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Biological Safety
Handbook
The University of Texas
Health Science Center
at San Antonio
April 2016
TABLE OF CONTENTS
1.0
Introduction ……………………………………………………………….
Purpose ………………………………………………………………
Responsibilities ….…………………………………………………..
1.2.1 Institutional Biosafety Committee (IBC) ……………………
1.2.2 Biological Safety Officer ……………………………………..
1.2.3 Environmental Health and Safety Department (EH&S) …
1.2.4 Institution / Management ……………………………………
1.2.5 Principal Investigator, Faculty, Supervisors ………………..
1.2.6 Employees, students, volunteers ………………………..…..
Reporting a Safety Concern ………………………………………..
Imminently Dangerous to Life and Health …………………………
Laboratory Evaluation Deficiencies ………………………………..
4
5
5
5
6
6
6
7
8
8
8
8
Rules, Regulations, and Guidelines ………………………………..
9
1.1
1.2
1.3
1.4
1.5
2.0
2.1
2.2
2.3
2.4
2.5
2.6
3.0
General Principles and Biosafety Levels …………………………
3.1
3.2
3.3
3.4
4.0
Background – Risk Group Classification …………………………
Definitions ……………………………………………………………..
Biohazard, Biosafety………………………………………………..
Risk Assessment …………………………………………………….
Containment ………………………………………………………….
Biosafety Levels 1 – 4 (BSL 1 – 4) …………………………………
Animal Biosafety Level (ABSL 1- 4) ………………………………..
Plant Biosafety Levels 1 –4 …………………………………………
Arthropod Biosafety ………………………………………………….
Work with clinical specimens ……………………………………….
Hygiene, Housekeeping and Personal Protective Equipment
4.1
4.2
4.3
5.0
NIH Guidelines for Research involving Recombinant or Synthetic
Nucleic Acid Molecules ……………………………………………..
Health and Human Services: CDC/NIH Guidelines:
Biosafety in Microbiological and Biomedical Laboratories ………
OSHA and Texas DSHS - Bloodborne Pathogens Standard …..
HHS(CDC) and USDA Select Agents and Toxins ……………….
Shipping/transportation of Dangerous goods …………………….
Import permits ……………………………………………………….
Lab practices and Personal Hygiene in the Laboratory ………….
Laboratory Housekeeping …………………………………………..
Personal Protective Equipment …………………………………….
General Guidelines for Handling Biological Agents …………….
5.1
5.2
Introduction……………………………………………………………
Training………………………………………………………………..
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9
9
10
11
11
12
13
13
13
13
14
15
16
16
16
16
17
17
18
19
22
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Practices and procedures …………………………………………
Laboratory requirements and security ……………………………
Posting and Labeling requirements ……………………………….
Procedures for Control of Biohazards in Clinical Laboratories …
Universal Precautions ………………………………………………
Aerosols: Risks, production and prevention ………………………
22
24
26
26
27
28
Disinfection, Sterilization, and Decontamination ………………...
30
30
30
30
31
5.3
5.4
5.5
5.6
5.7
6.0
6.1
6.2
6.3
6.4
7.0
Procedures for Spills of Biohazardous Materials ……………….
7.1
7.2
7.3
8.0
Definitions …………………………………………………………….
General Procedures …………………………………………………
Choosing a Method and Selecting a Disinfectant ………………..
Sterilization Methods ……………………………………………….
32
32
32
33
Immediate actions for a biohazardous spill ……………………….
Clean up and Decontamination after a spill ………………………
Procedure for spills in a corridor or common use area ………….
Laboratory Equipment ……………………………………………
8.1 Biological Safety Cabinets (BSC) …………………………………….....
8.1.1 Overview and types of Biological Safety Cabinets ………….
8.1.2 Certification of a Biological Safety Cabinet …………………
8.1.3 Decontamination of a Biological Safety Cabinet ……………
8.1.4 Flammable Gases in a Biological Safety Cabinet ………….
8.1.5 Ultraviolet Lamps in a Biological Safety Cabinet ……………
8.1.6 Procedures for Use of Class I and II cabinets ………………
8.2 – 8.13 Other Laboratory Equipment ……………………………………
9.0
Medical Surveillance, Exposure control …………………………
35
35
35
40
41
42
43
43
45
9.1 Overview of medical surveillance …………………………………………
9.2 Response for Injury/Exposures and Reporting ………………………….
9.3 Employee Health Records ………………………………………………..
9.4 Blood borne Pathogen Exposure Control Plan …………………………
9.5 TB Exposure Control Plan ………………………………………………
55
55
55
57
58
58
10.0 Medical Waste Management ……………………………………..
59
11.0 Transport, Packaging and Shipment Biohazardous Materials … 62
12.0 Recombinant and Synthetic Nucleic Acid Molecules (rDNA) …..
64
13.0 Work with Viruses and Viral Vectors ……………………………… 65
14.0 Toxins of Biological Origin ………………………………………………… 74
15.0 Integrated Pest Management ……………………………………… 76
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APPENDICES
Appendix A: Registration of work with recombinant and/or synthetic
nucleic acids, infectious agents and other potentially
biohazardous materials Forms ………………………………… 77
Appendix B: Risk Groups: Classification of Micro-organisms on the
Basis of Hazard as provided in the NIH Guidelines ……… 83
Appendix C: HHS/CDC Select Agents and USDA High
Consequence Livestock Pathogens …………………………..
91
Appendix D: Laboratory Biosafety Level Criteria (BSL 1-4) ……………
95
Appendix E: Vertebrate Animal Safety Level Criteria (ABSL 1-4) ……
107
Appendix F: Lab Specific Forms: ……………………………………….
121
1. Writing Lab Specific Standard Operating Procedures
2. Lab specific SOP for work with Lentivirus Template form
3. Biological Spill Response and Laboratory Specific Emergency
Plan – Template Form
Appendix G: Standard Operating Procedures (SOPs) and Plans …… 126
1. Biosafety Cabinet SOP
2. Transport of Biological Materials SOP
3. Biomedical Waste Management SOP
4. Lentivirus SOP
5. FACS Lentivirus SOP
6. Autoclave SOP
7. Bloodborne Pathogen Exposure Control Plan
8. TB Exposure Control Plan
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1.0 INTRODUCTION
1.1 Purpose. The University of Texas Health Science Center at San Antonio is committed to the
development, maintenance, and support of a comprehensive plan for the research and clinical
community that has as its primary purpose the safety of all individuals who deal with, control,
research, or come in contact with biological agents and toxins. The Health Science Center is
committed to ensuring that all activities of a potentially biologically hazardous nature are safely
conducted. All university faculty, staff and associated students will engage in prudent practices
necessary to protect people and the environment from biological hazards through conformance
with the federal, state, and local regulations and guidelines and Institutional policies.
The objective of the University of Texas Health Science Center at San Antonio’s Biological Safety
Program is to assist all levels of management in fulfilling the Health Science Center’s commitment to
provide an environment for high quality research, teaching, and learning while maintaining a safe
workplace, preventing environmental contamination and complying with applicable federal, state and
local requirements. It is vital that faculty, staff and students have enough information available to aid
them in the safe conduct of their daily work activities related to teaching and research.
The purpose of the Biological Safety Handbook is to provide university-wide safety guidelines,
policies and procedures for the use and manipulation of biohazardous and potentially
biohazardous materials. This handbook provides information on how to minimize the risk to
personnel from exposures to biohazards through the application of administrative, engineering,
and work practice controls and by increasing awareness of the biological hazards that may be
encountered in research, clinical, and teaching laboratories. The Biological Safety Handbook is not
intended to be an exhaustive or fully comprehensive reference, rather a guide for Principal
Investigators, staff, students and other technically qualified individuals. Each laboratory or clinic area
must have specific operating procedures, contingency and emergency plans that are specific for their
particular needs. Further advice concerning hazards associated with specific biological agents,
recombinant or synthetic nucleic acid molecules (rDNA), and the development of new or unfamiliar
activities should be obtained through consultation with the Institutional Biosafety Committee and
Environmental Health and Safety's Biological Safety Division.
All faculty, staff, students, and others who work with biological agents and rDNA must be familiar with
and conduct their operations in accordance with the requirements set forth in this manual and any
applicable regulations and guidelines.
Signature on file
__
Michael A. Charlton, Ph.D.
Assistant Vice President for Risk
Management and Safety
The University of Texas Health Science
Center at San Antonio
Signature on file
__
David Kolodrubetz, Ph.D.
Chair, Institutional Biosafety Committee
The University of Texas Health Science
Center at San Antonio
Signature on file
__
James D. Kazen
Executive Vice President
Facility Planning and Operations
The University of Texas Health Science
Center at San Antonio
Signature on file
__
William Henrich, M.D., MACP
President
The University of Texas Health Science
Center at San Antonio
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1.2 Responsibilities
1.2.1 Institutional Biosafety Committee (IBC). The Institutional Biosafety Committee (IBC) is
mandated by the National Institutes of Health (NIH), Office of Science Policy (OSP) for an
institution, The University of Texas Health Science Center at San Antonio, conducting or
sponsoring recombinant or synthetic nucleic acid molecule research in accordance with the NIH
Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (in
successive paragraphs, this document will be referred to as the NIH Guidelines). The Committee’s
responsibilities need not be restricted to recombinant or synthetic nucleic acid research and may
include review of other research of a biohazardous nature.
The IBC uses the most current versions of the NIH/CDC document “Guidelines for Research
Involving Recombinant and Synthetic Nucleic Acid Molecules,” and “Biosafety in Microbiological and
Biomedical Laboratories“ as well as other NIH/CDC agent specific and infection control guidance
documents and best management practices as a basis for review.
The committee membership as mandated by NIH/OSP consists of at least five (5) primary
members and appropriate ex-officio members including a Chair, Biological Safety Officer (if
applicable), plant expert (if applicable), animal expert (if applicable), human gene therapy expertise
or ad hoc consultant (if applicable) and two community members. Appropriate alternate members
may be assigned. For more information on the term of membership and their terms, consult the
Handbook of Operating Procedures (HOP), 1.7.1. http://uthscsa.edu/hop2000/1.7.1.pdf
The Institutional Biosafety Committee is charged to …
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advise the President, Executive Vice President for Facilities Planning and Operations, and the
Director of Environmental Health and Safety in matters pertaining to hazards of a biological
nature;
review and approve research projects involving recombinant or synthetic nucleic acid
molecules (rDNA) conducted at or sponsored by the institution
review and approved other projects of a biohazardous nature including assessment of the
containment levels, facilities, procedures, practices, training and expertise of personnel;
assess the risks involved in such projects and the measures proposed for their containment
advise on the safe receipt, use, storage, and disposal of potentially hazardous biological
agents;
assess the risks involved in such projects and the measures proposed for their containment;
review plans for areas designated to be constructed or remodeled for biohazardous work;
establish criteria and monitor adherence to these criteria for the use of rDNA and biohazardous
agents and facilities designed for use with such agents;
ensure that all aspects of Appendix M (Transfer of Recombinant or Synthetic Nucleic Acid
Molecules into human research participants) of the NIH Guidelines have been met;
serve as the Institutional Review Entity in accordance with Section 7.1 of the U.S. Government
Policy for Dual Use Research of Concern including toxins; and serve as an avenue of appeal in
cases of dispute and exception.
See Appendix A for Institutional Biosafety Committee procedures and registration for work
with biohazardous materials and recombinant or synthetic nucleic acid molecules.
1.2.2 Biological Safety Officer (BSO). An individual appointed by an institution to oversee
management of biosafety risks. The NIH Guidelines require that a BSO be appointed when the
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institution is engaged in large-scale research or production activities involving viable organisms
containing recombinant or synthetic nucleic acid molecules, or in research requiring containment
at BL-3 or BL-4. For purposes of NIH funding, the Biological Safety Officer will be the Director of
the Environmental Health and Safety department, or qualified staff member(s) so appointed by the
Director. The duties of the BSO are described in section IV-B-3 of the NIH Guidelines and include
but not limited to:
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being a member of the Institutional Biosafety Committee

conducting periodic inspections to ensure that laboratory standards are rigorously
followed;
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reporting to the Institutional Biosafety Committee and the institution any significant
problems, violations of the NIH Guidelines, and any significant research-related
accidents or illnesses of which the Biological Safety Officer becomes aware unless
the Biological Safety Officer determines that a report has already been filed by the
Principal Investigator;
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developing emergency plans for handling accidental spills and personnel
contamination and investigating laboratory accidents involving recombinant or
synthetic nucleic acid molecule research;
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providing advice on laboratory security;
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providing technical advice to Principal Investigators and the Institutional Biosafety
Committee on research safety procedures
1.2.3 Environmental Health and Safety Department (EH&S), Biological Safety Division. The
Environmental Health and Safety Office, Biological Safety Division will …
 establish general polices and standards for the use of biological materials at the
Health Science Center with the advice and guidance of the Institutional Biosafety
Committee (IBC) and the Infection Policy and Education Committee.
 provide consultation and technical information for handling biological agents.
 review proposals and protocols for the use of hazardous biological agents and
recombinant and/or synthetic nucleic acid molecules and submit these to the
Institutional Biosafety Committee with recommendations.
 oversee the biological safety cabinet certification program.
 present training in biosafety and bloodborne pathogens and other safety seminars
upon request
 provide consultation on the purchases of biological safety cabinets and other
specialized safety related equipment
 provide assistance or advice in the decontamination or disinfection of facilities and
equipment
 assist in the development of safety plans and training programs
 perform injury/accident investigations related to a potential biological exposure
 perform initial laboratory evaluations to review compliance with this handbook and
applicable federal, state and local regulations. Annual laboratory safety evaluations
may be delegated to other personnel.
 follow up on spills or releases of biohazardous agents, assist in containment,
decontamination and disinfection within the scope of their response training on
UTHSC property.
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1.2.4 Institution/Management. The institution is responsible for:
 ensuring that recombinant and synthetic nucleic acid research is conducted in
conformance with the NIH Guidelines.
 establishing and implementing policies that provide for safe conduct of research
 ensuring compliance with applicable federal, state and local regulations, which are
binding on all personnel and students within all divisions and departments at the
UTHSC working in research laboratories and clinical areas involving biohazardous
materials.
1.2.5 Principal Investigators/Faculty/and Supervisors. The Principal Investigator (PI) is the
individual who submits the application to employ biological agents and/or rDNA in his or her work or
is the responsible faculty supervisor over student labs. This individual has the primary responsibility
for adherence to all UTHSC policies, guidelines and federal, state and local regulations. The PI is
also fully responsible for the safe use of such agents by himself/herself and those under his or her
direction.
Principal Investigators/Faculty/and Supervisors will…
 comply with and enforce all UTHSC standards, policies, procedures and referenced
regulations regarding the use, handling, containment, and disposal of biological
agents as outlined in this handbook.
 develop laboratory safety procedures specific to that lab or protocol(s), placing a
priority on engineering controls and work practice (e.g. biosafety cabinets and
containment levels) to eliminate or minimize employee/student exposure.
 ensure that employees/students are aware of and trained in the hazards, safe
handling and disposal procedures of biological agents/rDNA.
 ensure that staff are supplied with the appropriate Personal Protective Equipment
(PPE) and medical surveillance for their job duties.
 ensure that safety equipment is available in the laboratory, used when necessary and
adequately maintained.
 encourage employees to report any changes or suspected changes in their health
status.
 complete necessary documentation to report potential exposures and encourage
employees to seek medical treatment and receive appropriate medical surveillance.
 inform EH&S of any significant changes in the protocol for the use of hazardous
biological agents.
 report potential exposures and spills of biological materials/rDNA to EH&S
 notify EH&S and complete the laboratory decommissioning form when leaving the
university to ensure proper disposition of biological agents, chemicals, equipment, etc.
Additionally for those Principal Investigators engaged in recombinant or synthetic nucleic
acid research, the PI will….
 be responsible for full compliance with the NIH Guidelines in the conduct of
recombinant or synthetic nucleic acid molecule research.
 if engaged in human gene transfer research, comply with all aspects of Appendix M.
The PI may delegate to another party, such as a corporate sponsor, the reporting
functions set forth in Appendix M (of the NIH Guidelines), with written notification to
the NIH/OSP of the delegation and of the name(s), address, telephone, and fax
numbers of the contact. The Principal Investigator is responsible for ensuring that the
reporting requirements are fulfilled and will be held accountable for any reporting
lapses.
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not initiate or modify any recombinant or synthetic nucleic acid molecule research
which requires Institutional Biosafety Committee approval prior to initiation until that
research or the proposed modification thereof has been approved by the Institutional
Biosafety Committee and has met all other requirements of the NIH Guidelines;
determine whether experiments are covered by Section III-E, Experiments that
Require Institutional Biosafety Committee Notice Simultaneous with Initiation, and
ensure that the appropriate procedures are followed. (Note: per institutional policy, the
Institutional Biosafety Committee will review all research involving recombinant and
synthetic nucleic acid molecules.)
report any significant problems, violations of the NIH Guidelines, or any significant
research-related accidents and illnesses to the Biological Safety Officer, Institutional
Biosafety Committee and other appropriate authorities within 30 days. (Note: The
BSO will make any necessary reports for spills and exposures to NIH.)
comply with shipping requirements for recombinant or synthetic nucleic acid
molecules.
1.2.6 Individual employees, students, volunteers, and other individuals (Visiting scientists,
residents, fellows, etc.).
It is the responsibility of each Individual to …
 comply with the established safety procedures and policies.
 maintain awareness of the risks associated with assigned duties.
 attend all required training classes to stay in compliance with relevant rules and
regulations.
 take all necessary and appropriate safety precautions relevant to performance of their
duties, including wearing the necessary personal protective equipment.
 inform the immediate supervisor, EH&S or Compliance office of any unsafe practices
or conditions in the work area.
 inform their immediate supervisor when an injury or exposure occurs.
 report to their supervisor any change or suspected change in the health status if there
is a possibility it may be work-related.
 report all biological/rDNA spills and accidents to the supervisor.
1.3
Reporting a Safety Concern. All safety concerns should be reported to Environmental
Health & Safety (1.343T DTL, (210) 567-2955). However, all employees, students and
volunteers are encouraged to talk to their immediate supervisors regarding unsafe
conditions. In the event that the supervisor is unable to resolve the issue, Environmental
Health & Safety can offer assistance.
1.4
Imminently Dangerous to Life and Health. If Environmental Health & Safety personnel
note any conditions where there is risk of imminent danger to life, health, or facilities, this
concern will be brought to the immediate attention of the appropriate Safety Manager(s)
and the Director of Environmental Health & Safety. Corrective action may include
immediate shut down of all operations as outlined in the January 1, 1995 memorandum
from the UTHSCSA President titled, Responsibilities and Authority of the Institutional
Safety Officer.
1.5
Laboratory Evaluation Deficiencies and Escalated Notifications.
Any noted deficiencies observed during routine laboratory evaluations will be corrected by
the responsible Principal Investigator (PI) within the required time frame (usually 30
business days). Deficiencies posing an unusual hazard, or those of a serious nature that
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have not been resolved after a 60 day period, will advance to an Escalated Deficiency.
Environmental Health & Safety will use the following sequential notification methods and
time line:
a) A letter from the Safety Manager to the PI/Supervisor – 30 business days to respond
b) A letter from the appropriate Safety Committee Chair to PI/Supervisor – 30 business
days to respond
c) A letter from the Director of Environmental Health & Safety to the Department Chair –
30 business days to respond
d) A letter from the Director of Environmental Health & Safety to the Dean of supervising
school – 30 business days to respond
e) A letter from the Director of Environmental Health & Safety to the Executive Vice
President – 30 business days to respond
f) A letter to the UTHSCSA President – 30 business days to respond
g) A letter to the UT Board of Regents – 30 business days to respond
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2.0 Rules, Regulations & Guidelines
The following is a brief summary of the regulatory authorities that either regulate or provide
guidelines for the use of biological materials (clinical specimens, cell lines, etc.), infectious agents
and recombinant or synthetic nucleic acid molecules.
2.1 NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid
Molecules (referred to as NIH Guidelines). In 1974, a recombinant DNA Advisory Committee
(RAC) was established to determine appropriate biological and physical containment practices
and procedures for experiments involving recombinant DNA that potentially posed risks to human
health and the environment. As a result of the committee’s activity, the initial version of the NIH
Guidelines was published in 1976. It has been amended and revised many times since then.
Included in the NIH Guidelines is a requirement for the institution to establish an Institutional
Biosafety Committee (IBC) with authority to approve or disapprove proposed rDNA research
using the NIH Guidelines as a minimum standard.
For more information, please refer to the Recombinant DNA Research section in this manual and
the website http://osp.od.nih.gov/office-biotechnology-activities/biosafety/nih-guidelines
2.2 Biosafety in Microbiological and Biomedical Laboratories (BMBL):
http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
This publication by the Department of Health and Human Services (HHS), Centers for Disease
Control (CDC) and the National Institutes of Health (NIH), originally published in 1984, is the
cornerstone of biosafety practice in the United States. The BMBL describes combinations of
standard and special microbiological practices, safety equipment, and facilities which are
recommended for working with a variety of infectious agents in various laboratory settings.
2.3 Texas Department of State Health Services (TDSHS) and Occupational Safety and
Health Administration (OSHA) Bloodborne Pathogens Standard. OSHA promulgated a rule
to deal with the occupational health risk caused by exposure to human blood and other potentially
infectious materials (OPIM). OSHA’s final rule (29CFR 1910.1030) became effective December
1991 and includes a combination of engineering and work practice controls, personal protective
clothing and equipment, training and medical follow-up of exposure incidents, vaccination, and
other provisions. The TDH rule, 25 TAC Part 1, Chapter 96, Bloodborne Pathogen Control,
became effective September 1, 2000 and is analogous to the OSHA Bloodborne Pathogen
Standard.
2.4 Department of Health and Human Services (HHS): Select Agents and toxins, 42 CFR 73
and United States Department of Agriculture (USDA) 9 CFR 121 and 7 CFR 331. In 1996,
HHS published a set of rules (Additional Requirements for Facilities Transferring or Receiving
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Select Agents, October 24,1996, Appendix A of 42 CFR Part 72.6) requiring facilities and
institutions to be registered and approved in order to transfer or receive select biological agents
and toxins. Subsequent to the enactment of Public Law 107-188, The Public Health Security and
Bioterrorism Preparedness and Response Act of 2002, additional requirements for facilities or
entities that possess, use, or transfer select agents and toxins have been published in 42 CFR
73; 9 CFR 121 and 7 CFR 331; December 13, 2002, interim final rule. The final rule became
effective March 18, 2005 and has been amended several times. More information on the federal
Select Agent Program can be found at:
http://www.selectagents.gov/
The most current list of select agents and toxins covered under this rule is included in
Appendix C.
2.5 Shipping/Transportation of Dangerous Goods. Packaging, shipment and transportation
requirements for infectious substances, diagnostic specimens and biological products as well as
other hazardous materials (ie. dry ice, formalin, etc.) are addressed in the following rules and
guidelines:
o
U.S. Department of Transportation (DOT):49 CFR Parts 171-178
o
International Air Transport Association (IATA): Dangerous Goods Regulations
o
International Civil Aviation Organization (ICAO):Technical Instructions for the Safe
Transport of Dangerous Goods by Air
o
United Nations: Recommendations of the Committee of Experts on the Transportation
of Dangerous Goods
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U.S. Public Health Service:42 CFR Part 72
o
U.S. Postal Service:39 CFR Part 111
o
U.S. Department of Labor, OSHA:29 CFR 1910.1030
2.6 Import Permits. CDC Import permit Program. Import permits are required for infectious
biological agent, infectious substance, vector, animals, arthropods, snails, and import or
transfer of live bats, non-human primate material as outlined in U.S. Public Health Service, 42
CFR Part 71, Foreign Quarantine. Information and permits may be obtained from the CDCs
Import Permit Program at their website:
http://www.cdc.gov/od/eaipp/importApplication/agents.htm
APHIS Permits. The US Department of Agriculture (USDA) Animal and Plant Health
Inspection Service (APHIS) issues permits for the import, transit and release of regulated
animals, animal products, veterinary biologics, plants, plant products, pests, organisms, soil,
and genetically engineered organisms. Tissue culture materials and suspensions of cell
culture containing growth stimulants of bovine or other livestock origins are controlled by the
USDA due to the potential risk of introduction of exotic animal diseases into the U.S.
Further information on import of live animals or animal products may be obtained by calling
the USDA/APHIS at (301) 851-3300 or at the website http://www.aphis.usda.gov/vs
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3.0 GENERAL PRINCIPLES AND BIOSAFETY LEVELS
3.1 Background and Risk Group Classification. Numerous governmental and international
agencies including the NIH and World Health Organization (WHO) have classified biological agents
(bacteria, fungi, viruses, parasites, prions, rDNA, biologically derived toxins, pathogens of plants and
animals) into Risk Groups (RG) based on the transmissibility, invasiveness, virulence and lethality of
the specific agent. The risk groups listing in the NIH Guidelines for Research involving Recombinant
DNA Molecules (NIH Guidelines) is one of the accepted standards in the United States, even when
recombinant DNA technology is not utilized. These Agent Risk Groups (RG-1 through RG-4) are
listed in Appendix B of this handbook. Note: These Agent Risk groups are not all-inclusive. Another
resource for Risk Group classifications is that published by the American Biological Safety
Association, which is available on their website at www.absa.org .
Classification of Infectious Microorganisms by Risk Group
Risk Group
Classification
NIH Guidelines for
Research involving
Recombinant DNA
Molecules
World Health Organization Laboratory
Biosafety Manual 3rd Edition
Risk Group 1
Agents not associated with
disease in healthy adult
humans.
(No or low individual and community risk)
A microorganism unlikely to cause human
or animal disease.
Risk Group 2
Agents associated with
human disease that is rarely
serious and for which
preventive or therapeutic
interventions are often
available.
(Moderate individual risk; low community
risk) A pathogen that can cause human or
animal disease but is unlikely to be a
serious hazard to laboratory workers, the
community, livestock or the environment.
Laboratory exposures may cause serious
infection, but effective treatment and
preventive measures are available and
the risk of spread of infection is limited.
Risk Group 3
Agents associated with
serious or lethal human
disease for which preventive
or therapeutic interventions
may be available (high
individual risk but low
community risk).
(High individual risk; low community risk)
A pathogen that usually causes serious
human or animal disease but does not
ordinarily spread from one infected
individual to another. Effective treatment
and preventive measures are available.
Risk Group 4
Agents 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).
(High individual and community risk)A
pathogen that usually causes serious
human or animal disease and can be
readily transmitted from one individual to
another, directly or indirectly. Effective
treatment and preventive measures are
not usually available.
The table is from Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th ed.
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3.2 Definitions.
General Definition of Biohazard
A biohazardous agent is one that is biological in nature, capable of replication in a host or
the environment, and capable of producing deleterious effects upon other biological
organisms, particularly humans. Biohazards are biological agents or biological substances
present in or arising from the work environment that present or may present a hazard to the
health or well-being of the worker or community.
Biological Safety or Biosafety
Biological safety or biosafety is the application of knowledge, techniques, equipment, and
facility design to prevent personal, laboratory and environmental exposure to potentially
infectious agents or biohazards.
3.3 Risk Assessment.
The BMBL states that “Risk assessment is an important responsibility for directors and principal
investigators of microbiological and biomedical laboratories. Institutional biosafety committees
(IBC), animal care and use committees, biological safety professionals, and laboratory animal
veterinarians share in this responsibility. Risk assessment is a process used to identify the
hazardous characteristics of a known infectious or potentially infectious agent or material, the
activities that can result in a person’s exposure to an agent, the likelihood that such exposure will
cause a Laboratory Acquired Infection (LAI), and the probable consequences of such an infection.
The information identified by risk assessment will provide a guide for the selection of appropriate
biosafety levels and microbiological practices, safety equipment, and facility safeguards that can
prevent LAIs.”
The NIH Guidelines state that, “In deciding on the appropriate containment for an experiment, the
first step is to assess the risk of the agent itself. Appendix B (Of the NIH Guidelines), Classification
of Human Etiologic Agents on the Basis of Hazard, classifies agents into Risk Groups based on an
assessment of their ability to cause disease in humans and the available treatments for such
disease. Once the Risk Group of the agent is identified, this should be followed by a thorough
consideration of how the agent is to be manipulated. Factors to be considered in determining the
level of containment include agent factors such as: virulence, pathogenicity, infectious dose,
environmental stability, route of spread, communicability, operations, quantity, availability of
vaccine or treatment, and gene product effects such as toxicity, physiological activity, and
allergenicity. Any strain that is known to be more hazardous than the parent (wild-type) strain
should be considered for handling at a higher containment level. Certain attenuated strains or
strains that have been demonstrated to have irreversibly lost known virulence factors may qualify
for a reduction of the containment level compared to the Risk Group assigned to the parent strain.”
Steps in a risk assessment:
1. Identify the hazards associated with the agent and assess the risk. Agent risk can include
capability to infect and cause disease, severity of disease, route of exposure, medical surveillance
and treatment availability.
2. Review laboratory procedures and hazards associated with those procedures. Procedural
hazards may include agent concentration, volume, equipment used, aerosol generating
procedures, use of sharps, and hazards associated with work with animals.
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3. Make a determination of the appropriate biosafety level. Additional precautions may be
necessary based on the risk.
4. Evaluate personnel regarding their technical expertise and the safety equipment.
5. Review the risk assessment with knowledgeable individuals and the Institutional Biosafety
Committee.
www.cdc.gov
3.4 Containment.
The purpose of containment is to confine biohazards and to reduce the potential exposure of the
laboratory worker, persons outside of the laboratory, and the environment to potentially infectious
agents. The three elements of containment include laboratory practice and technique, safety
equipment, and facility design.
3.4.1 Primary Containment:
Protection of personnel and the immediate laboratory environment through good
microbiological technique (laboratory practice) and the use of appropriate safety
equipment such as a biosafety cabinet and personal protective equipment (PPE).
Laboratory practice and technique
 strict adherence to standard microbiological practices and procedures
 be aware of any potential hazards and be trained and proficient in the practices and
techniques associated with the materials being handled
 use appropriate safety equipment for the specific procedure
 the laboratory supervisor must be trained in laboratory techniques, safety procedures,
and hazards associated with handling potentially infectious agents
Safety Equipment
 The primary barriers between the infectious agent and the worker includes biological
safety cabinets, safety centrifuge cups, personal protective clothing and is most
effective when used with good laboratory technique.
3.4.2 Secondary Containment.
Protection of the environment external to the laboratory from exposure to infectious
materials through a combination of facility design and operational practices.
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Facility design. Secondary barrier against potential exposure includes engineering
features which allow handling of hazardous materials without endangering laboratory
personnel, the work area, or the environment and is most effective when combined with
good laboratory technique and safety equipment. An example of facility design is negative
airflow into the laboratory with respect to the corridor.
3.4.3 Biosafety Levels 1-4.
Four biosafety levels of containment are described in the current edition of Biosafety in
Microbiological and Biomedical Laboratories (BMBL). Combinations of laboratory practices,
safety equipment, and laboratory design can be made to achieve different levels of physical
containment for work with biohazardous agents. Currently four Biosafety Levels (BSL1-4)
define the level of containment necessary to protect personnel and the environment. A
Biosafety Level 1 (BSL-1) is the least restrictive, while Biosafety Level 4 (BSL-4) requires a
special containment laboratory or facility. Most of the research at the UTHSC is conducted at
Biosafety Levels 1 and 2. The NIH Guidelines also details four biosafety levels of containment
(BL1-4) for work with rDNA.
A summary of the different biosafety level requirements (BSL-1 to 4) can be found
below. Specific criteria for each biosafety level can be found in Appendix D of this handbook.
Biosafety Level 1 (BSL-1) precautions are appropriate for facilities that work with wellcharacterized agents not consistently know to cause disease in healthy adult humans; of
minimal potential hazard to laboratory personnel and the environment. Level 1 precautions
rely on good microbiological technique without special primary or secondary barriers.
Biosafety Level 1 is appropriate for undergraduate and secondary teaching laboratories.
Biosafety Level 2 (BSL-2) is appropriate for agents that pose a moderate potential hazard to
personnel and the environment. Biohazardous agents that are handled at a BSL-2
containment level are associated with human disease for which preventative or therapeutic
interventions are often available. The primary hazards to personnel include accidental,
ingestion, and skin or mucous membrane exposure to infectious materials. Biosafety Level 2
precautions are appropriate for facilities that work with a broad range of indigenous
moderate-risk agents known to cause human disease (e.g., Hepatitis B virus, and
Toxoplasma spp.). Level 2 precautions are necessary when working with human blood, body
fluids, tissues or human cell lines where the presence of an infectious agent is unknown but
possible. Biosafety Level 2 is recommended for clinical, diagnostic, research or teaching
laboratories involving moderate risk agents associated with human disease.
Biosafety Level 3 (BSL-3) applies to clinical, diagnostic, research or teaching laboratories
working with microorganisms whose primary route of transmission is via inhalation and the
disease may be fatal. Autoinoculation and ingestion may also represent hazards to personnel
working with these agents. Biosafety Level 3 precautions apply to facilities that work with
indigenous or exotic agents with the potential for aerosol transmission and lethal infection.
Biosafety Level 3 precautions emphasize primary and secondary barriers. For primary
protection, all laboratory manipulations should be performed in a biological safety cabinet or
other enclosed equipment. Secondary protection should include controlled access to the
laboratory and specialized ventilation.
Biosafety Level 4 (BSL-4) represents maximum containment and is required for dangerous
and exotic agents which pose a high risk of life-threatening disease, the possibility of aerosol
transmission, and no known vaccine or therapy (e.g., Marburg or Congo-Crimean viruses).
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supplied positive-pressure safety suits are necessary when working with level 4 agents. In
addition, isolated facilities, specialized ventilation, and waste management systems are
required.
Animal Biosafety.
Four animal biosafety levels are described in the BMBL and NIH Guidelines for infectious disease
and synthetic/recombinant nucleic acids work with laboratory animals. Safety practices, equipment,
and facilities are designated by Animal Biosafety Levels, BMBL assigns ABSL1-4 and the NIH
Guidelines assigns BL1-N through BL4-N.
Plant Biosafety.
The NIH Guidelines also describes general plant biosafety levels for research involving genetically
modified plants. Safety practices, equipment, and facilities are designated by Plant Biosafety Levels
BL1-P through BL4-P.
Arthropod Biosafety.
The American Committee Medical Entomology (CME), a subcommittee of the American Society of
Tropical Medicine and Hygiene (ASTMH), drafted guidelines for work with arthropods “Arthropod
Containment Guidelines”. Most uses of Drosophila are excluded from these guidelines. Work with
transgenic drosophila does fall under the NIH Guidelines and work is registered with and reviewed by
the Institutional Biosafety Committee.
Work with Clinical Specimens.
Specimens from either humans or animals may contain infectious agents. These specimens can
include tissues, organs, blood or other body fluids. Personnel in clinical laboratories shall practice
universal precautions, treating all specimens as if they are infectious. Personnel working with
specimens of human origin must follow the Bloodborne Pathogen standard (Texas Dept. of State
Health Services; OSHA standard 29 CFR 1910.1030) and the Health Science Center’s Bloodborne
Pathogen Exposure Control Plan.
Non-human primate specimens can also harbor infectious agents and shall be treated as potentially
infectious. These specimens should be handled with the same general precautions as work with
human specimens.
Human and other primate specimens and cells derived from those specimens should be handled
using a minimum of BSL-2 practices and containment.
Specimens from other animals are handled based on the risk, which includes the presence of
possible zoonotic agents and animals infected with a biohazardous agent as part of a research
protocol.
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4.0 Laboratory Hygiene and Work Practices, Housekeeping and Personal
Protective Equipment
4.1 Personal Hygiene and Work Practices. Personal hygiene in the laboratory is directed mainly
toward the prevention of occupationally-acquired disease or physical injury. However, it can raise the
quality of laboratory work by reducing possibilities for contamination of experimental material.
Adherence to good work practices provides a margin of safety in situations where the hazard may be
unrecognized. The history of occupational injury is replete with examples of hazards unrecognized
until too late. The following guidelines are standard operating procedures for working in a research
or clinical laboratory. Clinical operations may have additional standard operating procedures for work
practices and infection control.
1. Do not eat, drink, chew gum, use tobacco, apply cosmetics, or handle contact lenses in
the laboratory or in animal rooms.
2. Food and drinks, including water, shall not be stored in the laboratories. Laboratory
sinks shall not be used as a source of drinking water. Refrigerators in laboratories are
for experimental materials only. Food and drinks for human consumption shall be
stored outside the laboratory and only in refrigerators specifically designated for
that purpose.
3. Do not use laboratory equipment for food preparation. Do not use empty food
containers for laboratory materials or samples. Microwave ovens in the laboratory shall
only be used for research purposes.
4. Smoking is not permitted in laboratories or animal rooms, or anywhere on
UTHSCSA campuses.
5. Shaving or brushing teeth is not permitted in laboratories. Toothbrushes, razors,
toiletry supplies, and cosmetics should only be used in designated areas outside the
laboratory after thoroughly washing the hands and face or showering.
6. Facial hair is discouraged in areas where there is the potential for airborne hazardous
materials. Facial hair retains particulates more persistently than clean-shaven skin. A
clean-shaven face is essential to the adequate fit of a face mask or respirator when
the work requires respiratory protection.
7. Keep hands away from the mouth, nose, eyes, face and hair when working in the
laboratory.
8. Books and journals should be used only in clean areas if possible. Care must be taken
not to contaminate papers, research notes, computer keyboards, etc.
9. Personal handkerchiefs should not be used in the laboratory.
should be available in laboratories and change rooms.
Disposable tissues
10. Appropriate clothing is required when working in UTHSCSA laboratories.
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



Wear closed toe shoes and long pants or long skirt. Open toe sandals are not allowed
in the laboratory.
Remove loose jewelry and jewelry that may compromise or tear gloves or get caught
up in equipment.
Secure loose hair and clothing to avoid contact with flames, equipment, and potential
contamination.
Do not wear potentially contaminated protective clothing out of the laboratory area.
Dispose of contaminated clothing into biohazard bags or hampers designated for
collecting contaminated clothing.
Wear the appropriate personal protective equipment in the laboratory.
11. Wash hands frequently after handling biological materials, after removing gloves
and other personal protective equipment and before leaving the laboratory.
12. Decontaminate work surfaces at the end of the experiment or at the
end of the day with an appropriate disinfectant.
13. Decontaminate all potentially contaminated equipment used in an experiment.
4.2 Laboratory Housekeeping. All UTHSC laboratories must be managed and well-maintained as a
safe work environment. General housekeeping is important when it comes to safety. The UTHSC
Housekeeping staff provides limited services such as emptying the regular trash receptacles.
Therefore, it is the responsibility of the Principal Investigator and lab workers to practice good
housekeeping techniques on a regular basis. The following guidelines will keep laboratories
organized and provide a safe workplace.
1. Keep laboratory benches clear of unnecessary clutter.
2. Keep all containers, tubes, flasks and reagents clearly labeled and stored in appropriate
areas.
3. Keep large boxes of supplies off of floors and bench tops.
4. Perform routine clean-outs to remove all unwanted biologicals, chemicals, equipment, or
supplies. Broken, unwanted or laboratory equipment requiring service or repair must be
cleared by Environmental Health & Safety. Requests for equipment clearances can be
submitted from the EH&S website (http://research.uthscsa.edu/safety/equip-clearance.shtml).
5. Immediately clean up small spills of biological material using an appropriate disinfectant.
If the spill is large or of an infectious nature, immediately notify personnel in the lab,
evacuate the lab, and close the door. Notify EH&S at (210)567-2955. All spill of recombinant
biological agents must be reported to the Biological Safety Officer in EH&S. For additional
information on spill response, see section 7.
6. Ensure all waste materials (chemical, biological, and radiological) are disposed
of according to UTHSC policies. Dispose of biohazardous wastes according
to the UTHSC Biomedical Waste Management SOP (Appendix F of this manual).
7. Broken laboratory glassware that is not biohazardous must be placed in a sturdy
cardboard box (do not use a regulated medical waste box). Once full, the box should be
closed securely and marked “Broken Glassware”. Housekeeping staff will collect these
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boxes, which are examined by Environmental Health & Safety staff prior to placing in the
compactor, located outside General Stores.
8. Transport biological materials outside of the laboratory in a closed leak-proof container as
per the Transport of Biological Materials SOP, Appendix F of this manual.
4.3 Personal Protective Equipment (PPE)
Protective clothing will be provided whenever it is necessary by reason of hazards, processes or
environmental conditions. Protective clothing shall be used when biological, chemical, radiological
hazards, or mechanical irritants are encountered in a manner capable of causing injury or impairment
through inhalation, absorption, or physical contact. It is the intent of the UTHSCSA to follow the
requirements set forth in 20 CFR 1910.134 Subpart 1- Personal Protective Equipment.
Responsibilities
Principal Investigator (PI)
The principal Investigator or designated supervisor is responsible for identifying all
procedures that required personal protective equipment and ensuring that individuals
(employees, students, fellows, residents, volunteers, etc.) under their supervision are
provided with the PPE appropriate for the job. The PI ensures that employees are trained in
the usage of PPE and that the employee uses and maintains the PPE as needed to provide
effective protection against hazards.
Individual
Each individual shall inspect, use and maintain all the PPE issued and immediately
report to the supervisor if the PPE is defective or needs to be replaced. It is the responsibility
of each worker to wear the appropriate PPE when necessary and to talk with your supervisor
if questions arise regarding additional PPE that may be needed.
Environmental Health and Safety
Environmental Health and Safety department will, upon request, provide a hazard
assessment and recommendations on appropriate PPE.
Procedures
a. Personal Protective Equipment (PPE) may include lab coats, closed front gowns, surgical
caps, masks, respirators, gloves, scrubs, uniforms, etc. As a minimum, a laboratory coat is
furnished to all personnel working in a laboratory. Safety glasses and gloves should also be
available in the laboratory. Gloves should be appropriate for the work with biological, chemical
or radioactive materials. Safety goggles and/or face shield should be worn when there is a
splash hazard. Other PPE (surgical masks, respirators, shoe covers, hair bonnets) may be
required in the laboratory or in an animal room based on the agent risk assessment, the type
of animal, and Department of Laboratory Animal Services standard operating procedures.
b. Personal Protective Equipment shall be provided in sizes appropriate for
personnel.
c. Some procedures may require additional Personal Protective Equipment such as face
shields, respirators, hearing protection. Additional information on respiratory protection can be
found in the UTHSC Respiratory Protection Plan and hearing protection in the Physical
Safety Manual.
d. Personal Protective Equipment shall be removed before leaving the laboratory and should
not be worn in the corridors or other public areas.
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Training for Personal Protective Equipment (PPE).
Training on use and maintenance of PPE is the responsibility of the PI/supervisor. Use of most PPE
requires very minimal instruction and maintenance. However, specialized equipment such as
respirators requires additional training including a medical evaluation, fit test and instruction on how
to don and doff (remove) a respirator, and routine cleaning as well as choosing the right type of filter.
A fit test will identify the proper size for the worker, and ensure that the respirator will provide
maximum protection from exposure to hazardous materials. Do not wear any respirator, such as a
N95, half-face, or full- face respirator, without completing a fit test. Under the UTHSC Respiratory
Protection Program, Environmental Health & Safety will assist all employees who are required to
wear a respirator as part of his or her job duties.
UTHSC Policy on Wearing Gloves.
It is mandatory that gloves be worn during situations where exposure to hazardous materials
(biological, chemical, radiological) is imminent. Protection of the employees from hazards is a top
priority. Latex or nitrile gloves offer protection when working with biological agents. In some cases,
double gloving may be appropriate based on the risk. Gloves should be visually inspected for tears or
holes prior to use. Additionally, one should regularly change gloves when contamination has
occurred. One should always take steps to avoid spread of contamination. Therefore, gloves must
always be removed upon exit from any laboratory or other areas where hazardous materials are
used, processed, or stored. Disposable gloves shall not be reused. Dispose of all used gloves in the
approved medical waste containers. It is extremely important that all public areas are kept clean at
all times. It is everyone’s responsibility to eliminate unnecessary contamination throughout UTHSC
property.
Remove gloves when:

Exiting laboratories
 Opening doors (labs,
restrooms, stairwells,
lecture halls, etc.)
 Walking through public
hallways
 Using elevators
 Using telephones
 Using water fountains
Non-compliance with this policy should be reported to the Environmental Health & Safety
Department. All violations will be reviewed by Environmental Health & Safety. The first offense will
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result in a visit from Environmental Health & Safety for remedial training. A letter from the director of
Environmental Health & Safety documenting such offense will be sent to the employee and their
supervisor.
The table below shows information about different types of personal protective equipment (PPE)
and the hazards for which they may be used.
TYPES of PPE
Applications
Protection of eyes when working with biological agents where there is a
potential splash hazard.
Safety glasses Chemical goggles
Provides protection when working with animals.
Provides impact protection when needed.
Face shields provide protection of entire face.
Face shield
Closed toe shoes
Shoe covers
Protect feet from exposure to hazardous agents, especially during
accidental spills or breakage.
Shoe covers are used prevent spread of contamination (from shoes to
clean areas like animal rooms or clinical areas (surgery suites).
Shoe covers prevent shoes from becoming contaminated and are
appropriate when an individual is cleaning up a spill.
Lab coats and disposable gowns offer protection from contamination and
splashes. Lab coats should be buttoned closed prior to working with
hazardous agents.
Lab Coat
N95 respirator
Chemical resistant
Apron
Full face respirator
Half face respirator
Chemically-resistant aprons provided heavy duty protection when working
with hazardous chemicals.
N95 respirators provide protection against infectious agents transmitted by
aerosol route.
Respirators also provide protection from organic vapors, dusts, and non-oil
containing mists.
Full face respirators provide protection against organic vapors. Different
types of cartridges available for various chemical applications. Also
provides added face protection.
Half face respirators provide protection from organic vapors. Various
cartridge types for different chemical applications. Acceptable when face
protection is not needed.
* Require fit testing and medical evaluation prior to use. Contact
Environmental Health & Safety,
567-2955
Noise levels above recommended action level of 85 dB for prolonged
periods of time. Noise reduction rates range from 20 to 33 dB.
Earplugs
Earmuffs
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5.0 General Guidelines for Handling Biological Agents
5.1 Introduction.
Laboratory acquired infections have been documented for personnel working in research and clinical
laboratories. An infection occurs when disease-causing organisms enter the human body in sufficient
numbers and by a particular route and overcome the body’s defense mechanisms. Routes of
exposure to infectious agents have been documented through the mouth, skin, eyes, and lungs.
Most laboratory acquired infections point to accidents such as spillages or splashes which can
aerosolize an agent, or through accidental autoinoculation with a needle or other contaminated sharp
during work with some type of infectious agent.
Therefore, the most important element in maintaining a safe work environment is strict adherence to
good microbiological techniques and laboratory practices. Every individual working with infectious
agents, potentially infected materials, and recombinant agents must be aware of the potential risks
and must be trained and proficient in the practices and techniques required for handling these
agents. The following work practices help to provide a safe working environment in the laboratory.
5.2 Training.
An initial determination of an employee’s required training is assessed during New Employee
Orientation. Based on this initial assessment, employees and their supervisors are notified as to
which courses that the newly hired individual is required to take. Initial and refresher training for
laboratory and clinical safety is provided by EH&S through online and classroom courses.
Courses include:
 Basic Biological Safety – Required for the PI and all individuals working in the
research laboratory. This course includes an overview of the NIH Guidelines for
Research Involving Synthetic or Recombinant Nucleic acid Molecules and the BMBL.
This is an annual requirement.
 Basic Bloodborne Pathogens Safety – Required of the PI and all individuals in
research and clinical areas who work with or have a potential exposure to human and
non-human-primate blood, body fluids, tissues, primary cells, immortal human cell
lines.
 Infectious Substance Shipping and Dry Ice - Required of all individuals in research
and clinical areas who package infectious substances/clinical specimens/dry ice for
shipment and/or complete the shipping documents.
Personnel including students, who work in laboratories, shall be trained and proficient in basic
microbiological practices. Instruction concerning specific laboratory procedures including the risks
and hazards associated with the agents worked with in the lab are the responsibility of the PI. The PI
should document lab specific training and Standard Operating Procedures (SOPs).
5.3 Practices and Procedures.
This section provides information regarding hazards involved with certain laboratory practices and
methods for preventing them. The following work practices should be utilized when working at each
Biosafety Level. The NIH/CDC document “Guidelines for Research Involving Recombinant and
Synthetic Nucleic Acid Molecules,” and “Biosafety in Microbiological and Biomedical Laboratories“
(BMBL) are used to develop standard operating procedures for work at each biosafety level. For
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more specific information on practices, procedures, safety equipment and facility requirements for
each Biosafety Level, refer to Appendix D.
Biosafety Level 1
Biosafety level 1 (BSL-1) is suitable for work involving well-characterized agents not known to
consistently cause disease in immune-competent adult humans and which present minimal potential
hazard to laboratory personnel and the environment.
 Control access to the laboratory.
 Use procedures that minimize creation of splashes and aerosols.
 Do not smoke, eat, drink or store food in the laboratory.
 Wear laboratory gowns or coats, gloves and protective eyewear when appropriate. Do not
wear PPE outside of the laboratory
 Do not mouth pipette. Use mechanical pipetting devices.
 Safe handling of sharps. Dispose of sharps into puncture resistant containers.
 Wash hands after completing experimental procedures and before leaving laboratory.
 Decontaminate work surfaces after completion of work and immediately after a spill.
 Decontaminate all cultures, stocks and other potentially infectious materials before disposal.
Decontaminate other contaminated materials before washing or reuse.
 For off-site decontamination, package contaminated materials in closed, durable, leakproof
containers.
 Control insect and rodent infestations.
 Keep areas neat and clean.
 Report spills, accidents, near misses and disease symptoms related to laboratory acquired
infection to the PI.
Biosafety Level 2
Biosafety Level 2 (BSL-2) builds upon the practices outlined in BSL-1. BSL-2 is suitable for work
involving biological agents that pose moderate hazards to personnel and the environment.
 Control access to the laboratory and keep laboratory doors closed.
 Laboratory personnel have specific training in handling pathogenic agents and are supervised
by scientists competent in handling those agents and associated procedures.
 Allow only persons informed of the potential hazards to enter BSL2 areas.
 Post a universal biohazard label on equipment where infectious agents are used/stored.
 Keep animals and plants, not used in the experiment, out of the laboratory.
 Do not smoke, eat, drink, store food or apply cosmetics in BSL2 areas.
 Wear PPE when appropriate; do not wear PPE outside of the laboratory.
 Wash hands after removing PPE as well as before leaving laboratory.
 Change PPE when soiled or compromised.
 Do not mouth pipette. Use mechanical pipetting devices.
 Use procedures that minimize splashing and aerosol formation.
 Safe handling of sharps. Dispose of sharps into puncture resistant containers. Avoid using
needles whenever possible.
 Substitute plastic for glass where feasible.
 Use biological safety cabinets for aerosol generating procedures and to contain aerosolproducing equipment.
 Disinfect work surfaces daily and immediately after a spill.
 Maintain a biological spill kit within the laboratory.
 Report spills, accidents, near misses and disease symptoms related to laboratory acquired
infection to the PI.
 Ensure that all biomedical waste containers are labeled with the biohazard symbol.
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Decontaminate all cultures, stocks and other potentially infectious materials before disposal.
Decontaminate other contaminated materials before washing or reuse.
For off-site decontamination, package contaminated materials in closed, durable, leakproof
containers.
Control insect and rodent infestations.
Keep areas neat and clean.
Note: BSL2+ is not an official designation but generally refers to additional safety practices as
determined by the Institutional Biosafety Committee.
Biosafety Level 3:
Biosafety Level 3 (BSL-3) builds upon the practices outlined above. BSL-3 is suitable for work
involving agents that can cause serious or potentially lethal disease to personnel through the
inhalation route of exposure. A BSL-3 laboratory has special engineering and design features and all
procedures involving manipulation of infectious materials is done inside the biosafety cabinet. For
more specific information on practices, procedures, safety equipment and facility requirements for
Biosafety Level 3, refer to Appendix D.
Needles and Syringes:
Avoid using syringes and needles whenever possible. If the use of a syringe is necessary:
 use engineered sharps or safe sharps whenever possible
 use the needle-locking type (luer lock), or a disposable syringe-needle unit
 do not place syringes in pans containing pipets or other glassware requiring sorting
 do not cut, bend or recap needles
 dispose of needles in approved sharps containers in accordance with UTHSCSA ‘s
Waste Disposal Procedures and the Bloodborne Pathogens Exposure Control Plan.
Transport of Biological materials:
Individuals shall follow the Transport of Biological Materials SOP (Appendix G.2) when transporting
hazardous materials through the public corridors.
1. Use secondary containment (clean plastic tub or cart) to safely move these materials from
one area to another. Secondary containment not only provides protection for the worker, but
also provides a means of containment in case of accidental spills or breakage.
2. Lab carts should have a lip to protect containers from tipping over or sliding off of the cart
during transport.
3. Do not carry samples or hazardous materials directly in your hands.
4. If transporting infectious agents, a biohazard sticker should be placed on the outside of
the secondary container.
5.4. Laboratory facility requirements and Laboratory Security
5.4.1. Laboratory facility requirements are applicable to laboratories working at all biosafety levels.
Additional laboratory facility requirements may be necessary for higher containment laboratories.
1. Post a warning sign at the entrance to the laboratory, which includes the universal biohazard
symbol with the word “BIOHAZARD” when infectious materials or infected animals are present in
the laboratory or animal room. This warning sign should include the name of the agent, the
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biosafety level, medical surveillance requirements, entry/exit information, personal protective
equipment to be used, and the name and emergency contact phone number of the Principal
Investigator/supervisor or person(s) responsible for the laboratory.
2. Laboratory doors should be self-closing.
3. Doors to the laboratory are kept closed.
3. Doors must be locked when personnel are away from the laboratory.
4. Limit access into the laboratory during procedures involving biohazardous agents. Allow entry
only to persons informed of the potential hazards.
5. Each laboratory shall contain a sink for hand washing and an area eye wash station which is
readily available within the work area.
6. The laboratory is designed so that it is easily cleaned and decontaminated. Carpets and
rugs are not allowed.
7. Laboratory benchtops must be impervious to water, resistant to heat and chemicals.
8. Chairs in the lab must be covered with an impermeable covering so that it is easily cleaned
and decontaminated with an appropriate disinfectant.
9. Laboratory windows that open to the outside are not recommended.
10. The laboratory should have inward directional airflow which is not recirculated to other
areas outside of the lab.
5.4.2. Laboratory Security
All personnel working in laboratories should be mindful that theft and misuse of biological agents,
chemicals, radiological materials, equipment and data are realistic possibilities. All laboratories,
even those working with low risk biological agents at biosafety level 1 must maintain a basic level
of security. For hazardous materials stored outside of the laboratory or in a common room, the
equipment shall be kept locked.
Personnel shall wear visible identification while at UTHSC. Doors to laboratories shall be locked
when no one is present in the lab. Personnel shall report suspicious activity, threats, or other
emergencies to UTPD at 7-8911 from a university phone or 567-8911 from a cell phone.
5.5. Posting and Labeling Requirements
5.5.1 BIOHAZARD SYMBOL. Areas of the facility where blood or other potentially infectious
materials are handled, processed, or stored shall have the biohazard label posted at the
entrance and the agent(s) being used listed. Additionally, labels shall be affixed to
equipment and containers used with these potentially infectious materials as listed below.
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These labels shall be affixed to containers of potentially infectious waste; refrigerators, and
freezers containing blood and other potentially infectious materials; and other containers
used to store or transport blood or other potentially infectious materials.
a. Labels shall include the biohazard symbol and the word “Biohazard.” These labels shall
be fluorescent orange or orange-red, or predominantly so, with lettering and
biohazard symbol in a contrasting color. Written wording shall be provided in
English, and also in Spanish where required i.e. – medical waste containers as per
30 TAC Part 1, Subchapter Y 330.1004(i)(4).
b. Labels shall either be an integral part of the container or shall be affixed as close as
safely possible to the container by string, wire, adhesive, or other method that
prevents their loss or unintentional removal.
c. Labels required for contaminated equipment shall be in accordance with this paragraph
and shall also state which portions of the equipment remain contaminated.
d. Regulated medical waste that has been properly decontaminated (i.e. autoclaved,
sterilized, etc.) must have any biohazard labels defaced, covered, or removed prior
to disposal. The waste must be labeled as “treated medical waste” in accordance
with the provisions of 25 TAC §1.136(a).
5.5.2 Spill Plan. Post a spill plan in the lab (see Appendix F for template spill plan.).
5.6 Procedures for Control of Biohazards in Clinical Laboratories.
Each clinical laboratory will have on hand, in the laboratory, an exposure control plan. The
department will make arrangements to provide those services, and equipment that are needed to
comply with the Texas Department of State Health Services (TDSHS) and OSHA Bloodborne
Pathogens Standard and the UTHSCSA’s Exposure Control Plan, which are available from the
Environmental Health and Safety Department, 1.343T and on the Environmental Health and Safety
website.
STANDARD BLOOD AND BODY FLUID, OR UNIVERSAL PRECAUTIONS
Clinical Procedures in healthcare facilities must handle patient specimens without full knowledge of
the diagnosis. Specimens may contain multiple infectious etiologic agents. To minimize personal
exposure to clinical specimens of an unknown nature, all personnel in clinical laboratories should
observe the Centers for Disease Control (CDC) guidelines for universal precautions when handling
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all specimens of blood, body fluids, or tissues. This means that all clinical material should be
considered to be infectious and should be handled as potentially hazardous. Procedures for working
in clinical laboratories, research, or teaching laboratories that handle human blood, tissues, body
fluids are outlined in the UTHSCSA’s Bloodborne Pathogen Exposure Control Plan (Appendix G.7).
In addition to the provisions of the preceding section, Procedures for Control of Biohazards in
Research Laboratories, the following procedures are to be followed:
1. Utilize Standard or Universal Precautions – Consider all human blood, body fluids and tissue from
patients are infectious. See below for more information.
2. All persons processing blood and body-fluid specimens (e.g., removing tops from vacuum tubes)
must wear gloves. Masks and protective eyewear must be worn if mucous-membrane contact
with blood or body fluids is anticipated. Gloves must be changed and hands washed with soap
and water after completion of specimen processing.
3. For routine procedures, such as histologic and pathologic studies or microbiologic culturing, a
biological safety cabinet is not necessary. However, biological safety cabinets must be used
whenever procedures are conducted that have a high potential for generating aerosols. These
include activities such as blending, sonicating, and vigorous mixing.
4. Mechanical pipetting devices must be used for manipulating all liquids in the laboratory. Mouth
pipetting is prohibited.
5. Use of needles and syringes must be limited to situations in which there is no alternative, and all
precautions must be taken to prevent injuries such as sticks with needles. Recapping, cutting,
bending a needle should not be done.
6. Laboratory work surfaces must be decontaminated with an appropriate chemical germicide after
a spill of blood or other body fluids and when work activities are completed.
7. Contaminated materials used in laboratory tests must be decontaminated before reprocessing or
be placed in bags and disposed of in accordance with Institutional Polices for disposal of
infectious waste.
8. Scientific equipment that has been contaminated with blood or other body fluids must be
decontaminated and cleaned before being repaired in the laboratory or transported outside the
laboratory for repairs.
9. All persons must wash their hands after completing laboratory activities and remove protective
clothing before leaving the laboratory.
5.7 AEROSOLS.
Definition:
The term “aerosol” refers to the physical state of liquid or solid particles suspended in air. The
production of aerosols while handling infectious agents may present a serious risk of exposure.
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Risks
Exposure to infectious aerosols via the respiratory route are capable of transmitting diseases and
poses a serious laboratory hazard. The individual working with the infectious agent and other
individuals in the laboratory are at risk from a laboratory acquired infection (LAI). This hazard
requires special precautions since the infectious aerosols may not be a recognized route of
transmission for the natural disease. Infective dose and agent stability are particularly important in
establishing the risk of airborne transmission of disease. Experimental animals can shed zoonotic
agents and other infectious agents (worked with in a research project) in their saliva, urine, or feces.
Procedures such as cage changing, washing down equipment/rooms can create aerosols which
pose a hazard to the animal care staff and research personnel. A review of past LAIs indicate that
the majority of these incidents are probably due to an aerosol exposure.
Aerosol particles one to five microns in size present the greatest hazard to the laboratory worker
because:
 small particles readily penetrate and remain in the respiratory tract if inhaled
 many routine laboratory procedures create aerosols in this size range
 aerosols are usually undetected
 they may remain suspended in air for long periods of time
Aerosols can settle on equipment normally considered to be clean. Skin contamination from
aerosols or from handling contaminated equipment may result in infection through ingestion, contact,
or skin abrasions. Risks associated with aerosols can be reduced or eliminated by the use of good
technique in a biological safety cabinet.
Aerosol Production
Aerosols may be produced in the use of:













centrifuge
blender
shaker
magnetic stirrer
sonicator
pipet
vortex mixer
syringe and needle
opening a vacuum-sealed ampoule
grinder, mortar, and pestle
heated inoculating loop
freeze-dried sample
opening test tubes and culture tubes
To reduce the potential for infection during aerosol production:
 perform activities that may produce aerosols in a biological safety cabinet (BSC)

keep tubes stoppered when vortexing or centrifuging

allow aerosols to settle for one to five minutes before opening centrifuge, blender, or
tubes that have been mixed
place a cloth soaked with disinfectant over the work surface to deactivate possible spills
or droplets of biohazardous agents

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




reconstitute or dilute contents of an ampoule slowly
when mixing two solutions, discharge the secondary fluid down the side of the
container or as close as possible to the surface of the primary solution
allow inoculating loop or needle to cool before touching biological specimens,
wrap with disinfectant-soaked gauze when removing the needle from the rubber stopper
of a test tube or vial, breaking the cap on an ampoule, removing stoppers or plugs from
tubes
Work in a BSC when re-suspending sedimented material. Use a swirling rotary motion
rather than shaking. If shaking is necessary, wait a few minutes to permit the aerosol to
settle before opening the tube.
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6.0 Disinfection, Sterilization and Decontamination
This chapter describes basic methodology for reducing the possibility of transmission of infectious
agents to laboratory workers (Laboratory Acquired Infections, LAI), to other workers in the university,
the public and the environment. Environmentally associated laboratory infections can be transmitted
directly or indirectly from air, contaminated fomites, laboratory equipment, and aerosols to laboratory
personnel. Proper selection and use of disinfectants and decontamination and sterilization
methodology is critical in reducing the transmission of infectious agents.
6.1 Definitions.
 Disinfection-reduction in the number of pathogenic organisms by the direct application of
physical or chemical agents; spores may be resistant.
 Decontamination-removal of organisms to a level near zero. Decontamination reduces
the level of microbial contamination so that infection transmission is eliminated and the
material (equipment, etc.) is safe to handle.
 Sterilization-the total destruction of all living organisms and viruses, including bacterial
endospores.
6.2 Choosing a Method and Selecting a Disinfectant.
The method of choice for sterilization or disinfection will depend on several factors:
 the target organism (the biological agent) and the number of organisms present,
 the amount of organic matter present (blood, soil, etc.),
 the characteristic(s) of the materials or areas to be disinfected,
 the temperature
Selecting a Disinfectant
Use the following table to aid in the selection of an appropriate disinfectant. The disinfectant should
be an EPA registered disinfectant effective against the biological agent that you are working with.
Clinics use a broad spectrum disinfectant that is effective against viruses (e.g. HIV, HBV, HCV, etc.),
bacteria (e.g. Mycobacterium, Listeria, etc.) and fungi.
http://www.epa.gov/oppad001/chemregindex.htm
DESCRIPTIONS OF COMMONLY USED DISINFECTANTS
SUBSTANCE
Alcohols
Phenolics
Formaldehyde
Quaternary
Ammonium
Compounds
Chlorine
Iodine
DESCRIPTION
Ethyl or isopropyl alcohol at 70-80% concentration is a good general purpose
disinfectant; not effective against bacterial spores
Effective against vegetative bacteria, fungi, and lipid-containing viruses;
irritating odor
At a concentration of 5-8% formalin, good disinfectant properties against
vegetative bacteria, spores, and viruses; irritating odor
Cationic detergents which are strongly surface active; extremely effective
against lipoviruses; not effective against bacterial spores; can be neutralized
by anionic detergents (soaps)
Low concentration (50-500 ppm) active against vegetative bacteria and most
viruses; higher concentration (2500 ppm) required for bacterial spores;
corrosive to metal surfaces; must be prepared fresh; laundry bleach (5.25%
chlorine) may be freshly diluted and used as a disinfectant
Recommended for general use; effective against vegetative bacteria and
viruses; poor activity against bacterial spores.
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6.3 Sterilization Methods.
Wet Heat (Steam)
Decontamination by this method requires a properly functioning autoclave that is monitored on a
routine basis. The cycle requires approximately 15 psi pressure with a chamber temperature of at
least 250oF (121oC). The cycle time begins when the materials being sterilized reach the
predetermined temperature. The length of time required for complete sterilization is dependent upon
the volume size of the load (usually 30-60 minutes) and the type of material being autoclaved.
Monitor steam sterilization effectiveness with a biological indicator, Bacillus stearothermophilus.
Autoclave tape is not an indicator of sterility; it simply indicates that the proper temperature has been
achieved on the surface. See Autoclave Recordkeeping in Appendix F.
Dry Heat
This is less effective than steam, and requires more time (two to four hours) and a higher
temperature (320-338oF or 160-170oC). Monitor dry heat sterilization with a B. subtilis biological
indicator.
Ethylene Oxide Gas (EtO) – Contact the Safety office before purchasing
This gas is lethal for all known microorganisms and is best used to sterilize heat-resistant organisms
or heat-sensitive equipment.
EtO sterilization efficiency may be affected by:

temperature-- microbiocidal activity of EtO increases by a factor of 2.7 with each 10oC rise in
temperature (range of 5 to 37oC and concentration 884 mg/L EtO). Normal EtO sterilization
temperature is 49 to 60oC (120-140oF).

EtO concentration-- required sterilization time decreases with increased EtO concentration.
Normal EtO sterilization concentration is 500-1000 mg/L at 40 to 60oC.

humidity-- moisture is required; relative humidity of 30-60% is necessary to ensure proper
conditions.

exposure time-- this is affected by the variable noted above. Follow the manufacturer’s
recommendations and monitor with biological indicators (b. subtilis var. niger).
Precautions for Using EtO
When using ethylene oxide, you should be aware of these two facts:
 a 3% mixture of EtO in air may be explosive. Commercially available mixtures of EtO in freon
or CO2 are not explosive
 exposure to ethylene oxide gas may result in harmful physical effects. The current exposure
level limit for EtO is 1 ppm for an eight-hour time-weighed average and an excursion limit of 5
ppm averaged over a 15 minute sampling period. Levels exceeding 1000 ppm have been
observed when the sterilizer door is opened for aeration. Install an automatic door opener
and allow 15 minutes aeration before removing the articles to the aeration basket.
6.4 General Procedures.
 Frequently disinfect frequently all floors, bench tops, and equipment where biohazardous
materials are used.
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






Sterilize all infectious materials and contaminated equipment prior to being washed, stored,
or discarded.
Use autoclavable or disposable materials whenever possible. Keep reusable and disposable
items separate.
Minimize the amount of materials and equipment present when working with infectious
agents.
Sterilize or properly store all biohazardous materials at the end of each day.
Be aware that agar and other materials may interfere with the germicidal actions of chemical
disinfectants, thus requiring higher concentrations or longer contact time.
Ensure sterilization by reviewing autoclave printout of cycle completion. Autoclave tape does
not ensure that the cycle has been completed. Use suitable indicators to verify autoclave
performance on a monthly basis. See autoclave recordkeeping in Appendix H.
Use clearly marked holding containers such as “NON-INFECTIOUS” or
“BIOHAZARDOUS TO BE AUTOCLAVED”
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7.0 Procedures for Spills of Biohazardous Materials
For spills of infectious materials outside a primary containment device like the biosafety cabinet,
contact Environmental Health and Safety at 210-567-2955 or after hours contact UTPD at 567-2800.
The following procedure is general in nature. Each lab should have a lab specific spill plan.
A template emergency and spill response plan is found in Appendix H.
STEP
1
2
3
4
5
IMMEDIATE ACTION NEEDED IN CASE OF SPILL
PROCEDURES
Alert people in the area of the spill
a. hold your breath
b. leave the room
c. close the door behind you
Remove and containerize contaminated protective garments immediately at
the door after exiting
Isolate the area. Place a warning sign on door
Wash hands thoroughly with germicidal soap
Contact EH&S at 7-2955 and provide information about the spill.
If a highly infectious agent is involved, it may be necessary to turn off the
building ventilation to avoid circulating any aerosols.
HOW TO CLEAN UP AND DECONTAMINATE AFTER A SPILL
STEP
PROCEDURE
1
2
3
4
5
6
STEP
1
2
3
Wait 30 minutes to allow settling of aerosols before re-entering the area
Put on appropriate PPE before entering the area to be cleaned
Apply disinfectant soaked paper towels or pads over the spill.
Allow 20 minutes contact time. Begin at the perimeter and work inward. Wipe
up all liquids and media involved. Place items in a biohazard bag.
Use forceps, tongs or broom to remove broken items or glass into a sharps
container. Autoclave all material used in the cleaning process, including the
PPE.
Wash or mop the spill area with a disinfectant solution
Thoroughly decontaminate with disinfectant any contaminated equipment that
cannot be autoclaved
PROCEDURE FOR SPILLS IN HALLWAYS
PROCEDURE
Restrict traffic through the area and notify EH&S at 7-2955 and UTPD to
assist in traffic control
Shut any room doors adjacent to the spill area
EH&S will proceed with the decontamination procedure
Note: If the spilled biohazardous material is labeled/tagged with a radionuclide,
refer to the Radiation Safety Manual and seek assistance from the Radiation
Safety Division.
Re-occupancy of a Spill Area
Before reoccupying any area where a spill has occurred:
 Environmental Health and Safety and the supervisor of the area must determine that the
decontamination has been effective.
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

stringent decontamination measures must have been executed if the spilled agents were
of a highly infectious nature
surface swab sampling may be necessary
Reporting – Any potential exposures must be reported to the supervisor and Environmental Health
and Safety. The individual should seek medical treatment and the appropriate forms must be
completed and submitted to the Worker’s Compensation Coordinator in EH&S.
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8.0 Laboratory Equipment
The following laboratory equipment may be found in a research or clinical laboratory. This chapter
covers some of the special hazards that laboratory equipment presents and offers guidance on the
appropriate and safe use of this equipment. The following equipment is covered in this section.
Biological Safety Cabinets (BSC) and Laminar Flow Clean Benches
Refrigerators, Freezers
Microwave ovens
Vacuum Systems
Centrifuges
Blenders and sonicators
Lyophilizers and ampoules
Pipettes and Pipet Aids
Syringes and Needles
Loop sterilizers and Bunsen burners
Autoclaves
Laboratory furniture
Flow Cytometry
Cryostat
Equipment repair and disposal
8.1 BIOLOGICAL SAFETY CABINETS
Overview
Biological safety cabinets (BSC) are the principle equipment used as a primary barrier to protect
personnel against exposure to biohazardous or infectious agents and to prevent releases to the
environment. The BSC controls the aerosols that may be created by experimental procedures.
Primary barriers are important because many laboratory techniques are known to produce aerosols
that can be readily inhaled by the laboratory worker. The majority of laboratory acquired infections
have been attributed to aerosol exposure (Pike 1967).
However, BSCs are only one part of the biosafety program. A PRIMARY BARRIER SUCH AS
BIOLOGICAL SAFETY CABINET MERELY COMPLEMENTS GOOD MICROBIOLOGICAL
PRACTICES AND CAREFUL WORK PRACTICES.
The BSC is designed to protect personnel and the environment from exposure to potentially
infectious aerosols but is dependent on both the abilities of the laboratory worker to use the BSC
correctly and the adequate functioning of the BSC. A BSC should never be used with infectious
materials until it has been certified.
8.1.1. Selecting and placement of the Biological Safety Cabinet
Principal Investigators should consult with Environmental Health and Safety when ordering a new
biosafety cabinet and for placement in the laboratory. EH&S can provide guidance on the type of
BSC to purchase based on the use of the BSC.
1. Factors influencing BSC selection
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There are several types of biological safety cabinets, each with their own advantages and
limitations; the Principal Investigator must carefully assess their research program and
match specific requirements to the appropriate contamination control cabinet. Pertinent
factors are:
a. Proposed activity. Procedures that may cause aerosols are of particular concern.
b. Risk of the biological agent. All known characteristics of the agent should be
evaluated, e.g., infectivity, hazards of pathogenicity, concentration of viable agent,
history of known laboratory acquired infections, classification of risk group.
c. Toxins, chemicals or radioactive materials. These materials should be evaluated
when used in conjunction with the biological agent work or in support of clinical
operations.
d. Control Objectives. The control protection desired should be determined from the
risk assessment including the proposed activity, the specific biological agent and any
toxins, chemicals, radioactive materials used in the work.

Product Protection only.

Personnel and Environmental Protection only.

Personnel, Product Protection, and Environmental Protection
Selection of a Safety Cabinet through Risk Assessment
Biological risk &
biosafety Level
Personnel
Protection
Product
Protection
Environmental
Protection
BSC Class
BSL 1 – 3
Yes
No
Yes
I
BSL 1 – 3
Yes
Yes
Yes
II (A1, A2, B1,
B2)
BSL – 4
Yes
Yes
Yes
III;
II—When used
in suit room with
suit
2. Three basic types of Biosafety Cabinets (BSCs)
o Class I BSC: The class I BSC is an open fronted (no sash) cabinet that provides
personnel and environmental protection but no product protection. These BSCs are
typically used to house aerosol generating equipment like centrifuges, harvesters,
etc. or aerosol generating procedures like animal cage changing. Personnel
protection is provided by inward airflow at a minimum of 75 linear feet per minute
(lfm). Some class 1 BSCs have an integral exhaust fan and allow recirculation of air
into the room after HEPA filtration but some class I BSC are hard ducted (direct
connection with the building exhaust).
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Class I BSC, photo illustration – BMBL 5th ed. Appendix A.
o Class II BSC: The Class II Biosafety Cabinet (Types A1, A2, B1 and B2) provide
personnel, environmental and product protection. Airflow is drawn into the front grille
of the cabinet, providing personnel protection. In addition, the downward flow of
HEPA-filtered air provides product protection by minimizing the chance of crosscontamination across the work surface of the cabinet. Because cabinet exhaust air is
passed through a certified HEPA filter, it is particulate-free (environmental
protection), and may be recirculated to the laboratory (Type A1 and A2 BSCs) or
discharged from the building via a canopy or “thimble” connected to the building
exhaust. Exhaust air from Types B1 and B2 BSCs must be discharged directly to the
outdoors via a hard connection. HEPA filters are effective at trapping particulates
and thus infectious agents but do not capture volatile chemicals or gases. Class II
BSCs provide the microbe-free work environment necessary for cell culture
propagation and also may be used for the formulation of nonvolatile antineoplastic or
chemotherapeutic drugs.
Class II, A1 biosafety cabinet. photo illustration – BMBL 5th ed. Appendix A.
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o Class III BSC: The Class III BSC is a totally enclosed exhaust protective cabinet.
These are a completely enclosed system with negative pressure of gas tight
construction, including glove boxes. The Class III BSC was designed for work with
highly infectious microbiological agents and for the conduct of hazardous operations
and provides maximum protection for the environment and the worker. Long, heavyduty rubber gloves are attached in a gas-tight manner to ports in the cabinet to allow
direct manipulation of the materials isolated inside. Although these gloves restrict
movement, they prevent the user’s direct contact with the hazardous materials.
o Laminar Flow hood clean benches and vertical Flow clean benches are
not a biosafety cabinet. It only provides product protection and does not protect the
individual. The airflow is typically outward toward the user. Clean benches should
never be used when handling cell culture materials, drug formulations, potentially
infectious materials, or any other potentially hazardous materials.
Horizontal laminar flow clean bench, photo illustration – BMBL 5th ed. Appendix A.
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B
S
Class,
Type
L
1-3
I
1-3
II, A1
Non-volatile
toxics, organics &
radio-nuclides
Fa c e
Velocity
(fpm)
Airflow pattern
75
In at front.
Exhausted
through HEPA to
room or ducted
outside
YES
7 5 to
7 0 % ai r
recirculated to
cabinet ; 30%
exhausted
through HEPA to
room, or ducted
out
YES (minute
amounts)
< 100
Volatile toxics, organics
& radio-nuclides
1
YES
NO
1-3
II, A2
100
Plenums under
negative pressure
to room. 70% air
recirculated to
cabinet. Exhaust
to room, or
ducted out
YES
1-3
II, B1
100
3 0 % ai r
recirculated; 70%
HEPA exhausted
through hard duct
outside
YES
1-3
II, B2
100
100% air HEPA
exhausted
through hard duct
outside
YES
YES (minute amounts)
III
N/A
Supply air inlets
and hard-duct
exhaust through 2
HEPA filters
YES
YES (minute amts)
4
2
YES (minute amounts)
2
YES (minute amounts)
3. High Efficient Particulate Air (HEPA) Filter
One of the most critical components of any biological safety cabinet is the high
efficiency particulate air (HEPA) filter. HEPA filters consist of a continuous sheet of
borosillicate fiber paper pleated over rigid corrugated separators and mounted in a
wooden frame. They are extremely delicate and the filter media should never be
touched. Most BSCs have HEPA filters in the exhaust and supply system, with the
exception of the Class I BSC which does not have a HEPA- filtered air supply.
The HEPA filter has an efficiency of 99.97% for particles 0.3 microns in diameter.
This size particle is used as the basis for filter definition because theoretical studies
have shown that filtration efficiency should be at minimum for particles of this
diameter with efficiency increasing for particles either larger or smaller.
HEPA filters are not effective, however, against chemicals in gaseous state that
readily pass through these particulate barriers. Since most BSCs are partially
recirculating, gaseous build up to a point of equilibrium may occur.
Therefore, chemicals that readily vaporized, should not be used in recirculating
cabinets if they meet one of the following conditions:
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
Chemicals that alone or in combination attack filter components or stainless
steel. This must be ascertained for each chemical used in the cabinet

Chemicals that alone or in combination are explosive or flammable. In
recirculating BSCs, a build-up of vapors may cause an explosion due to
ignition by a motor spark or burner in the work zone. Such explosions have
occurred in the past. It is the policy of this institution to not allow use of gas
in a recirculating BSC. Gas may be allowed only in a ducted, 100%
exhausted class II B2 upon consultation with EH&S.

Chemicals that may become toxic to the operator, or any combination of two
or more chemicals that may react and the resultant product becomes toxic
to the operator. If the cabinet is being correctly used, i.e., operator's arm
only inside the unit with the view screen in a safe position and the blowers
functioning; toxicity or irritation should only occur through skin penetration,
either directly or through wounds. A proper evaluation of the chemical
toxicity should include not only information on single exposure, effects of
concentration, but the effect of many small exposures over time.

If any one of your experimental chemicals fits any one of the above
categories, you should either be working in a hard ducted BSC or a fume
hood.
Life Span of the HEPA Filter
The life span of a HEPA filter is governed by how you use your cabinet and where it
is located. Under normal laboratory conditions, a filter will last from three to five
years, but misuse of the BSC and/or a heavy dust load within the laboratory will
shorten the filter's lifespan. If your laboratory is next to a construction area or any
area that creates large amounts of dust, the filter life will be shortened significantly.
Animal bedding and dander can also reduce the life of the filter.
4. Placement of the BSC
The ideal location for the biological safety cabinet is remote from the entry (i.e., the rear of the
laboratory away from traffic), since people walking parallel to the face of a BSC can disrupt the air
curtain at the front of the cabinet. Open windows, air supply registers, portable fans or laboratory
equipment that create air movement (e.g., centrifuges, vacuum pumps) should not be located near
the BSC. Similarly, chemical fume hoods must not be located close to BSCs. Disruption of the air
curtain can lead to product contamination and the release of infectious aerosols outside of the
cabinet through the front opening.
Whenever possible, adequate clearance should be provided behind and on each side of the
cabinet to allow easy access for maintenance and to ensure that the cabinet air re-circulated to the
laboratory is not hindered. A 12 to 14 inch clearance above the cabinet may be required to provide
for accurate air velocity measurement across the exhaust filter surface and for exhaust filter
changes.
8.1.2. Certification of the Biological Safety Cabinet
Biological Safety Cabinets must be maintained and routinely inspected and tested by qualified
personnel. The Environmental Health and Safety office contracts with an outside vendor to certify the
cabinets on an annual basis. Depending on the use, some cabinets are certified every six months.
Never use a biological safety cabinet with hazardous materials unless it has been certified to meet
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minimum safety specifications (e.g., ANSI/NSF Standard No. 49 or NIH-03-112). Every biological
safety cabinet should be certified by qualified personnel whenever:

New cabinets has been purchased and installed, but before it is used. (Notify
Environmental Health and Safety office of new BSC installation and to schedule
certification of the BSC). A cabinet leak test is also to be performed.

After cabinet has been moved or re-located.

After HEPA filter replacement or maintenance repairs that require the BSC to be opened,
(a cabinet leak test is also to be performed).

At least annually.
A certification sticker will be placed on the BSC with the certification date and next certification due
date. Other information on the sticker includes the manufacturer/model number, the serial number
and the certifier.
Laminar flow benches are not included on the EH&S BSC contract and certification is the
responsibility of the Investigator/department. The use of this type of equipment is usually for product
protection. If there are external agencies that requires certification of laminar flow clean benches for
your work, i.e., Food and Drug Administration and EPA-Pesticide, it will be the responsibility of the PI
to meet these requirements.
8.1.3 Decontamination of the Biological Safety Cabinet
If biohazardous agents (Risk group 2, 3, or 4) are used in the biosafety cabinet, then the biosafety
cabinets must be decontaminated before:
 being moved or relocated.
 being decommissioned and going to the warehouse.
 before a change in principal investigator and agents worked with.
 when repairs are needed that involves opening the panel of the BSC housing the HEPA
filter.
Decontamination of biosafety cabinets may be performed by Environmental Health and Safety staff
or by a qualified contractor.
The Environmental Health and Safety Department maintains an inventory (location and biohazardous
agents used) of biological safety cabinets and schedules the annual certification inspections. Call
567-2955 for more information.
Biosafety Cabinet decontamination guidance is available on the Environmental Health and Safety
website at: http://research.uthscsa.edu/safety/cabinet-decon.shtml
Environmental health and Safety personnel will provide BSC decontamination services using a
paraformaldehyde decontamination protocol. For paraformaldehyde decontamination, the following
guidance is for laboratory personnel:

Once Biosafety Cabinet Decontamination has been scheduled, lab personnel must leave
laboratory by 3pm. Personnel cannot enter laboratory while decontamination is in
progress.
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
This is an OVERNIGHT decontamination. Decontamination should be completed by 10am
the following morning.

After decontamination, the Biosafety Cabinet SHOULD NOT be used prior to being moved
or repaired.

After the Biosafety Cabinet has been repaired or moved, cabinet MUST be re-certified
before being used again. Contact EH&S at 567-2955 for further information
Alternatively, Vaporized Hydrogen Peroxide decontamination can be requested from an authorized
vendor. The cost of this decontamination is the PIs responsibility.
8.1.4 Flammable Gases in a Biological Safety Cabinet (BSC)
Overview
The use of natural gas or other flammable gases are not allowed in a recirculating
BSC.
The only approved BSC for use of natural gas is a fully ducted (100% exhausted),
class II B2 biosafety cabinet. Although the use of natural gas in a class II B2
biosafety cabinet is discouraged, the EH&S department will review requests for use
of natural gas in these BSCs . Contact the EH&S department at 567-2955.
The use of natural gas or other flammable gases within recirculating BSCs may allow
flammable gases to concentrate, potentially leading to an explosive atmosphere that upon
ignition, it could explode. The use of flammable gases within a BSC may alter the airflow
pattern used to protect product and personnel.
New projects and maintenance requests will no longer add house natural gas to
recirculating biological safety cabinets. Natural gas lines to recirculating BSCs will be
turned off as they are discovered. When applicable, natural gas lines to BSCs will be
physically disconnected and capped during laboratory renovation projects.
Open flames should not be necessary in the near microbe-free environment of a biological
safety cabinet. Alternatives to the use of flammable gases to disinfect include:
a. The use of an electric Bunsen burner.
b. The use of a bact-cinerator. Safely sterilizes loops and needles by infrared heat
Complete sterilization occurs in only 5 to 7 seconds at optimum temperature of 1500°F
(815°C). Heat is contained in deep ceramic tube to safeguard laboratory personnel—no
open flame. No need to wait for instruments to turn red.
Bacti-cinerator
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Alternatives that avoid the need to disinfect instruments within a BSC include:
a. the use of pre-sterilized inoculating loops and needles.
b. pre-autoclaving forceps, scalpels, etc. in covered autoclavable plastic containers or the
special sleeves supplied for this use by various companies. These can be taken into the
BSC and used individually and placed in an autoclavable discard tray located in the BSC
for used/contaminated utensils.
c. the use of a Bunsen burner outside the BSC (> 2 feet away from the BSC) for some
applications.
Flaming the necks of bottles is not necessary due to the protective airflow in the BSC.
Using a flame for this purpose would disturb the airflow and may result in contamination of
the tissue culture flasks.
8.1.5 Ultraviolet Lamps
Ultraviolet (UV) lamps are not recommended in BSCs nor are they necessary. If installed,
UV lamps must be cleaned weekly to remove any dust and dirt that may block the
germicidal effectiveness of the ultraviolet light. The lamps should be checked weekly with a
UV meter to ensure that the appropriate intensity of UV light is being emitted. UV lamps
must be turned off when the room is occupied to protect eyes and skin from UV exposure,
which can burn the cornea and cause skin cancer. If the cabinet has a sliding sash, close
the sash when operating the UV lamp. Newer BSCs have the UV interlocked with closing
the sash.
.
A warning sign should be posted on the BSC reminding personnel to turn off the UV lamp.
These signs may be obtained through the EH&S office.
8.1.6. Safe Use of a Biological Safety Cabinet:
The safe use of a BSC is described in an SOP in Appendix F of this manual.
SAFETY RULES FOR USE OF CLASS I AND II CABINETS
Preparations
It is recommended that a biological safety cabinet be left on at all times. However, if the unit is not left
running continuously, turn the blower on and air purge for at least ten minutes to remove airborne
contamination.
NOTE: Never turn off the blower or lower the sash on a biological safety cabinet that is
vented to the outside of the building.


Turn off the UV light. Never work in the unit with the UV light illuminated. (UV light will
damage the human eye very quickly).
The work surface should be wiped down with the appropriate disinfectant (70% alcohol is
usually suitable). Do not depend on the UV germicidal lamp to provide a sterile work surface
(see “Disinfection and Sterilization” section of this chapter).
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



Everything needed to complete the particular procedure should be placed inside the cabinet
prior to beginning work. Arrange implements in a logical manner to segregate clean and dirty
materials.
Remember to provide a container for wastes on the inside of the cabinet. Nothing should
pass in or out through the air barrier until the procedure is complete.
Never place any items on the intake grilles as this will block the air supply and could
compromise your safety and the product’s sterility
Laboratory doors should remain closed during an experiment, especially when working with
infectious agents. Post a sign stating, “Cabinet in Use or Experiment in Progress.” Every
effort should be made to restrict the opening and closing of doors and walking traffic in the
work area, as these activities will disturb the cabinet’s air flow.
Use of the Biosafety Cabinet (BSC)
Follow these guidelines when using the safety cabinet:
 always wear a lab coat
 conduct your work at least 4“ back from the glass view panel. The middle third of the
work surface is the ideal area to be used. Never cover the air intake grille at the front of
the cabinet.
 set up your work area so that you are working from clean to dirty.
 limit arm movements and do not make fast, pumping motions
 natural gas burners shall not be used in recirculating biosafety cabinets. If a burner is
required, use an electric Bunsen burner. (since a burner will produce air turbulence,
place it to the rear of the workspace). Most procedures when combined with good
aseptic technique and proper cabinet use, should not require use of a flame.
 place a disinfectant-soaked towel on the work surface to contain any splatters or small
spills that may occur during the procedure
 minute amounts of volatile toxics, organics & radio-nuclides may be used in biosafety
cabinets class II, A2 and above; the chemical concentration must remain well below the
LEL (Lower Explosive Limit) of the compound. Never use flammable solvents in a
biological safety cabinet.
Completion of a Job
When finished using a safety cabinet, make sure that:
 all equipment which has been in direct contact with the research agent is enclosed or the
surface decontaminated
 waste containers are covered
 the cabinet is allowed to operate for five minutes with no activity in order to purge
airborne contaminants from the work area
 all equipment is removed from the cabinet
 interior work surfaces are decontaminated
 you thoroughly wash your hands and arms with warm, soapy water.
Biohazardous Spills In the Biosafety Cabinet
In case a biohazardous spill occurs inside the cabinet:
 decontamination steps should be taken while the cabinet is operating to prevent the
escape of contaminants
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

spray or wipe walls, work surface, and all apparatus that is affected with an appropriate
disinfectant detergent. (Make sure to wear gloves while doing this.)
if the spill is large, flood the work surface with disinfectant and allow to stand 10-15
minutes before removing it
Remember
 the biological safety cabinet is not a substitute for good laboratory practice
 aerosols can escape when the airflow is disrupted by:
a rapid movement of hands or arms,
opening doors to the room,
persons walking past the cabinet
 hands, arms, and the front of the user may be contaminated
 decontaminate the working surfaces of the biosafety cabinet before and after each use
8.2 Vacuum Systems
Vacuum Systems that connect directly to a building's central vacuum or a motorized pump must be
protected from aspiration of fluids or other hazardous materials into those vacuum systems.
Protection of these vacuum systems allows the equipment to be safely serviced. Contamination of
the equipment can result in costly repairs.
A simple means to achieve protection against pathogens is illustrated in this diagram.
Dual vacuum flasks (A and B) are placed in series and a disposable in-line HEPA filter (C) is
placed between the flasks and the source valve (D). Flask A typically is used to collect the
potentially infectious liquids into a disinfectant such as bleach. Flask B serves as a fluid overflow
collection vessel. Extending the tube to the bottom of the flask helps to minimize splatter. The filter
should be replaced whenever there is evidence of failure or blockage. When this arrangement is
used for work that is done inside a biological safety cabinet (BSC), the flasks also should be
located inside the BSC.
Vacuum flasks used on the benchtop for BSL-1 work should be protected against breakage by
being positioned in a leak-proof secondary container. These vacuum flasks should also have an
inline HEPA filter to protect the house vacuum line.
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8.3 Centrifuges
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 the
manufacturer's instructions. Users should be properly trained and operating instructions
including safety precautions should be prominently posted on or by the unit.
Aerosols are created by practices such as filling centrifuge tubes, removing supernatant, and resuspending sediment 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:

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.

Fill and open centrifuge tubes, rotors and accessories in a BSC. Avoid overfilling of
centrifuge tubes so that closures do not become wet. After tubes are filled and sealed, wipe
them down with disinfectant.

Always balance buckets, tubes and rotors properly before centrifugation.

When working with infectious agents, a safety centrifuge cup/bucket is used. The safety
centrifuge cup must be loaded and unloaded in the BSC. The safety centrifuge cup is
disinfected prior to being taken out of the BSC and placed back in the centrifuge. (Check
with the centrifuge manufacturer about which disinfectants can be used on the equipment.)
Safety centrifuge cups/sealed rotor

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 fatiguing resulting in disintegration
of rotors and to use proper cleaning techniques and centrifuge components.
Manufacturer's recommendations must be meticulously followed to avoid metal fatigue,
distortion and corrosion.

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,
appropriate chemical disinfectants are necessary for decontamination.

Promptly clean up spills using an appropriate disinfectant.

Periodic cleaning of the centrifuge and rotors is recommended.
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8.4 Blenders, Sonicators, Lyophilizers
The use of any of these devices results in considerable aerosol production. Blending, cell
disrupting and grinding equipment should be used in a BSC when working with biohazardous
materials.
Safety Blenders
Safety blenders, although expensive, are designed to prevent leakage from the bottom of the
blender jar, provide a cooling jacket to avoid biological inactivation, and to 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.
Lyophilizers and Ampoules
Depending on 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.
Opening ampoules containing liquid or lyophilized infectious culture material should be performed
in a BSC to control the aerosols produced. Gloves must be worn. When recovering the contents of
an ampoule, care should be taken not to cut the gloves or hands or disperse broken glass into
eyes, face, or laboratory environment. 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. (Some ampoules
are pre-scored.) Reconstitute the contents of the ampoule by slowly adding liquid to avoid
aerosolization of the dried material. Mix the contents of the ampoule slowly, withdraw contents in
to a new container. Discard the towel and ampoule top and bottom as biohazardous waste into
sharps container.
Ampoules used to store biohazardous material in liquid nitrogen have exploded causing eye
injuries and exposure to the infectious agent. The use of polypropylene tubes eliminates this
hazard. These tubes are available dust free or pre-sterilized and are fitted with polyethylene caps
with silicone washers. Heat sealable polypropylene tubes are also available.
8.5 Pipettes and Pipet Aids
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Mouth pipetting is strictly prohibited. Mechanical pipetting aids must be used. Confine pipetting
of biohazardous or toxic fluids to a biosafety cabinet. If pipetting of biohazardous/toxic materials
can’t be performed in a biosafety cabinet, alternative engineering and/or procedural controls must
be used. Consult with EH&S. Pipetting on the open bench should be confined to low risk agents
(i.e. lab strains of E. coli).
Use the following precautions:

Always use cotton-plugged pipettes and pipet tips when pipetting biohazardous or toxic
fluids.

Never prepare any kind of biohazardous mixtures by suction and expulsion through a
pipette.

Biohazardous materials should not be forcibly discharged from pipettes. Use "to deliver"
pipettes rather than those requiring "blowout."

Do not discharge biohazardous material from a pipette at a height. Whenever possible
allow the discharge to run down the container wall.

Place contaminated, reusable pipettes horizontally in a pan containing enough liquid
disinfectant to completely cover them.

Autoclave the pan and pipettes as a unit before processing them for reuse.

Discard contaminated Pasteur pipettes in an appropriate size sharps container.

When work is performed inside a biosafety cabinet, all pans or sharps containers for
contaminated glassware should be placed inside the cabinet as well while in use.
8.6 Syringes and Needles
Syringes and hypodermic needles need to be handled with extreme caution to avoid accidental
injection and aerosol generation. Generally, the use of syringes and needles should be restricted
to procedures for which there is no alternative. Do not use a syringe and needle as a substitute for
a pipette.
Use needle locking syringes (luer-lock tip) or whenever possible use disposable syringe-needle
units in which the needle is an integral part of the syringe.
When using syringes and needles with biohazardous or potentially infectious agents:

Work in a biosafety cabinet whenever possible.
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
Wear gloves.

Fill the syringe carefully to minimize air bubbles.

Expel air, liquid and bubbles from the syringe vertically into a cotton pad moistened with a
disinfectant.

Do not re-cap needles
Needles should not be bent, sheared, replaced in the sheath or guard (capped), or removed from
the syringe following use. If it is essential that a contaminated needle not be recapped or removed
from a syringe. If the syringe (Hamilton syringe, anesthesia syringe, etc.) is one that is reused,
then a mechanical device or the one-handed scoop method or recapping station must be used to
remove the needle. Always dispose of needle and syringe unit promptly into an approved sharps
container. (See the Biological Waste Management SOP and the Bloodborne Pathogen Exposure
Control Plan)
Do not overfill sharps containers. Sharps containers should be filled to approximately 2/3 full and
the lid closed. Closed sharps containers can be placed in the regulated medical waste box.
8.7 Loop Sterilizers and Bunsen Burners
Sterilization of inoculating loops or needles in an open flame generates small particle aerosols that
may contain viable microorganisms. The use of a shielded electric incinerator or hot bead sterilizer
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 or
recommended. Wear eye protection.
Continuous flame gas burners shall not be used in recirculating BSCs and may only be used in a
100% ducted class II B2 BSC upon consultation with EH&S. These burners can produce
turbulence which disturbs the protective airflow patterns of the cabinet. Additionally, the heat
produced by the continuous flame may damage the HEPA filter.
The use of alcohol lamps are strictly discouraged and maybe prohibited by fire code.
Bacti-cinerator
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Bacti-cinerator. Sterilizes needles, loops, and culture tube mouths in five to seven seconds at
optimum sterilizing temperature of 815.6°C (1500°F). Eliminates microorganism spattering
associated with flame sterilization. Ceramic funnel tube enclosed in stainless-steel perforated
guard and cast-aluminum support stand.
Electric burner. Electric Bunsen burner. Temperature ranges from 800 - 1000ºC (1472 - 1832ºF).
Replaces heating elements. Corrosion resistant metal housing.
Combine the advantages of the gas burner with the clean operation and control ease of electric
heating. Conical shaped heating elements are easy to replace. Since the radiation emitted from
the heater is directed upwards only, it can be used for such purposes as heating test tubes,
crucibles, small flasks and beakers independent of their shape. Air circulation from the vented
housing keeps the base cool enough to hold during operation. The top cowl deflects heat away
from your hand.
Glass Bead Sterilizers
Glass Bead sterilizer

Glass Beads Sterilizers are not FDA approved sterilizers and is not intended for medical,
clinical or other patient oriented applications. However, they can be used in non-health care
facilities. Glass Beads sterilizers are a quick, easy and accurate alternative to traditional
methods of sterilization. Once switched 'ON', the units are ready to use within 30 minutes
and ensure total sterilization. The constant temperature of 250° C ensures the total
destruction of microorganisms and spores in a few seconds. Glass bead sterilizers are
suitable for sterilizing scissors and other instruments, inoculating loops, etc.

Most Glass Beads Sterilizers are incorporated with a thermostat, which maintains the set
temperature with an accuracy of ± 1% of the preset range. These units, having a stainless
steel body, are compact enough to be placed in a BSC, clean bench or workside table.
8.8 Autoclaves and Pressure Vessels
a. Steam sterilization of infectious waste utilizes saturated steam within a pressure vessel at
temperatures sufficient to kill infectious agents potentially present in the wastes.
b. In treatment by steam sterilization, decontamination of the waste occurs primarily from
steam penetration. Heat conduction provides a secondary source of heat transfer.
Therefore, for effective and efficient treatment, the degree of steam penetration is the
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critical factor. For steam to penetrate throughout the waste load, the air must be completely
displaced from the treatment chamber.
c. The presence of residual air within the sterilizer chamber can prevent effective sterilization.
d. The principal factors that should be considered when treating infectious wastes are:

Type of wastes,

Packaging and Containers,

Volume of wastes and

Its arrangement in the treatment chamber
Many infectious wastes that have multiple hazards should not be steam sterilized because of the
potential for exposure of equipment operators to toxic, radioactive, or other hazardous chemicals.
Infectious wastes that should not be steam sterilized include those that contain antineoplastic
drugs, toxic chemicals, or chemicals that could be volatilized by steam.
a. Persons involved in steam sterilizing of infectious wastes should be educated in proper
techniques to minimize personal exposure to the hazards posed by these wastes. These
techniques include use of personnel protective equipment, minimization of aerosol
formation, and prevention of spillage of waste during autoclave loading.
b. An autoclave tape record should be used to ensure that a sufficiently high temperature is
maintained for an adequate time during the cycle. The tape record should be verified after
the run to ensure that the cycle ran properly. Failure to attain or maintain operating
temperature may indicate mechanical failure.
c. All steam sterilizers should be routinely inspected and serviced. Monitoring the steam
sterilization process is required to ensure effective treatment. The process should be
monitored periodically to check that proper procedures are being followed and that the
equipment is functioning properly.
d. Each autoclave should be checked on a routine basis by using biological indicators (spores
of Bacillus stereotherophilus) to determine that the autoclaved biological wastes have been
sterilized at the proper temperature, pressure and time. Temperature indicator autoclave
tape that changes color indicate only the outside of the container was exposed to a certain
temperature, they do not indicate that the waste has been sterilized.
e. All material to be autoclaved must be accompanied by a time, thermal, biological and color
indicator and be must be placed in the autoclave by a member of the responsible research
group who will see that temperature and pressure are up to desired levels.
f.
Biohazardous waste of animals housed at ABSL-2 or ABSL-3 including carcasses, their
tissues, bedding and cages must be placed in biohazard autoclavable bags that are tied
closed. These bags are placed in an autoclavable tray prior to autoclaving. The animal
waste is disposed of per Department of Laboratory Animal Resources (DLAR) SOP.
g. To assure that sterilization is being accomplished sterilization procedures and equipment
must be certified before routine use and periodically thereafter (preferably during each
cycle). Certification is usually accomplished utilizing instrumentation, chemical indicators,
and biological indicators to verify that appropriate physical parameters are satisfactory to
kill resistant commodities.
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h. Records of the disposition of Regulated Medical Waste (RMW) should be kept on file for
three years. Disposition of RMW is handled through a contractor. These disposition records
are kept on file in the Environmental Health and Safety office.
8.9 Laboratory Furniture
The laboratory furniture must be easily cleanable and able to withstand decontamination
procedures should they become contaminated. Cloth chairs, carpets and rugs are not appropriate
for laboratories.
Bench tops should be impervious to water and resistant to acids, alkalis, organic solvents
(disinfectants and sterilants) and moderate heat (Bunsen burners, alcohol lamps and microbe
incinerators). Wood furniture is not appropriate for the laboratory.
Laboratory furniture should be of sturdy construction with spaces between benches, cabinets and
equipment accessible for cleaning. Laboratory furniture should be capable of supporting
anticipated loading and uses.
Chairs and stools should be covered with a non-fabric and non-absorbent material. Chairs
should be covered in a material like plastic, vinyl or leather finish that can be easily cleaned in the
event of contamination or spill.
8.10 Flow Cytometry
For infectious, pathogenic, and/or toxic materials, flow cytometry must be conducted in a certified,
totally ducted biological safety cabinet or the flow cytometer must have aerosol management.
For sorting of Lentivirus transduced cells, the PI and technicians must follow the FACS Lentivirus
SOP.
Flow cytometry is a method of quantifying structural or biochemical features of cells or other small
particles by using a laser as an excitation light source and photodetectors for measurement. This
is generally accomplished by either light scatter and/or fluorescence. Flow Cytometry may further
be defined as a technology to measure properties of particles as they move, or flow, in liquid
suspension.
In some flow (or sorting) cytometers, the liquid containing the particles is broken into droplets by
the reciprocating motion of a nozzle. Individual particles are captured in small droplets, and then
those droplets are electrostatically charged and deflected through a high potential. If a plug or
other obstruction (bubbles, clumps of cells, etc.) occurs, then this stream of droplets can go awry,
generating aerosols. There is a potential for aerosol exposure to individuals near a flow cytometer
because modern sorting units operate with 14 to 40 pounds per square inch of pressure (some
units can operate at 100 psi). Even non-sorting flow cytometers, which do not suspend cells in
droplets, operate under pressures near 5 psi and can develop leaks and generate aerosols.
The following requirements must be followed when using a flow cytometer due to the potential for
aerosol exposure to individuals:
1. Used only in a negative pressure laboratory.
2. Performed by trained individuals.
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3. Operated with proper personal protective equipment (PPE) including gloves, lab coats, and
safety goggles.
4. Cleaned and properly disinfected after each use.
5. The catch basin (traps) should have an adequate disinfectant (i.e. bleach) added when the
unit is in use. Must have proper contact time prior to disposal down the drain.
6. When possible, biological samples should be fixed (usually with 1% paraformaldehyde)
before being run through the flow cytometer.
8.11 Cryostat
Frozen sectioning done on unfixed tissue poses a high risk because of the knife blade and
techniques involved. Freezing of tissue does not inactivate infectious agents. Freezing propellants
under pressure should not be used for frozen sections as they may cause the splattering of
droplets of infectious material. Gloves and safety goggles should be worn while working with
frozen sections.
The contents of the cryostat should be considered to be contaminated and should be
decontaminated frequently with an appropriate disinfectant. The wastes trimmings and sections of
tissue that accumulate in the cryostat should be considered to be contaminated, should be
removed during decontamination and preferably autoclaved. The cryostat should be defrosted and
decontaminated once a week with a tuberculocidal hospital disinfectant with a label claim for
tuberculocidal disinfection.
Stainless steel mesh gloves should be worn when changing knife blades. Solutions used for
staining frozen sections should be considered to be contaminated. The wastes stain should be
disposed of as required by Hazardous Materials Management Program.
8.12 Refrigerators, Freezers and Microwaves
Refrigerators and freezers
Appropriate use of refrigerators and freezers in the laboratory are essential for the health and
safety of laboratory personnel. The following stringent procedures must be implemented and
maintained for safety use of refrigerators.

Laboratory refrigerators and freezers must never be used for the storage of food or
beverages (non-laboratory use).

Items stored inside the refrigerators and freezers must be properly labeled.

Uncapped containers are not permitted in refrigerators.

Containers capped with aluminum foil, corks, corks wrapped in aluminum foil, or glass
stoppers are not appropriate.

The refrigerator and freezer must be labeled with the appropriate hazard warning label. A
biohazard sticker must be placed on the door of any unit storing biohazardous materials
(infectious agents, human/non-human primate blood, tissue and body fluids, etc.).

Scheduled inventory and disposal of outdated or unnecessary materials must be
maintained. Dispose of any materials that are not essential immediately.
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
Where appropriate, purchase a unit that has been designated for "flammable storage" by
the manufacturer for flammable materials that require cold storage. There must be no
source of electrical sparks within the refrigerator compartment. Some units have been
modified by removing any interior light that is activated by a switch mounted on the door
frame; moving the contacts of the thermostat controlling the temperature to a position
outside the refrigerated compartment; and removing the contacts for any thermostat
present that controls fans within the refrigerated compartment to the outside of the
refrigerated compartment. An alternative to modification is to place a prominent sign
warning against storage of flammable substances within the unit.

Potentially explosive or highly toxic substances in laboratory refrigerators are discouraged.
Prominent warning signs are displayed on the outside of the unit. Laboratories using such
materials should purchase and use an explosion-proof refrigerator.
Microwave
Microwave ovens have become prominent laboratory instrumentation and standard equipment
used in the laboratory. When microwave ovens are in use, laboratory personnel shall be available
to monitor their use.

NEVER LEAVE MICROWAVE OVENS UNATTENDED WHILE IN OPERATION!

Microwave ovens should be labeled for “Laboratory use only”.

Containers used for heating must be microwavable.

Care must be exercised in handling hot containers and heated liquids.

Laboratory microwaves shall not be used to heat food and drinks for human consumption.
A. Potential health hazards
Several points must be addressed when operating a "household-type" microwave in the laboratory.
These potential hazards include the following:

Airborne concentrations of formalin vapors may be increased when using the microwave
oven for fixation purposes (vapor concentration may well exceed the allowable PEL).

Unvented microwave ovens may release concentrated vapors when door is opened.
(Chamber ventilation systems prevent buildup of toxic or flammable vapors and any vented
air from the oven should be passed through the laboratory ventilation system or charcoal
filter, or the oven should be enclosed in a fume hood).

Procedures using alcohols and xylene are flammable and potentially volatile. Potential
ignition of volatile solution vapors may occur by a spark (switch) that runs off the
magnetron when the door is opened (unless switch is explosion-proof).

Solvent vapors may cause deterioration of door gaskets (if present) resulting in radiation
leakage.
B. Controls
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Because of the aforementioned potential hazards, microwave units have been developed for
laboratory use. These units are costly and for laboratories that use non laboratory (household)
units, some solutions are proposed to address these problems.

The use of zip-type bags, particularly those made for biological purposes, or at least,
freezer-weight kitchen type, can be used to contain the fumes created when solutions are
heated. (Thinner gauge bags will melt in the oven).

After heating, the container and the bag are taken to a fume hood and safely opened, thus
reducing exposure to the lab tech.

Enclosing containers in bags also contains any spill that might occur, protecting the lab
person from solution contact and preserving the surface of the oven interior.

Minimization of the amount of solution used for many procedures, such as the use of
micro-containers in place of coplin jars, further reduces the amount of vapor concentrations
that may be produced.

Control of volatile solutions is accomplished by quality control calibration of the oven,
knowledge of boiling points of solvents, and controlled heating of such solutions when
microwaved.

The non-laboratory or household microwave ovens manufactured currently do not have
rubber-type gaskets at the door edge. If the oven does have gasket, a routine inspection by
a radiation safety officer should be able to detect if the gasket is no longer sealing the door
properly.
8.13 Equipment Repair/Disposal

Equipment / Instrumentation used for storage of biological commodities:
1. All laboratory equipment must be cleared by EH&S prior to moving, repair or
decommissioning.
2.
For disinfection, remove all items, defrost if applicable and disinfect with
appropriate disinfectant for the biological agents stored or processed in that
equipment. Typical disinfection is to wipe the equipment with a 10% bleach solution
(1 part bleach to 9 parts water) or 70% Ethanol.
3. An online request is then submitted to EH&S for equipment clearance.

Combination of chemicals/drugs and biological agents:
1. Follow the aforementioned protocols. Be careful not to combine incompatible
substances such as bleach and ammonia. Contact EH&S if further guidance is
needed.
2. Radioactive material and any combination of radioactive material with chemicals,
drugs, or biological agents: Follow the reporting, surveying and tagging procedures
as established by Radiation Safety.
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9.0 Medical Surveillance and Exposure Control
Overview of Medical Surveillance and Exposure Control
The UTHSC Employee Health and Wellness Clinic (EWHC) and the Student Health Center, located in
the Nursing School on main campus, serve as vital components of the Biological Safety Program by
helping to protect the health and safety of employees, students and authorized visitors in clinics,
research and teaching laboratories. The primary objective of the program is prevention, and early
detection of work-related health effects achieved through training, periodic evaluations and
immunizations. The major services provided include: pre-employment immunizations for designated
personnel, immunizations for designated personnel, periodic evaluations, maintenance of employee
occupational health records, response to exposures, epidemiology, and considerations for other special
medical situations.
Employee Health and Wellness Clinic, http://ehwc.uthscsa.edu
Phone: 210-567-2788, FAX: 210-567-2779
Student Health Center, http://shc.uthscsa.edu/
Phone: 210-567-9355; FAX: 210-567-5903; Email: [email protected]
9.1 Pre-Employment Immunizations and Medical Surveillance
Pre-Employment Immunizations:
UT Health Science Center at San Antonio policy on pre-employment immunizations is found in the
Handbook of Operating Procedures, 8.5.8 Pre-Employment Immunizations. The complete policy is
found at the following link.
http://uthscsa.edu/hop2000/8.5.8.pdf
The UT Health Science Center at San Antonio (Health Science Center) is committed to protecting
the health and safety of patients, employees, patient and employee family members, and the
community as a whole by providing a consistent testing and immunization standard for all
individuals who are offered positions of employment or training within the Health Science Center.
This policy is applicable to faculty, employees, fellows, post-docs, visiting scientists or clinicians
and volunteers of the Health Science Center who are offered a position or are participating in a
program that requires:
1. Any patient or research subject contact;
2. Animal contact; or
3. Potential exposure to human or animal blood or body fluids as part of an applicant’s
involvement with research or patient care.
Medical Surveillance:
U.S. Department of Health and Human Services (HHS) describes medical surveillance as the
systematic assessment of employees exposed or potentially exposed to occupational hazards. This
assessment monitors individuals for adverse health effects and determines the effectiveness of
exposure prevention strategies. A medical surveillance program includes the analysis of both
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individual and aggregate surveillance data over time, with the goal of reducing and ultimately
preventing occupational illness and injury.
Medical surveillance is an ongoing process based on the individual’s exposure assessment which
may include the following:
 the biological agents worked with and procedures performed in a laboratory,
 work with recombinant or synthetic nucleic acid molecules
 work at BSL3 containment,
 engagement in large-scale research or production activities (work with more than 10L),
 if an individual has patient contact,
 if an individual works with animals,
 work with chemicals, toxins
 work with radiological materials and radiation producing devices.
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 the institution’s healthcare provider s for appropriate counseling and
guidance.
Other Special Medical Considerations:
 Allergies: Individuals with histories of allergies, specifically animal allergies, will be evaluated
and advised of the potential health risk of animal exposure.
 Immuno-suppressive disorders: Individuals with Immunosuppressive disorders or undergoing
treatment with steroids, immunosuppressive drugs, or antibiotics will be counseled on work
related risks and can consider work assignment modification to avoid exposure to specific
hazards.
 Reproductive Concern: Employees and authorized visitors will be advised to inform UTHSC
EHWC or Student Health Services staff if they are pregnant or are considering pregnancy,
so that they can be counseled on work related risks and can consider work assignment
modification to avoid exposure to specific hazards.
 Disease Outbreak Evaluations: In the event of a disease outbreak, the exposure will be
documented. Physical examinations and lab tests will be done as necessary. Appropriate
treatment will be given or referral made as needed.
Job Hazard Analysis:
EH&S can conduct a job hazard analysis on request and as a result of an exposure follow-up.
9.2 Response to Injury/Exposures and Reporting
Exposures to infectious agents can arise from a variety of incidents, including aerosols, splashes of
liquid into mucous membranes or broken skin, percutaneous injury and animal scratches or bites. In
the event of any incident of this type, the immediate response should be directed towards saving an
individual’s life. In the event of a life-threatening injury, call extension 7-8911 to reach the UT Police
Department (UTPD) dispatch from an UTHSC phone or 567-8911 from a cell phone. UTPD will
dispatch the necessary emergency medical service.
Procedure for percutaneous injury/bloodborne exposure/infectious agent exposure, animal bite.
 Wash the injured area immediately with soap and water. If splash to the eyes/face, go to the
closed eyewash and flush eyes with water for a minimum of 15 minutes.
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








Notify the supervisor.
The individual should seek post exposure care / medical treatment at the Employee Health
and Wellness Clinic or Student Health Center during the hours of 8:00am to 5:00pm (210567-2788).
If exposure occurs after 5:00 p.m. or on a weekend, or holiday, the employee/student should
go to the University Hospital System (UHS) Emergency triage (210-358-2488), which is open
24 hours/7days a week or to the UHS Express Med clinic (210-358-5510) which is open until
10:00pm.
If available, bring the following documents and forms, which can be found on the
EH&S/Workers compensation website at
http://research.uthscsa.edu/safety/workerscomp.shtml
1. UTHSCSA Notification of on the Job injury
2. Employee Exposure Notification and medical evaluation option form.
3. Relevant medical records.
4. Information on the source patient/individual.
The employee should report to UTHSC EHWC or students report to the Student Health clinic
as soon as possible on the next working day.
Contact EH&S at 210-567-2955 to report the injury.
Complete the Employer’s First Report of Injury Form, which must also be completed for
reporting to Workers’ Compensation and Insurance. FAX to EH&S at 210-567-2965.
For Sharps injuries, complete the Sharps injury form and FAX to EH&S at 210-567-2965.
For select agents and toxins, the CDC/USDA has defined an occupational exposure as: “Any
event which results in any person in a registered entity facility or lab not being appropriately
protected in the presence of an agent or toxin. This may include reasonably anticipated skin,
eye, mucous membrane, or parenteral contact with blood or other potential infectious
materials that may result from the performance of a person’s duties. For example, a sharps
injury from a needle being used in select agent or toxin work would be considered an
occupational exposure.” These exposures should be reported immediately to EHS for
reporting to the CDC/USDA as required by law.
9.3 Employee Health Records
Employee Health Records Health records obtained from employees, students and authorized
visitors will be kept confidential and maintained in a secure location by the UT Employee/Student
Health Services. Access to records will be limited to authorized personnel per state and federal law.
Records will not be released to anyone without the individual's written consent, except in situations
required by law.
9.4 Bloodborne Pathogen Exposure Control Plan (ECP)
The Bloodborne Pathogens Exposure Control Plan (ECP) provides guidelines and procedures to
avoid or minimize occupational exposure to bloodborne pathogens and implement procedures and
processes for exposure management. The ECP includes information on medical surveillance and
the Hepatitis B Virus (HBV) vaccination, post-exposure procedures, recordkeeping, training
requirements, and other topics in compliance with the Texas Dept. of State Health Services and
OSHA Bloodborne Pathogen Standards.
The Bloodborne Pathogens ECP applies to all Health Care Personnel (HCP) and employees at the
university. It includes clinical laboratories, research laboratories, dental clinics, and other health
care clinics and facilities operated by UT Health Science Center faculty and staff. UT Health
Science Center faculty advisors should also use the ECP to ensure that students and NonEmployees (as defined in the UT Health Science Center’s Handbook of Operating Procedures,
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HOP 4.5.15) under their charge, exposed to blood or other potentially infectious materials, adhere
to the guiding principles and policies of the ECP.
The Bloodborne Pathogen ECP is available on the EH&S website at
http://research.uthscsa.edu/safety/BBPExposureControlPlan.pdf
9.5 Tuberculosis Exposure Control Plan (ECP)
The TB Exposure Control Plan (ECP) contains guidelines and procedures that should be used in
conjunction with standard healthcare or research techniques to minimize exposure to
Mycobacterium tuberculosis.
This plan should be used by supervisors to develop patient and employee procedures to minimize
the exposure to Mycobacterium tuberculosis and to assist all levels of management in
implementing effective policies for providing safe patient care in the clinical environment and safe
laboratory practices during the course of employment at University of Texas Health Science
Center San Antonio.
The Tuberculosis ECP contains information on medical surveillance including the Tuberculin Skin
Test (TST).
The Tuberculosis ECP is available on the EH&S website at
http://research.uthscsa.edu/safety/TBplan.shtml
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10.0 Medical Waste Management
The UT Health Science Center at San Antonio must comply with all applicable regulations
regarding the collection, transportation and treatment of regulated medical waste (RMW). Federal
Department of Transportation (DOT) regulations require a generator (UT Health Science Center at
San Antonio) of regulated medical waste to certify that the packaging and documentation of
transported regulated medical waste complies with DOT regulations regarding waste classification,
packaging, labeling and shipping. It is imperative that all UT Health Science Center at San Antonio
departments understand the rules regarding proper identification, classification, segregation and
packaging of regulated medical waste.
The term "medical waste" includes biohazardous, biomedical, infectious or regulated medical
wastes as defined under federal, state or local laws, rules, regulations and guidelines. This
EXCLUDES pharmaceuticals, chemotherapeutic wastes, radioactive wastes, wastes containing
mercury or other heavy metals, chemicals such as solvents, reagents, corrosives or ignitable
materials classified as hazardous under Federal EPA regulations.
Management of Regulated Medical Waste and other wastes is administered through the
Environmental Health and Safety department, Environmental Protection Division. For more
information, see the website: http://research.uthscsa.edu/safety/epdisposal.shtml
For specific information on laboratory waste procedures, see the Biomedical Waste Management
SOP in appendix F of this manual.
The following information highlights the package requirements for medical waste and what is
accepted and not accepted.
These regulated medical waste instructions are provided to assist your compliance with the
packaging requirements of 49 CFR 173.197, which require that the waste be packaged in a
container that is:
1. rigid;
2. leak resistant;
3. impervious to moisture;
4. of sufficient strength to prevent tearing or busting under normal conditions of use and
handling;
5. sealed to prevent leakage during transport;
6. puncture resistant for sharps and sharps with residual fluids; and
7. break-resistant and tightly lidded or stoppered for fluid in quantities greater than 20 cubic
centimeters.
Accepted Waste
 Sharps
Needles and syringes, scalpel blades, glass pipettes, slides, dental wires, etc. provided
they are placed in "sharps" container before being placed into the Regulated Medical
Waste (RMW) red bag liner and the medical waste box.
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
Laboratory Wastes
Cultures and stocks of Biohazard Level (Risk Group) 1, 2, and 3 infectious agents, test
tubes, etc.
NOTE: All materials originating in BSL-3 facilities must be autoclaved prior to removal from
the BSL-3 facility and then be identified and labeled for incineration.

Contaminated Disposal Material, Equipment, and Instruments
Includes but is not limited to: blood administration sets, drainage collection devices, gloves
and gowns, dressings.

Blood, Blood Products, Body Fluids
Accepted Waste Which Must be Identified and Labeled for Incineration

Trace-contaminated Chemotherapy Waste
Empty drug vials, syringes and needles, IV tubing, gowns and gloves. See 40 CFR, Part
261:7.

Pathological Waste
Human or animal body parts, organ, tissues and surgical specimens (exclusive of
formaldehyde or other preservatives). Human body parts under the control of the
Anatomical Board of the State of Texas must be disposed of in accordance with Texas
Administrative Code, Title 25, Chapter 479, Rule 479.4. Animal carcasses must be
disposed of according to Department of Laboratory Animal Resources (DLAR) procedures.

BSL-3 Waste
Laboratory wastes as mentioned above originating from a BSL-3 facility. (BSL-3 waste is
autoclaved before it leaves the BSL-3 facility.)
Wastes NOT Accepted.

Pharmaceutical Waste
Please request assistance. EH&S can characterize some items as non-RCRA hazardous
materials. RCRA listed or characteristic hazardous pharmaceutical waste cannot be
disposed of through sewers, landfills, or through RMW. It must be treated as hazardous
chemicals (see below) and shipped to a RCRA facility.

Chemicals
Formaldehyde, acids, alcohols, waste oil, solvents, reagents, fixer, developer, etc. Virtually
all chemicals should be treated as chemical waste and not disposed of with RMW. Please
request a chemical pick up.

Bulk Chemotherapy Waste
Antineoplastic and cytotoxic materials including the non-conforming U-listed chemotherapy
wastes Chlorambucil, cyclophosphamide, Daunomycin, Melphalan, Mitomycin C,
Streptozptocin, and Uracil Mustard.

Hazardous Waste
Drums, pails, bottles, cans or other containers with hazardous warning labels or symbols,
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batteries, heavy metals, etc. Please request a chemical pick up for these materials or
suspected materials.

Radioactive Waste
Any container with a radioactivity level that exceeds regulatory or permitted limits; leadcontaining materials. Please request a radioactive materials pick up for any radioactive
materials or suspected radioactive materials.

Compressed Gas Cylinders, canisters, Inhalers and aerosol cans
All of these items must be treated as RCRA hazardous waste. Please request a chemical
pick up from EH&S.

Glass thermometers, sphygmomanometers, and other devices or solutions
containing mercury or mercury compounds.
Please request assistance from EH&S.

Any Overweight Container
Do not overfill the regulated medical waste containers. The boxes should be properly
closed when 2/3 full. These containers have a 40 lb. maximum weight limit.
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11.0 Transport, Packaging and Shipment of Biohazardous Materials
This section of the Handbook provides an overview of the requirements for transport of biological
materials throughout the university and shipment of infectious agents, biological products, and dry
ice. For specific information, personnel must review the Biological Materials Transport SOP in
Appendix G of this manual.
Transport of biological agents within a laboratory or building should be done in sturdy secondary
containment as to prevent any spilling and/or leakage. If the material to be transported could
puncture the primary container, a secondary, puncture-resistant container should be used. Any
equipment that is suspected of being contaminated must also be contained or decontaminated prior
to movement or service work.
Shipping Biological Materials:
For the purposes of shipping, biological materials may be classified as either infectious substances
(Category A or B), exempt patient specimens, genetically modified organisms, or biological products.
Shipments of biological materials must comply with the U.S. DOT 49 CFR Part 173. If the biological
material is shipped by air, then the shipment must comply with IATA/ICAO requirements. Some
carriers (i.e. FEDEX, etc.) may have additional requirements.
Division 6.2 Infectious substance: A material known or are reasonably suspected to contain a
pathogen. A pathogen is a micro-organism (including bacteria, viruses, rickettsiae, parasites, fungi),
or other agent such as proteinaceous infectious particle (prion) that can cause disease in humans or
animals. (U.S. DOT Hazardous Materials Regulations (HMR; 49 CFR Part 173.134.)
Category A: An infectious substance in a form capable of causing permanent disability,
life threatening or fatal disease in humans or animals when exposure to it occurs.
Category A infectious substances are shipped as infectious substances, affecting humans
(UN2814), or infectious substances affecting animals (UN2900). Indicative examples of
Category A infectious substances (i.e. Bacillus anthracis, African Swine Fever Virus, etc.) can
be found listed in the IATA Dangerous Goods Regulations. Category B infectious substances
are materials that are infectious, but do not meet the standard for inclusion in Category A.
 Category A Infectious Substances Requirements: Triple layer packaging materials used
for shipping must be tested to ensure the sample will not leak. Absorbent material must
be used and an itemized contents list must be enclosed. The outer package must bear a
Class 6.2 Infectious Substance label. A Shipper’s Declaration is also required.
Category B: An infectious substance that is not in a form generally capable of causing
permanent disability or life threatening or fatal disease in otherwise healthy humans or
animals when exposure to it occurs. This would include unscreened human blood or
human tissue. Category B infectious substances are shipped with the proper shipping name
“biological substance, category B” and are assigned to UN3373. An exempt human specimen
that has minimal likelihood of containing pathogens is exempt from many shipping
requirements. Professional judgment is used to determine if a specimen contains pathogens
and must be based on the patient’s medical history, symptoms, local conditions, and
individual circumstances. If there is more than minimal likelihood that a patient specimen
contains pathogens, it must be shipped as an infectious substance (either Category A under
UN2814 or UN2900, or Category B under UN3373).
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
Category B Infectious Substances Requirements: Triple layer packaging materials used
for transport must be tested to ensure sample will not leak. Outer package and air
waybill must bear “UN3373” and “Biological substance, category B” statement. No
Shipper’s Declaration required
Biological product is defined as products derived from living organisms that are known not to
produce viruses, toxins, etc. and are manufactured and distributed in accordance with requirements
of national government authorities. These include, but are not limited to, finished or unfinished
products such as vaccines. Biological products are not currently regulated for the purposes of
shipping.
Infectious substance or patient specimen shipments with dry ice:
The following requirements are required in addition to the above requirements for Infectious
substances or patient specimens shipped with dry ice.
Requirements:
 Never place dry ice in a sealed air tight container! The outer package must be approved
to hold dry ice. A class 9, UN 1845 Dry Ice label, including an estimated weight of the dry
ice, is affixed to the package.
Training:
U.S. DOT Hazardous Material Regulations (HMR) apply to anyone who handles, offers for transport,
transports hazardous materials, causes hazardous materials to be transported or performs any
hazmat functions related to transport in commerce (49 CFR 171.1). The US variations published in
the ICAO technical instructions are also followed in 49 CFR 171.22.
Laboratory personnel must be properly trained on transportation and shipment regulations before
shipping an infectious substance and dry ice. If you are involved in the shipment of any hazardous
materials or substances, you must receive training on the regulations and requirements prior to
shipping each class or division of hazardous materials.
Infectious Substance Shipping will need to be repeated every two years or as often as the
regulations change. This training can be taken online.

For more information, contact the Environmental Health and Safety office at 567-2955 or
visit the EH&S website, http://research.uthscsa.edu/safety/transport.shtml .
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12.0 Recombinant and Synthetic Nucleic Acid Molecules (rDNA)
Recombinant DNA (rDNA) is widely used in research, biotechnology and medicine and is used at the
UT Health Science Center. Recombinant DNA is the general name for taking a piece of one DNA
and combining it with another strand of DNA. Molecular cloning is the laboratory process used to
create recombinant DNA.
The NIH Office of Biotechnology Activities document NIH Guidelines for Research Involving
Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) specifies practices for work with
recombinant and synthetic nucleic acid molecules (rDNA). Since the UT Health Science Center
receives NIH funding, as a condition of funding, the institution complies with the NIH Guidelines.
For more information, please refer to the NIH/OSP website at:
http://osp.od.nih.gov/office-biotechnology-activities/biosafety/nih-guidelines
Definition per the NIH Guidelines Recombinant and synthetic nucleic acids are defined as:
(i) molecules that a) are constructed by joining nucleic acid molecules and b) that can replicate in a
living cell, i.e. recombinant nucleic acids;
(ii) 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
(iii) molecules that result from the replication of those described in (i) or (ii) above.
Committee Review:
All rDNA research is reviewed by the Institutional Biosafety Committee. The review includes rDNA
used in vitro and in vivo.
See the Biological Safety Policy in the Appendix A for notification, review and approval procedures.
Reporting Lab Incidents or illness, spills, noncompliance:
Lab incidents or illness, spills, and significant problems involving recombinant or synthetic nucleic
acid molecules or violations/noncompliance with the NIH Guidelines should be promptly reported
to the Biological Safety Officer in EH&S (210-567-2955) for investigation and reporting of the
incident to the National Institutes of Health Office of Science Policy (NIH/OSP), the Institutional
Biosafety Committee and other appropriate authorities.
Examples of things that should be reported include needlesticks containing recombinant or
synthetic nucleic acid molecules, the escape or improper disposition of a transgenic animal, or
spills of high-risk recombinant or synthetic materials occurring outside of a biosafety cabinet.
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13.0 Work with Viruses and Viral Vectors
Overview:
Viral vectors are increasingly being used in molecular biology as a tool to introduce a gene of
interest into human, animal and insect cells to provide transient or permanent transgene
expression. It is important to understand how to safely use these tools and the potential safety
implications of their use.
Registration for work with viruses and viral vectors
The UTHSC Institutional Biosafety Committee will review and approve all work with viral vectors
and viruses prior to commencement of the research. Additionally, approval is required for research
involving viral vectors/viruses introduced into animals. Because UTHSC receives funding from NIH
grants for research involving recombinant nucleic acids, ALL research conducted at the UTHSC
must comply with the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic
Acid Molecules (NIH Guidelines) and University policies.
Evaluating Viral vectors safety:
When evaluating the safe use of viral vectors, a number of factors need to be considered
including:
 Risk Group (RG) of the organism (virus);
 Tropism (host range);
 Route of transmission;
 Can the virus integrate into the host genome;
 Replication deficiency or replication competency
 The specific gene(s) being introduced (is the gene an oncogene or encode for a toxic
protein/product)
Virus and Viral Vectors
The most common virus and viral vectors used are:
 Baculovirus
 Adeno-associated virus (AAV)
 Adenovirus
 Epstein-Barr Virus (EBV)
 Retrovirus (MMLV)
 Lentivirus
 Poxvirus
 Herpes virus
General Containment:
Biological safety containment levels are provided for each viral vector system below. It is important
to note that in some cases, the use of a higher-containment level may be required, depending on
the specific properties of the vector and/or insert. The summary below includes information on
virology, laboratory hazards, biological safety containment procedures, Personal Protective
Equipment (PPE), disinfection, and animal use procedures.
Baculovirus:
Baculovirus is a lytic virus, primarily pathogenic for insects. Baculovirus vector systems are often
used to obtain a high level of expression of a desired protein in insect cells (Sf9 cells).
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Potential Health Hazards
Non-genetically modified wild type baculoviruses are not capable of infecting vertebrate cells and
thus do not pose any inherent hazards to laboratory workers. However, the use of mammalian
specific promoters have achieved expression of foreign genes in a wide variety of mammalian cell
lines and primary cell cultures.
LABORATORY HAZARDS:
There have been no reported lab acquired infections.
BIOSAFETY CONTAINMENT:
Work with Baculovirus may be performed at BSL-1. (BSL-2 may be needed for some work with
recombinant Baculovirus.)
PPE required for manipulation at BSL-1: lab coat, disposable gloves, eye protection for procedures
with potential splash hazard.
Adeno-Associated Virus (AAV)
AAV are parvoviridae, icosahedral, 20-25 nm in diameter, single-stranded DNA viruses with a
protein capsid. Adeno-associated viruses (AAV) require a helper virus (typically adenovirus,
herpesvirus or vaccinia) to replicate. When there is no helper virus, AAV can insert its DNA
into the host chromosome stably and remain latent. These viruses are infectious to humans with
no known disease association.
Potential Health Hazards
AAV are infectious human viruses but they are not known to cause direct disease in humans.
However, AAV may be associated with insertional mutagenesis and cancer, thereby making AAV
possibly not as safe as previously thought. The NIH Guidelines (Appendix B), state that “adeno‐
associated virus (AAV-all serotypes); and recombinant or synthetic AAV constructs, in which the
transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and
are produced in the absence of a helper virus can in most cases, be handled safety at BSL 1.
Using AAV in conjunction with Adenovirus or other helper viruses will be performed at BSL-2.
LABORATORY HAZARDS:
Inhalation of aerosolized droplets, mucous membrane contact, parenteral injection, or ingestion.
There is no specific treatment for infection with AAV.
BIOSAFETY CONTAINMENT:
Construction of AAV, which uses helper viruses, should be performed at BSL-2 within a BSC.
Once constructed, AAV may be manipulated at BSL-1.
PPE required for manipulation at BSL-1 or BSL-2: lab coat, disposable gloves, eye protection for
procedures with potential splash hazard.
ANIMAL BIOSAFETY CONTAINMENT:
Animal housing must be maintained at ABSL-1. ABSL-2 if helper virus is present.
DISINFECTION:
Adeno-Associated virus (AAV) susceptible to: 0.5% Sodium hypochlorite, 2% Glutaraldehyde,
0.25% sodium dodecyl sulfate, or Autoclave for 30 minutes at 121˚C under 15 lbs. per square inch
of steam pressure.
Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) is recommended.
Alcohol/ethanol is NOT an effective disinfectant against AAV.
Additional Information: NIH Guidelines Appendix B-I: Risk Group 1 Agents
Adenovirus
Recombinant Adenoviruses used for biomedical research are based on Adenovirus type 5. These
are medium-sized (90-100 nm), non-enveloped, icosohedral, double-stranded DNA viruses.
Virus packaged via transfection of HEK 293 cells with adenoviral-based vectors are capable of
infecting human cells. The probability of producing replication competent adenovirus (RCA)
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increases with each successive amplification. RCA is produced when adenoviral DNA recombines
with E1-containing genomic DNA in HEK 293 cells.
Potential Health Hazards
Adenoviruses often cause respiratory illness, including the common cold, bronchitis, and
pneumonia. Additional clinical symptoms include conjunctivitis (“pink eye”), cystitis, gastroenteritis
(stomach flu), tonsillitis, rash-associated illness, and rare cases of severe disease, especially in
those with compromised immune systems.
NOTE: Adenoviral vectors do not have to be replication competent to cause corneal and
conjunctival damage.
LABORATORY HAZARDS:
Inhalation of aerosolized droplets, mucous membrane contact, parenteral inoculation, or ingestion.
Adenovirus is unusually stable in the environment. Adenovirus can still be infectious following
extraction with ether and/or chloroform.
BIOSAFETY CONTAINMENT:
BSL-2
NO open-bench work
Biological Safety Cabinet (BSC) required
Eye protection for procedures with slash potential, disposable gloves, laboratory coat required in
addition to BSC.
When centrifuging adenovirus, rotors/buckets must be loaded/unloaded within the BSC and wiped
down with appropriate disinfectant prior to being removed from BSC.
ANIMAL BIOSAFETY CONTAINMENT:
Adenovirus must be administered under ABSL-2 containment with the use of a BSC.
Animals may shed/excrete adenovirus post-administration. Animals must be housed under ABSL-2
containment.
DISINFECTION:
Adenovirus susceptible to: 0.5% Sodium hypochlorite, 2% Glutaraldehyde, 5% Phenol, or
Autoclave for 30 minutes at 121˚C under 15 lbs. per square inch of steam pressure.
Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended.
Alcohol/ethanol is NOT an effective disinfectant against adenovirus.
Additional Information: Public Health Agency of Canada—Adenovirus MSDS
Epstein-Barr Virus
Epstein-Barr virus (EBV) is a member of the herpesvirus family and one of the most common
human viruses. EBV is found worldwide and most people become infected with the virus sometime
during their lives. In the United States, as many as 95% of adults between 35 and 40 years of age
have been infected. It is an icosahedral, lipid envelope double-stranded linear DNA virus sized
120-150nm in diameter. EBV has been found in the tumor cells of a heterogeneous group of
malignancies (i.e. Burkitt’s lymphoma, lymphomas associated with immunosuppression, other nonHodgkin’s lymphomas, Hodgkin’s disease, nasopharyngeal carcinoma, gastric adenocarcinoma,
lymphoepithelioma-like carcinomas, and immunodeficiency-related leiomyosarcoma). EBV is a
transforming virus and is often used to produce immortalized cell lines.
Potential Health Hazards
Infectious mononucleosis—acute viral syndrome with fever, sore throat, splenomegaly and
lymphadenopathy; lasting one to several weeks; rarely fatal.
Burkitt’s lymphoma—monoclonal tumor of B cells; typically involving children; jaw involvement also
common; hyperdemic in highly malarial areas.
Nasopharyngeal carcinoma—malignant tumor of epithelial cells of the nasopharynx; usually
involving adults between 20 and 40 years of age.
LABORATORY HAZARDS:
Inhalation of aerosolized droplets, mucous membrane contact, parenteral inoculation, or ingestion.
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BIOSAFETY CONTAINMENT:
BSL-2
NO open-bench work
Biological Safety Cabinet (BSC) required
Lab coat, disposable gloves, eye protection for procedures with potential splash hazard
required in addition to BSC.
When centrifuging EBV, rotors/buckets must be loaded/unloaded within the BSC and wiped down
with appropriate disinfectant prior to being removed from BSC.
ANIMAL BIOSAFETY CONTAINMENT:
EBV vector must be administered under BSL-2 containment. Animals must be housed as ABSL-2
containment.
DISINFECTION:
Epstein-Barr Virus is susceptible to: 0.5% sodium hypochlorite, 70% ethanol, glutaraldehyde,
formaldehyde Freshly prepared 10% household bleach (0.5% Sodium hypochlorite)
recommended.
Additional Information:
Public Health Agency of Canada—Epstein-Barr Virus MSDS
Retroviruses
Murine Leukemia Virus (MLV) Vectors or Moloney murine leukemia virus (MMLV)
Murine Leukemia Virus (MLV) is an enveloped, icosahedral, single-stranded virus with a linear
RNA genome, approximately 100nm in diameter. MLV integrates into the host genome and is
present in infected cells as a DNA provirus. Cell division is required for infection.
The host range of recombinant MLV is dependent on the specificity of the viral envelope.
 Ecotropic: The ecotropic env gene produces particles that infect only rodent cells.
 Amphotropic: Amphotropic env gene allows infection of both murine and non-murine cells,
including human cells. VSV-G envelope allows infection in a wide range of mammalian and
non-mammalian cells. Amphotropic MLV integrates into the host’s genome – this
translates into stable expression of introduced genes and the potential for insertional
mutagenesis of host genes.
LABORATORY HAZARDS:
In mice, virus is transmitted via blood from infected mother to offspring; may also occur via germ
line infection. Infection in humans appears to require direct parenteral injection with amphotropic or
pseudo typed MLV.
BIOSAFETY CONTAINMENT:
BSL-1 containment for ecotropic MLV demonstrated to be replication incompetent. PPE required
for BSL-1 work: lab coat, disposable gloves, eye protection for procedures with potential splash
hazard.
BSL-2 containment for amphotropic or pseudotyped MLV. Biological Safety Cabinet (BSC)
required.
Lab coat, disposable gloves, eye protection for procedures with potential splash hazard required in
addition to BSC.
When centrifuging MLV, rotors/buckets must be loaded/unloaded within the BSC and wiped down
with appropriate disinfectant prior to being removed from BSC.
ANIMAL BIOSAFETY CONTAINMENT:
MLV vector must be administered under BSL-2 containment.
Animals administered ecotropic MLV may be housed under ABSL-1 conditions .
Animals administered amphotropic/pseudotyped MLV must be housed under ABSL-2 conditions
for 72-hours post ad-ministration, after which animals may be moved to ABSL-1 housing.
DISINFECTION:
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MLV is susceptible to: 0.5% sodium hypochlorite, 70% ethanol, glutaraldehyde, formaldehyde,
iodine solutions containing ethanol.
Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended.
Additional Information: NIH Guidelines Appendix B-V: Animal Viral Etiologic Agents in Common
Use
Lentivirus Vectors
For work with Lentivirus, refer to the Lentivirus SOP.
http://research.uthscsa.edu/safety/Lentivirus.shtml
Lentiviruses are a subset of retroviruses that produce multi-organ diseases characterized by long
incubation periods and persistent infection. Lentivirus vector systems can include viruses of nonhuman origin as well and simian and human viruses. There are five (5) serotypes recognized,
based upon the mammalian hosts with which they are associated.
Bovine lentiviruses: Bovine immunodeficiency virus, Jembrana disease virus
Equine lentiviruses: Equine infectious anemia virus
Feline lentiviruses: Feline immunodeficiency virus
Ovine/caprine lentiviruses: Caprine arthritis-encephalitis virus, Ovine lentivirus, Visna virus
Primate lentivirus group: Human immunodeficiency virus (HIV) types 1-3, Simian AIDS retrovirus
(SRV-1), Simian immunodeficiency virus (SIV)
Unlike simple retroviruses, lentiviruses, such as HIV, are capable of integrating into host
chromosomes and infecting non-dividing cells. This feature was the major impetus for the
development of various lentiviral vector-based gene delivery systems, which can deliver large
genetic cargo to non-dividing cells in vitro and in vivo. The more recent generations of lentiviral
vectors have been designed to sufficiently diminish the possibility that recombination might occur
in a host to produce wildtype-potentially infectious virus.
Lentiviral plasmids are based on the genomes of lentiviruses. In most cases, the plasmids have
been derived from the HIV-1 genome. HIV lentiviral vectors however have been modified so they
are safe to use in research labs. When producing lentiviral particles, three components are
typically required:
1. A lentiviral vector or “transfer vector” containing the shRNA or transgene and the
flanking LTRs
2. A packaging vector or set of packaging plasmids
3. An envelope vector
The second generation system has one packaging plasmid which includes all the important
packaging components: Gag, Pol, Rev, and Tat. To produce virus, a single packaging plasmid, an
envelope plasmid, and a transfer vector are required.
The main difference in the 3rd generation system is that cotransfection with 4 plasmids, 2
packaging plasmids, an envelope plasmid, and a transfer plasmid, is required to generate
recombinant virus. The 3rd generation packaging system offers maximal biosafety but is more
cumbersome to use, as it involves the transfection of four different plasmids in the producer cells.
Potential Health Hazards
Lentiviruses are transmitted via direct exposure to infected bodily fluids. Lentiviruses persist
lifelong— being both a function of their ability to integrate into the host chromosome and ability to
evade host immunity. Lentiviruses replicate, mutate and undergo selection by host immune
responses. The clinical manifestation of infection includes non-specific symptoms such as
lymphadenopathy, anorexia, chronic diarrhea, weight loss, fever, and fatigue.
LABORATORY HAZARDS:
Direct contact with skin and mucous membranes, parenteral inoculation, ingestion.
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BIOSAFETY CONTAINMENT:
BSL-2 for generating and manipulating lentivirus vectors. BSL-2 for Viruses (with gene of interest)
ordered from company.
NO open-bench work
Biological Safety Cabinet (BSC) required
Disposable gloves, laboratory coat, eye protection for procedures with splash hazard required in
addition to BSC.
When centrifuging lentivirus, rotors/buckets must be loaded/unloaded within the BSC and wiped
down with appropriate disinfectant prior to being removed from BSC.
ANIMAL BIOSAFETY CONTAINMENT:
Lentiviral vector(s) and transduced cell lines are administered under BSL-2 containment.
Animals must be housed under ABSL-2 conditions for 72 hours during this period, after which
animals may be moved to ABSL-1 housing.
DISINFECTION:
Susceptible to: 0.5% sodium hypochlorite, 2% glutaraldehyde, formaldehyde, ethanol
Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended
Additional Information: NIH Biosafety Considerations for Research with Lentiviral Vectors
Poxvirus
Poxviruses are the largest known DNA viruses and are distinguished from other viruses by their
ability to replicate entirely in the cytoplasm of infected cells. The core contains a 200 -kilobase
(kb), double-stranded DNA genome and is surrounded by a lipoprotein core membrane.
Poxviruses do not require nuclear factors for replication, and thus, can replicate with little
hindrance in enucleated cells.
Recombinant vaccinia vectors-Vaccinia virus can accept as much as 25kb of foreign DNA, making
it useful for expression large eukaryotic and prokaryotic genes. Foreign genes can be stably
integrated into the viral genome, resulting in efficient replication and expression of biologically
active molecules.
Vaccinia is used to generate live recombinant vaccine for the treatment of other illnesses.
Potential Health Hazards
Vaccinia virus normally has no serious health effects in humans, although it can cause disease of
the skin when used to inoculate against the smallpox virus . Accidental infection with the virus can
occur through contact between the vaccination lesion and broken skin (inadvertent inoculation).
Serious complications such as ocular vaccinia, myopericarditis, eczema vaccinatum (a papular,
vesicular and pustular rash that is very infectious, 38 cases per million doses), progressive
vaccinia (progressive necrosis at the vaccination site, 3 cases per million doses), post-vaccinial
CNS disease (headache, lethargy, seizures and coma, 12 cases per million doses), fetus
malformations and abortion (very rare) sometimes occur after vaccination.
LABORATORY HAZARDS:
Direct contact with skin and mucous membranes, parenteral inoculation, ingestion, inhalation of
aerosols
BIOSAFETY CONTAINMENT:
BSL-2
NO open-bench work
Biological Safety Cabinet (BSC) required
Disposable gloves, laboratory coat, eye protection for procedures with splash hazard required in
addition to BSC.
When centrifuging poxvirus, rotors/buckets must be loaded/unloaded within the BSC and wiped
down with appropriate disinfectant prior to being removed from BSC.
ANIMAL BIOSAFETY CONTAINMENT:
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Poxvirus vectors can be administered under BSL2 containment.
Animals must be housed under ABSL-2 conditions for 72 hours following infection, after which
animals may be moved to ABSL-1 housing.
DISINFECTION:
Susceptible to: 0.5% sodium hypochlorite, 2% glutaraldehyde, formaldehyde, ethanol.
Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended.
Herpesvirus Vectors
Herpes Simplex Virus (Types I and II) are icosahedral, lipid enveloped, double-stranded linear
DNA viruses approximately 110-200nm in diameter. HSV types I and II can be differentiated
immunologically. HSV-I causes herpes gingivostomatitis; whereas HSV-II causes herpes genitalis,
or genital herpes. HSV-derived vectors are unique in that the vectors have a broad host cell range
and cell tropism in dividing and non-dividing cells, able to infect almost every cell type in most
vertebrates that have been examined.
Potential Health Hazards
Oral herpes—primary infection is typically mild and occurs in early childhood; reactivation of latent
infection results in fever blisters or cold sores, usually on the face and lips, which crust and heal
within a few days; Possible CNS involvement (meningoencephalitis), 70% mortality rate if left
untreated; causes approximately 2% of acute pharyngotonsillitis.
Genital herpes—sexually transmitted, associated with aseptic meningitis; vaginal delivery may
pose risk to newborn (encephalitis and death).
Both HSV-I and HSV-II are capable of infecting the genital tract or oral mucosa.
LABORATORY HAZARDS:
Inhalation of aerosolized droplets, mucous membrane contact, parenteral inoculation, or ingestion.
Only treatment available is anti-viral drug therapy for symptoms.
BIOSAFETY CONTAINMENT:
BSL-2
NO open-bench work
Biological Safety Cabinet (BSC) required
Lab coat, disposable gloves, eye protection for procedures with potential splash hazard
required in addition to BSC.
When centrifuging HSV, rotors/buckets must be loaded/unloaded within the BSC and wiped down
with appropriate disinfectant prior to being removed from BSC.
ANIMAL BIOSAFETY CONTAINMENT:
HSV vector must be administered under BSL-2 containment. Animals must be housed at ABSL-2
containment.
DISINFECTION:
HSV is susceptible to: 0.5% sodium hypochlorite, 70% ethanol, glutaraldehyde, formaldehyde,
iodine solutions containing ethanol
Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended.
Additional Information: Public Health Agency of Canada—
Replication Competency Testing
A majority of viral vectors used today are disabled such that replication competent viruses are not
readily formed by any biological process that might occur in normal hosts. EH&S and the IBC
encourages the use of such vectors in all relevant applications. In particularly sensitive
applications, demonstrating that the viral stock used has no apparent contamination with
replication competent vectors is essential. Of course, assays for replication competence will never
be perfect or absolute, so the IBC asks that researchers use a current procedure of demonstrated
sensitivity and specificity.
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If ordering a pre-made viral vector with your gene of interest, the company or university vector core
should have quality control procedures and testing in place to ensure replication incompetent virus.
It is the responsibility of the PI to check with the company on these quality control measures.
Below is a summary guide of the current IBC recommendations for common classes of vectors.
If another procedure or reference method is used to accomplish the same conclusion, researchers
are asked to submit that procedure and published article with their IBC registration. Even more
rigorous testing may be required in some instances, such as a vector bearing a pathogenic gene
or in human gene therapy, or in any materials that could be released in the environment.
When completing your IBC application, you should not check off that your vector has been tested
for replication competency, unless you have documentation for this assay.
If working with:
Adenovirus
Adeno-associated virus (with
adenovirus helper)
Adeno-associated virus
(Adenovirus-free)
Lentivirus
Retrovirus
(ecotrophic and amphotrophic)
Vaccinia
Environmental Health and Safety Department
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Test for replication competency by:
PCR for E1a prior to use. Confirmation of absence of RCV
must be documented by researcher prior to use in animals.
PCR prior to use. Confirmation of absence of RCV must be
documented by researcher prior to use in animals.
Testing not required.
ELISA assay for p24 antigen.
Confirmation of absence of RCV must be documented by
researcher prior to use in animals.
Amplification in a permissive cell line followed by screening
by appropriate detection assay. Confirmation of absence of
RCV must be documented by researcher prior to use in
animals.
Not applicable due to its use as a replicating vector.
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Viral Vector
Risk
Group
Biosafety
Level
Special
Requirements
In
Vivo
ABSL-2
Disinfectant
2
BSL-2
Adeno-Associated
virus (AAV)
2
BSL-1 /
BSL-2
ABSL-1 / ABSL-2
2
BSL-2
ABSL-2
Herpes Virus HSVI and HSV-II
2
BSL-2
ABSL-2
Retroviruses /
Murine Leukemia
virus (MLV)
1/2
BSL-1 /
BSL-2
ABSL-1 / ABSL-2
Lentivirus
2
BSL-2
ABSL-2
AAV must be
constructed under BSL-2
containment; once
constructed, AAV may
be handled at BSL-1
containment.
Animals must be housed
under ABSL-1
containment; if helper
virus is present, ABSL-2
containment.
Epstein-Barr viral vectors
must be administered to
animals under ABSL-2
containment.
Animals must be housed
under ABSL-2
containment.
HSV vectors must be
administered to animals
under ABSL-2
containment.
Animals must be housed
under ABSL-2
containment.
Ecotropic murine
retroviruses may be
handled at BSL-1 /
ABSL-1.
Amphotrophic or pseudo
typed murine
retroviruses must be
handled at BSL-2 /
ABSL-2 for 72-hours
post administration.
Lentiviral vector must be
administered to animals
under ABSL-2
containment.
Animals housed under
ABSL-2 containment for
72-hours post
administration.
Epstein-Barr virus
In
vitro
Adenovirus
Adenoviral vector must
be administered to
animals under ABSL-2
containment.
Animals housed under
ABSL-2 containment.
Freshly prepared 10%
sodium hypochlorite
recommended
disinfectant.
Alcohol not effective
disinfectant against
adenovirus.
Freshly prepared 10%
sodium hypochlorite
recommended
disinfectant.
Alcohol not effective
disinfectant against AAV.
Freshly prepared 10%
sodium hypochlorite
recommended
disinfectant.
Freshly prepared 10%
sodium hypochlorite
recommended
disinfectant.
Freshly prepared 10%
sodium hypochlorite
recommended
disinfectant.
Freshly prepared 10%
sodium hypochlorite
recommended
disinfectant.
References:
UNC- Chapel Hill,
Univ. of Wisconsin,
Stanford University
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14.0 Toxins of Biological Origin
Biological toxins are toxins derived of biological origin. Biological toxins are produced by certain
bacteria, fungi, protozoa, plants, reptiles, amphibians, fish, echinoderma, mollusks, and insects but
can also be produced by synthetic means. Some biological toxins are extremely toxic and can
cause death or incapacitation from exposure at very low levels.
Select Agent Toxins:
Some biological toxins are regulated as Select Agent toxins under federal regulations, HHS and
USDA Select Agents and Toxins 7 CFR Part 331, 9 CFR part 121, and 42 CFR Part 73.
(http://www.cdc.gov/od/sap/ or http://www.aphis.usda.gov/vs/ncie/bta.html )
These toxins are regulated due to their potential to pose a severe threat to public health and
safety. Possession, use, and transfer of these toxins are highly regulated.
A complete list of select agent toxins can be found at http://www.cdc.gov/od/sap/docs/salist.pdf.
In small quantities, some of these toxins are exempt from select agent registration. See the table
below.
Note: The possession, use, transfer or destruction of ANY select agent toxin, IN
ANY QUANTITY, must be approved through the Environmental Health and Safety
office. For questions regarding work with toxins, call 567-2955.
The following toxins are not regulated if the amount under the control of a Principal Investigator,
treating physician or veterinarian, or commercial manufacturer or distributor does not exceed, at any
time, the amounts indicated in the table below.
However, Principal Investigators working with exempt amounts are still required to register with the
Environmental Health and Safety office prior to ordering the toxin. Work with exempt quantities of
toxin will be reviewed by the Chemical Safety Committee and depending on the use also by the
Institutional Biosafety Committee.
Exempt Amounts of Select Agent Toxins Permissible per Principal Investigator
HHS Toxins
Abrin
Botulinum neurotoxins
Short paralytic alpha Conotoxins
Diacetoxyscirpenol (DAS)
Ricin
Saxitoxin
Staphylococcal enterotoxins (Subtypes A, B, C, D, and E)
T-2 toxin
Tetrodotoxin
Environmental Health and Safety Department
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Amount
100 mg
0.5 mg
100 mg
1000 mg
100 mg
100 mg
5 mg
1,000 mg
100 mg
Page 75
Work with Select Agent Toxins exceeding the exempt amount.
Investigators that want to work with toxin amounts exceeding the listed exempt amounts must be
registered and approved with the Select Agent Program, the Responsible Official for the
UTHSCSA and the Institutional Biosafety Committee prior to work with the toxin.
Working with Biological Toxins
Work with most toxins, in quantities routinely used in research, can be safely performed by the
researcher with minimal risk to the worker and the environment by following established
procedures for work with hazardous chemicals. Since toxins do not replicate, it is difficult for
person to person transmission.
Because some toxins can be extremely hazardous, even in minute quantities, biological toxins
require strict safeguards against their inhalation, absorption through skin or mucous membranes
(typically due to a splash), ingestion, or percutaneous injury. A chemical hygiene plan and Safety
Data Sheet (SDS) specific for the toxin must be on hand in the laboratory for inspection.
Key points of the guidelines are:
1. Written safety protocols to cover the use of the specific toxin(s) in use.
2. Specific training of personnel including the risk, work practices and post-exposure
procedures.
3. Security measures in place to protect against unauthorized access to toxin(s)
4. Inventory control system in place
5. Written plan for toxin-related emergencies (spill, exposure, etc) posted
6. BSL-2 or BSL-3 containment and practices in use.
Disposal of Biological Toxins
Specific inactivation and disposal requirements should be in place for acute biological toxins.
Some toxins are quite resistant to conventional methods of inactivation. These agents cannot
simply be placed in an autoclave.
All unused toxin or waste must be collected by the Environmental Health and Safety office,
Division of Environmental Protection for disposal as hazardous waste. Call 567-2955 for more
information or complete an online disposal request.
Toxins classified as select agents, even if in exempt amounts, require that someone from EH&S
observe and document the destruction of these agents.
For toxins introduced into animals, the PI must follow the disposal procedures approved by the
Chemical Safety Committee, the IACUC and DLAR.
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15.0 Integrated Pest Management (IPM)
Integrated Pest Management Overview.
Pest management is an integral part of managing the research laboratories, clinical facilities, and
laboratory animal facilities at the UT health Science Center. Pests such as cockroaches, flies, and
other pests can compromise the integrity of the research and clinical environment and transmit
diseases. The Health Science Center has adopted an integrated pest management (IPM) planning
process. This process utilizes internal and external expertise and laboratory engineering
considerations to monitor, abate, and control pest related issues in laboratories.
An integrated process involves the researcher/clinician, Lab Animal Resources, housekeeping
services, facilities management maintenance, and contracted pest control services. The primary
goal of IPM is to prevent pest problems by managing the facility environment in such a way as to
make it less conducive to pest infestation. IPM is site specific based on the type of facility and
what the needs and goals are of that space. IPM also considers the cost and effectiveness of the
program.
A contracted pest management company in consultation with Facilities Management staff and the
investigator assists, monitors, and develops strategies and practices to abate pest related issues
in the research laboratory. The most common approach to pest control has been the application of
pesticides, either as a preventive or remedial measure.
The Health Science Center has contracted services to provide pest management through the
application of pesticides on a routine basis. The monitoring of pest issues through the use of traps,
visual inspections, and staff interviews are used to identify areas and conditions that may foster
pest activity.
Many pest problems can be prevented or corrected by using proper sanitation, reducing clutter and
pest habitat, and by performing repairs that exclude pests and reduce pest habitat. Pest control
methods such as trapping, exclusion, caulking, washing, and freezing can be applied safely and
effectively when used in conjunction with proper sanitation and structural repair.
Maintaining records of structural deficiencies and housekeeping conditions can help to track
problems and determine if corrective actions are completed in a timely manner.
Monitoring is the central activity of an IPM program and is used in place of preventive pesticide
treatments. If the laboratory or clinical area is experiencing a pest problem, it should be reported to
Facilities Management at 567-2885.
Recordkeeping: Facilities Management/Custodial Services will maintain a logbook to record pest
activity and conditions pertinent to the IPM program. The logbook may contain protocols and
procedures for IPM services in that facility, Safety Data Sheets on the pesticides used, pesticide
labels, treatment records, floor plans and survey reports.
Environmental Health and Safety Department
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REFERENCES
1. Bodily, H.L. “Diagnostic Procedures for Bacterial, Mycotic and Parasitic Infections.” General
Administration of the Laboratory. Eds. H.L. Bodily, H.L., E. L. Updyke, and J.O. Mason. New
York: American Public Health Association, 1970. pp. 11-28.
2. Collins, C. H., E.G. Hartley and R. Pilsworth. The Prevention of Laboratory Acquired Infection.
Public Health Laboratory Service, Monograph Series No. 6, 1974.
3. Darlow, H.M. “Methods in Microbiology.” Safety in the Microbiological Laboratory. Eds. J.R.
Norris and D.W. Robbins. Academic Press, Inc., 1969. pp. 169-204.
4. Hellman, A., M. N. Oxman, and R. Pollack (ed.). Biohazards in Biological Research.
Cold Spring Harbor Laboratory, 1973.
5. Steere, N.V. (ed.). CRC Handbook of Laboratory Safety. Second Edition, The Chemical Rubber
Company, Cleveland, 1971.
6. United States. Department of Health, Education and Welfare. Laboratory Safety at the Center
for Disease Control. Publication No. CDC 75-8118. Washington: GPO, September 1974
7. United States. Department of Health, Education and Welfare, Public Health Service,
National Institutes of Health, National Institutes of Health Biohazards Safety Guide.
U.S. Government Printing Office, Stock No. 1740-00383. Washington GPO, 1974.
8. United States. U.S. Department of Health and Human Services, Public Health Service. Center
for Disease Control and Prevention and National Institutes of Health. Biosafety in
Microbiological and Biomedical Laboratories, 5th edition. Dec. 2009.
9. United States. Federal Register, April 15, 2016 (81 FR 22286). NIH Guidelines for
Research Involving Recombinant or Synthetic Nucleic Acid Molecules. April 2016.
Environmental Health and Safety Department
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Page 78
APPENDIX A
Institutional Biosafety Committee (IBC) Procedures and Registration of
Biohazardous Materials and Recombinant and Synthetic Nucleic Acid Molecules
Principal Investigators (PIs) proposing research projects involving recombinant or synthetic nucleic
acid molecules (rDNA) or potentially biohazardous agents, clinical specimens, etc., must submit
appropriate forms to Environmental Health and Safety/Biological Safety Program. All projects
conducted with rDNA and/or Biohazardous agents at the UTHSCSA must be registered with the
IBC; there are no exempt experiments or activities.
It is strongly suggested that the PI or faculty member consult with the EH&S/ Biological Safety
Division during the planning stage of a proposal involving biological agents.
Note: Core laboratories (Flow cytometry, sequencing, etc.) may submit their procedures for
review by the IBC. These laboratories are expected to comply with the current BMBL. PIs
submitting biohazardous materials or rDNA to the core labs shall be approved through the
IBC.
A. Projects requiring review:
1. Recombinant or Synthetic Nucleic Acid Molecules (rDNA) Projects (Genetically Modified,
Engineered, Transgenic, Cloned, etc.)
The PI (researcher or instructor) of each research project or the faculty member for each
course proposing to carry out projects involving rDNA molecules must complete an
Application to Use Biohazardous Materials. Project may begin after approval from IBC.
2. Projects involving Biohazardous agents (Chlamydia, Fungi, Bacteria, Prions, Virus, Protozoan,
Rickettsia, etc.)
All PIs must register their activities with the Institutional Biosafety Committee by completing an IBC
application. Activity may start only after authorization from the IBC.
Note: Work with laboratory strains of E. coli must also be registered as this typically involves rDNA.
Select Agents: Certain restricted biological agents and toxins have been designated as "Select
Agents" by the Centers for Disease Control and Prevention and US Department of Agriculture.
Possession, use and transfer of these restricted agents must be registered with the Select Agent
Program with the CDC or the USDA and be approved by the Institutional Biosafety Committee and
the Responsible Official prior to obtaining these agents. The Biological Safety Program is responsible
for coordinating the federal registration process for all UTHSCSA investigators.
3. Plant and Plant Tissue Cultures
All proposals involving the use of genetically modified plants or plant tissue cultures must be
registered and approved by the IBC.
Note: Certain plant or plant tissue cultures may require APHIS-USDA, State and international
permits. No work can be done with DOA Prohibited Listed Plant species.
4. Projects involving Animals
Projects involving the introduction of a biological agent, rDNA, cells/cell lines, or tissue into animals
must register with the IBC.
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Some work with transgenic animals (purchasing and breeding) are exempt from the NIH Guidelines.
However, PIs must still complete the Transgenic/Knockout Animal exemption form and return the
form to EH&S for review.
All work with animals must be approved by the Institutional Animal Care and Use Committee
(IACUC).
Any experiment involving both animals and biological agents will be classified and reviewed at the
appropriate level. The Vertebrate Animal Biosafety Level recommendations as given in the latest
edition of “Biosafety in Microbiological and Biomedical Laboratories” will be used as a guide for the
Investigator. The committee will cooperate with the Institutional Animal Care and Use Committee
(IACUC) on all experiments involving both animals and biological agents/rDNA.
5. Human Subjects
Projects involving the use of human subjects must be registered with the Institutional Review Board
(IRB).
Human clinical trials that involve the introduction of an infectious or potentially infectious agent and/or
genetically modified biological agent into human subjects is reviewed by the IBC and the IRB.
PIs who want to work with existing specimens including de-identified specimens should also go
through the IRB to verify that the work is considered “non-human research” according the
regulations. Examples of “non-human research” include repository research, tissue banking and
databases, anonymous pre-existing data sets or specimens, coded pre-existing coded prospective
data or specimens.
Work with human tissues, organs, blood and body fluids, and cells should also be registered with
the IBC.
Note: Work with primary cells ordered from a company is also reviewed by the IBC but does not
require IRB review.
6. Human Cell Lines
Work with immortalized human cell lines must be registered with the IBC.
7. Mixed Use
Investigators conducting research involving the concurrent use of radioactive or other toxic materials
(i.e., toxins, chemo agents etc.) or environmental release of biological agents must register their
proposal with Environmental Health and Safety and be approved by the appropriate committees.
a. Use of Radioactive materials: Call the Radiation Safety Division at 567-2955.
b. Use of toxic/carcinogenic chemicals: Contact the Chemical Safety Division at 567-2955.
c. Production of Hazardous Wastes: Contact the Environmental Protection Division at 5672955.
d. Release into the Environment: Call Environmental Health and Safety office at 567-2955.
8. Human Gene Transfer
Human Gene Transfer/Therapy Research involves the deliberate transfer of recombinant DNA, or
DNA or RNA derived from recombinant DNA, into human subjects. Both Institutional Review Board
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(IRB) and the Institutional Biosafety Committee (IBC) must authorize the protocol. Consult the NIH
Guidelines, Appendix M for more information.
9. Human Embryonic Stem Cell, Germ Cells and Stem Cell-Derived Test Articles
All research using human embryonic stem cells, germ cells and pluripotent cells must be authorized
by the Institutional Review Boards - Committee on Human Subjects (primary compliance) and IBC
(review for import, training, and containment).
10. Soils
The importation, movement, transportation, or exportation of soil requires an USDA permit.
11. Toxins
All use of biological toxins must be approved by the Chemical Safety Committee. Use of toxins that
are select agents must also be reviewed by the Biological Safety Division. Quantities of select agent
toxins that exceed the exempted amount (see chapter 14.0) must be registered through the Select
Agent Program and be approved by the Institutional Biosafety Committee and Responsible Official.
12. Dual Use Research
Dual use research encompasses biological research with legitimate scientific purpose, the results
of which may be misused to pose a biologic threat to public health and/or national security. The
National Science Advisory Board for Biosecurity (NSABB) will assist in the development of
guidance for identifying and overseeing dual use research and research results.
The oversight system for dual use research is likely to be complementary to that of Select Agents
in that it will be based on principles and practices for reducing the likelihood that biological
organisms, knowledge, and technologies are intentionally misused to pose a risk to public health
and national biosecurity.
Dual use research is reviewed by the Institutional Review Entity (IRE) and the Institutional
Biosafety Committee.
B. Eligibility
The PI of a research protocol must be a UTHSCSA faculty member. If the PI is not a faculty
member, a Co-Investigator shall be listed who is a faculty member. Under special circumstances a
scientist from another institution may be eligible to use biological agents at the UTHSCSA with the
approval of the Dean or Department Chair. All applicable approvals must be obtained prior to
starting their research. Graduate students’ proposal are under the authority of the PI.
C. Submission of IBC Applications
Registration forms are submitted to the Environmental Health and Safety office, Biological Safety
Division, for pre-review a minimum of three weeks prior to the next IBC meeting. The IBC
application is sent to the IBC approximately 1-2 weeks prior to the meeting to allow sufficient time
for review by the committee members.
D. Accession Number
All protocols are assigned an accession number.
E. IBC Review
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All protocols submitted to the Biological Safety Division are reviewed.
As per UTHSCSA policy, there are no exempt protocols with the exception of some
transgenic/knockout mice purchases/breeding.
The IBC will review each application for:

Research for conformity with the NIH Guidelines (if applicable) and BMBL, 5th ed.

Potential risk to environment and public health (risks to close contacts, health care
workers, and the community, as well as to individual research participants)

Containment levels per NIH Guidelines (if applicable) and BMBL, 5th ed.

Adequacy of facilities, equipment, personal protective equipment, and procedures

PI and other personnel experience and training, medical surveillance

Dual use

For Gene Transfer protocols, trial design, biosafety and containment, and compliance with
NIH Guidelines, Appendix M.
F. Expedited Review
The Institutional Biosafety Committee does not use expedited review since the NIH Guidelines
requires a formal vote at a convened meeting.
G. Committee Meeting and Authorization
A quorum of members are required to be in attendance to vote on items requiring a vote such as
approval of the meeting minutes and IBC application approval/approval with clarifications/tabled.
Per the NIH Guidelines, the meetings are open meetings.
A quorum is defined as 50% or more of the voting committee membership.
In some cases, the Principal Investigators may be asked to attend the IBC meeting to answer
questions about their respective protocols. This is highly recommended especially for NIH
regulated environmental release, and moderate-high risk protocols.
Email is an important tool to aid the IBC in conducting certain activities. For example, it is
acceptable for institutions to use email for distribution of protocol materials, to conduct pre-meeting
reviews, to poll members about particular matters, and other similar tasks.
Conflict of interest: Individuals with a conflict of interest in the application presented must leave the
room during discussion and voting on the application.
The IBC reviews each application and votes to approve, approve pending clarification or tabled
(not approved). A simple majority of the voting committee members in attendance is necessary to
take action. Investigators listed on the protocol may be present for presentation of the application
but must leave the room for the final discussion and vote.
H. Executive Session
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At the discretion of the IBC, discussions may be held in closed sessions to protect the intellectual
property of the investigator. In such cases the principal investigator, location of the facility and
biological commodities used may be considered as confidential.
J. Minutes
As prescribed by NIH Office of Biotechnology Activities, minutes of the convened IBC meeting will
reflect:
a. Date and time of the meeting,
b. Individuals who attended,
c. Whether the minutes of the prior meeting where approved,
d. All major motions (including motions to approve, approve with clarification, or table an
application)
e. Major points of order
f.
Time of adjournment.
The minutes will contain sufficient detail to serve as a record of major points of discussion and the
committee's rationale for particular decisions, documenting that the IBC has fulfilled its review and
oversight responsibilities as outlined in Section IV-B-2B of the NIH Guidelines.
Minutes will be redacted to protect proprietary or private information (trade secrets, confidential
commercial information, personal telephone numbers, home addresses or other information that
may compromise institutional or national security.)
K. Final Authorization
Once an application is approved, the IBC sends a Letter of Authorization to the Principal
Investigator stating what they are approved for and any conditions/stipulations for approval. IBC
approvals are for five years. However, additional audits and inspections of the facility may be
required in addition to mandated annual inspections. If a protocol is tabled, an email will be sent to
the PI detailing why the application was tabled (not approved).
If a protocol is approved pending clarifications, the Chairperson or BSO may be authorized to
provide final approval once the revised protocol incorporates the suggested conditions.
Copies of the Letter of Authorization may be forwarded to the Institutional Animal Care Program
(IACP) or IRB as appropriate.
L. Application Modifications (amendments)
1. Rather than submitting a new application during the five year approval period, a PI with an
approved application may submit an amendment to make changes such as but not limited to:

Addition or deletion of personnel,

Addition or deletion of laboratory facilities,

Addition or deletion of biological agents,

Addition or deletion of recombinant or synthetic nucleic acids (host, plasmids, genes)
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
Addition or deletion of animal protocols where biological agents and/or recombinant or
synthetic nucleic acids are introduced into animals

Addition/deletion/modification of procedures
Depending upon the type of modification, an amendment may be administratively approved
or may go to full committee review.
M. Continuing Review of Applications
Every five years, each PI will submit a renewal application. The renewal application will be
reviewed by the IBC.
N. Suspension
An authorized application can be suspended with just cause by action of the Biological Safety
Officer (BSO) or Institutional Biosafety Committee. A just cause may include any significant
problems or violations of the NIH Guidelines or significant research related accidents or illnesses,
which are reportable to NIH/OSP. Suspended applications will remain suspended until an
investigation has been completed, the deficiencies have been corrected and the IBC votes to lift
the suspension.
O. Appeal
An appeal to an IBC decision must be in writing and addressed to the IBC. The appeal must be
specific for the concerns addressed in the "approve pending modification", "suspension" or "denial"
memorandum. Appeals will be reviewed and a decision will be rendered at the next scheduled IBC
meeting.
Any investigator who believes that a biological agent has been improperly classified may request a
reconsideration of the classification. The request should be submitted to the BSO or the Chair of the
Institutional Biosafety Committee and should contain a suggestion for alternative classification and
documentation in support of that suggestion. After considering the new information, the IBC will then
inform the investigator of its decision. If the investigator does not agree with the decision of the full
Committee, the matter will be referred for final decision to the Executive Vice President for Facilities
Planning and Operations.
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APPENDIX B
RISK GROUPS:
CLASSIFICATION OF BIOHAZARDOUS AGENTS ON THE BASIS OF HAZARD
This appendix reflects the current state of knowledge and should be considered a resource
document. Included are the more commonly encountered agents and is not meant to be allinclusive. Classification of Risk Groups is provided in the NIH Guidelines for Research involving
Recombinant and Synthetic Nucleic Acid Molecules (NIH Guidelines).
http://osp.od.nih.gov/office-biotechnology-activities/biosafety/nih-guidelines
Information on agent risk assessment may be found in the Agent Summary Statements of the
current CDC/NIH publication, Biosafety in Microbiological and Biomedical Laboratories.
Risk Group 1 (RG1) Agents
RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents include
asporogenic Bacillus subtilis or Bacillus licheniformis (see Appendix C-IV-A, Bacillus subtilis or Bacillus
licheniformis Host-Vector Systems, Exceptions); adeno- associated virus (AAV – all serotypes); and recombinant
or synthetic AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene
product or a toxin molecule and are produced in the absence of a helper virus. A strain of Escherichia
coli(see Appendix C-II-A, Escherichia coli K-12 Host Vector Systems, Exceptions) is an RG1 agent if it (1) does
not possess a complete lipopolysaccharide (i.e., lacks the O antigen); and (2) does not carry any active virulence
factor (e.g., toxins) or colonization factors and does not carry any genes encoding these factors.
Those agents not listed in Risk Groups (RGs) 2, 3 and 4 are not automatically or implicitly classified
in RG1; a risk assessment must be conducted based on the known and potential properties of the
agents and their relationship to agents that are listed.
Risk Group 2 (RG2) Agents
BACTERIAL AGENTS including Chlamydia
--Acinetobacter baumannii (formerly Acinetobacter calcoaceticus)
--Actinobacillus
--Actinomyces pyogenes (formerly Corynebacterium pyogenes)
--Aeromonas hydrophila
--Amycolata autotrophica
--Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum)
--Arizona hinshawii - all serotypes
--Bacillus anthracis
--Bartonella henselae, B. quintana, B. vinsonii
--Bordetella including B. pertussis
--Borrelia recurrentis, B. burgdorferi
--Burkholderia (formerly Pseudomonas species) except those listed in Appendix B-III-A (RG3))
--Campylobacter coli, C. fetus, C. jejuni
--Chlamydia psittaci, C. trachomatis, C. pneumoniae
--Clostridium botulinum, C. chauvoei, C. haemolyticum, C. histolyticum, C. novyi, C. septicum, C. tetani
--Coxiella burnetii – specifically the Phase II, Nine Mile strain, plaque purified, clone 4
--Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale
--Dermatophilus congolensis
--Edwardsiella tarda
--Erysipelothrix rhusiopathiae
--Escherichia coli - all enteropathogenic, enterotoxigenic, enteroinvasive and strains bearing K1 antigen,
including E. coli O157:H7
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--*Francisella tularensis specifically *F. tularensis subspecies novicida [aka F. novicida], strain Utah
112; *F. tularensis subspecies holarctica LVS; *F. tularensis biovar tularensis strain ATCC 6223 (aka strain
B38)
*For research involving high concentrations, BL3 practices should be considered (see Appendix G-II-C2. Special Practices (BL3)).
--Haemophilus ducreyi, H. influenzae
--Helicobacter pylori
--Klebsiella - all species except K. oxytoca (RG1)
--Legionella including L. pneumophila
--Leptospira interrogans - all serotypes
--Listeria
--Moraxella
--Mycobacterium (except those listed in Appendix B-III-A (RG3)) including M. avium complex, M. asiaticum,
M. bovis BCG vaccine strain, M. chelonae, M. fortuitum, M. kansasii, M. leprae, M. malmoense, M.
marinum, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi
--Mycoplasma, except M. mycoides and M. agalactiae which are restricted animal pathogens
--Neisseria gonorrhoeae, N. meningitidis
--Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. transvalensis
--Pseudomonas aeruginosa
--Rhodococcus equi
--Salmonella including S. arizonae, S. choleraesuis, S. enteritidis, S. gallinarum-pullorum, S. meleagridis, S.
paratyphi, A, B, C, S. typhi, S. typhimurium
--Shigella including S. boydii, S. dysenteriae, type 1, S. flexneri, S. sonnei
--Sphaerophorus necrophorus
--Staphylococcus aureus
--Streptobacillus moniliformis
--Streptococcus including S. pneumoniae, S. pyogenes
--Treponema pallidum, T. carateum
--Vibrio cholerae, V. parahaemolyticus, V. vulnificus
--Yersinia enterocolitica
--Yersinia pestis specifically pgm(–) strains (lacking the 102 kb pigmentation locus) and lcr(–) strains (lacking
the LCR plasmid)
FUNGAL AGENTS
Blastomyces dermatitidis
--Cladosporium bantianum, C. (Xylohypha) trichoides
--Cryptococcus neoformans
--Dactylaria galopava (Ochroconis gallopavum)
--Epidermophyton
--Exophiala (Wangiella) dermatitidis
--Fonsecaea pedrosoi
--Microsporum
--Paracoccidioides braziliensis
--Penicillium marneffei
--Sporothrix schenckii
--Trichophyton
PARASITIC AGENTS
Ancylostoma human hookworms including A. duodenale, A. ceylanicum
--Ascaris including Ascaris lumbricoides suum
--Babesia including B. divergens, B. microti
--Brugia filaria worms including B. malayi, B. timori
--Coccidia
--Cryptosporidium including C. parvum
--Cysticercus cellulosae (hydatid cyst, larva of T. solium)
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--Echinococcus including E. granulosis, E. multilocularis, E. vogeli
--Entamoeba histolytica
--Enterobius
--Fasciola including F. gigantica, F. hepatica
--Giardia including G. lamblia
--Heterophyes
--Hymenolepis including H. diminuta, H. nana
--Isospora
--Leishmania including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana, L. peruviana, L. tropica
--Loa loa filaria worms
--Microsporidium
--Naegleria fowleri
--Necator human hookworms including N. americanus
--Onchocerca filaria worms including, O. volvulus
--Plasmodium including simian species, P. cynomolgi, P. falciparum, P. malariae, P. ovale, P. vivax
--Sarcocystis including S. sui hominis
--Schistosoma including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi
--Strongyloides including S. stercoralis
--Taenia solium
--Toxocara including T. canis
--Toxoplasma including T. gondii
--Trichinella spiralis
--Trypanosoma including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cruzi
--Wuchereria bancrofti filaria worms
VIRUSES
Adenoviruses, human - all types
Alphaviruses (Togaviruses) - Group A Arboviruses
--Chikungunya vaccine strain 181/25
--Eastern equine encephalomyelitis virus
--Venezuelan equine encephalomyelitis vaccine strains TC-83 and V3526
--Western equine encephalomyelitis virus
Arenaviruses
--Junin virus candid #1 vaccine strain
--Lymphocytic choriomeningitis virus (non-neurotropic strains)
--Tacaribe virus complex
--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I
through IV)
Bunyaviruses
--Bunyamwera virus
--Rift Valley fever virus vaccine strain MP-12
--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I
through IV)
Caliciviruses
Coronaviruses
Flaviviruses - Group B Arboviruses
--Dengue virus serotypes 1, 2, 3, and 4
--Japanese encephalitis virus strain SA 14-14-2
--Yellow fever virus vaccine strain 17D
--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I
through IV)
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Hepatitis A, B, C, D, and E viruses
Herpesviruses - except Herpesvirus simiae (Monkey B virus) (see Appendix B-IV-D, Risk Group 4 (RG4) Viral Agents)
--Cytomegalovirus
--Epstein Barr virus
--Herpes simplex types 1 and 2
--Herpes zoster
--Human herpesvirus types 6 and 7
Orthomyxoviruses
--Influenza viruses types A, B, and C (except those listed in Appendix B-III-D, Risk Group 3 (RG3) - Viruses
and Prions)
--Tick-borne orthomyxoviruses
Papilloma viruses
--All human papilloma viruses
Paramyxoviruses
--Newcastle disease virus
--Measles virus
--Mumps virus
--Parainfluenza viruses types 1, 2, 3, and 4
--Respiratory syncytial virus
Parvoviruses
--Human parvovirus (B19)
Picornaviruses
--Coxsackie viruses types A and B
--Echoviruses - all types
--Polioviruses - all types, wild and attenuated
--Rhinoviruses - all types
Poxviruses - all types except Monkeypox virus (see Appendix B-III-D, Risk Group 3 (RG3) - Viruses and
Prions) and restricted poxviruses including Alastrim, Smallpox, and Whitepox (see Section V-L, Footnotes
and References of Sections I through IV)
Reoviruses - all types including Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus)
Rhabdoviruses
--Rabies virus - all strains
--Vesicular stomatitis virus non exotic strains: VSV-Indiana 1 serotype strains (e.g. Glasgow, MuddSummers, Orsay, San Juan) and VSV-New Jersey serotype strains (e.g. Ogden, Hazelhurst)
Rubivirus (Togaviruses)
--Rubella virus
Risk Group 3 (RG3) Agents
RG3 agents are associated with serious or lethal human disease for which preventive or
therapeutic interventions may be available.
BACTERIAL AGENTS Including Rickettsia
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--Bartonella
--Brucella including B. abortus, B. canis, B. suis
--Burkholderia (Pseudomonas) mallei, B. pseudomallei
--Coxiella burnetii (except the Phase II, Nine Mile strain listed in Appendix B-II-A, Risk Group 2 (RG2) Bacterial Agents Including Chlamydia)
--Francisella tularensis (except those strains listed in Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents
Including Chlamydia)
--Mycobacterium bovis (except BCG strain, see Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents
Including Chlamydia), M. tuberculosis
--Pasteurella multocida type B -"buffalo" and other virulent strains
--Rickettsia akari, R. australis, R. canada, R. conorii, R. prowazekii, R. rickettsii, R, siberica, R.
tsutsugamushi, R. typhi (R. mooseri)
--Yersinia pestis (except those strains listed in Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents
Including Chlamydia)
FUNGAL AGENTS
--Coccidioides immitis (sporulating cultures; contaminated soil)
--Histoplasma capsulatum, H. capsulatum var. duboisii
PARASITIC AGENTS
None
VIRUSES AND PRIONS
Alphaviruses (Togaviruses) - Group A Arboviruses
--Chikungunya virus (except the vaccine strain 181/25 listed in Appendix B-II-D Risk Group2 (RG2) – Viruses)
--Semliki Forest virus
--St. Louis encephalitis virus
--Venezuelan equine encephalomyelitis virus (except the vaccine strains TC-83 and V3526, see Appendix BII-D (RG2) – Viruses)
--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I
through IV)
Arenaviruses
--Flexal
--Lymphocytic choriomeningitis virus (LCM) (neurotropic strains)
Bunyaviruses
--Hantaviruses including Hantaan virus
--Rift Valley fever virus
Coronaviruses
--SARS-associated coronavirus (SARS-CoV)
--Middle East respiratory syndrome coronavirus (MERS-CoV)
Flaviviruses - Group B Arboviruses
--Japanese encephalitis virus (except those strains listed in Appendix B-II-D Risk Group2 (RG2) - Viruses)
--West Nile virus (WNV)
--Yellow fever virus
--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I
through IV)
Orthomyxoviruses
-- Influenza viruses 1918-1919 H1N1 (1918 H1N1), human H2N2 (1957-1968), and highly pathogenic avian
influenza H5N1 strains within the Goose/Guangdong/96-like H5 lineage (HPAI H5N1).
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Poxviruses
--Monkeypox virus
Prions
--Transmissible spongiform encephalopathies (TSE) agents (Creutzfeldt-Jacob disease and kuru
agents)(see Section V-C, Footnotes and References of Sections I through IV, for containment instruction)
Retroviruses
--Human immunodeficiency virus (HIV) types 1 and 2
--Human T cell lymphotropic virus (HTLV) types 1 and 2
--Simian immunodeficiency virus (SIV)
Rhabdoviruses
--Vesicular stomatitis virus (except those strains listed in Appendix B-II-D Risk Group2 (RG2) - Viruses)
Risk Group 4 (RG4) Agents
RG4 agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions
are not usually available.
BACTERIAL AGENTS
None
FUNGAL AGENTS
None
PARASITIC AGENTS
None
VIRAL AGENTS
Arenaviruses
--Guanarito virus
--Lassa virus
--Junin virus (except the candid #1 vaccine strain listed in Appendix B-II-D Risk Group2 (RG2) – Viruses)
--Machupo virus
--Sabia
Bunyaviruses (Nairovirus)
--Crimean-Congo hemorrhagic fever virus
Filoviruses
--Ebola virus
--Marburg virus
Flaviruses - Group B Arboviruses
--Tick-borne encephalitis virus complex including Absetterov, Central European encephalitis, Hanzalova,
Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer
encephalitis viruses
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Herpesviruses (alpha)
--Herpesvirus simiae (Herpes B or Monkey B virus)
Paramyxoviruses
--Equine Morbillivirus (Hendra virus)
Hemorrhagic fever agents and viruses as yet undefined
Animal Viral Etiologic Agents in Common Use
The following list of animal etiologic agents is appended to the list of human etiologic agents. None of these
agents is associated with disease in healthy adult humans; they are commonly used in laboratory
experimental work.
A containment level appropriate for RG1 human agents is recommended for their use. For agents that are
infectious to human cells, e.g., amphotropic and xenotropic strains of murine leukemia virus, a containment
level appropriate for RG2 human agents is recommended.
Baculoviruses
Herpesviruses
--Herpesvirus ateles
--Herpesvirus saimiri
--Marek's disease virus
--Murine cytomegalovirus
Papilloma viruses
--Bovine papilloma virus
--Shope papilloma virus
Polyoma viruses
--Polyoma virus
--Simian virus 40 (SV40)
Retroviruses
--Avian leukosis virus
--Avian sarcoma virus
--Bovine leukemia virus
--Feline leukemia virus
--Feline sarcoma virus
--Gibbon leukemia virus
--Mason-Pfizer monkey virus
--Mouse mammary tumor virus
--Murine leukemia virus
--Murine sarcoma virus
--Rat leukemia virus
Murine Retroviral Vectors
Murine retroviral vectors to be used for human transfer experiments (less than 10 liters) that contain less than
50% of their respective parental viral genome and that have been demonstrated to be free of detectable
replication competent retrovirus can be maintained, handled, and administered, under BL1 containment.
Exemption and Exceptions
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There are exemption and exceptions listed in Appendix C of the NIH Guidelines. However, the
UTHSC Institutional Biosafety Committee reviews all rDNA research including experiments that the
NIH Guidelines consider exempt with the exception of the purchase, breeding and transfer of
transgenic animals of the following conditions are met. The PI shall request an exemption.
NIH Guidelines, Appendix C-VIII.
Generation of BL1 Transgenic Rodents via Breeding
The breeding of two different transgenic rodents or the breeding of a transgenic rodent and a non-transgenic
rodent with the intent of creating a new strain of transgenic rodent that can be housed at BL1 containment will
be exempt from the NIH Guidelines if:
(1) Both parental rodents can be housed under BL1 containment; and
(2) neither parental transgenic rodent contains the following genetic modifications: (i) incorporation of more
than one-half of the genome of an exogenous eukaryotic virus from a single family of viruses; or (ii)
incorporation of a transgene that is under the control of a gammaretroviral long terminal repeat (LTR); and
(3) the transgenic rodent that results from this breeding is not expected to contain more than one-half of an
exogenous viral genome from a single family of viruses.
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APPENDIX C
HHS/CDC Select Agents and USDA High Consequence Livestock
Pathogens and Toxins
Background
Department of Health and Human Services (HHS): Select Agents and toxins, 42 CFR 73 and
United States Department of Agriculture (USDA) 9 CFR 121 and 7 CFR 331.
In 1996, HHS published a set of rules (Additional Requirements for Facilities Transferring or
Receiving Select Agents, October 24,1996, Appendix A of 42 CFR Part 72.6) requiring facilities and
institutions to be registered and approved in order to transfer or receive select biological agents and
toxins.
On June 12, 2002 President Bush signed the "Public Health Security and Bioterrorism Preparedness
and Response Act of 2002" (Public Law 107-188). The law is designed to improve the ability of the
United States to prevent, prepare for, and respond to bioterrorism and other public health
emergencies. The Law requires that all persons possessing biological agents or toxins deemed a
threat to public health notify the Secretary, Department of Health and Human Services (HHS).
Subsequent to the enactment of Public Law 107-188, The Public Health Security and Bioterrorism
Preparedness and Response Act of 2002, additional requirements for facilities or entities that
possess, use, or transfer select agents and toxins have been published in 42 CFR 73; 9 CFR 121
and 7 CFR 331; December 13, 2002, interim final rule. The final rule became effective March 18,
2005 and has been amended several times. More information on the federal Select Agent Program
can be found at:
http://www.selectagents.gov/
Definitions
•
Select Agent and Toxin
–
•
Any biological agent or its toxin, as defined in 42 CFR 73.4 or §73.5, and
capable of being used to inflict mass casualties, death, or other biological
malfunction on human, animal, plant, or other environmental populations,
food, or water source
Toxin
–
The toxic material or product of plants, animals, microorganisms, infectious
substances, or a recombinant or synthesized molecule, however derived or
produced.
Responsible Official
A Responsible Official (RO) is appointed for the Select Agent Program. Alternate
Responsible Official(s) (ARO) can also be appointed as necessary.
Work with Select Agents
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No access to or work with a select agent can be initiated until the entity (UTHSCSA)
and the PI are registered and approved by the HHS or USDA for that agent and the
agent/work is approved by the Institutional Biosafety Committee (IBC). No work with
select agent in animals can be started until approved by the HHS/USDA, the IBC and
the IACUC.
Transfers
•
All transferring parties must be registered with CDC
–
Whether inter or intra-state
–
Exception: exempt status
•
Both sender & receiver CDC approved & registered for the select agent or toxin
•
Transfer form completed as per CDC procedure
•
All transfers approved & signed by RO, or Alternate RO
•
Must be shipped as per DOT, ICAO / IATA requirements
Security Risk Assessments
Employees with unrestricted access to select agents and toxins must be authorized
by the HHS or USDA and the RO.
Each Individual must:

Pass the routine UTHSC-PD security check

Complete the UTHSCSA Annual Statement of Eligibility to Handle Select
Biological Agents or Toxins (HOP 8.10.1)

Complete the FBI FD-961 form, complete the FBI-258 fingerprint cards and
pass the U.S. Dept. of Justice background check.
The following biological agents and toxins have been determined to have the potential to pose a severe
threat to both human and animal health, to plant health, or to animal and plant products. An attenuated
strain of a select agent or an inactive form of a select toxin may be excluded from the requirements of
the Select Agent Regulations.
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HHS and USDA Select Agents and Toxins
7CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73
HHS SELECT AGENTS AND TOXINS
Abrin
Botulinum neurotoxins*
Botulinum neurotoxin producing species
of Clostridium*
Conotoxins (Short, paralytic alpha conotoxins
containing the following amino acid sequence
X1CCX2PACGX3X4X5X6CX7)1
Coxiella burnetii
Crimean-Congo haemorrhagic fever virus
Diacetoxyscirpenol
Eastern Equine Encephalitis virus3
Ebola virus*
Francisella tularensis*
Lassa fever virus
Lujo virus
Marburg virus*
Monkeypox virus3
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
Rickettsia prowazekii
SARS-associated coronavirus (SARS-CoV)
Saxitoxin
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*
OVERLAP SELECT AGENTS AND TOXINS
Bacillus anthracis*
Bacillus anthracis Pasteur strain
Brucella abortus
Brucella melitensis
Brucella suis
Burkholderia mallei*
Burkholderia pseudomallei*
Hendra virus
Nipah virus
Rift Valley fever virus
Venezuelan equine encephalitis virus3
USDA SELECT AGENTS AND TOXINS
African horse sickness virus
African swine fever virus
Avian influenza virus3
Classical swine fever virus
Foot-and-mouth disease virus*
Goat pox virus
Lumpy skin disease virus
Mycoplasma capricolum3
Mycoplasma mycoides3
Newcastle disease virus2,3
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
Synchytrium endobioticum
Xanthomonas oryzae
*Denotes Tier 1 Agent
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1
C = Cysteine residues are all present as disulfides, with the 1st and 3rd Cysteine, and the 2nd and 4th Cysteine
forming specific disulfide bridges; The consensus sequence includes known toxins α-MI and α-GI (shown
above) as well as α-GIA, Ac1.1a, α-CnIA, α-CnIB; X1 = any amino acid(s) or Des-X; X2 = Asparagine or
Histidine; P = Proline; A = Alanine; G = Glycine; X3 = Arginine or Lysine; X4 = Asparagine, Histidine,
Lysine, Arginine, Tyrosine, Phenylalanine or Tryptophan; X5 = Tyrosine, Phenylalanine, or Tryptophan; X6 =
Serine, Threonine, Glutamate, Aspartate, Glutamine, or Asparagine; X7 = Any amino acid(s) or Des X and;
“Des X” = “an amino acid does not have to be present at this position.” For example if a peptide sequence
were XCCHPA then the related peptide CCHPA would be designated as Des-X.
2
A virulent Newcastle disease virus (avian paramyxovirus serotype 1) has an intracerebral pathogenicity index
in day-old chicks (Gallus gallus) of 0.7 or greater or has an amino acid sequence at the fusion (F) protein
cleavage site that is consistent with virulent strains of Newcastle disease virus. A failure to detect a cleavage
site that is consistent with virulent strains does not confirm the absence of a virulent virus.
3
Select agents that meet any of the following criteria are excluded from the requirements of this part: Any low
pathogenic strains of avian influenza virus, South American genotype of eastern equine encephalitis virus ,
west African clade of Monkeypox viruses, any strain of Newcastle disease virus which does not meet the
criteria for virulent Newcastle disease virus, all subspecies Mycoplasma capricolum except subspecies
capripneumoniae (contagious caprine pleuropneumonia), all subspecies Mycoplasma mycoides except
subspecies mycoides small colony (Mmm SC) (contagious bovine pleuropneumonia), and any subtypes of
Venezuelan equine encephalitis virus except for Subtypes IAB or IC, provided that the individual or entity can
verify that the agent is within the exclusion category. 9/10/13
Select Agent and Toxin Exclusions
The select agent regulations (7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73) established a
procedure by which an attenuated strain of a select biological agent or toxin that does not pose a severe
threat to public health and safety, animal health, or animal products may be excluded from the
requirements of the select agent regulations.
The list of excluded agents and toxins can be found at:
http://www.selectagents.gov/SelectAgentsandToxinsExclusions.html
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APPENDIX D:
LABORATORY BIOSAFETY LEVEL CRITERIA
This Appendix contains a summary of the four Biosafety Levels from CDC/NIH the Biosafety
in Microbiological and Biomedical Laboratories, 5th edition. The latest guidelines for
Etiological Agents, rDNA, or Oncogenic Viruses, must be reviewed for complete up-to-date
recommendations.
Summary of Biosafety Levels
Safety
Levels
1
2
Agent Characteristics
Not known to cause
disease in healthy adults.
Standard
Microbiological
Practices
Associated with human
disease.
Routes of transmission
include percutaneous
injury, ingestion, mucous
membrane exposure
BSL 1 practice plus:
- Limited access
-Biohazard warning
signs
-Sharps precautions
-Biosafety manual
defining any needed
waste decontamination
of all infectious wastes
Or medical surveillance
policies
Indigenous or exotic agent
with the potential for
aerosol transmission.
3
4
Safety Practices
Known to cause disease
with
serious or lethal
consequences.
Dangerous/exotic agents
which pose high risk of
life-threatening disease
and
aerosol transmitted
infection.
Related agents with
unknown risk of
transmission.
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BSL 2 practice plus:
-Decontamination of all
waste
-Decontamination of
laboratory
clothing before
laundering
-Medical surveillanceBaseline serum collected
and stored.
Level 3 precautions plus:
-Clothing change before
entering
-Shower upon exit
-All material
decontaminated
upon exit from facility
Primary Barriers and
Safety Equipment
Secondary
Barriers
No primary barriers; PPE:
gloves, labcoats; eye, face
protection as needed
Laboratory bench
and
Sink required
Primary barriers -Biological
safety cabinet or other physical
containment devices used for all
manipulations of agents that
cause splashes or aerosols of
infectious agents.
PPE: Laboratory coats, gloves,
face and eye protection, as
needed
BSL 1 plus:
-Autoclave
available
-Class I or II biological
safety cabinet or other physical
containment devices.
-Protective clothing
-Respiratory protection.
BSL 2 precautions
plus:
-Physical
separation from
access corridors.
-Self-closing,
double door
access.
-Exhausted air not
recirculated
-Negative airflow
into laboratory.
All procedures conducted in
Class III biological safety
cabinets
or in Class I or II safety cabinets
with full-body, air-supplied,
positive pressure personnel
suits.
Level 3
precautions plus:
-Separate building
or isolated zone.
-Dedicated
supply/exhaust,
vacuum, and
decontamination
systems.
-Other
requirements, as
necessary.
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BIOSAFETY LEVEL I
Biosafety Level 1 is suitable for work involving well-characterized agents not known to consistently cause
disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel
and the environment. BSL-1 laboratories are not necessarily separated from the general traffic patterns in the
building. Work is typically conducted on open bench tops using standard microbiological practices. Special
containment equipment or facility design is not required, but may be used as determined by appropriate risk
assessment. Laboratory personnel must have specific training in the procedures conducted in the laboratory
and must be supervised by a scientist with training in microbiology or a related science.
The following standard practices, safety equipment, and facility requirements apply to BSL-1.
A. Standard Microbiological Practices
1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory.
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 punctureresistant 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.
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
when infectious agents are present. The sign may include the name of the agent(s) in use, and the
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name and phone number of the laboratory supervisor or other responsible personnel. Agent
information should be posted in accordance with the institutional policy.
10. An effective integrated pest management program is required.
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 the institution’s healthcare provider for appropriate
counseling and guidance.
B. Special Practices
None required.
C. Safety Equipment (Primary Barriers and Personal Protective Equipment)
1. Special containment devices or equipment, such as BSCs, are not generally required.
2. Protective laboratory coats, gowns, or uniforms are recommended to prevent contamination of
personal clothing.
3. Wear protective eyewear when conducting procedures that have the potential to create splashes of
microorganisms or other hazardous materials. Persons who wear contact lenses in laboratories
should also wear eye protection.
4. Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should
be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Wash
hands prior to leaving the laboratory. In addition, BSL-1 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.
D. Laboratory Facilities (Secondary Barriers)
1. Laboratories should have doors for access control.
2. Laboratories must have a sink for hand washing.
3. The laboratory should be designed so that it can be easily cleaned. Carpets and rugs in laboratories
are not appropriate.
4. Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between
benches, cabinets, and equipment should be accessible for cleaning.
a. Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and
other chemicals.
b. Chairs used in laboratory work must be covered with a non-porous material that can be easily
cleaned and decontaminated with appropriate disinfectant.
5. Laboratories windows that open to the exterior should be fitted with screens.
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BIOSAFETY LEVEL 2
Biosafety Level 2 builds upon BSL-1. BSL-2 is suitable for work involving agents that pose moderate hazards
to personnel and the environment. It differs from BSL-1 in that: 1) laboratory personnel have specific training
in handling pathogenic agents and are supervised by scientists competent in handling infectious agents and
associated procedures; 2) access to the laboratory is restricted when work is being conducted; and 3) all
procedures in which infectious aerosols or splashes may be created are conducted in BSCs or other physical
containment equipment.
The following standard and special practices, safety equipment, and facility requirements apply to BSL-2.
A. Standard Microbiological Practices
1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory.
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 punctureresistant 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.
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 when
infectious agents are present. Posted information must include: the laboratory’s biosafety level, the
10. An effective integrated pest management program is required. (See Appendix G.)
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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 the institution’s healthcare provider for appropriate
counseling and guidance.
B. Special Practices
1. All persons entering the laboratory must be advised of the potential hazards and meet specific
entry/exit requirements.
2. Laboratory personnel must be provided medical surveillance, as appropriate, and offered available
immunizations for agents handled or potentially present in the laboratory.
3. Each institution should consider the need for collection and storage of serum samples from at-risk
personnel.
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.
a. Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff
properly trained and equipped to work with infectious material.
b. Equipment must be decontaminated before repair, maintenance, or removal from the laboratory.
8. Incidents that may result in exposure to infectious materials must be immediately 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.
9. Animal and plants not associated with the work being performed must not be permitted in the
laboratory.
10. All procedures involving the manipulation of infectious materials that may generate an aerosol should
be conducted within a BSC or other physical containment devices.
C. Safety Equipment (Primary Barriers and Personal Protective Equipment)
1. 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.
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b. High concentrations or large volumes of infectious agents are used. Such materials may be
centrifuged in the open laboratory using sealed rotor heads or centrifuge safety cups.
2. Protective laboratory coats, gowns, smocks, or uniforms designated for laboratory use must be
worn while working with hazardous materials. Remove protective clothing before leaving for
non-laboratory areas, e.g., cafeteria, library, and administrative offices). Dispose of protective
clothing appropriately, or deposit it for laundering by the institution. It is recommended that
laboratory clothing not be taken home.
3. Eye and face protection (goggles, mask, face shield or other splatter guard) is used for
anticipated splashes or sprays of infectious or other hazardous materials when the microorganisms
must be handled outside the BSC or containment device. Eye and face protection must be disposed of
with other contaminated laboratory waste or decontaminated before reuse. Persons who wear contact
lenses in laboratories should also wear eye protection.
4. Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should
be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Gloves
must not be worn outside the laboratory. In addition, BSL-2 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.
5. Eye, face and respiratory protection should be used in rooms containing infected animals as
determined by the risk assessment.
D. Laboratory Facilities (Secondary Barriers)
1. Laboratory doors should be self-closing and have locks in accordance with the institutional policies.
2. Laboratories must have a sink for hand washing. The sink may be manually, hands-free, or automatically
operated. It should be located near the exit door.
3. The laboratory should be designed so that it can be easily cleaned and decontaminated. Carpets and
rugs in laboratories are not permitted.
4. Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between
benches, cabinets, and equipment should be accessible for cleaning.
a. Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and
other chemicals.
b. Chairs used in laboratory work must be covered with a non-porous material that can be easily
cleaned and decontaminated with appropriate disinfectant.
5. Laboratory windows that open to the exterior are not recommended. However, if a laboratory does
have windows that open to the exterior, they must be fitted with screens.
6. BSCs must be installed so that fluctuations of the room air supply and exhaust do not interfere with
proper operations. BSCs should be located away from doors, windows that can be opened, heavily
traveled laboratory areas, and other possible airflow disruptions.
7. Vacuum lines should be protected with liquid disinfectant traps.
8. An eyewash station must be readily available.
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9. There are no specific requirements for ventilation systems. However, planning of new facilities should
consider mechanical ventilation systems that provide an inward flow of air without recirculation to
spaces outside of the laboratory.
10. HEPA filtered exhaust air from a Class II BSC can be safely recirculation back into the laboratory
environment if the cabinet is tested and certified at least annually and operated according to
manufacturer’s recommendations. BSCs can also be connected to the laboratory exhaust system by
either a thimble (canopy) connection or directly exhausted to the outside through a hard connection.
Provisions to assure proper safety cabinet performance and air system operation must be verified.
11. A method for decontaminating all laboratory wastes should be available in the facility (e.g., autoclave,
chemical disinfection, incineration, or other validated decontamination method).
BIOSAFETY LEVEL 3
Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities where work is
performed with indigenous or exotic agents that may cause serious or potentially lethal disease through the
inhalation route of exposure. Laboratory personnel must receive specific training in handling pathogenic and
potentially lethal agents, and must be supervised by scientists competent in handling infectious agents and
associated procedures.
All procedures involving the manipulation of infectious materials must be conducted within BSCs or other
physical containment devices.
A BSL-3 laboratory has special engineering and design features.
The following standard and special safety practices, equipment, and facility requirements apply to BSL-3.
A. Standard Microbiological Practices
1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory.
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 punctureresistant 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.
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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. A method for decontaminating all laboratory wastes should be available in the facility,
preferably within the laboratory (e.g., autoclave, chemical disinfection, incineration, or other validated
decontamination 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
when infectious agents are present. Posted information must include the laboratory’s biosafety level,
the supervisor’s name (or other responsible personnel), telephone number, and required procedures
for entering and exiting the laboratory. Agent information should be posted in accordance with the
institutional policy.
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 the institution’s
healthcare provider for appropriate counseling and guidance.
B. Special Practices
1. All persons entering the laboratory must be advised of the potential hazards and meet specific
entry/exit requirements.
2. Laboratory personnel must be provided medical surveillance and offered appropriate immunizations for
agents handled or potentially present in the laboratory.
3. Each institution should consider the need for collection and storage of serum samples from at-risk
personnel.
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-3 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.
a. Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff
properly trained and equipped to work with infectious material.
b. Equipment must be decontaminated before repair, maintenance, or removal from the laboratory.
8. Incidents that may result in exposure to infectious materials must be immediately evaluated and
treated according to procedures described in the laboratory biosafety manual. All such incidents must
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be reported to the laboratory supervisor. Medical evaluation, surveillance, and treatment should be
provided and appropriate records maintained.
9. Animals and plants not associated with the work being performed must not be permitted in the
laboratory.
10. All procedures involving the manipulation of infectious materials must be conducted within a BSC, or
other physical containment devices. No work with open vessels is conducted on the bench. When a
procedure cannot be performed within a BSC, a combination of personal protective equipment and
other containment devices, such as a centrifuge safety cup or sealed rotor must be used.
C. Safety Equipment (Primary Barriers and Personal Protective Equipment)
1. All procedures involving the manipulation of infectious materials must be conducted within a BSC
(preferably Class II or Class III), or other physical containment devices.
2. Workers in the laboratory where protective laboratory clothing with a solid-front, such as tie-back or
wrap-around gowns, scrub suits, or coveralls. Protective clothing is not worn outside of the laboratory.
Reusable clothing is decontaminated before being laundered. Clothing is changed when contaminated.
3. Eye and face protection (goggles, mask, face shield or other splash guard) is 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 must also wear eye protection.
4. Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should
be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Gloves
must not be worn outside the laboratory. In addition, BSL-3 laboratory workers:
a. Changes gloves when contaminated, glove integrity is compromised, or when otherwise
necessary. Wear two pairs of gloves when appropriate.
b. Remove gloves and wash hands when work with hazardous materials has been completed and
before leaving the laboratory.
c. Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated
laboratory waste. Hand washing protocols must be rigorously followed.
5. Eye, face, and respiratory protection must be used in rooms containing infected animals.
D. Laboratory Facilities (Secondary Barriers)
1. Laboratory doors must be self-closing and have locks in accordance with the institutional policies. The
laboratory must be separated from areas that are open to unrestricted traffic flow within the building.
Laboratory access is restricted. Access to the laboratory is through two self-closing doors. A clothing
change room (anteroom) may be included in the passageway between the two self-closing doors.
2. Laboratories must have a sink for hand washing. The sink must be hands-free or automatically
operated. It should be located near the exit door. If the laboratory is segregated into different
laboratories, a sink must also be available for hand washing in each zone. Additional sinks may be
required as determined by the risk assessment.
3. The laboratory must be designed so that it can be easily cleaned and decontaminated. Carpets and rugs
are not permitted. Seams, floors, walls, and ceiling surfaces should be sealed. Spaces around doors and
ventilation openings should be capable of being sealed to facilitate space decontamination.
a. Floors must be slip resistant, impervious to liquids, and resistant to chemicals. Consideration
should be given to the installation of seamless, sealed, resilient or poured floors, with integral
cove bases.
b. Walls should be constructed to produce a sealed smooth finish that can be easily cleaned and
decontaminated.
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c. Ceilings should be constructed, sealed, and finished in the same general manner as walls.
Decontamination of the entire laboratory should be considered when there has been gross contamination of
the space, significant changes in laboratory usage, for major renovations, or maintenance shut downs.
Selection of the appropriate materials and methods used to decontaminate the laboratory must be based on
the risk assessment.
4. Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between
benches, cabinets, and equipment must be accessible for cleaning.
a. Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and
other chemicals.
b. Chairs used in laboratory work must be covered with a non-porous material that can be easily
cleaned and decontaminated with appropriate disinfectant.
5. All windows in the laboratory must be sealed.
6. BSCs must be installed so that fluctuations of the room air supply and exhaust do not interfere with
proper operations. BSCs should be located away from doors, heavily traveled laboratory areas, and
other possible airflow disruptions.
7. Vacuum lines must be protected with HEPA filters, or their equivalent. Filters must be replaced as
needed. Liquid disinfectant traps may be required.
8. An eyewash station must be readily available in the laboratory.
9. A ducted air ventilation system is required. This system must provide sustained directional airflow by
drawing air into the laboratory from “clean” areas toward “potentially contaminated” areas. The
laboratory shall be designed such that under failure conditions the airflow will not be reversed.
a. Laboratory personnel must be able to verify directional airflow. A visual monitoring device, which
confirms directional airflow, must be provided at the laboratory entry. Audible alarms should be
considered to notify personnel of air flow disruption.
b. The laboratory exhaust air must not re-circulate to any other area of the building.
c. The laboratory building exhaust air should be dispersed away from occupied areas and from
building air intake locations or the exhaust air must be HEPA filtered.
HEPA filter housings should have gas-tight isolation dampers, decontamination ports, and/or bagin/bag-out (with appropriate decontamination procedures) capability. The HEPA filter housing should
allow for leak testing of each filter and assembly. The filters and the housing should be certified at
least annually.
10. HEPA filtered exhaust air from a Class II BSC can be safely re-circulated into the laboratory environment
if the cabinet is tested and certified at least annually and operated according to manufacturer’s
recommendations. BSCs can also be connected to the laboratory exhaust system by either a thimble
(canopy) connection or directly exhausted to the outside through a hard connection. Provisions to assure
proper safety cabinet performance and air system operation must be verified. BSCs should be certified at
least annually to assure correct performance. Class III BSCs must be directly (hard) connected up through
the second exhaust HEPA filter of the cabinet. Supply air must be provided in such a manner that prevents
positive pressurization of the cabinet.
11. A method for decontaminating all laboratory wastes should be available in the facility, preferably
within the laboratory (e.g., autoclave, chemical disinfection, or other validated decontamination
method).
12. Equipment that may produce infectious aerosols must be contained in primary barrier devices that
exhaust air through HEPA filtration or other equivalent technology before being discharged into the
laboratory. These HEPA filters should be tested and/or replaced at least annually.
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13. Facility design consideration should be given to means of decontaminating large pieces of equipment
before removal from the laboratory.
14. Enhanced environmental and personal protection may be required by the agent summary statement, risk
assessment, or applicable local, state, or federal regulations. These laboratory enhancements may include,
for example, one or more of the following: an anteroom for clean storage of equipment and supplies with
dress-in, shower-out capabilities; gas tight dampers to facilitate laboratory isolation; final HEPA filtration of
the laboratory exhaust air; laboratory effluent decontamination; and advanced access control devices, such
as biometrics.
15. The BSL-3 facility design, operational parameters, and procedures must be verified and documented
prior to operation. Facilities must be re-verified and documented at least annually.
Biosafety Level 4 (UT Health Science Center does not have BSL-4 laboratories)
Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of
aerosol-transmitted laboratory infections and life-threatening disease that is frequently fatal, for which there are
no vaccines or treatments, or a related agent with unknown risk of transmission. Agents with a close or identical
antigenic relationship to agents requiring BSL-4 containment must be handled at this level until sufficient data
are obtained either to confirm continued work at this level, or re-designate the level.
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APPENDIX E
VERTEBRATE ANIMAL BIOSAFETY LEVEL CRITERIA
These guidelines represent four levels of practices, safety equipment, and facilities for
experiments on animals infected with agents which are known or believed to produce
infections in humans.
Animal Biosafety Levels 1-4 describe increasing stages of
containment and are recommended as minimal standards for activities involving infected
laboratory animals. Animal Biosafety Levels 1-4 list facilities and practices applicable to
work on animals infected with agents classified into corresponding Biosafety Levels 1-4.
The following guidelines are reprinted from those published in “Biosafety in Microbiological
and Biomedical Laboratories,” HHS/CDC/NIH 5th edition, Dec. 2009.
ANIMAL BIOSAFETY LEVEL 1
The following standard practices, safety equipment, and facility requirements apply to ABSL-1.
A.
Standard Microbiological Practices
1. The animal facility director establishes and enforces policies, procedures, and protocols for institutional policies
and emergencies.
Each institute must assure that worker safety and health concerns are addressed as part of the animal protocol
review.
Prior to beginning a study animal protocols must also be reviewed and approved by the Institutional Animal
Care and Use Committee (IACUC)5 and the Institutional Biosafety Committee.
2. A safety manual specific to the animal facility is prepared or adopted in consultation with the animal facility
director and appropriate safety professionals. The safety manual must be available and accessible. Personnel
are advised of potential hazards and are required to read and follow instructions on practices and procedures.
3. The supervisor must ensure that animal care, laboratory and support personnel receive appropriate training
regarding their duties, animal husbandry procedures, potential hazards, manipulations of infectious agents,
necessary precautions to prevent exposures, and hazard/exposure evaluation procedures (physical hazards,
splashes, aerosolization, etc.). Personnel must receive annual updates and additional training when
procedures or policies change. Records are maintained for all hazard evaluations, employee training sessions
and staff attendance.
4. An appropriate medical surveillance program is in place, as determined by risk assessment. The need for an
animal allergy prevention program should be considered.
Facility supervisors should ensure that medical staff is informed of potential occupational hazards within the animal
facility, to include those associated with research, animal husbandry duties, animal care and manipulations.
Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic
interventions. Therefore, all personnel and particularly women of childbearing age should be provided information regarding immune
competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to selfidentify to the institution’s healthcare provider for appropriate counseling and guidance.
Personnel using respirators must be enrolled in an appropriately constituted respiratory protection program.
5. A sign incorporating safety information must be posted at the entrance to the areas where infectious materials
and/or animals are housed or are manipulated. The sign must include the animal biosafety level, general
occupational health requirements, personal protective equipment requirements, the supervisor’s name (or other
responsible personnel), telephone number, and required procedures for entering and exiting the animal areas.
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Identification of specific infectious agents is recommended when more than one agent is being used within an
animal room.
Security-sensitive agent information should be posted in accordance with the institutional policy.
Advance consideration should be given to emergency and disaster recovery plans, as a contingency for man-made
or natural disasters.
6. Access to the animal room is limited. Only those persons required for program or support purposes are
authorized to enter the facility.
All persons including facility personnel, service workers, and visitors are advised of the potential hazards (natural or
research pathogens, allergens, etc.) and are instructed on the appropriate safeguards.
7. Protective laboratory coats, gowns, or uniforms are recommended to prevent contamination of personal clothing.
Gloves are worn to prevent skin contact with contaminated, infectious and hazardous materials, and when handling
animals.
Gloves and personal protective equipment should be removed in a manner that minimizes transfer of infectious
materials outside of the areas where infectious materials and/or animals are housed or are manipulated.
Persons must wash their hands after removing gloves, and before leaving the areas where infectious materials and/or animals are housed
or are manipulated.
Eye and face and respiratory protection should be used in rooms containing infected animals, as dictated by the risk
assessment.
8. 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 of the laboratory in cabinets or
refrigerators designed and used for this purpose.
9. All procedures are carefully performed to minimize the creation of aerosols or splatters of infectious materials and
waste.
10. Mouth pipetting is prohibited. Mechanical pipetting devices must be used.
11. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be
developed and implemented.
When applicable, laboratory supervisors should adopt improved engineering and work practice controls that reduce
the risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items.
These include:
a. Use of needles and syringes or other sharp instruments in the animal facility is limited to situations where there is no alternative.
for such procedures as parenteral injection, blood collection, or aspiration of fluids from laboratory animals and
diaphragm bottles.
b. Disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or
otherwise manipulated by hand before disposal. Used disposable needles must be carefully placed in
puncture-resistant containers used for sharps disposal. Sharps containers should be located as close to
the work site as possible.
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.
e. Equipment containing sharp edges and corners should be avoided.
12. Equipment and work surfaces are routinely decontaminated with an appropriate disinfectant after work with an
infectious agent, and after any spills, splashes, or other overt contamination.
13. Animals and plants not associated with the work being performed must not be permitted in the areas where
infectious materials and/ or animals are housed or are manipulated.
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14. An effective integrated pest management program is required. (See Appendix G.)
15. All wastes from the animal room (including animal tissues, carcasses, and bedding) are transported from the animal room in
leak-proof, covered containers for appropriate disposal in compliance with applicable institutional, local and state requirements.
Decontaminate all potentially infectious materials before disposal using an effective method.
B. Special Practices
None required.
C. Safety Equipment (Primary Barriers and Personal Protective Equipment)
1. A risk assessment should determine the appropriate type of personal protective equipment to be utilized.
2. Special containment devices or equipment may not be required as determined by appropriate risk assessment.
3. Protective laboratory coats, gowns, or uniforms may be required to prevent contamination of personal clothing.
Protective outer clothing is not worn outside areas where infectious materials and/or animals are housed or
manipulated. Gowns and uniforms are not worn outside the facility.
4. Protective eyewear is worn when conducting procedures that have the potential to create splashes of
microorganisms or other hazardous materials. Persons who wear contact lenses should also wear eye
protection when entering areas with potentially high concentrations or airborne particulates.
Persons having contact with NHPs must assess risk of mucous membrane exposure and wear protective
equipment (e.g., masks, goggles, face shields, etc.) as appropriate for the task to be performed.
5. Gloves are worn to protect hands from exposure to hazardous materials.
A risk assessment should be performed to identify the appropriate glove for the task and alternatives to latex gloves
should be available.
Change gloves when contaminated, glove integrity is compromised, or when otherwise necessary.
Gloves must not be worn outside the animal rooms.
Gloves and personal protective equipment should be removed in a manner that prevents transfer of infectious
materials.
Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated waste.
6. Persons must wash their hands after handling animals and before leaving the areas where infectious materials and/or
animals are housed or are manipulated. Hand washing should occur after the removal of gloves.
D. Laboratory Facilities (Secondary Barriers)
1. The animal facility is separated from areas that are open to unrestricted personnel traffic within the building.
External facility doors are self-closing and self-locking.
Access to the animal facility is restricted.
Doors to areas where infectious materials and/or animals are housed, open inward, are self-closing, are kept closed
when experimental animals are present, and should never be propped open. Doors to cubicles inside an animal
room may open outward or slide horizontally or vertically.
2. The animal facility must have a sink for hand washing.
Sink traps are filled with water, and/or appropriate liquid to prevent the migration of vermin and gases.
3. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior
surfaces (walls, floors and ceilings) are water resistant. Floors must be slip resistant, impervious to liquids, and
resistant to chemicals.
It is recommended that penetrations in floors, walls and ceiling surfaces be sealed, including openings around
ducts, doors and doorframes, to facilitate pest control and proper cleaning.
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4. Cabinets and bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and
other chemicals. Spaces between benches, cabinets, and equipment should be accessible for cleaning.
Chairs used in animal area must be covered with a non-porous material that can be easily cleaned and
decontaminated. Furniture must be capable of supporting anticipated loads and uses. Sharp edges and
corners should be avoided.5. External windows are not recommended; if present windows must be resistant to
breakage. Where possible, windows should be sealed. If the animal facility has windows that open, they are
fitted with fly screens. The presence of windows may impact facility security and therefore should be assessed
by security personnel.
6. Ventilation should be provided in accordance with the Guide for Care and Use of Laboratory Animals.1 No
recirculation of exhaust air may occur. It is recommended that animal rooms have inward directional airflow.
Ventilation system design should consider the heat and high moisture load produced during the cleaning of animal
rooms and the cage wash process.
7. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize
horizontal surface areas to facilitate cleaning and minimize the accumulation of debris or fomites.
8. If floor drains are provided, the traps are filled with water, and/or appropriate disinfectant to prevent the migration of vermin and
gases.
9. Cages are washed manually or preferably in a mechanical cage washer. The mechanical cage washer should
have a final rinse temperature of at least 180°F. If manual cage washing is utilized, ensure that appropriate
disinfectants are selected.
10. Illumination is adequate for all activities, avoiding reflections and glare that could impede vision.
11. Emergency eyewash and shower are readily available; location is determined by risk assessment.
ANIMAL BIOSAFETY LEVEL 2
Animal Biosafety Level 2 builds upon the practices, procedures, containment equipment, and facility requirements of ABSL-1. ABSL-2
is suitable for work involving laboratory animals infected with agents associated with human disease and pose moderate hazards to
personnel and the environment. It also addresses hazards from ingestion as well as from percutaneous and mucous membrane exposure.
A.
Standard Microbiological Practices
1. The animal facility director establishes and enforces policies, procedures, and protocols for institutional policies
and emergencies.
Each organization must assure that worker safety and health concerns are addressed as part of the animal
protocol review.
Prior to beginning a study, animal protocols must also be reviewed and approved by the IACUC 5 and the
Institutional Biosafety Committee.
2. A safety manual specific to the animal facility is prepared or adopted in consultation with the animal facility director and
appropriate safety professionals.
The safety manual must be available and accessible. Personnel are advised of potential hazards, and are required
to read and follow instructions on practices and procedures.
Consideration should be given to specific biohazards unique to the animal species and protocol in use.
3. The supervisor must ensure that animal care, laboratory, and support personnel receive appropriate training
regarding their duties, animal husbandry procedure, potential hazards, manipulations of infectious agents,
necessary precautions to prevent hazard or exposures, and hazard/exposure evaluation procedures (physical
hazards, splashes, aerosolization, etc.). Personnel must receive annual updates or additional training when
procedures or policies change. Records are maintained for all hazard evaluations, employee training sessions
and staff attendance.
4. An appropriate medical surveillance program is in place, as determined by risk assessment. The need for an
animal allergy prevention program should be considered.
Facility supervisors should ensure that medical staff is informed of potential occupational hazards within the animal
facility, to include those associated with research, animal husbandry duties, animal care and manipulations.
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Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or
prophylactic interventions. Therefore, all personnel and particularly women of childbearing age should be
provided information regarding immune competence and conditions that may predispose them to infection.
Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider
for appropriate counseling and guidance.
Personnel using respirators must be enrolled in an appropriately constituted respiratory protection program.
5. A sign incorporating the universal biohazard symbol must be posted at the entrance to areas where infectious materials and/ or animals
are housed or are manipulated when infectious agents are present. The sign must include the animal biosafety level, general occupational
health requirements, personal protective equipment requirements, the supervisor’s name (or names of other responsible personnel),
telephone number, and required procedures for entering and exiting the animal areas. Identification of all infectious agents is necessary
when more than one agent is being used within an animal room.
Security-sensitive agent information and occupational health requirements should be posted in accordance with the
institutional policy.
Advance consideration should be given to emergency and disaster recovery plans, as a contingency for man-made
or natural disasters.1,3,4
6. Access to the animal room is limited. Only those persons required for program or support purposes are
authorized to enter the animal facility and the areas where infectious materials and/or animals are housed or
manipulated.
All persons including facility personnel, service workers, and visitors are advised of the potential hazards (physical,
naturally occurring, or research pathogens, allergens, etc.) and are instructed on the appropriate safeguards.
7. Protective laboratory coats, gowns, or uniforms are recommended to prevent contamination of personal clothing.
Gloves are worn to prevent skin contact with contaminated, infectious and hazardous materials and when handling
animals.
Gloves and personal protective equipment should be removed in a manner that prevents transfer of infectious
materials outside of the areas where infectious materials and/or animals are housed or are manipulated.
Persons must wash their hands after removing gloves, and before leaving the areas where infectious materials
and/or animals are housed or are manipulated.
Eye, face and respiratory protection should be used in rooms containing infected animals, as dictated by the risk
assessment.
8. 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 of the laboratory in cabinets or
refrigerators designated and used for this purpose.
9. All procedures are carefully performed to minimize the creation of aerosols or splatters of infectious materials and
waste.
10. Mouth pipetting is prohibited. Mechanical pipetting devices must be used.
11. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed and implemented.
When applicable, laboratory supervisors should adopt improved engineering and work practice controls that reduce the risk of sharps
injuries. Precautions must always be taken with sharp items. These include:
a. The use of needles and syringes or other sharp instruments in the animal facility is limited to situations
where there is no alternative such as parenteral injection, blood collection, or aspiration of fluids from
laboratory animals and diaphragm bottles.
b. Disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or
otherwise manipulated by hand before disposal. Used, disposable needles must be carefully placed in
puncture-resistant containers used for sharps disposal. Sharps containers should be located as close to
the work site as possible.
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; it should be removed using a brush and dustpan, tongs, or
forceps. Plastic ware should be substituted for glassware whenever possible.
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e. Use of equipment with sharp edges and corners should be avoided.
12. Equipment and work surfaces are routinely decontaminated with an appropriate disinfectant after work with an
infectious agent, and after any spills, splashes, or other overt contamination.
13. Animals and plants not associated with the work being performed must not be permitted in the areas where
infectious materials and/ or animals are housed or manipulated.
14. An effective integrated pest management program is required.
15. All wastes from the animal room (including animal tissues, carcasses, and bedding) are transported from the
animal room in leak-proof containers for appropriate disposal in compliance with applicable institutional, local
and state requirements.
Decontaminate all potentially infectious materials before disposal using an effective method.
B.
Special Practices
1. Animal care staff, laboratory and routine support personnel must be provided a medical surveillance program as
dictated by the risk assessment and administered appropriate immunizations for agents handled or potentially
present, before entry into animal rooms.
When appropriate, a base line serum sample should be stored.
2. Procedures involving a high potential for generating aerosols should be conducted within a biosafety cabinet or
other physical containment device. When a procedure cannot be performed within a biosafety cabinet, a
combination of personal protective equipment and other containment devices must be used.
Restraint devices and practices that reduce the risk of exposure during animal manipulations (e.g., physical
restraint devices, chemical restraint medications) should be used whenever possible.
3. Decontamination by an appropriate method (e.g. autoclave, chemical disinfection, or other approved
decontamination methods) is necessary for all potentially infectious materials and animal waste before
movement outside the areas where infectious materials and/or animals are housed or are manipulated. This
includes potentially infectious animal tissues, carcasses, contaminated bedding, unused feed, sharps, and
other refuse.
A method for decontaminating routine husbandry equipment, sensitive electronic and medical equipment should be
identified and implemented.
Materials to be decontaminated outside of the immediate areas where infectious materials and/or animals are
housed or are manipulated must be placed in a durable, leak proof, covered container and secured for
transport. The outer surface of the container is disinfected prior to moving materials. The transport container
must have a universal biohazard label.
Develop and implement an appropriate waste disposal program in compliance with applicable institutional, local and
state requirements. Autoclaving of content prior to incineration is recommended.
4. Equipment, cages, and racks should be handled in a manner that minimizes contamination of other areas.
Equipment must be decontaminated before repair, maintenance, or removal from the areas where infectious
materials and/or animals are housed or are manipulated.
5. Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained
and equipped to work with infectious material.
6. Incidents that may result in exposure to infectious materials must be immediately evaluated and treated according to procedures
described in the safety manual. All such incidents must be reported to the animal facility supervisor or personnel designated by the
institution. Medical evaluation, surveillance, and treatment should be provided as appropriate and records maintained.
C.
Safety Equipment (Primary Barriers and Personal Protective Equipment)
1. Properly maintained BSCs, personal protective equipment (e.g., gloves, lab coats, face shields, respirators, etc.)
and/or other physical containment devices or equipment, are used whenever conducting procedures with a
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potential for creating aerosols, splashes, or other potential exposures to hazardous materials. These include
necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, and intranasal
inoculation of animals.
When indicated by risk assessment, animals are housed in primary biosafety containment equipment appropriate
for the animal species, such as solid wall and bottom cages covered with filter bonnets for rodents or other
equivalent primary containment systems for larger animal cages.
2. A risk assessment should determine the appropriate type of personal protective equipment to be utilized.
Scrub suits and uniforms are removed before leaving the animal facility. Reusable clothing is appropriately
contained and decontaminated before being laundered. Laboratory and protective clothing should never be
taken home.
Gowns, uniforms, laboratory coats and personal protective equipment are worn while in the areas where infectious
materials and/or animals are housed or manipulated and removed prior to exiting. Disposable personal
protective equipment and other contaminated waste are appropriately contained and decontaminated prior to
disposal.
3. Eye and face protection (mask, goggles, face shield or other splatter guard) are used for manipulations or
activities that may result in splashes or sprays from infectious or other hazardous materials and when the
animal or 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 should also wear eye protection when entering areas with potentially high concentrations
or airborne particulates.
Persons having contact with NHPs should assess risk of mucous membrane exposure and wear protective
equipment (e.g., masks, goggles, face shields) appropriate for the task to be performed. Respiratory protection
is worn based upon risk assessment.
4. Gloves are worn to protect hands from exposure to hazardous materials. A risk assessment should be performed to identify the
appropriate glove
Gloves are changed when contaminated, glove integrity is compromised, or when otherwise necessary.
Gloves must not be worn outside the animal rooms.
Gloves and personal protective equipment should be removed in a manner that prevents transfer of infectious
materials.
Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated waste.
Persons must wash their hands after handling animals and before leaving the areas where infectious materials and/or animals are
housed or are manipulated. Hand washing should occur after the removal of gloves.
D.
Laboratory Facilities (Secondary Barriers)
1. The animal facility is separated from areas that are open to unrestricted personnel traffic within the building.
External facility doors are self-closing and self-locking.
Doors to areas where infectious materials and/or animals are housed, open inward, are self-closing, are kept closed
when experimental animals are present, and should never be propped open. Doors to cubicles inside an animal
room may open outward or slide horizontally or vertically.
2. A hand-washing sink is located at the exit of the areas where infectious materials and/or animals are housed or
are manipulated. Additional sinks for hand washing should be located in other appropriate locations within the
facility.
If the animal facility has segregated areas where infectious materials and/or animals are housed or manipulated, a
sink must also be available for hand washing at the exit from each segregated area.
Sink traps are filled with water, and/or appropriate disinfectant to prevent the migration of vermin and gases.
3. The animal facility is designed, constructed, and maintained to facilitate cleaning and housekeeping. The interior
surfaces (walls, floors and ceilings) are water resistant.
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Penetrations in floors, walls and ceiling surfaces are sealed, including openings around ducts, doors and
doorframes, to facilitate pest control and proper cleaning.
Floors must be slip-resistant, impervious to liquids, and resistant to chemicals.
4. Cabinets and bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and
other chemicals. Spaces between benches, cabinets, and equipment should be accessible for cleaning.
Furniture should be minimized. Chairs used in animal area must be covered with a non-porous material that can be
easily cleaned and decontaminated. Furniture must be capable of supporting anticipated loads and uses.
Sharp edges and corners should be avoided.
5. External windows are not recommended; if present, windows must be sealed and resistant to breakage. The
presence of windows may impact facility security and therefore should be assessed by security personnel.
6. Ventilation should be provided in accordance with the Guide for Care and Use of Laboratory Animals.1 The
direction of airflow into the animal facility is inward; animal rooms maintain inward directional airflow compared
to adjoining hallways. A ducted exhaust air ventilation system is provided. Exhaust air is discharged to the
outside without being recirculated to other rooms.
Ventilation system design should consider the heat and high moisture load produced during the cleaning of animal
rooms and the cage wash process.
7. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize
horizontal surface areas, to facilitate cleaning and minimize the accumulation of debris or fomites.
8. Floor drains must be maintained and filled with water, and/or appropriate disinfectant to prevent the migration of
vermin and gases.
9. Cages should be autoclaved or otherwise decontaminated prior to washing. Mechanical cage washer should
have a final rinse temperature of at least 180°F. The cage wash area should be designed to accommodate the
use of high-pressure spray systems, humidity, strong chemical disinfectants and 180°F water temperatures
during the cage/equipment cleaning process.
10. Illumination is adequate for all activities, avoiding reflections and glare that could impede vision.
11. If BSCs are present, they must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper
operations. BSCs should be located away from doors, heavily traveled laboratory areas, and other possible airflow disruptions.
HEPA filtered exhaust air from a Class II BSC can be safely re-circulated back into the laboratory environment if the
cabinet is tested and certified at least annually and operated according to manufacturer’s recommendations.
BSCs can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or
directly to the outside through an independent, hard connection. Provisions to assure proper safety cabinet
performance and air system operation must be verified. BSCs should be recertified at least once a year to
ensure correct performance.
All BSCs should be used according to manufacturer’s specifications to protect the worker and avoid creating a
hazardous environment from volatile chemicals and gases.
12. If vacuum service (i.e., central or local) is provided, each service connection should be fitted with liquid
disinfectant traps and an in-line HEPA filter placed as near as practicable to each use point or service cock.
Filters are installed to permit in-place decontamination and replacement.
13. An autoclave should be present in the animal facility to facilitate decontamination of infectious materials and
waste.
14. Emergency eyewash and shower are readily available; location is determined by risk assessment.
ANIMAL BIOSAFETY LEVEL 3
Animal Biosafety Level 3 involves practices suitable for work with laboratory animals infected with indigenous or exotic
agents, agents that present a potential for aerosol transmission, and agents causing serious or potentially lethal
disease. ABSL-3 builds upon the standard practices, procedures, containment equipment, and facility requirements of
ABSL-2.
The ABSL-3 laboratory has special engineering and design features.
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A.
Standard Microbiological Practices
1. The animal facility director establishes and enforces policies, procedures, and protocols for institutional policies
and emergencies.
Each institute must assure that worker safety and health concerns are addressed as part of the animal protocol
review.
Prior to beginning a study, animal protocols must be reviewed and approved by the IACUC 5 and the Institutional
Biosafety Committee.
2. A safety manual specific to the animal facility is prepared or adopted in consultation with the animal facility
director and appropriate safety professionals.
The safety manual must be available and accessible. Personnel are advised of potential and special hazards, and
are required to read and follow instructions on practices and procedures.
Consideration must be given to specific biohazards unique to the animal species and protocol in use.
3. The supervisor must ensure that animal care, laboratory and support personnel receive appropriate training
regarding their duties, animal husbandry procedures, potential hazards, manipulations of infectious agents,
necessary precautions to prevent hazard or exposures, and hazard/exposure evaluation procedures (physical
hazards, splashes, aerosolization, etc.). Personnel must receive annual updates or additional training when
procedures or policies change. Records are maintained for all hazard evaluations, employee training sessions
and staff attendance.
4. An appropriate medical surveillance program is in place, as determined by risk assessment. The need for an
animal allergy prevention program should be considered.
Facility supervisors should ensure that medical staff is informed of potential occupational hazards within the animal
facility, to include those associated with the research, animal husbandry duties, animal care, and
manipulations.
Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or
prophylactic interventions. Therefore, all personnel and particularly women of childbearing age should be
provided information regarding immune competence and conditions that may predispose them to infection.
Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider
for appropriate counseling and guidance.
Personnel using respirators must be enrolled in an appropriately constituted respiratory protection program.
5. A sign incorporating the universal biohazard symbol must be posted at the entrance to areas where infectious
materials and/or animals are housed or are manipulated. The sign must include the animal biosafety level,
general occupational health requirements, personal protective equipment requirements, the supervisor’s name
(or other responsible personnel), telephone number, and required procedures for entering and exiting the
animal areas. Identification of specific infectious agents is recommended when more than one agent is used
within an animal room.
Security-sensitive agent information and occupational health requirements should be posted in accordance with the
institutional policy.
Advance consideration should be given to emergency and disaster recovery plans, as a contingency for man-made
or natural disasters.1,3,4
6. Access to the animal room is limited to the fewest number of individuals possible. Only those persons required
for program or support purposes are authorized to enter the animal facility and the areas where infectious
materials and/or animals are housed or are manipulated.
All persons, including facility personnel, service workers, and visitors, are advised of the potential hazards (natural
or research pathogens, allergens, etc.) and are instructed on the appropriate safeguards.
7. Protective laboratory coats, gowns, or uniforms are recommended to prevent contamination of personal clothing.
Gloves are worn to prevent skin contact with contaminated, infectious/hazardous materials and when handling
animals. Double-glove practices should be used when dictated by risk assessment.
Gloves and personal protective equipment should be removed in a manner that prevents transfer of infectious
materials outside of the areas where infectious materials and/or animals are housed or are manipulated.
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Persons must wash their hands after removing gloves and before leaving the areas where infectious materials
and/or animals are housed or are manipulated.
Eye, face and respiratory protection should be used in rooms containing infected animals, as dictated by the risk assessment.
8. 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.
9. All procedures are carefully performed to minimize the creation of aerosols or splatters of infectious materials and
waste.
10. Mouth pipetting is prohibited. Mechanical pipetting devices must be used.
11. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be
developed and implemented.
When applicable, laboratory supervisors should adopt improved engineering and work practice controls that
reduce the risk of sharps injuries. Precautions must always be taken with sharp items. These include:
a. Use of needles and syringes or other sharp instruments in the animal facility is limited to situations where
there is no alternative such as parenteral injection, blood collection, or aspiration of fluids from laboratory
animals and diaphragm bottles.
b. Disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or
otherwise manipulated by hand before disposal. Used, disposable needles must be carefully placed in
puncture-resistant containers used for sharps disposal. Sharps containers should be located as close to
the work site as possible.
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; it should be removed using a brush and dustpan, tongs, or
forceps. Plastic ware should be substituted for glassware whenever possible.
e. Use of equipment with sharp edges and corners should be avoided.
12. Equipment and work surfaces are routinely decontaminated with an appropriate disinfectant after work with an
infectious agent, and after any spills, splashes, or other overt contamination.
13. Animals and plants not associated with the work being performed must not be permitted in the areas where
infectious materials and/ or animals are housed or are manipulated.
14. An effective integrated pest management program is required.
15. All wastes from the animal room (including animal tissues, carcasses, and bedding) are transported from the
animal room in leak-proof containers for appropriate disposal in compliance with applicable institutional, local
and state requirements.
Decontaminate all potentially infectious materials before disposal using an effective method.
B.
Special Practices
1. Animal care staff, laboratory and routine support personnel must be provided a medical surveillance program as
dictated by the risk assessment and administered appropriate immunizations for agents handled or potentially
present, before entry into animal rooms.
When appropriate, a base line serum sample should be stored.
2. All procedures involving the manipulation of infectious materials, handling of infected animals or the generation of
aerosols must be conducted within BSCs or other physical containment devices when practical.
When a procedure cannot be performed within a biosafety cabinet, a combination of personal protective equipment
and other containment devices must be used.
Restraint devices and practices are used to reduce the risk of exposure during animal manipulations (e.g., physical
restraint devices, chemical restraint medications).
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3. The risk of infectious aerosols from infected animals or their bedding also can be reduced if animals are housed
in containment caging systems, such as solid wall and bottom cages covered with filter bonnets, open cages
placed in inward flow ventilated enclosures, HEPA-filter isolators and caging systems, or other equivalent
primary containment systems.
4. Actively ventilated caging systems must be designed to prevent the escape of microorganisms from the cage.
Exhaust plenums for these systems should be sealed to prevent escape of microorganisms if the ventilation
system becomes static, and the exhaust must be HEPA filtered. Safety mechanisms should be in place that
prevent the cages and exhaust plenums from becoming positive to the surrounding area should the exhaust fan
fail. The system should also be alarmed to indicate operational malfunctions.
5. A method for decontaminating all infectious materials must be available within the facility, preferably within the areas
where infectious materials and/or animals are housed or are manipulated (e.g., autoclave, chemical disinfection, or
other approved decontamination methods).
Consideration must be given to means for decontaminating routine husbandry equipment, sensitive electronic and
medical equipment.
Decontaminate all potential infectious materials (including animal tissues, carcasses, contaminated bedding,
unused feed, sharps, and other refuse) by an appropriate method before removal from the areas where
infectious materials and/or animals are housed or manipulated.
It is recommended that animal bedding and waste be decontaminated prior to manipulation and before removal
from the areas where infectious materials and/or animals are housed or are manipulated, preferably within the
caging system.
Develop and implement an appropriate waste disposal program in compliance with applicable institutional, local and
state requirements.
6. Equipment, cages, and racks should be handled in a manner that minimizes contamination of other areas.
Equipment must be decontaminated before repair, maintenance, or removal from the areas where infectious
materials and/or animals are housed or are manipulated.
Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained
and equipped to work with infectious material.
7. Incidents that may result in exposure to infectious materials must be immediately evaluated and treated according to procedures
described in the safety manual. All such incidents must be reported to the animal facility supervisor or personnel designated by the
institution. Medical evaluation, surveillance, and treatment should be provided as appropriate and records maintained.
A.
Safety Equipment (Primary Barriers and Personal Protective Equipment)
1. Properly maintained BSCs and other physical containment devices or equipment should be used for all
manipulations for infectious materials and when possible, animals. These manipulations include necropsy,
harvesting of tissues or fluids from infected animals or eggs, and intranasal inoculation of animals.
The risk of infectious aerosols from infected animals or bedding can be reduced by primary barrier systems. These systems may
include solid
wall and bottom cages covered with filter bonnets, ventilated cage rack systems, or for larger cages placed in
inward flow ventilated enclosures or other equivalent systems or devices.
2. A risk assessment should determine the appropriate type of personal protective equipment to be utilized.
Personnel within the animal facility where protective clothing, such as uniforms or scrub suits. Reusable clothing is
appropriately contained and decontaminated before being laundered. Laboratory and protective clothing should
never be taken home. Disposable personal protective equipment such as non-woven olefin cover-all suits,
wrap-around or solid-front gowns should be worn over this clothing, before entering the areas where infectious
materials and/or animals are housed or manipulated. Front-button laboratory coats are unsuitable.
Disposable personal protective equipment must be removed when leaving the areas where infectious materials
and/or animals are housed or are manipulated. Scrub suits and uniforms are removed before leaving the
animal facility.
Disposable personal protective equipment and other contaminated waste are appropriately contained and
decontaminated prior to disposal.
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3. All personnel entering areas where infectious materials and/or animals are housed or manipulated wear appropriate eye, face and
respiratory protection. To prevent cross contamination, boots, shoe covers, or other protective footwear, are used where indicated.
Eye and face protection must be disposed of with other contaminated laboratory waste or decontaminated before
reuse. Persons who wear contact lenses should also wear eye protection when entering areas with potentially
high concentrations or airborne particulates.
4. Gloves are worn to protect hands from exposure to hazardous materials.
A risk assessment should be performed to identify the appropriate glove for the task and alternatives to latex gloves
should be available.
Procedures may require the use of wearing two pairs of gloves (double-glove).
Gloves are changed when contaminated, glove integrity is compromised, or when otherwise necessary.
Gloves must not be worn outside the animal rooms.
Gloves and personal protective equipment should be removed in a manner that prevents transfer of infectious
materials.
Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated waste.
Persons must wash their hands after handling animals and before leaving the areas where infectious materials and/or animals are
housed or are manipulated. Hand washing should occur after the removal of gloves.
D. Laboratory Facilities (Secondary Barriers)
1. The animal facility is separated from areas that are open to unrestricted personnel traffic within the building.
External facility doors are self-closing and self-locking.
Access to the animal facility is restricted.
Doors to areas where infectious materials and/or animals are housed, open inward, are self-closing, are kept closed
when experimental animals are present, and should never be propped open.
Entry into the containment area is via a double-door entry, which constitutes an anteroom/airlock and a change room. Showers may
be considered based on risk assessment. An additional double-door access anteroom or double-doored autoclave may be provided
for movement of supplies and wastes into and out of the facility.
2. A hand-washing sink is located at the exit of the areas where infectious materials and/or animals are housed or
are manipulated. Additional sinks for hand washing should be located in other appropriate locations within the
facility. The sink should be hands-free or automatically operated.
If the animal facility has multiple segregated areas where infectious materials and/or animals are housed or are
manipulated, a sink must also be available for hand washing at the exit from each segregated area.
Sink traps are filled with water, and/or appropriate liquid to prevent the migration of vermin and gases.
3. The animal facility is designed, constructed, and maintained to facilitate cleaning, decontamination and
housekeeping. The interior surfaces (walls, floors and ceilings) are water resistant.
Penetrations in floors, walls and ceiling surfaces are sealed, including openings around ducts and doorframes, to facilitate pest
control, proper cleaning and decontamination. Walls, floors and ceilings should form a sealed and sanitizable surface.
Floors must be slip resistant, impervious to liquids, and resistant to chemicals. Flooring is seamless, sealed resilient
or poured floors, with integral cove bases.
Decontamination of an entire animal room should be considered when there has been gross contamination of the
space, significant changes in usage, for major renovations, or maintenance shut downs. Selection of the
appropriate materials and methods used to decontaminate the animal room must be based on the risk
assessment.
4. Cabinets and bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and
other chemicals. Spaces between benches, cabinets, and equipment should be accessible for cleaning.
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Furniture should be minimized. Chairs used in animal areas must be covered with a non-porous material that can
be easily cleaned and decontaminated. Furniture must be capable of supporting anticipated loads and uses.
Equipment and furnishings with sharp edges and corners should be avoided.
5. External windows are not recommended; if present, all windows must be sealed and must be resistant to
breakage. The presence of windows may impact facility security and therefore should be assessed by security
personnel.
6. Ventilation of the facility should be provided in accordance with the Guide for Care and Use of Laboratory
Animals.1 The direction of airflow into the animal facility is inward; animal rooms maintain inward directional
airflow compared to adjoining hallways. A ducted exhaust air ventilation system is provided. Exhaust air is
discharged to the outside without being recirculated to other rooms. This system creates directional airflow,
which draws air into the animal room from “clean” areas and toward “contaminated” areas.
Ventilation system design should consider the heat and high moisture load produced during the cleaning of animal
rooms and the cage wash process. HEPA filtration and other treatments of the exhaust air may not be required,
but should be considered based on site requirements, specific agent manipulations and use conditions. The
exhaust must be dispersed away from occupied areas and air intakes.
Personnel must verify that the direction of the airflow (into the animal areas) is proper. It is recommended that a visual monitoring
device that indicates directional inward airflow be provided at the animal room entry. The ABSL-3 animal facility shall be designed
such that under failure
conditions the airflow will not be reversed. Alarms should be considered to notify personnel of ventilation and HVAC
system failure.
7. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize
horizontal surface areas, to facilitate cleaning and minimize the accumulation of debris or fomites.
8. Floor drains must be maintained and filled with water, and/or appropriate disinfectant to prevent the migration of
vermin and gases.
9. Cages are washed in a mechanical cage washer. The mechanical cage washer has a final rinse temperature of
at least 180°F. Cages should be autoclaved or otherwise decontaminated prior to removal from ABSL-3 space.
The cage wash facility should be designed and constructed to accommodate high-pressure spray systems,
humidity, strong chemical disinfectants and 180°F water temperatures during the cage cleaning process.
10. Illumination is adequate for all activities, avoiding reflections and glare that could impede vision.
11. BSCs (Class II, Class III) must be installed so that fluctuations of the room air supply and exhaust do not
interfere with proper operations. Class II BSCs should be located away from doors, heavily traveled laboratory
areas, and other possible airflow disruptions.
HEPA filtered exhaust air from a Class II BSC can be safely re-circulated into the laboratory environment if the
cabinet is tested and certified at least annually and operated according to manufacturer’s recommendations.
BSCs can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or
exhausted directly to the outside through a direct (hard) connection. Provisions to assure proper safety cabinet
performance and air system operation must be verified. BSCs should be certified at least annually to assure
correct performance.
Class III BSCs must supply air in such a manner that prevents positive pressurization of the cabinet or the
laboratory room.
All BSCs should be used according to manufacturers’ specifications.
When applicable, equipment that may produce infectious aerosols must be contained in devices that exhaust air
through HEPA filtration or other equivalent technology before being discharged into the animal facility. These
HEPA filters should be tested and/or replaced at least annually.
12. An autoclave is available which is convenient to the animal rooms where the biohazard is contained. The
autoclave is utilized to decontaminate infectious materials and waste before moving it to the other areas of the
facility. If not convenient to areas where infectious materials and/or animals are housed or are manipulated,
special practices should be developed for transport of infectious materials to designated alternate location/s
within the facility.
13. Emergency eyewash and shower are readily available; location is determined by risk assessment.
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14. The ABSL-3 facility design and operational procedures must be documented. The facility must be tested to
verify that the design and operational parameters have been met prior to use. Facilities should be re-verified at
least annually against these procedures as modified by operational experience.
15. Additional environmental protection (e.g., personnel showers, HEPA filtration of exhaust air, containment of other piped
services, and the provision of effluent decontamination) should be considered if recommended by the agent summary statement, as
determined by risk assessment of the site conditions, or other applicable federal, state or local regulations.
ANIMAL BIOSAFETY LEVEL 4
(There are no ABSL-4 Facilities at the UTHSCSA)
Animal Biosafety Level 4 is required for work with animals infected with dangerous and exotic agents that pose a high individual risk of
aerosol-transmitted laboratory infections and life-threatening disease that is frequently fatal, for which there are no vaccines or treatments;
or a related agent with unknown risk of transmission. Agents with a close or identical antigenic relationship to agents requiring ABSL-4
containment must be handled at this level until sufficient data are obtained either to confirm continued work at this level, or to redesignate the level. Animal care staff must have specific and thorough training in handling extremely hazardous, infectious agents and
infected animals. Animal care staff must understand the primary and secondary containment functions of standard and special practices,
containment equipment, and laboratory design characteristics. All animal care staff and supervisors must be competent in handling
animals, agents and procedures requiring ABSL-4 containment. The animal facility director and/or laboratory supervisor control access to
the animal facility within the ABSL-4 laboratory in accordance with institutional policies.
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APPENDIX F
Lab Specific Forms and Plans
1. Guidelines for Writing Standard Operating Procedures (SOP)
Purpose:
SOPs should be viewed as valuable research tools that are worth the time and effort required to
prepare them. Written SOPs document that a risk assessment has been performed and provide a
written means to inform and advise workers about hazards in their work place.
A well-written SOP can be used to satisfy several compliance requirements. For example,
CDC/NIH guidelines in the "Biosafety Level 2, Standard Microbiological Practices" section of
Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition states:
"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.”
SOPs should be written for all procedures that pose an identified potential risk to the health and
safety of the worker and of others present in the lab. A separate SOP does not need to be written
for each individual experiment, just for the handling of each organism. Organisms that pose the
same risks and are handled in the same way may be included in one SOP. Be sure to include
exposure controls and safety precautions for all identified potential risks of exposure associated
with the types of procedures the organisms may be used for, i.e. centrifugation, contaminated
sharps, homogenization, etc.
The process of writing SOPs requires an individual to think through all steps of a procedure and
perform a risk assessment before work is begun. This process allows for standardization of
materials and methods, resulting in quality research as well as identifying any safety issues
associated with the procedure.
General Guidelines:
Every SOP should include:

date written, dates of revisions, name of person that wrote the SOP

procedural methods/materials (detailed enough to allow someone to complete the procedure)

risk identification

exposure controls

waste disposal

spill procedures

accident procedures
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
any pertinent references

any required record keeping
SOPs must be lab specific. They should not consist only of copies of manufacturer's inserts,
manuals from other sources, or another lab's SOPs (unless work is collaborative and carried out in
the same research space). You may, however, wish to refer to other documents.
The template is for guidance only and is not meant to be all-inclusive or a required format.
Steps to Write a SOP:
Task
Review laboratory protocols and identify the
potential hazards associated with the
Step procedures performed in the protocols - Risk
Assessment.
1
Examples
Using Risk Group 2
microorganisms
Using biological
toxins
rDNA procedures
Aerosol production
Determine the types of exposure risk to the
identified hazards that each step could present. Use of sharps
Step
2
Exposure to infected
animals
Resource
Risk Group
Classifications
(basis for biosafety
level evaluation)
MSDS for Biological
Materials
Bloodborne
Pathogen Exposure
Control Plan
Department of
Laboratory Animal
Resources (DLAR)
Develop an exposure control plan that
workers must adhere to that will minimize
the risk of personal exposure and prevent
the release of infectious agents. The
exposure control plan must include each
exposure risk identified in Step 2. Include
personal protective equipment and work
practices in accordance with the Biosafety
Step Level determined by completing the Risk
3
Assessment process in Step 1.
Secondary
containment in
centrifuges
Working in a
certified biological
safety cabinet
Frequency and
method used to
clean work surfaces
Biosafety in
Microbiological and
Biomedical
Laboratories
(BMBL) - 5th
Edition
NIH Guidelines for
Research Involving
Recombinant DNA
Molecules
Personal protective
equipment
required; gloves,
face shields, lab
coat, etc.
Immunizations, if
needed
Identify the types of wastes that will be
Step generated and plan for how they will be
treated/disposed of.
4
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Sharps
Culture media
Biomedical Waste
Management SOP
Contaminated
disposables
Page 124
Chemical waste
Toxin waste
Develop a lab specific plan for how spills
and accidental exposures will be handled.
List emergency procedures including
location of emergency equipment,
emergency contact information with phone
numbers, spill clean-up/decontamination
Step
methods, and when and how to seek
5
emergency medical care. Include who the
worker should notify in case of an accident
and how to file an accident report.
Broken tubes in
centrifuge
Face/eye splash
Needle stick, cut or
contact with broken
skin
Contaminated liquid
spill
Injury reporting
procedures
Biological Spill and
Emergency
response plan
Include routine clean-up procedures and
materials in plan.
Completed SOPs should be:

brief, succinct, and usable.

used to train all new employees (remember to document training).

reviewed with employees as part of their annual laboratory-specific safety update training
(document training).

reviewed annually for accuracy and completeness by supervisor and workers.

available in the laboratory for worker reference.

may submitted with an IBC application.

used as a lab specific supplement to the department's Lab Safety Plan.

signed by the supervisor if written by someone other than P.I. or lab director.
2. Lab Specific Biological Spill and Emergency Response Template
This template is to be used for all laboratories that work with rDNA and/or biohazardous agents.
Specific information may include a description of the biohazardous agents used in the lab and the
type/brand of disinfectant used.
This biological spill and emergency response template complies with the NIH Guidelines, Section IVB-2-b-(6), Appendix G-I (Emergency Plan).
The lab specific emergency and spill response plan shall be posted in the laboratory.
SEE TEMPLATE ON NEXT PAGE:
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The University of Texas Health Science Center at San Antonio
Biological Spill Response and Laboratory Specific Emergency Plan
This template can be used in writing lab specific SOPs (Standard Operating Procedures) to comply with the NIH Guidelines, Section IV-B-2-b-(6),
Appendix G-I (Emergency Plan). Post this plan in the lab and review it with workers annually.
Complete the highlighted sections with Lab Specific Requirements.
P.I./Lab Supervisor:
Lab Location:
Biological Agent (s)
Emergency Contact Info:
(report all spills to P.I. or Lab Supervisor)
Type of Disinfectant/Notes on Use
Cleanup Procedures (bench tops,
centrifuges, etc.)
Section 1: Spill Response Equipment

Written spill procedure including emergency phone numbers

Disinfectant suitable for biological materials being used

Paper towels, gloves, shoe covers, safety glasses

Forceps to pick up sharps, including broken glass

Sharps container for broken glass, etc.

Autoclavable squeegee & dustpan

Biohazard bags (orange bags or autoclave clear bags for 60
minutes at 1210C)
Lab Specific Requirements:
Section 2: Small and moderate spills of low risk agents outside the
biological safety cabinet:

Notify other workers in the area of the spill and control traffic
through area.

Remove any contaminated clothing and put in autoclavable
bag. Be aware that autoclaving may damage fabric.

Wear shoe covers if spill is on floor, may be splashed beyond
immediate area of spill.

Put on gloves and cover spill area with paper towels.

Pour disinfectant over towels from edges of spill to center, be
careful not to splatter.

Decontaminate all objects in spill area.

Allow 20-30 minutes of contact time.

Use squeegee and dust pan to recover spill materials and put in
biohazard bag.

Pick up any sharps, including broken glass, with forceps and
place in sharps container.

Wipe area with disinfectant and clean towels, mop if spill on
floor.

Remove gloves and foot covers before leaving area of the spill,
put in biohazard bag, and wash hands.
Lab Specific Requirements:
Section 3: Large spills (greater than 100 mls) outside of the biological
safety cabinet:

Evacuate room, close doors, prevent others from entering, and wait 30
minutes for aerosols to settle.

Follow procedures for small and moderate spills or contact
Environmental Health and Safety at 567-2955 for assistance.
Lab Specific Requirements:
Section 4: For small to moderate spills
(less than 100 mls) in a biological safety cabinet,
follow Section 2 procedures plus:






Leave the cabinet running.
Wipe down all interior surfaces with appropriate disinfectant.
Determine if spill has gone beyond the work surface such as in the
grilles or side seams. Disassemble and decontaminate if necessary.
If the cabinet has a catch basin below the work surface that may be
involved in the spill, flood the basin with disinfectant. Do not use
alcohol as a large quantity of alcohol presents a flammable hazard.
Clean basin after 20 minutes.
Autoclave or wipe down all items in cabinet with disinfectant.
Let cabinet run for at least 10 minutes after cleanup.
Lab Specific Requirements:
Section 5: For major spills (greater than 100 mls) in a
biological safety cabinet:

Contact Environmental Health and Safety (567-2955) to determine if
professional decontamination is indicated.
For any spills of agents that are transmitted by inhalation, such as Mycobacterium tuberculosis, evacuate the lab immediately, close the door,
place a “Warning: Do Not Enter” sign on the door and do not allow anyone to enter the lab. Remove any contaminated clothing, wash
exposed skin with soap and water, call Environmental Health and Safety for assistance at 567-2955.
If Spill Results in a Hazard Exposure ( i.e. face and/or eye splash, cut or puncture with sharps, contact with non-intact skin):

Administer first aid (Wash wound with soap and water or flush eyes with water for 10 minutes) or call 911 or 567-2800 for serious
accidents (use of a university phone will contact UTPD and speed the dispatch process)

Report the incident to your supervisor as soon as possible.

Seek medical attention as soon as possible.
During business hours, employees may go to the UT Health Science Center Employee Health and Wellness Clinic
(EHWC) located in the Nursing School, room 1.445 (567-2788). Students may go to the Student Health Clinic (5679355). These clinics are familiar with occupational exposures (i.e. contaminated needlestick injury).
After business hours, EH&S suggests that you go to the Emergency Room at University Hospital (358-2488). The
Express Med clinic, 4150 Medical Drive (358-5510) is open until 10:00PM.
Note: Workers’ Compensation Insurance allows you to seek medical attention from alternate healthcare providers.

Complete the required forms including the Employer’s First Report of Injury form available on the UT Health Science Center’s
Environmental Health and Safety website under Worker’s Comp..

Report all biohazard incidents requiring medical treatment to Environmental Health and Safety at 567-2955 (FAX: 567-2965).
Note: It is important to fill out all of the appropriate paperwork in order to be eligible to collect workers compensation should any
illnesses arise from the hazardous exposure in the future. For rDNA exposures, the Biosafety Officer will make a report to NIH
OSP as required.
Lab Specific Requirements:
Environmental Health and Safety Department
Revised April 2016
Page 126
Appendix G
Standard Operating Procedures (SOPs) and Plans
1. Biosafety Cabinet SOP
2. Transport of Biological Materials SOP
3. Biomedical Waste Management SOP
4. Lentivirus SOP
5. FACS Lentivirus SOP
6. Autoclave SOP
7. Bloodborne Pathogen Exposure Control Plan
8. TB Exposure Control Plan
Environmental Health and Safety Department
Revised April 2016
Page 127
Training for all personnel (including students)
Principal Investigator: __________________________________
I acknowledge that I have read and understood the policies and procedures outlined in the Biological Safety
Handbook. I have received adequate training and am aware of the specific hazards of the biological agents
worked with in the laboratory/clinical area. All my questions have been answered to my satisfaction and I
understand that I should ask my supervisor/PI or Environmental Health & Safety if I have questions
regarding procedures and laboratory safety.
Date
Name
Environmental Health and Safety Department
Revised April 2016
HSC Badge #
Signature
Page 128