uniforms designated for laboratory use are
recommended for work with BSL-1 agents. Remove protective clothing before leaving the
laboratory. Dispose of protective clothing appropriately, or deposit it for laundering. It is
recommended that laboratory clothing not be taken home.
3. Safety glasses are required in the laboratory whenever anyone in the lab is working with
chemical or biological agents that present a splash hazard, or when there is the potential for
flying glass. Splash goggles (and mask, face shield or other splatter guard as needed) should be
used for anticipated splashes or sprays of infectious or other hazardous materials. Eye and face
protection must be disposed of with other contaminated laboratory waste or decontaminated
before reuse. Persons who wear contact lenses in laboratories should also wear eye protection.
48
4. Gloves should be worn to protect hands from exposure to hazardous materials. Glove selection
should be based on an appropriate risk assessment. Alternatives to latex gloves should be
available. Gloves must not be worn outside the laboratory. In addition, laboratory workers
should:
a. Change gloves when contaminated, glove integrity is 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.
Laboratory facilities (secondary barriers)
1. Laboratory doors must remain closed for access control when biohazardous material is being
used.
2. Each laboratory must contain at least one sink for handwashing.
3. The laboratory is designed so that it can be easily cleaned. Carpets, rugs and cloth chairs are not
appropriate in laboratories.
4. Bench tops are impervious to water and are resistant to moderate heat and the organic solvents,
acids, alkalis, and chemicals used to decontaminate the work surface and equipment.
5. Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between
benches, cabinets, and equipment are accessible for cleaning.
I have received and understand the biological safety training as outlined in this document.
WORKER SIGNATURE:
DATE:
PI SIGNATURE:
DATE:
LABORATORY LOCATION:
49
APPENDIX E-2
BIOSAFETY LEVEL 2 (BSL-2) LABORATORY WORKER TRAINING
BSL-2 is similar to BSL-1 and is suitable for work involving agents of moderate potential hazard to
personnel and the environment. It differs from BSL-1 in that (1) laboratory personnel have specific
training in handling pathogenic agents and are directed by competent scientists; (2) access to the
laboratory is limited when work is being conducted; (3) extreme precautions are taken with contaminated
sharp items; and (4) certain procedures in which infectious aerosols or splashes may be created are
conducted in biological safety cabinets (BSCs) or other physical containment equipment.
The following standard and special practices, safety equipment and facilities apply to agents assigned to
BSL- 2:
Standard microbiological practices
1. The laboratory supervisor must enforce UW-Stout policies that control access to the laboratory
(e.g. granting key card access only to authorized students and staff, keeping doors closed).
2. Persons must wash their hands after working with potentially hazardous materials and before
leaving the laboratory.
3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for
human consumption must not be permitted in laboratory areas. Food must be stored outside the
laboratory area in cabinets or refrigerators designated and used for this purpose.
4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.
5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware
must be developed and implemented. Whenever practical, laboratory supervisors should adopt
improved engineering and work practice controls that reduce risk of sharps injuries. Precautions,
including those listed below, must always be taken with sharp items. These include:
a. Careful management of needles and other sharps are of primary importance. Needles
must not be bent, sheared, broken, recapped (without specific training), 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. Plastic ware should be substituted for glassware
whenever possible.
6. Perform all procedures to minimize the creation of splashes and/or aerosols.
7. Decontaminate work surfaces after completion of work and after any spill or splash of potentially
infectious material with appropriate disinfectant.
50
8. 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.
9. A sign incorporating the universal biohazard symbol must be posted at the entrance to the
laboratory or classroom when infectious agents are present. The sign should include the name of
the agent(s) in use, the procedure required for entry and exit (e.g. PPE), special practices (e.g.
use of BSC, immunizations) and the name and phone number of the laboratory supervisor or
other responsible personnel.
10. An effective integrated pest management program is required (see Appendix G of the BMBL).
11. 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. Personal health status may impact an individual’s susceptibility to infection,
ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel
and particularly women of childbearing 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 their healthcare provider for appropriate
counseling and guidance.
Special practices
1. Access to the laboratory must be restricted when work is being conducted.
2. All persons entering the laboratory must be advised of the potential hazards.
3. Laboratory personnel must be provided medical surveillance, as appropriate, and offered
available immunizations for agents handled or potentially present in the laboratory.
4. A laboratory-specific biosafety manual must be prepared and adopted as policy. The biosafety
manual 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.
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 should be routinely decontaminated, as well as, after spills, splashes, or
other potential contamination.
51
8. Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff
properly trained and equipped to work with infectious material.
9. Equipment must be decontaminated before repair, maintenance, or removal from the laboratory.
10. Incidents that may result in exposure to infectious materials must be immediately evaluated and
treated according to procedures described in the laboratory biosafety manual. All such incidents
must be reported to the laboratory supervisor. Medical evaluation, surveillance, and treatment
should be provided and appropriate records maintained.
11. Animals and plants not associated with the work being performed must not be permitted in the
laboratory.
12. All procedures involving the manipulation of infectious materials that may generate an aerosol
should be conducted within a BSC or other physical containment devices.
Safety equipment (primary barriers and personal protective equipment)
1. For BSL-2 agents, properly maintained BSCs, 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 are
required for BSL-2 agents. Remove protective clothing before leaving the laboratory. Dispose of
protective clothing appropriately, or deposit it for laundering. It is recommended that laboratory
clothing not be taken home.
3. Safety glasses are required in the laboratory whenever anyone in the lab is working with
chemical or biological agents that present a splash hazard, or when there is the potential for
flying glass. Splash goggles (and mask, face shield or other splatter guard as needed) are 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 should also wear eye protection.
4. Gloves should be worn to protect hands from exposure to hazardous materials. Glove selection
should be based on an appropriate risk assessment. Alternatives to latex gloves should be
available. Gloves must not be worn outside the laboratory. In addition, laboratory workers
should:
a. Change gloves when contaminated, glove integrity is compromised, or when otherwise
necessary.
52
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. A risk assessment should be used to determine if eye, face and/or respiratory protection is
needed in rooms containing infected animals.
Laboratory facilities (secondary barriers)
1. Laboratory doors must remain closed for access control.
2. Each laboratory contains at least one sink for handwashing.
3. The laboratory is designed so that it can be easily cleaned. Carpets, rugs and cloth chairs are not
appropriate in laboratories.
4. Bench tops are impervious to water and are resistant to moderate heat and the organic solvents,
acids, alkalis, and chemicals used to decontaminate the work surface and equipment.
5. Laboratory furniture is capable of supporting anticipated loading and uses. Spaces between
benches, cabinets, and equipment are accessible for cleaning.
6. An eyewash station must be readily available.
7. HEPA-filtered exhaust air from a Class II BSC can be safety recirculated back into the laboratory
environment if the cabinet is tested and certified at least annually and operated according to
manufacturer’s recommendations.
8. A method for decontaminating all laboratory waste is available.
I have received and understand the biological safety training as outlined in this document.
WORKER SIGNATURE:
DATE:
PI SIGNATURE:
DATE:
LABORATORY LOCATION:
53
APPENDIX E-3
BIOSAFETY LEVEL 2 (BSL-2) LAB TRAINING
This training provides a basic understanding of biosafety hazards and practices required to enter UWStout’s designated BSL-2 laboratory spaces. If you will be working with biological agents that
require BSL-2 containment, you must receive additional training (such as bloodborne
pathogens training, safe use of biosafety cabinets, standard microbiological practices) from
your instructor/laboratory supervisor.
 The “biosafety level” defines the laboratory practices and techniques, safety equipment and
laboratory facilities recommended for work with biohazardous materials.
 Biohazardous materials are infectious agents or hazardous biological materials that present a
potential risk to the health of humans, animals or the environment.
 BSL-2 is for work with moderate-risk agents that are associated with human disease of varying
severity.
 All doors to laboratory or classroom spaces designated as BSL-2 labs have a sign with a large
orange “biohazard” symbol and entry and exit requirements for everyone who enters the space.
 What are the potential hazards?
o
Exposure is most likely to occur via accidental needle stick; splash into the eyes, nose or
mouth; or accidental ingestion  people working with the material.
o
The risk of exposure by being in the room and inhaling the material is extremely low.
 What are the rules?
o
Lab coats are mandatory for everyone who enters.
o
No open-toed shoes.
o
Safety glasses must be worn by everyone who enters if anyone in the room is working
with a liquid chemical or biohazardous material that presents a splash hazard.
o
Doors must not be propped open.
o
No eating, drinking, applying cosmetics or storing food for human consumption at any
time.
o
Wash hands with soap and water before leaving the room.
54
APPENDIX F
TREATMENT, HANDLING AND DISPOSAL OF BIOLOGICAL MATERIALS
Proper treatment, handling and disposal of cultures and items contaminated by potentially biohazardous
materials are a vital step toward protection of laboratory and hazardous waste personnel from infectious
disease. This waste handling process is also necessary to prevent the release of potentially infectious
agents into the community at large. Treatment and disposal of biological waste is regulated by several
federal and state agencies.
An exposure occurs when potentially infectious materials are permitted to enter a person’s bloodstream
through a break in the skin or contact with the eyes, nose, or mouth. Examples of exposure related to
biohazardous waste handling include incidents such as:

Splashing liquid biological waste into the eye during pour-off for disposal

Puncturing the skin with a biologically contaminated needle

Spilling liquids from a ruptured biohazardous bag onto broken, unprotected skin.
In the event of an exposure to potentially infectious materials, take the following actions:

Wash the exposed skin or flush the mucous membrane for 10-15 minutes.

Notify your lab director or supervisor and the Biological Safety Officer or Safety and Risk
Management..

Contact your health care provider to determine the need for evaluation and/or possible
treatment.
A waste is considered to be an infectious waste if it falls in one of the following categories:
1. Sharps are potentially the most hazardous items in the infectious waste stream. A high degree
of caution should always be used when handling any sharp object, contaminated or not.
a. Contaminated sharps (contacted blood or a potentially infectious material) that are both
infectious and may easily cause punctures or cuts in the skin, including but not limited to
hypodermic needles, syringes with needles attached, scalpel blades, lancets, broken glass
vials, broken rigid plastic vials and laboratory slides, must be disposed of as infectious
waste.
b. Unused or disinfected sharps that are being discarded, including hypodermic needles,
scalpel blades, lancets and syringes with needles attached must also be disposed of as
infectious waste.
c.
Note: Only "contaminated" broken glass, plastic vials, laboratory slides, etc. are
considered infectious waste. However, all discarded sharps (contaminated or not) such
as hypodermic needles, scalpel blades, lancets and syringes with needles attached are
considered infectious waste.
55
2. Bulk blood and body fluids from humans means drippable or pourable quantities or items
saturated with blood or other potentially infectious materials. In making this determination ask
whether blood or other potentially infectious materials are dripping from the item or can be
squeezed, poured or flaked from the item.
3. Human tissue.
4. Microbiological laboratory waste means cultures derived from clinical specimens or
laboratory equipment that has come in contact with these cultures.
5. Tissue (including whole carcasses), bulk blood or body fluids from an animal that is carrying an
infectious agent or has been used in recombinant or synthetic nucleic acid molecule research.
6. Mixed wastes are potentially infectious waste contaminated with other types of waste (e.g.
radioisotopes or toxic/carcinogenic compounds). Because of the difficulty in disposal of wastes
regulated by more than one set of requirements and regulatory agencies, it is critical that
provision be made for proper management prior to the initiation of any research that might result
in mixed waste. Mixed wastes may require special containers, labeling, storage, etc. Contact
Safety and Risk Management prior to initiation of any research that might result in potentially
infectious waste with multiple hazards.
7. Microorganism cultures should be inactivated, using appropriate procedures, before disposal.
This includes cultures of Risk Group 1 and 2 organisms. This is considered good laboratory
practice.
Items that generally are not considered infectious waste include the following:
1. Items soiled but not saturated with blood of body fluids from humans (application of the
drippable, squeezable, pourable, flakeable rule).
2. Tissue, blood, body fluids or cultures from an animal that is not known to be carrying or
experimentally infected with a zoonotic infectious agent.
3. Animal manure and bedding from uninfected animals.
More than one treatment option is available for infectious waste. Sharps are collected and sent off
campus for incineration. The remaining infectious waste is autoclaved (steam sterilization) and then
disposed of as normal trash.
1. All non-sharp laboratory materials utilized in experiments with biological materials (e.g.
microorganisms, recombinant or synthetic nucleic acid molecules, cell cultures) must be treated
prior to disposal by an approved decontamination method such as autoclaving. These wastes
should be stored in bags bearing the biohazard symbol prior to decontamination. While in use for
waste storage, biohazard bags must be secured in a manner that will eliminate spillage. If a bag
is used primarily for disposal items that are not likely to release liquids (e.g. contaminated pipette
tips or kim wipes), a wire bag rack or rigid container is an acceptable means of securing the bag
to eliminate spillage. If the bag is used for storage of items that are likely to release liquids and
possible result in leakage, the bag should be stored in a leak proof container such as a trash can
with a lid that is also labeled with the biohazard symbol.
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2. When transporting waste bags to the autoclave for treatment, secure the bags closed with a
rubber band, twist tie or other closure device that can be easily removed and place the bags in
secondary container such as a pan or bucket. Always use a cart to move the bags if possible.
3. After autoclaving, any bag displaying the biohazard symbol should be placed in a non-transparent
plastic bag or other secondary non-transparent container (e.g. closed cardboard box, dark
colored trash bag) prior to disposal into the normal trash. Bags with the biohazard symbol,
regardless of use, must not be placed into the regular waste stream without defacing the symbol
or over-bagging.
Collection and handling:
1. Infectious waste should be segregated and contained in an enclosed area until it is treated.
2. Sharps should be placed in a puncture-proof and leak-proof container with a sealable lid. The
outside container must be labeled with a visible biohazard emblem (fluorescent orange
background with contrasting color - typically black - biohazard symbol). Red sharps containers
are commercially available.
3. Other infectious waste should be placed in an infectious waste bag (leak proof) with a biohazard
label.
4. Uncontaminated plastic pipette tips and uncontaminated glass are not restricted waste and can
be disposed in the regular waste stream after placement in a container, such as a designated
cardboard box, that will eliminate the potential of punctures and cuts to solid waste handlers or
the public. When it is time to dispose of the box, tape it shut and label it with “uncontaminated
plastic pipette tips” or “uncontaminated glass.”
5. Contact University Police (x2222) for the transportation of infectious waste to Student Health
Services.
Type of Waste
Animal bedding
from infected
animals
Animal carcasses
and tissue
Liquid infectious
waste including all
recombinant or
synthetic nucleic
acid molecule
material
Container for Disposal
or Treatment
Place cage, water bottle
and bedding in an
autoclave bag
Red biohazard bag; store
carcasses in freezer
Local Treatment
Suitable liquid handling
container
Disinfect with 1 part bleach
to 10 parts liquid, or other
appropriate disinfectant per
an RSC-approved protocol,
and sewer. Can also
autoclave and sewer. (Do
not sewer agar; handle as
solid waste.)
Autoclave
None
Who to Call
for Disposal
Place autoclaved
bedding in
regular trash
Safety & Risk
Management
(contractor
incineration)
N/A
57
Type of Waste
Contaminated nonsharps (including
recombinant or
synthetic nucleic
acid molecules)
Glass or other
non-sharps (e.g.
pipette tips) not
contaminated with
hazardous material
Sharps,
contaminated or
not
Biological toxins
Container for Disposal
or Treatment
Autoclave bag
Local Treatment
Autoclave
Who to Call
for Disposal
Place autoclaved
materials in
regular trash
Dedicated box closed and
taped shut
None required
Regular trash
Sharps container
None
Suitable treatment
container
Treat with 2N NaOH for at
least 1 hr, neutralize the
NaOH solution with acid
until the pH is between 5
and 8, sewer if toxin is not
in a hazardous chemical
mixture
Campus Police
(x2222) for
transport to
Student Health
If toxin in a
hazardous
chemical
mixture, place in
hazardous waste
container (no
longer a toxin
hazard) and call
Safety & Risk
Management
58
APPENDIX G
SELECT AGENTS AND TOXINS
The following list is taken from the joint APHIS/CDC National Select Agent Registry website
(http://www.selectagents.gov) and is dated September 10, 2013. Select agents and toxins are agents
that HHS considers to have the potential to pose a severe threat to human health. High Consequence
Livestock Pathogens and Toxins are agents that the USDA considers to have the potential to pose a
severe threat to animal or plant health, or to animal or plant products. The plant pathogens listed by
USDA have been deemed a threat to plant health or products. Agents that pose a severe threat to animal
health, animal products, and public health are referred to as “overlap agents.”
HHS Select Agents And Toxins
Abrin
Botulinum neurotoxins
Botulinum neurotoxin producing species of
Clostridium
Conotoxins
Coxiella burnetii
Crimean-Congo haemorrhagic fever virus
Diacetoxyscirpenol
Eastern Equine Encephalitis virus
Ebola virus
Overlap Select Agents And Toxins
Bacillus anthracis
Bacillus anthracis Pasteur strain
Brucella abortus
Brucella melitensis
Brucella suis
Burkholderia mallei
Burkholderia pseudomallei
Francisella tularensis
Hendra virus
Nipah virus
Rift Valley fever virus
Venezuelan Equine Encephalitis virus
Rickettsia prowazekii
USDA Select Agents And Toxins
African horse sickness virus
African swine fever virus
Avian influenza virus
Classical swine fever virus
Foot-and-mouth disease virus
Goat pox virus
Lumpy skin disease virus
Lassa fever virus
Marburg virus
Monkeypox virus
Reconstructed replication competent forms of the
1918 pandemic influenza virus containing any
portion of the coding regions of all eight gene
segments (Reconstructed 1918 Influenza virus)
Ricin
SARS-associated corona virus (SARS-CoV)
Saxitoxin
Shiga-like ribosome inactivating proteins
Shigatoxin
South American Haemorrhagic Fever viruses
(Chapare, Guanarito, Junin, Machupo, Sabia)
Staphylococcal enterotoxins A,B,C,D, E subtypes
T-2 toxin
Tetrodotoxin
Tick-borne encephalitis complex (flavi) viruses
(Far Eastern subtype, Siberian subtype)
Kyasanur Forest disease virus
Omsk Hemorrhagic fever virus
Variola major virus (Smallpox virus)
Variola minor virus (Alastrim)
Yersinia pestis
Mycoplasma capricolum
Mycoplasma mycoides
Newcastle disease virus
Peste des petits ruminants virus
Rinderpest virus
Sheep pox virus
Swine vesicular disease virus
USDA Plant Protection and Quarantine
(PPQ) Select Agents And Toxins
Peronosclerospora philippinensis
(Peronosclerospora sacchari)
Phoma glycinicola (formerly Pyrenochaeta
glycines)
Ralstonia solanacearum
Rathayibacter toxicus
Sclerophthora rayssiae var zeae
Synchytrium endobioticum
Xanthomonas oryzae
59
APPENDIX H
ACRONYMS
APHIS
Animal and Plant Health Inspection Service (USDA)
BMBL
Biosafety in Microbiological and Biomedical Laboratories
BSC
Biological safety cabinet
BSL
Biosafety level
BSO
Biological Safety Officer
CDC
Centers for Disease Control and Prevention
HEPA
High efficiency particulate air
HHS
U.S. Department of Health and Human Services
IACUC
Institutional Animal Care and Use Committee
IRB
Institutional Review Board
NIH
National Institutes of Health
OBA
Office of Biotechnology Activities (NIH)
PI
Principal investigator
PPE
Personal protective equipment
RAC
Recombinant DNA Advisory Committee (NIH)
RG
Risk Group
RSC
Research Safety Committee
USDA
U.S. Department of Agriculture
UV
Ultraviolet
60