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Johns Hopkins Institutions Biosafety Manual

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Abstract

This Biosafety Manual was prepared to provide laboratory and clinical investigators current information on biohazard control. The manual will be updated as our knowledge of the risks involved in the handling of biological hazards increases and as methods for containment of these hazards improve.
JOHNS HOPKINS
INSTITUTIONS
BIOSAFETY MANUAL
June 1995
To obtain additional copies of this manual call the Office of Safety and
Environmental Health (OSEH), (410) 955-5918.
EMERGENCY TELEPHONE NUMBERS
EAST BALTIMORE CAMPUS
FIRE, pull alarm then dial..................................................................................................................................................... 5-4444
MEDICAL EMERGENCY .................................................................................................................................................. 5-4444
SECURITY EMERGENCY ................................................................................................................................................ 5-5585
OFFICE OF SAFETY & ENVIRONMENTAL HEALTH (OSEH) .................................................................................. 5-5918
STIX HOTLINE for bloodborne pathogens ........................................................................................................................ 5-7849
BIOSAFETY OFFICER ....................................................................................................................................................... 5-5918
RADIATION SAFETY OFFICER ...................................................................................................................................... 5-3712
COMPENSATION CLINIC ................................................................................................................................................ 5-6433
OCCUPATIONAL HEALTH SERVICES ......................................................................................................................... 5-6211
MARYLAND POISON CENTER ................................................................................................................................... 528-7701
HOMEWOOD CAMPUS
FIRE, pull alarm then dial.......................................................................................................................................................... 911
MEDICAL EMERGENCY ....................................................................................................................................................... 911
SECURITY EMERGENCY ................................................................................................................................................... 7777
OTHER EMERGENCY .......................................................................................................................................................... 7777
STUDENT HEALTH SERVICES .......................................................................................................................................... 8270
OFFICE OF SAFETY & ENVIRONMENTAL HEALTH (OSEH) ..................................................................................... 8798
BIOSAFETY OFFICER ................................................................................................................................................... 955-5918
RADIATION SAFETY OFFICER ......................................................................................................................................... 7308
OCCUPATIONAL HEALTH SERVICES ..................................................................................................................... 516-7701
MARYLAND POISON CENTER ................................................................................................................................... 528-7701
ASTHMA AND ALLERGY CENTER (BAYVIEW CAMPUS)
FIRE, pull alarm then dial..................................................................................................................................................... 0-2424
MEDICAL EMERGENCY .................................................................................................................................................. 0-2424
SECURITY EMERGENCY ................................................................................................................................................ 0-2424
FOREWARD
The Johns Hopkins Hospital and the Johns Hopkins University have high standards in
teaching and research. The University and Hospital are committed to provide an academic
and working environment as free as possible from all hazards. If we are to honor our
commitment we must have the continued cooperation of faculty, administration, research
and supervisory personnel for the establishment of safe procedures and for providing good
training and adequate facilities for the protection of employees, students, patients and
visitors.
The administration of the safety program has been placed with the Office of Safety and
Environmental Health which offers a wide range of technical assistance, training aids and
reference materials in biohazard control. Please use these resources to implement your
safety program.
Edward J. Bernacki, M.D., M.P.H., Chair
Joint Committee on Health, Safety and Environment
PREFACE
This Biosafety Manual was prepared to provide laboratory and clinical investigators current
information on biohazard control. The manual will be updated as our knowledge of the risks
involved in the handling of biological hazards increases and as methods for containment of these
hazards improve.
The Biosafety Manual brings together information which will assist supervisors in carrying out their
responsibilities in the management of biohazards. The Manual provides suggested procedures,
techniques and equipment to protect personnel, animals and experiments from unintentional
infection or contamination in addition to pertinent policy generated by Hopkins committees and
standards resulting from federal, state and local legislation.
The manual represents a compendium of biohazards control information that can serve as a base for
specific programs. All personnel using potentially biohazardous materials should become familiar
with the Biosafety Manual and should conduct their operation in accordance with the level of risk
appropriate for the procedure and the potentially biohazardous agent or materials they handle.
The success of the biohazardous control program depends on investigators who are motivated
toward a safe working environment and who have knowledge of safe operational procedures. The
Biosafety Office will endeavor to do its part by providing accurate information and technical
assistance to assist in the establishment of adequate biohazards controls for the protection of both the
investigators and their experiments.
Richard W. Gilpin, Ph.D., R.B.P.
Biosafety Officer
Byron S. Tepper, Ph.D., C.S.P.
Director, Office of Safety & Environmental Health
TABLE OF CONTENTS
PAGE
SECTION I -- SAFETY RESPONSIBILITIES
Administrative Responsibilities ............................................................................................................... 1
Administrative Officers .......................................................................................................................... 1
Department Chairman and/or Director .................................................................................................. 1
Principal Investigator and/or Supervisor ............................................................................................... 1
Individual Researchers and/or Technicians ........................................................................................... 2
Periodic Reviews and Updates of Registered Research .......................................................................... 3
Biological Agents and/or Materials ....................................................................................................... 3
Recombinant DNA ................................................................................................................................. 3
Registration of Research with
Pathogenic and/or Oncogenic Materials ................................................................................................ 4
Background ............................................................................................................................................. 4
Definition ................................................................................................................................................ 4
Responsibility ......................................................................................................................................... 4
Assessment of Hazard ............................................................................................................................ 5
Purpose of Registration .......................................................................................................................... 5
Procedure for Registration ...................................................................................................................... 6
Standard Operating Procedure (SOP) Manual
for Biosafety Level 3 Laboratories ...................................................................................................... 7
Vaccinia Virus Vaccination ...................................................................................................................... 8
Registration of Research with HBV and/or HIV ..................................................................................... 9
Requirements .......................................................................................................................................... 9
Informed Consent for Use of Postexposure Zidovudine (AZT) ........................................................... 9
Registration of Research with Sheep or Goats ...................................................................................... 12
Background ........................................................................................................................................... 12
Recommended Control Measures for Q Fever .................................................................................... 12
Registration of Research with Recombinant DNA ............................................................................... 14
Registration Form ................................................................................................................................. 14
Review Procedure ................................................................................................................................. 14
Review of Recombinant DNA Research With Moderate Risk
Biological Agents or Materials at Biosafety Level 1 or 2 ................................................................. 15
Review of Recombinant DNA Research Involving Biosafety Level 3
for High Risk Biological Agents or Materials or Recombinant
DNA Involving Human Subjects ....................................................................................................... 15
Registration of Research with Non-Human Primates ........................................................................... 17
Tuberculosis .......................................................................................................................................... 17
Herpesvirus-B in Macaque Monkeys .................................................................................................. 18
TABLE OF CONTENTS
PAGE
SECTION II -- SAFETY COMPLIANCE
Standard Practices, Training and Precautions ....................................................................................... 21
Universal Precautions Guidelines for Research Laboratories .............................................................. 23
Definitions ............................................................................................................................................ 23
Precautions ............................................................................................................................................ 23
HBV and/or HIV Agent Summary Statements ..................................................................................... 27
Q Fever Precautions ............................................................................................................................... 28
Background ........................................................................................................................................... 28
Rationale ............................................................................................................................................... 28
Registration ........................................................................................................................................... 28
Medical Surveillance ............................................................................................................................ 29
Transport of Animals ........................................................................................................................... 29
Personnel Protection ............................................................................................................................. 30
Waste Disposal ..................................................................................................................................... 31
Decontamination .................................................................................................................................. 31
Biosafety Level 1 Guidelines ................................................................................................................. 32
Biosafety Level 2 Guidelines ................................................................................................................. 33
Biosafety Level 3 Guidelines ................................................................................................................. 34
Animal Biosafety Levels ........................................................................................................................ 35
Biosafety References .............................................................................................................................. 36
Biosafety Level 3 Facility Specifications .............................................................................................. 38
Performance .......................................................................................................................................... 38
Location ................................................................................................................................................ 38
Floors .................................................................................................................................................... 38
Walls ..................................................................................................................................................... 38
Ceiling ................................................................................................................................................... 38
Access Zone .......................................................................................................................................... 38
Doors ..................................................................................................................................................... 38
Windows ............................................................................................................................................... 39
Plumbing ............................................................................................................................................... 39
Vacuum System .................................................................................................................................... 39
Electrical ............................................................................................................................................... 39
Light Fixtures ....................................................................................................................................... 39
Lab Furniture ........................................................................................................................................ 39
Hand Washing Sink .............................................................................................................................. 40
Eye, Face and Body Spray Fixture ...................................................................................................... 40
Autoclave .............................................................................................................................................. 40
Ventilation ............................................................................................................................................ 40
Biological Safety Cabinets ................................................................................................................... 41
Chemical Fume Hood .......................................................................................................................... 41
Air Pressure Differential ...................................................................................................................... 41
Directional Airflow .............................................................................................................................. 42
Air Supply Vents .................................................................................................................................. 42
TABLE OF CONTENTS
PAGE
Fire Protection ....................................................................................................................................... 42
Summary of Biosafety Levels ................................................................................................................ 43
Classification of Etiologic Agents on the Basis of Hazard ................................................................... 44
Classification of Ongogenic Viruses on the Basis of Hazard ............................................................... 45
Criteria for Moderate Risk Oncogenic Viruses ................................................................................... 45
Criteria for High Risk Oncogenic Viruses ........................................................................................... 45
General Laboratory Practices and Procedures ....................................................................................... 46
Employee Action .................................................................................................................................. 46
Food, Drink, and Cosmetics ................................................................................................................. 46
Hand Washing ....................................................................................................................................... 46
Beards and Long Hair ........................................................................................................................... 47
Books and Journals ............................................................................................................................... 47
Pipettes .................................................................................................................................................. 47
Syringes ................................................................................................................................................. 48
Disposal of Research Laboratory Waste ................................................................................................ 49
Definition of Sharps .............................................................................................................................. 49
Disposal of Laboratory Waste .............................................................................................................. 49
Approved Laboratory Waste Containers ............................................................................................. 49
Utilization of Six or Ten Quart Plastic Containers .............................................................................. 50
Utilization of the Biohazard Box ......................................................................................................... 50
Additional Containers ........................................................................................................................... 51
Safe Disposal of Sharps Containers ..................................................................................................... 51
Disposal of General Laboratory Glass ................................................................................................. 52
Summary of Waste Disposed ............................................................................................................... 52
Centrifuge Containment ......................................................................................................................... 53
Centrifuge Precautions ......................................................................................................................... 53
Centrifuge Tubes ................................................................................................................................... 53
High-Speed Centrifuges ....................................................................................................................... 54
Containment Centrifugation ................................................................................................................. 54
Blenders, Sonicators, Mills, Grinders & Cell Sorters ........................................................................... 57
Water Bath Disinfectants ........................................................................................................................ 58
Laboratory Vacuum Traps ...................................................................................................................... 59
Refrigerators, Freezers and Dry Ice Chests ........................................................................................... 61
Transportation of Biohazardous Material .............................................................................................. 62
Test Tube and/or Vacutainer Techniques .............................................................................................. 63
Membrane Filters .................................................................................................................................... 64
Hazardous Operations (Two Person Rule) ............................................................................................ 65
Use of Dry Powders ................................................................................................................................ 66
Laboratory Construction and Renovation .............................................................................................. 67
Animal Care and Handling ..................................................................................................................... 68
Decontamination and Disinfection Procedures ..................................................................................... 69
TABLE OF CONTENTS
PAGE
Steam Autoclaving ................................................................................................................................. 71
Autoclave Decontamination Procedures ............................................................................................... 72
Background ........................................................................................................................................... 72
Indicators .............................................................................................................................................. 72
Containers ............................................................................................................................................. 74
Autoclave Operation ............................................................................................................................ 75
Processing Times .................................................................................................................................. 76
Odor Control ......................................................................................................................................... 77
Training ................................................................................................................................................. 77
Record Keeping .................................................................................................................................... 77
Autoclave Log Book Samples ............................................................................................................. 78
Dry Heat Sterilization ............................................................................................................................ 83
Ethylene Oxide Sterilization .................................................................................................................. 84
Formaldehyde Gas Decontamination of Equipment ............................................................................. 85
Ultraviolet Light Decontamination ........................................................................................................ 86
UV Lamp Operation ............................................................................................................................. 86
Training ................................................................................................................................................. 87
Chemical Disinfectants .......................................................................................................................... 88
Phenolic Compounds ........................................................................................................................... 88
Quaternary Ammonium Compounds .................................................................................................. 88
Iodophors .............................................................................................................................................. 89
Alcohols ................................................................................................................................................ 90
Aldehydes ............................................................................................................................................. 90
Chlorine Compounds ........................................................................................................................... 91
Mercurials ............................................................................................................................................. 91
Clean-up of Biohazardous Spills ........................................................................................................... 93
Biohazard Spill Inside a Biological Safety Cabinet ............................................................................ 93
Biohazard Spill Outside a Biological Safety Cabinet ......................................................................... 93
Radioactive Biohazard Spill Outside a Biological Safety Cabinet .................................................... 95
Shipment of Biological Materials .......................................................................................................... 97
SECTION III -- RECOMBINANT DNA GUIDELINES
Introduction ........................................................................................................................................ 101
Table of Contents ............................................................................................................................... 101
Section I. Scope of the NIH Guidelines ......................................................................................... 104
Section II. Containment ................................................................................................................... 106
Section III. Experiments Covered by the NIH Guidelines .............................................................. 107
Section IV. Roles and Responsibilities ............................................................................................ 114
Section V. Footnotes and References of Sections I Through IV .................................................... 124
Appendix A. Exemptions Under Section III-E-5
Sublists of Natural Exchangers ...................................................................................... 126
TABLE OF CONTENTS
PAGE
Appendix B. Classification of Etiologic Agents and Oncogenic Viruses
on the Basis of Hazard .................................................................................................... 127
Appendix C. Exemptions Under Section III-E-6 ............................................................................. 133
Appendix D. Major Actions Taken Under the NIH Guidelines ...................................................... 136
Appendix E. Certified Host-Vector Systems .................................................................................... 136
Appendix F. Containment Conditions for Cloning of Genes
Coding for the Biosynthesis of Molecules Toxic for Vertebrates ................................. 138
Appendix G. Physical Containment .................................................................................................. 139
Appendix H. Shipment ...................................................................................................................... 151
Appendix I. Biological Containment ............................................................................................... 152
Appendix J. Biotechnology Research Subcommittee ..................................................................... 155
Appendix K. Physical Containment for Large Scale Uses of Organisms
Containing Recombinant DNA Molecules ..................................................................... 155
Appendix L. Release into the Environment of Certain Plants ......................................................... 165
Appendix M. Points to Consider in the Design and Submission
of Protocols for the Transfer of Recombinant DNA Molecules
into the Genome of One or More Human Subjects .................................................. 165
Appendix P. Physical and Biological Containment for
Recombinant DNA Research Involving Plants ............................................................ 176
Appendix Q. Physical and Biological Containment for
Recombinant DNA Research Involving Animals ........................................................ 187
SECTION IV -- BIOHAZARD CABINETRY
Biohazard Containment Equipment ..................................................................................................... 202
Types of Containment Equipment ..................................................................................................... 202
Use of Containment Equipment ......................................................................................................... 202
Biohazard Cabinet Containment Equipment ..................................................................................... 203
Biological Safety Cabinet Categories .................................................................................................. 205
Use of Biological Safety Cabinets ....................................................................................................... 208
Operation of Biological Safety Cabinets ............................................................................................. 210
General Suggestions ........................................................................................................................... 210
Starting Up Your BSC ........................................................................................................................ 210
Cleaning Your BSC ............................................................................................................................ 213
Flammable or Explosive Materials within BSC's .............................................................................. 214
BSC Assignment for Specific Biosafety Levels .................................................................................. 215
Biosafety Cabinet Training Materials .................................................................................................. 216
Characteristics of BSC'S and other Airflow Equipment ..................................................................... 218
Class 1 Biological Safety Cabinets .................................................................................................... 219
Class II, Type A Biological Safety Cabinets ..................................................................................... 221
Class II, Type B1 Biological Safety Cabinets ................................................................................... 223
TABLE OF CONTENTS
PAGE
Class II, Type B2 Biological Safety Cabinet .................................................................................... 226
Class II, Type B3 Biological Safety Cabinets ................................................................................... 228
Class III Biological Safety Cabinets .................................................................................................. 230
Other Cabinets .................................................................................................................................... 232
Horizontal Laminar Flow Clean Air Benches (CAB's) .................................................................... 232
Chemical Fume Hoods ....................................................................................................................... 234
Biological Safety Cabinet Use Guidelines .......................................................................................... 236
Purchasing Procedures for Biological Safety Cabinets ...................................................................... 237
Vendors of Biological Safety Cabinets ............................................................................................... 242
Biological Safety Cabinet Installation Guidelines .............................................................................. 244
Set-upLocation ................................................................................................................................... 244
Gas and Vacuum Utility Connections ............................................................................................... 246
Electrical Service Connections .......................................................................................................... 246
New Installations ................................................................................................................................ 247
Working Height .................................................................................................................................. 247
Thimble Connection Exhaust Air Venting .......................................................................................... 248
Hard Connection Exhaust Air Venting ............................................................................................... 255
Frequency of Cabinet Certification ..................................................................................................... 257
Annual Cabinet Labor and Service Contract ...................................................................................... 258
Scheduling Cabinet Service and Certification .................................................................................... 259
Moving Your Cabinet .......................................................................................................................... 261
Certification Tests ................................................................................................................................ 262
SECTION V -- HAZARD WARNING SIGNAGE
Hazard Signage ..................................................................................................................................... 265
Hazard Warning Labels ....................................................................................................................... 269
Caution Signage
Biohazard, Infectious Agents, BL2 ................................................................................................... 278
Biohazard, Potentially Infectious Material, BL2 .............................................................................. 280
Biohazard, Infectious Agents, BL3 ................................................................................................... 282
Biohazard, Infected Animals ............................................................................................................. 284
Radioactive Materials ......................................................................................................................... 286
Radiation Area .................................................................................................................................... 288
High Radiation Area .......................................................................................................................... 290
Hazardous Chemical, Cancer Suspect Agent .................................................................................... 292
Cancer Hazard .................................................................................................................................... 294
Eye Protection Required .................................................................................................................... 296
Danger, Do Not Enter, Contaminated Area ...................................................................................... 298
Multiple Hazards ................................................................................................................................ 300
INDEX .................................................................................................................................................. 303
Section I -- Safety Responsibilities
1
ADMINISTRATIVE RESPONSIBILITIES
A. ADMINISTRATIVE OFFICERS
The Administrative Officers of the Johns Hopkins Institutions have the responsibility to
ensure that all research and investigative activities under their control are conducted in a
manner that presents the least possible hazard to employees, patients, students, visitors, and
to the surrounding community.
B. DEPARTMENT CHAIRMAN and/or DIRECTOR
Responsibility for the health and safety of employees, visitors and students ultimately rests
with each department head. They interpret institutional policies and recommendations and
assure compliance with their provisions. Each department head is responsible for approving
all research and all protocols for research involving hazardous biological agents prior to their
initiation.
C. PRINCIPAL INVESTIGATOR and/or SUPERVISOR
Supervisors at every level are responsible for biohazard control. They are responsible for
assuring appropriate training of employees on safe practices, for correcting errors and
defective conditions which could result in personal injury and/or property damage, and for
developing a positive attitude among employees toward biohazard safety and accident
prevention.
Other specific biohazard control responsibilities of Principal Investigators and Supervisors
are to:
1. Develop specific protocols to ensure safe practices and procedures in the use of
biological agents.
2. Obtain appropriate approvals of protocols for biohazardous research from the
Department Chairman and Institutional Biosafety Committee prior to the initiation of
research.
3. Implement policies, procedures and methods approved by the Joint Committee on
Health, Safety and Environment.
4. Properly register all potentially infectious agents or materials and recombinant DNA
projects with the Biosafety Officer.
5. Properly register all Biological Safety Cabinets and Clean Air Benches with the
Biosafety Officer and participate in the annual or semi-annual certification program.
Section I -- Safety Responsibilities
2
D. INDIVIDUAL RESEARCHERS and/or TECHNICIANS
Each employee is responsible for complying with all safety rules, regulations, and
procedures required for the task assigned. Each employee is responsible for reporting all
incidents resulting in or having the potential for personal injury, illness and/or property
damage or any action or existing condition which may result in such accidents.
Section I -- Safety Responsibilities
3
PERIODIC REVIEWS AND UPDATES OF REGISTERED RESEARCH
The Biosafety Officer will make periodic inspections of all projects involved with moderate
or high risk biological agents or materials (Biosafety Level 2 or higher) to assure continued
compliance with biohazard control requirements. All recombinant DNA research programs
covered by the NIH Guidelines will be inspected for adherence to the provisions of the NIH
Guidelines.
A. BIOLOGICAL AGENTS AND/OR MATERIALS
Every project involving biological agents or materials, sheep, goats, non-human primates,
and other potentially pathogenic materials will be reviewed annually by the Biosafety
Officer.
B. RECOMBINANT DNA
All research involving recombinant DNA shall be updated annually by the Biosafety Officer.
Section I -- Safety Responsibilities
4
REGISTRATION OF RESEARCH WITH PATHOGENIC AND/OR ONCOGENIC
MATERIALS
It is the responsibility of each principal investigator or laboratory director, with the advice of
the Biosafety Officer, to identify all biohazardous agents or materials in use or in stock and
file a Potentially Pathogenic and/or Oncogenic Agent or Material Registration Form with the
Office of Safety and Environmental Health. The Biosafety Officer should be notified
whenever a new etiological agent or biohazardous material is identified or introduced into
use.
A. BACKGROUND
From 1970 through 1982 the Johns Hopkins Institutions Joint Commission on the Use of
Infectious Agents and Other Biohazardous Materials investigated the potential hazards
associated with the use of biological agents and materials. A survey by the committee
identified over 130 laboratories in which one or more infectious agents were used in
investigative research. The committee recognized the necessity for updating and expanding
the database of the initial survey and recommended the establishment of a central registry of
biological agents and materials in use.
B. DEFINITION
For the purpose of this registry, pathogenic or oncogenic agents or materials shall include
known human and animal pathogens and oncogenic viruses; potential or suspect etiologic
agents; tissue cultures, tissues, blood and body fluids, specimens and samples presumed to
contain etiologic agents; microbial toxins; and organisms involved in genetic manipulation
experiments without consideration of the degree of infectivity, pathogenicity or public health
risk. A degree of hazard or risk will be assigned to the registered agents or materials by the
Biosafety Officer and/or the Institutional Biosafety Committee based on the information
supplied by the investigator, current classifications of etiologic agents, and current
publications.
C. RESPONSIBILITY
1. The Johns Hopkins Institutions Biosafety Officer, Office of Safety and
Environmental Health, is responsible for the maintenance of the registry.
2. It is the responsibility of each principal investigator to furnish information for all
biohazardous agents and materials presently in use in investigative research and for all
agents maintained in stock culture collections for research or teaching purposes.
3. It is the responsibility of each principal investigator to provide this information
whenever a new biological agent or material is identified or introduced into use.
Section I -- Safety Responsibilities
5
D. ASSESSMENT OF HAZARD
The level of competence and physical containment required for work with biological agents
or materials is dependent on an evaluation of the degree of hazard associated with each
research protocol. It should be noted that the criteria for establishing levels are not absolute.
The degree of hazard depends on the agent and the risk of exposure which may vary
depending on the nature and kind of study in which the biological agent or material is used.
Aerosol studies, passage in animals, and infection of vectors are examples of procedures
which markedly increase the hazard, whereas strict adherence to in vitro experiments or the
use of attenuated agents decrease the hazard. The hazard/risk levels will be evaluated for
each protocol to determine appropriate biohazard safety controls.
E. PURPOSE OF REGISTRATION
The purpose of completing the registration form is to ensure adequate review to determine
compliance with the biohazards safety regulations regarding the safe handling, storage, and
disposal of hazardous agents and to provide emergency information.
The registration form must be submitted when:
1. An investigator plans a laboratory project or procedure involving:
a. Biological agents requiring Biosafety Level 2, 3, 4, or 5 (Class 2, Class 3, Class 4
and Class 5).
b. Agents classified as low-risk, moderate-risk and high-risk oncogenic viruses.
c. Agents involved in research with recombinant DNA molecules.
d. Materials associated with potentially infectious bloodborne pathogens such as; all human
and nonhuman primate blood, body fluids containing visible blood, tissue specimens,
cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic
fluid, semen, vaginal secretions, and breast milk. This includes fresh human specimens
removed at surgery and submitted to the Department of Pathology.
e. Tissue cultures, tissues, body fluids, specimens and samples presumed to contain etiologic
or oncogenic agents.
2. The form may also be used when an investigator wants the Biosafety Officer to review
safety and containment procedures, e.g.:
a. A new technique or procedure is to be used.
Section I -- Safety Responsibilities
6
b. An existing technique is to be modified and the investigator believes that the
modification may increase the risk to laboratory personnel or animal caretakers.
c. An antigen, whose risks are well understood by the investigator, is to be highly
concentrated with possible increased risk.
d. When an infectious agent is to be used which makes vaccination of personnel
desirable, prior to the onset of work.
e. When the containment equipment or laboratory facility is altered, moved,
modified, replaced, or supplemented.
F. PROCEDURE FOR REGISTRATION
1. Preliminary Discussion
Contact the Biosafety Officer to determine if submission of a registration form will be
necessary for the proposed work and, if so, obtain a copy from the Office of Safety and
Environmental Health.
2. Submission of Form
Submit the completed form to the Biosafety Officer. The form should be submitted for
committee review one (1) week prior to deadline. For new grants, in particular, it is to the
advantage of the investigator to allow enough time to permit inclusion of the Institutional
Biosafety Committee's action on the protocol with the grant application.
3. Processing of Form
When the Biosafety Officer receives the protocol, the agent will be classified according to
guidelines and the requirement for further review will be determined. If necessary, the
investigator and the Biosafety Officer or appropriate designee together will evaluate the
project protocol, equipment, work space and other pertinent information.
The Biosafety Officer will present projects requiring Biosafety level 3 and above to the
Institutional Biosafety Committee and/or the Joint Committee on Health, Safety and
Environment, when applicable, and will write a report on their actions defining the
conditions for project approval. A copy of the report will be sent to all personnel who need
information about the work, (e.g.; the investigator, the department chairman / director, and
the dean of the appropriate university division).
4. Protocols
The principal investigator or laboratory director in charge of all projects or procedures
involving the use of moderate or high-risk biological agents or materials shall prepare a
written protocol including, but not limited to, the following information to be submitted to
Section I -- Safety Responsibilities
7
the Biosafety Officer along with the Potentially Pathogenic / Oncogenic Agent or Material
Registration Form:
a. Identification of the etiologic or oncogenic agent or material (genus, species, type),
pathogenicity of the agent, and host range if known.
b. Description of the experiment and techniques to be used and quantities of organisms to
be used.
c. Techniques, facilities and methods to assure containment.
d. Method for terminal inactivation of the agent or material.
e. Special requirements for handling experimentally infected animals.
G. STANDARD OPERATING PROCEDURE (SOP) MANUAL FOR BIOSAFETY
LEVEL 3 LABORATORIES
In addition to the items listed in "4" above, a Standard Operating Manual shall be prepared
by the principal investigator or laboratory director and submitted to The Johns Hopkins
Institutions Biosafety Officer for review. A hard copy and computer disk "template manual"
are available from the Biosafety Officer. The pages of this manual shall be numbered
consecutively and dated. The manual shall contain a:
1. "Table of Contents" page
2. List of personnel permitted to work in the laboratory
3. List of chemicals and materials kept in the laboratory
4. Floor plan of the laboratory
5. List of emergency contact numbers and procedures to be followed by personnel
during an emergency
6. Discussion of operating hours and procedures for admittance of housekeeping or
maintenance personnel for routine or special functions
7. Discussion of decontamination and waste disposal procedures to be followed by
laboratory personnel, including autoclave guidelines and log sheets.
8. Discussion of the types, locations and uses of available personnel protective
equipment
Section I -- Safety Responsibilities
8
9. Specific experimental procedures (SOP's) to be followed in the laboratory
10. Biosafety Level 3 laboratory specifications from OSEH
11. Signature pages signed by each researcher indicating successful completion of required
JHI and laboratory-specific training for work to be performed in the BSL 3 laboratory.
H. VACCINIA VIRUS VACCINATION
Vaccinia vaccination should not be given for international travel. Vaccinia vaccination is
indicated only for laboratory and health care workers directly involved with smallpox or
human orthopoxviruses, e.g., monkeypox, vaccinia, cowpox, and others. Principal hazards to
laboratory and animal care personnel are ingestion, accident parenteral inoculation, or
exposure of mucous membranes or broken skin to droplets or aerosols of infectious fluids or
tissues. The ACIP recommends administration of vaccinia vaccine to protect against these
hazards.
The following individuals should receive vaccinia within every ten (10) years:
1. Individuals who work directly with non-smallpox human orthopoxviruses or infectious
recombinants derived from the human orthopoxviruses including cowpox, vaccinia or
vaccinia subspecies; e.g., buffalopox, rabbitpox. No human infections seem to be caused by
certain others; e.g., mousepox, camelpox, racoonpox, skunkpox or Uasin gishu virus.
2. All animal care personnel who enter rooms housing animals infected with human
orthopoxvirus, or who touch cage litter or cages (prior to decontamination) which have
housed infected animals.
3. Certain health care workers involved in human trials of recombinant vaccinia vaccines.
4. Vaccinia vaccination is not required for visitors or service personnel who enter other
laboratories where work is conducted only with vaccinia, recombinant vaccinia or cowpox
viruses, provided they do not enter the laboratory while work with live viruses is actually in
progress.
REGISTRATION OF RESEARCH WITH HBV AND/OR HIV
A. REQUIREMENTS
1. Registration is required for investigators using human blood, tissue, body fluids, and
other materials potentially containing HBV or HIV.
Section I -- Safety Responsibilities
9
2. All research laboratories working with peripheral blood leukocyte cultures, other
tissue cultures containing HBV, HIV-I, -II, (HTLV-I, II, III/LAV) suspected HBV,
HIV or AIDS-associated retrovirus(es) must register and be inspected by the Office
of Safety & Environmental Health.
3. Experimental animal studies involving the use of human HBV or HIV, suspected
HIV, AIDS-associated retrovirus(es) or SIV will not be initiated without prior
approval from the Biosafety Officer and the Animal Use and Care Committee.
4. Growth and maintenance of HBV or HIV in permissive cell lines must be carried out
under work practices described in this document.
5. Large-scale production (one liter quantities or greater) of HBV or HIV and
concentrating virus-producing cells must be performed in an approved Biosafety
Level 3 facility with HEPA filtration of facility exhaust, when appropriate.
B. INFORMED CONSENT FOR USE OF POSTEXPOSURE ZIDOVUDINE (AZT)
1. The Johns Hopkins Institutions offers the drug zidovudine (AZT) to employees who
have a serious exposure to Human Immunodeficiency Virus (HIV, the AIDS virus)
while on the job. However, the Institutions do not actively recommend that anyone
take AZT for this purpose. The reason is that while AZT works to prolong the lives
of patients who are already infected with HIV, it has not been shown that AZT works
to prevent HIV infection after an exposure on the job. The scientific reasons for
offering AZT, the possible side effects of AZT, and the plan to watch for side effects
of AZT and for any signs of HIV infection are outlined below.
2. Several studies show that the chance of becoming infected with HIV following a
needlestick injury involving blood from an HIV-infected patient is about 0.4%, or 4
infections for every 1,000 needlestick injuries. The risk from exposure of the mouth,
eye, or broken skin is unknown and is probably much lower than the 0.4% figure for
needle sticks.
3. AZT works by preventing HIV from reproducing itself inside the cells of the human
body. AZT prolongs the lives of patients with AIDS and it slows the course of HIV
disease in patients with earlier stages of HIV infection. There are no studies showing
that AZT works in preventing HIV infection after an exposure on the job. There are
some reports of failure of AZT to prevent HIV infection after such exposures,
although the number of reported failures is small. Studies in animals infected with
viruses like HIV have shown that AZT may change the course of these infections
when AZT is given before or very soon after virus infection. Most animals stayed
well longer, but still were infected with the virus.
Section I -- Safety Responsibilities
10
4. The reason for offering AZT soon after exposures on the job is that it is possible that
AZT might prevent HIV infection, although the studies mentioned above do not
show that AZT will work when given in this way. The Food and Drug
Administration (FDA) has approved AZT for patients already infected with HIV.
The FDA has not approved AZT as a treatment to prevent infection after an exposure
on the job. Therefore, the use of AZT in this way is considered experimental. We
recommend that if the drug is to be given at all, AZT treatment should be started as
quickly as possible, within one to four hours of exposure (AZT will not be offered if
72 hours or more has passed from the time of exposure). The AZT dose offered is
200 mg every four hours for 72 hours and then 100 mg every four hours while awake
(five times per day) for the following 25 days.
5. There are side effects and other possible risks that should be known. The most
common side effects in patients with AIDS who receive AZT are headache, nausea,
muscle aches, anemia and low numbers of white blood cells in the blood. Rarely,
patients taking AZT develop numbness or tingling in the hands and feet or develop
liver damage. Most of these side effects are seen with prolonged use in patients with
advanced HIV infection and would not apply to a healthy person taking a short
course of the drug.
6. The most common complications in health care workers given AZT for on-the-job
exposures are fatigue, trouble sleeping, headaches, nausea and flu-like symptoms.
These side effects generally go away quickly when the drug is stopped or if the dose
is lowered.
7. The long term effects of AZT in humans are not known. Long term use of the drug
in rats and mice produces an increased risk for developing cancer of the vagina. It is
not known if AZT can cause harm to unborn children in pregnant women or can
influence a woman's ability to have children.
8. Due to these possible side effects and unknown effects on the unborn child, women
of childbearing age should have a negative pregnancy test before starting AZT
treatment. Also, health care workers who take AZT must agree to not become
pregnant or to breast feed while taking the drug and for four weeks after it is stopped
(AZT is passed into breast milk). It is possible that taking AZT would not prevent
HIV infection, but would instead delay the blood test turning positive. Therefore,
those who take AZT should continue to take precautions for one year after exposure.
9. Those who receive AZT treatment must be seen in the Workers' Compensation
Clinic as soon as can be scheduled after exposure and at weeks 2, 4 and 6, and at
months 3, 6 and 12 after exposure. Blood will be taken every two weeks during
AZT treatment and twice more after this has been stopped to check blood counts and
liver function tests. If side effects occur, the dose of AZT may be lowered or the
Section I -- Safety Responsibilities
11
drug may be stopped. Blood tests for HIV will be done at the first visit, and at
months 3, 6 and 12.
10. The choice of whether or not to be treated with AZT is up to each individual. The
drug will be provided for students of Johns Hopkins University Schools of Medicine
and Nursing, for employees of Johns Hopkins Schools of Medicine and Hygiene &
Public Health and for employees of The Johns Hopkins Hospital. If you choose to
take AZT, you may decide to stop it at any time, however, the Workers'
Compensation Clinic should be told of this decision. Your decision to take or to stop
AZT will have no effect on your current position or on any other treatment or follow-
up that you would receive as a result of this exposure.
11. All your records will be kept confidential within the limits of the law. Documents
which identify an individual by name will be kept in a secured file by your physician.
12. If an individual is found to be infected with HIV, they and their sexual partner(s) will
be offered information on the test results and how to prevent the spread of the
infection. Individuals should also know that it is necessary to report certain
communicable diseases, including AIDS, to appropriate state and federal government
agencies.
Section I -- Safety Responsibilities
12
REGISTRATION OF RESEARCH WITH SHEEP OR GOATS
A. BACKGROUND
The Q fever policy was formulated by a special Johns Hopkins committee in 1981 and
revised to include the suggestions of The JHU General Counsel, in March 1982. Q fever is a
zoonosis caused by the rickettsia, Coxiella burnetii. Domestic ungulates such as sheep,
cattle, and goats serve as the reservoir of infection for humans and shed the
desiccation-resistant organism in urine, feces, milk, and especially in birth products. In man,
the illness is generally mild and can be treated with the tetracycline family of antimicrobial
agents. However, Q fever hepatitis is often seen and Q fever endocarditis is an uncommon
but frequently fatal complication.
Q fever is an occupational hazard among persons working with animals or animal products,
and in laboratories working with C. burnetii. Recently, Q fever outbreaks have occurred in
medical research facilities using sheep or goats as research animals.
B. RECOMMENDED CONTROL MEASURES FOR Q FEVER
1. All investigators using sheep or goats in their experiments shall register with The Johns
Hopkins Institutions Biosafety Officer. The user shall describe;
a. the plan to protect employees during use of sheep and goats, including the
handling of products of conception and excrement,
b. the room(s) where sheep or goats will be housed,
c. the room(s) where sheep or goat related biological samples will be used, and
d. a list of all professional personnel, employees and students involved in the
project who will come into contact with the sheep or goat products.
2. The Biosafety Officer will send a "To Whom It May Concern" letter to the principal
investigator for forwarding to the granting agency when appropriate.
3. All professional personnel, employees and students coming into direct contact with
sheep and goats or sheep and goat products of conception will be included in the
medical surveillance program at Occupational Health Services.
4. Employee serum samples shall be collected by Occupational Health Services for
future use as a baseline reference. Serum samples shall be collected at the new
employment physical.
Section I -- Safety Responsibilities
13
5. Occupational Health Services will take an additional serum sample upon termination
of employment or transfer to a position which does not involve direct contact with
sheep or goats or their products. It is the responsibility of the principal investigator
to notify Occupational Health Services that an employee is leaving and to make
arrangements for the post-employment medical examination.
6. All employees and students entering sheep and goat holding areas, laboratories and
surgical facilities shall be informed of the Q fever hazard and shall receive written
instructions on safe work practices to minimize the Q fever hazard.
7. All personnel involved with sheep and goats or products of conception from sheep
and goats shall be informed of the symptoms of Q fever and shall be instructed to
contact the Workers' Compensation Clinic if they experience the symptoms.
8. In the event of a failure or breakdown in any of the systems or procedures described
herein, investigators shall report incidents to the Workers' Compensation Clinic as
soon as possible. The report must be in writing on the JHU Report of Incident Form.
Section I -- Safety Responsibilities
14
REGISTRATION OF RESEARCH WITH RECOMBINANT DNA
Research involving recombinant DNA requires strict adherence to the most current NIH
Guidelines for Recombinant DNA Research as published in the Federal Register, and any
local, state or federal regulation promulgated for Recombinant DNA activities.
A. REGISTRATION FORM
All principal investigators conducting recombinant DNA research are required to file the JHI
Recombinant DNA Registration Form.
B. REVIEW PROCEDURE
This information is required for evaluation of each proposal for compliance with NIH
Recombinant DNA guidelines.
The Biosafety Officer will review all proposals to identify those covered by the NIH
Guidelines and those which are exempt. The Recombinant DNA Committee, a
subcommittee of the Institutional Biosafety Committee, will review all non-exempt
proposals. An annual survey of recombinant DNA activity, by month of last annual
registration, will be used to update these files. The investigator will be informed of the
containment conditions established by the Recombinant DNA Committee and/or the
Biosafety Officer.
The review the of recombinant DNA proposals will provide:
1. Documentation that the proposed project is exempt from the NIH Guidelines, or
2. Documentation that the proposed project covered by the NIH Guidelines is in
compliance with the Guidelines, and
3. Assurance that the facilities will be in compliance with the NIH Guidelines.
4. A "To Whom It May Concern" letter of will be sent to the principal investigator for
forwarding to the granting agency when appropriate.
5. No project involving recombinant DNA research may be implemented without
registration with the Biosafety Officer.
6. No major changes in recombinant DNA experiments may be implemented without
notification of the Biosafety Officer. Minor changes need not be reported until the
annual update of recombinant DNA activity.
Section I -- Safety Responsibilities
15
7. A new or revised application is required for major changes in the recombinant DNA
aspects of an ongoing project, such as a change in the;
a. hosts or vectors.
b. donor species or nature of the DNA segment.
c. location of experiments, or
d. responsible investigator.
C. REVIEW OF RECOMBINANT DNA RESEARCH WITH MODERATE RISK
BIOLOGICAL AGENTS OR MATERIALS AT BIOSAFETY
LEVEL 1 or 2
1. Complete the Recombinant DNA Form and the Potentially Pathogenic / Oncogenic
Agent or Material Registration Form and forward them to the Biosafety Officer.
2. Principal investigators are to submit a research protocol, including a construct of the
recombinant molecule(s), to the Biosafety Officer.
3. Arrange with the Biosafety Officer for an on-site inspection of practices and
containment facilities.
4. The Biosafety Officer will:
a. Register the research protocol and send a letter containing a unique
registration number, recombinant DNA Guideline reference, and assigned
Biosafety Level for the research.
b. Submit the information for review by the Recombinant DNA Committee.
D. REVIEW OF RECOMBINANT DNA RESEARCH INVOLVING BIOSAFETY
LEVEL 3 FOR HIGH RISK BIOLOGICAL AGENTS OR MATERIALS
OR RECOMBINANT DNA RESEARCH INVOLVING HUMAN
SUBJECTS
1. Complete the Recombinant DNA Form and the Potentially Pathogenic / Oncogenic Agent or
Material Registration Form and forward them to the Biosafety Officer.
2. Principal investigators must submit a research protocol, including a construct of the
recombinant molecule(s), to the Biosafety Officer.
Section I -- Safety Responsibilities
16
3. Principal investigators must submit a Standard Operating Procedure Manual to the
Biosafety Officer for all research involving biosafety level 3 containment.
4. Principal investigators must submit a Standard Operating Procedure Manual for
research involving human gene therapy to the Biosafety Officer.
5. Arrange with the Biosafety Officer for an on-site inspection of practices and
containment facilities.
6. The Biosafety Officer will:
a. Register the research protocol and send a letter containing a unique
registration number, recombinant DNA Guideline reference, and assigned
Biosafety Level for the research.
b. Submit a report for review by the Recombinant DNA Committee and/or the
Institutional Biosafety Committee.
7. The protocol and inspection report will be reviewed and approved by the Joint
Committee on Health, Safety and Environment.
8. Results of review will be forwarded to; the investigator, the department director and
the dean of the appropriate university division.
Section I -- Safety Responsibilities
17
REGISTRATION OF RESEARCH WITH NON-HUMAN PRIMATES
INTRODUCTION
Research with non-human primates is registered with the Office of Safety and Environmental Health
to ensure that individuals working with the animals receive appropriate medical surveillance,
particularly the PPD skin test. If primates acquire tuberculosis, the disease may be transmitted to
humans, or visa versa.
A. TUBERCULOSIS
Tuberculosis is a chronic disease caused by bacteria of the genus Mycobacterium. The most
common form of the disease affects the lungs. Infection of other organs is much less
common. The main source of infection for humans in the United States is other infected
humans. In under-developed countries cattle may also be commonly infected and serve as a
source of infection for humans. The practice of milk pasteurization coupled with test and
slaughter programs for cattle have eliminated this source of infection in this country.
Non-human primates (monkeys) are very susceptible to tuberculosis, and most commonly
die if infected. The most common source of infection for monkeys is from infected humans
or other monkeys. Since many of the monkeys used in this institution are caught in the wild,
there is the possibility that they may catch tuberculosis from the humans with which they
come into contact.
Tuberculosis in Non-Human Primates: While tuberculosis is a potential problem for humans
in the hospital or research setting, this disease can also be highly infectious in a non-human
primate colony. All monkeys coming into the institution are quarantined for a minimum of
42 days to allow for three tuberculin tests at two week intervals. After the quarantine period,
all monkeys are periodically tuberculin tested, usually at 3 month intervals.
1. Tuberculin Testing
In order to protect the humans who work or come into contact with non-human
primates; and to protect the monkeys who come into contact with humans, each is
tuberculin tested on a routine basis.
At six month intervals all individuals who work with non-human primates will be
notified of the time and place for tuberculin testing.
2. Personal Protective Equipment
What can be done to minimize the possibility of catching tuberculosis from a
monkey? The simplest preventive measure is to wear a face mask and gloves when
handling, or working with monkeys.
B. HERPESVIRUS-B IN MACAQUE MONKEYS
Section I -- Safety Responsibilities
18
1. Background
All macaque monkeys are potential carriers of herpesvirus-B, also called herpes
simiae. In this institution, the two macaque species commonly used are rhesus
monkeys (Macaca-mulatta) and cynomolgus monkeys (Macaca fasicularis). This
virus is not found in non-macaca species of monkeys such as the african green,
squirrel, or baboon
2. Human Infection With Herpesvirus-B
Herpesvirus simplex is a human virus that commonly causes oral cold sores in
people. Herpesvirus-B acts similarly in the monkey, it sometimes causes a cold sore-
like lesion but generally causes no obvious physical problem for the monkey. The
problem comes when a human becomes infected with the macaque virus. While
human infection is very rare, the unfortunate consequence of such an infection is
usually death or severe and lasting neurologic disease.
Humans become infected with Herpesvirus-B usually by receiving a bite or a scratch
from an infected monkey who is shedding virus, from contact with tissues from an
infected monkey, or by injury from a cage or similar item that has been contaminated
with material from an infected monkey.
3. Antibody testing
Monkeys are often tested for the presence of antibody to Herpesvirus-B. However,
animals with negative titers are still capable of being infected and shedding virus.
Therefore, the safe thing to do is to treat all macaques as though they are
infected with this virus.
4. Personal Protective Equipment
The best way to prevent infection with this agent is to use common sense when
working with monkeys and to utilize protective devices.
a. Always wear face masks, long-sleeved laboratory coats or other clothing, and
rubber gloves when working with monkeys.
b. Unless absolutely necessary, never handle an awake macaque. Use a squeeze
cage and a restraint drug such as ketamine to catch an animal.
c. If you have to work with an awake animal, always use care when working
near the head or close enough for the monkey to grab you.
Section I -- Safety Responsibilities
19
5. Response to a Bite or Scratch
a. If you do receive a bite or scratch wound from a monkey; or from a piece of
equipment with which the animal or animal tissues has had contact, STOP
WORK, return the monkey to its cage, institute first aid immediately. Each
primate housing area, and laboratory has been equipped with a "Monkey
Injury Kit". Follow the instructions that are inside the kit. You should
familiarize yourself with the instructions in advance.
b. Notify Animal Services of the identification number of the monkey and its
location so that a veterinarian can examine the animal and take appropriate
steps to ascertain the potential hazard. Following initial first aid, go to the
Workers' Compensation Clinic.
6. First-Aid for Macaque Associated Wound
Each facility housing macaque monkeys, and each laboratory using such animals is
equipped with a "Monkey Injury Kit". Following a monkey associated wound
immediately open the kit.
Inside you will find the following items:
two screw-top culture tubes numbered "1" and "2"
two packages of sterile, cotton topped applicators
one, 1 ounce brown bottle of bleach
one, sterile povidone-iodine impregnated scrub brush
4x4 inch gauze sponges
one roll of adhesive tape
a. IF YOUR EYES, NOSE OR MOUTH HAVE BEEN EXPOSED,
IRRIGATE THE SITE WITH STERILE SALINE OR RAPIDLY
FLOWING WATER FOR 15 MINUTES and go to the Workers'
Compensation Clinic.
b. ONLY FOLLOW THESE INSTRUCTIONS FOR WOUNDS OF THE
SKIN. DO NOT USE FOR EYE, NOSE OR MOUTH CONTAMINATION
1) Open the Injury Kit and remove the contents.
2) If possible, take one sterile cotton swab, insert into wound and rotate
gently. Remove top from screw-top tube "1", and insert swab part
way inside. Break off wooden applicator so that the handled portion
stays outside of the tube. Replace top on tube.
Section I -- Safety Responsibilities
20
3) Dump the contents of the brown bottle into the Kit container, and fill
with water to the black line. Then using the 4x4 gauze sponges, soak
the wound with the prepared solution for at least five minutes.
4) Open the scrub brush package and gently but thoroughly scrub the
wound with the sponge brush and running water for 15 minutes.
5) After washing the wound, cover with gauze, secure with adhesive
tape, and go to the Workers' Compensation Clinic. Take the tube
labeled "2" and the second package of sterile applicators with you.
Section II -- Safety Compliance
21
STANDARD PRACTICES, TRAINING AND PRECAUTIONS
The objective of physical containment of biohazardous materials is to confine biological
agents or material and/or organisms containing recombinant DNA molecules, and thus
reduce the potential of injury to laboratory workers and persons outside of the laboratory.
Physical containment is achieved through the use of appropriate laboratory practices,
containment equipment and special laboratory design. Emphasis must be placed on primary
containment which is provided by laboratory practices and containment equipment. Special
laboratory design provides secondary containment against accidental release of organisms
or materials outside the laboratory or to the environment.
The combination of laboratory practices, containment equipment and special laboratory
design used to achieve physical containment includes approaches to containment of
pathogenic or oncogenic organisms and organisms containing recombinant DNA. The
standards are based on the four levels of containment identified by the Centers for Disease
Control (CDC) and the National Institutes of Health (NIH) in their publication, Biosafety in
Microbiological and Biomedical Laboratories, which encompass the elements of physical
containment described in the NIH Guidelines for Recombinant DNA Research with some
editorial changes.
A. All personnel directly or indirectly involved in experiments with biological agents and
materials or recombinant DNA must receive adequate instruction including, but not limited
to;
1. The biology of the organism(s) or material(s) used in experiments with emphasis on
potential biohazards,
2. Microbiological aseptic techniques, and
3. Proper techniques for decontamination / disinfection.
B. There should be strict adherence to good microbiological practice in all experiments.
C. All research groups must have a written emergency plan which describes the procedures to
be followed if an accident contaminates personnel, the laboratory, or the environment.
D. Vaccination may be appropriate for research with selected disease agents.
E. Serological monitoring and periodic medical examinations, where appropriate, will be
provided by Occupational Health Services.
F. All accidents and illnesses must be reported promptly to the Workers' Compensation Clinic.
Section II -- Safety Compliance
22
G. The selection of an appropriate biosafety level for work with a particular agent and/or
animal is dependent upon a number of factors. The most important of these include:
1. The virulence, pathogenicity, biological stability, and communicability of the agent;
2. The nature or function of the laboratory or activity;
3. The quantity and concentration of the agent;
4. The endemicity of the agent; and,
5. The availability of effective vaccines or therapeutic measures.
If a combination of increasingly stringent primary and secondary containment procedures
and facilities are used, laboratory studies and manipulations can be safely conducted on
agents that are correspondingly more hazardous.
H. In general, the biosafety level used for activities with infectious agents or infected animals
should be commensurate with that required for the agent of highest virulence known or
likely to be encountered in the course of contemplated work.
Section II -- Safety Compliance
23
UNIVERSAL PRECAUTIONS GUIDELINES FOR RESEARCH LABORATORIES
A. DEFINITIONS
The Centers for Disease Control (CDC) / National Institutes of Health (NIH) Universal
Precautions Guidelines cover all blood, tissue, body fluids from inside the body, and any
material which contains blood that can be seen. These materials must be considered
infectious for Hepatitis B and AIDS and must be handled under conditions which
reasonably preclude cutaneous, oral, or parenteral exposure to personnel.
Universal Precautions, sometimes termed Blood and Body Fluid Precautions, must be used
for handling ALL specimens covered by the guidelines from ALL patients.
Universal Precautions Guidelines state that all blood, tissue, body fluids from inside the
body, and any material which contains blood you can see are considered potentially
infectious for hepatitis and AIDS and must be handled under conditions which reasonably
preclude cutaneous, oral, and parenteral exposure to personnel. Work with these materials
is often registered at biosafety level 2.
Sputa should be considered potentially infectious for tuberculosis and should be handled
under conditions which reasonably preclude generation of aerosols and also contain
previously generated aerosols. Work with sputum is often registered at biosafety level 3.
B. PRECAUTIONS
1. If in the course of diagnostic or other laboratory examinations there is evidence that
the materials being studied contain an agent of higher or lower risk than expected,
the biosafety level should be raised or lowered accordingly, with the advice of the
Biosafety Officer.
2. The following recommended precautions are advised for persons performing
laboratory tests or studies on clinical specimens or other potentially infectious
materials such as tissue culture, embryonated eggs, or tissue from animals
inoculated with human blood, tissue, or body fluids:
Biosafety Level 2 (BSL 2) standards and special practices, containment equipment,
and facilities are recommended for activities involving all clinical specimens, body
fluids, and tissues from humans or from infected or inoculated laboratory animals.
3. Use of syringes, needles and other sharp instruments shall be avoided if possible.
Used needles and disposable cutting instruments must be discarded into an
approved puncture-resistant container. Needles shall not be re-sheathed, bent,
broken, removed from disposable syringes, or otherwise manipulated by hand.
Section II -- Safety Compliance
24
4. Protective gloves shall be worn by all personnel engaged in activities that may
involve direct contact of their skin with potentially infectious specimens, cultures,
or tissues.
5. Gloves shall be carefully removed and changed when they are visibly contaminated.
6. Personnel who have dermatitis or other lesions on their hands and who may have
indirect contact with potentially infectious material shall also wear protective
gloves.
7. Hand washing with mild soap and water immediately after infectious materials are
handled, after work is completed, after contaminated gloves are removed, and after
removal of gloves for any reason shall be a routine practice.
8. Generation of aerosols, droplets, splashes, and spills shall be avoided. A biological
safety cabinet shall be used for all procedures that might generate aerosols or
droplets and for all infected cell-culture manipulations.
9. Fluorescence-activated cell sorters generate droplets that could potentially result in
infectious aerosols. Translucent plastic shielding between the droplet-collecting
area and the equipment operator should be used to reduce this risk. HEPA-filtered
air exhaust systems may also be required when sorting live cells. Please contact the
Biosafety Officer for further information.
10. The use of plastic labware is encouraged to reduce breakage and minimize the risk
of puncture wounds.
11. Activities such as producing research laboratory scale amounts of HIV,
manipulating concentrated virus preparations, and conducting procedures that may
produce aerosols or droplets shall be performed in a BSL 2 facility with the
additional practices and containment equipment recommended for BSL 3.
12. Activities involving industrial-scale, large-volume production (volumes of one liter
or greater) or high concentrations or manipulations of concentrated HIV shall be
conducted in a BSL 3 facility using BSL 3 practices and equipment.
13. BSL 2 practices, containment equipment, and facilities for animals are
recommended for activities involving non-human primates and any animals
experimentally infected or inoculated with HIV. Because laboratory animals may
bite, throw feces or urine, or expectorate, all animal services personnel,
investigators, technical staff, students, and other persons who enter these animal
rooms shall wear coats, protective gloves, coveralls or uniforms, and when
appropriate, face shields or surgical masks and eye protection to protect their skin
and mucous membranes of their eyes, nose, and mouth. Only non-human primates
Section II -- Safety Compliance
25
directly involved in the experiments being conducted are permitted in the
laboratory.
14. All laboratory glassware, disposable material, and waste material known to have
come into contact with HIV culture, or any other microbial culture, should be
decontaminated, preferably in an autoclave, before it is washed, discarded, etc. A
method of disposing of other solid wastes is the biohazard box.
15. Laboratory workers shall wear laboratory coats, gowns, or uniforms when working
with HIV or with material known or suspected to contain HIV. Clothing that
becomes contaminated with HIV preparations should be decontaminated before
being laundered or discarded. Laboratory personnel must remove laboratory
clothing before leaving the laboratory.
16. Work surfaces shall be decontaminated with an appropriate chemical germicide
when procedures are completed, when surfaces are overtly contaminated, and at the
end of each work day. Many commercially available chemical disinfectants can be
used for decontaminating laboratory work surfaces, laboratory instrument surfaces,
spot cleaning of contaminated laboratory clothing, and for spills of infectious
materials. Prompt decontamination of spills should be standard practice.
17. If the outside of the specimen container is visibly contaminated, it should be cleaned
with a disinfectant such as a 1:10 dilution of liquid household bleach with water.
(Household bleach contains 5.25% or 52,500 ppm sodium hypochlorite). All blood
specimens should be placed in a secondary container, such as a portable cooler, for
transport. The container and bag should be examined carefully for breaks or cracks
and should be routinely decontaminated.
18. Blood spills should be cleaned up promptly with a disinfectant solution, such as
sodium hypochlorite (see above) and detergent. Articles soiled with blood should
be discarded into a red bag-lined biohazard box.
19. Universal precautions are recommended for handling all human blood specimens
for hematologic, microbiologic, chemical, and serologic testing; these are the same
precautions for preventing transmission of all bloodborne infections including
hepatitis B. It is not certain how effective 56-60o C heat is in destroying HIV in
serum, but heating small volumes of serum for 30 minutes at 56o C before serologic
testing may, under some circumstances, reduce residual infectivity. Such treatment
causes some false-positive results in HIV enzyme immunoassays and may also
affect some biochemical assays performed on serum.
20. Human serum from any source that is used as a control or reagent in a test
procedure should be handled at BSL 2.
Section II -- Safety Compliance
26
21. Medical surveillance programs should be in place in all laboratories that test
specimens, do research, or produce reagents involving HIV. The nature and scope
of a surveillance program needed for a particular laboratory or project should be
discussed with Occupational Health Services.
22. Other primary and opportunistic pathogenic agents may be present in the body
fluids and tissues of persons infected with HIV. Laboratory workers shall follow
accepted biosafety practices to ensure maximum protection against inadvertent
laboratory exposure to agents that may also be present in clinical specimens.
23. The laboratory director or designated laboratory supervisor is responsible for
carrying out the biosafety program in the laboratory. The laboratory director or
designated supervisor, in consultation with the Biosafety Officer, shall establish the
biosafety level for each component of the work to be done and shall ensure that
facilities and equipment are adequate and in good working order. The laboratory
director or designated laboratory supervisor shall ensure that appropriate initial and
periodic training is provided to the laboratory staff, and that recommended practices
and procedures are strictly followed.
24. Access to the laboratory must be limited by the principal investigator. The door to
the laboratory must be closed and posted with the universal biohazard symbol.
Only persons who have been advised of the potential hazards of the work and meet
specific entry requirements may enter the laboratory. Persons who are at increased
risk of acquiring infection are not permitted in the laboratory.
Section II -- Safety Compliance
27
HBV and/or HIV AGENT SUMMARY STATEMENTS
These publications, available at the Office of Safety & Environmental Health, are good
reference sources:
A. 1988 Agent Summary Statement for Human Immunodeficiency Virus and Report on
Laboratory Acquired Infection With Human Immunodeficiency Virus. MMWR, April 1,
1988/Vol.37/No. S-4 Supplement.
B. Guidelines for Prevention of Transmission of Human Immunodeficiency Virus and
Hepatitis B Virus to Health-Care and Public-Safety Workers. MMWR, June 23, 1989/Vol.
38/No. S-6.
C. H.I.V. Infection Control Guidelines, Maryland Governor's Advisory Council on Aids 5/89.
D. Occupational Exposure to Bloodborne Pathogens; Final Rule. Occupational Safety and
Health Administration. 29 CFR Part 1910.1030. Federal Register, vol. 56, No. 235,
December 6, 1991, p. 64175-64182.
E. Enforcement Procedures for the Occupational Exposure to Bloodborne Pathogens
Standard, 29 CFR 1910.1030. OSHA Instruction CPL 2-2.44C, March 6, 1992.
F. Guidelines for Protecting the Safety and Health of Health Care Workers, U.S. Department
of Health & Human Services, National Institute for Occupational Safety & Health, DHHS
(NIOSH) Publication No. 88-119, September, 1988.
G. Public Health Service Statement on Management of Occupational Exposure to Human
Immunodeficiency Virus, Including Considerations Regarding Zidovudine Postexposure
Use, Morbidity & Mortality Weekly Report, vol. 39, no. RR-1, January 26, 1990.
H. Working Safely With HIV in the Research Laboratory; Biosafety Level 2/3, Occupational
Safety and Health Branch, NIH Division of Safety, June, 1988.
I. Guidelines for Preventing the Transmission of Tuberculosis in Health-Care Settings, with
Special Focus on HIV-Related Issues, Morbidity & Mortality Weekly Report, vol. 39, no.
RR-17, December 7, 1990.
J. Johns Hopkins Institutions Bloodborne Pathogens Professional Study Guide, February,
1994.
Section II -- Safety Compliance
28
Q FEVER PRECAUTIONS
A. BACKGROUND
The Q fever policy was formulated by a special committee in 1981 and revised to include
the suggestions of The JHU General Council, in March 1982. Q fever is a zoonosis caused
by the rickettsia, Coxiella burnetti. Domestic ungulates such as sheep, cattle, and goats
serve as the reservoir of infection for humans and shed the desiccation-resistant organism in
urine, feces, milk, and especially in birth products. In man, the illness is generally mild and
may be treated with the tetracycline family of antimicrobial agents. However, Q fever
hepatitis is often seen and Q fever endocarditis is an uncommon but frequently fatal
complication. Q fever is an occupational hazard among persons working with animals or
animal products, and in laboratories working with C. burnetti. Recently, Q fever outbreaks
have occurred in medical research facilities using sheep as research animals.
B. RECOMMENDATIONS
1. The site of choice for conduct of sheep research should be an isolated setting apart
from hospital research and patient care areas.
2. It is possible to conduct research with sheep which are known or suspected to be
infected provided such research is performed in a containment facility or in an
isolated setting where only at-risk workers are admitted. When appropriate,
selected workers could be skin tested.
C. RATIONALE
These recommendations suggest measures for reducing the risk of human infection with Q
fever in facilities where sheep are used for research. They can neither encompass every
possible circumstance in which personnel might be exposed to Q fever-infected sheep, nor
can their implementation guarantee that transmission will not occur. Depending on the
specific research facility, more intensive or less intensive control measures may be
appropriate. Although some of the recommendations are not supported by controlled
epidemiologic trials, they do reflect the current understanding of the transmission and
control of Q fever.
D. REGISTRATION
All investigators using sheep or goats in their experiments shall register with the Biosafety
Officer, Office of Safety and Environmental Health, using the form entitled "Registration of
research with sheep and goats". The user shall describe; a) the plan for the use of sheep or
goats, including the handling of products of conception and excrement, b) the room(s)
where sheep or goats will be housed, c) the room(s) where sheep or goat related biological
Section II -- Safety Compliance
29
samples will be handled, and d) a list of all professional personnel, employees and students
involved in the project who will come into contact with the sheep or goat products.
E. MEDICAL SURVEILLANCE
1. All professional personnel, employees and students coming into direct contact with
sheep or goats or with sheep or goat products of conception will be included in the
medical surveillance program. Employee serum samples shall be collected by
Occupational Health Services as a baseline reference. Serum samples shall be
collected at the employment physical to serve as a baseline reference if needed. A
post-employment serum sample shall be taken by Occupational Health Services at
termination of employment or transfer to a position which does not involve direct
contact with sheep or goats or their products. It is the responsibility of the principal
investigator to notify Occupational Health Services that an employee is leaving and
to make arrangements for the post-employment medical examination and serum
sampling.
2. All employees and students entering sheep and goat holding areas, laboratories and
surgical facilities shall be informed of the Q fever hazard and shall receive written
instructions on safe work practices to minimize the Q fever hazard.
3. All personnel involved with sheep and goats or products of conception from sheep
and goats shall be informed about the symptoms of Q fever and shall be instructed
to contact the Occupational Health Services if they experience the symptoms.
Accidents and incidents shall be reported to the Workers' Compensation Clinic.
4. In the event of a failure or breakdown in any of the systems or procedures described
herein, investigators shall report incidents to the Biosafety Officer, Office of Safety
and Environmental Health as soon as possible. Any such report must be in writing
and shall include; a) the nature of the incident, b) the date and location of the
incident, c) the names of all professional personnel, employees and students
involved in the incident, and d) what measures, if any, were taken to prevent further
incidents.
F. TRANSPORT OF ANIMALS
Transport of all sheep and goats, pregnant or not, outside of posted Q fever hazard areas
shall be in enclosed, ventilated carts which will contain any Q fever rickettsia agents within
the cart enclosure. Air intakes and exhaust ports shall be fitted with an appropriate filter.
These carts shall be labeled and reserved for the exclusive transport of sheep and goats.
Carts shall not be opened or left unattended outside of posted Q fever hazard areas.
Transport carts shall be decontaminated after use. Transport of sheep and goats within
buildings shall be by established routes. Sheep and goats shall not be transported, under
any conditions, on elevators designated for patient or visitor use.
Section II -- Safety Compliance
30
G. PERSONNEL PROTECTION
1. Personal protective equipment shall be provided and worn by all personnel working
in animal rooms, laboratories, and surgical facilities.
2. The clothing for animal caretakers shall include full body protective clothing, head
cover, gloves, boots, and an approved respirator or mask. Protective clothing must
remain in the containment facility. Disposable protective clothing must be
decontaminated before disposal or properly red-bagged and incinerated.
3. Laboratory or surgical personnel must wear wrap-around or solid-front gowns or
uniforms when entering posted areas (Front-button laboratory coats are not
suitable). The use of respirators or masks and gloves is recommended in posted
areas. Protective clothing must remain in the facility and must be decontaminated
before being laundered. Disposable protective equipment shall be autoclaved and
then discarded into a red bag-lined biohazard box.
4. The use of disposable personal protective equipment, i.e. surgical gowns, TyvekTm
suits, face masks, etc., is recommended. It is recommended that animal handlers
and technical operations personnel be provided a separate clean clothing change for
use with sheep or goats. Clothing may be reused if personnel are not involved with
sheep or goat products of conception on a full time basis. Obviously soiled or
overtly contaminated clothing should be removed, wetted down with a disinfectant
and placed in a container for autoclaving and incineration.
5. All personal protective equipment shall be distinguished by a unique color or visible
marking to be immediately recognizable as equipment that should not be used
outside of a containment facility.
6. An appropriate closed container should be provided to hold discarded protective
clothing prior to autoclaving and incineration.
7. Personnel should use soap to wash their hands for ten to fifteen seconds, particularly
after removing gloves and before leaving containment areas. Washing with mild
soap is sufficient if good handwashing technique is used.
H. WASTE DISPOSAL
1. All sheep or goat related debris from animal pens and laboratories - litter, sheep
carcasses, excrement and products of conception shall be disposed of safely. Some
materials shall be discarded into red bag-lined biohazard boxes for incineration.
Other materials may be autoclaved in appropriate containers and discarded into a
red bag-lined biohazard box for incineration.
Section II -- Safety Compliance
31
2. The clean-up of animal debris shall be done in a manner which will produce the
least amount of airborne materials (aerosols).
I. DECONTAMINATION
1. All areas and equipment involving any contact with sheep or goats or products of
conception from sheep or goats shall be cleaned and disinfected on a regular basis
and immediately after each operation.
2. Special cleaning and disinfecting is required for machines used in Q fever hazard
areas which are shared with other areas. The manufacturer should be consulted if
high tech machines in involved, but in general, a good cleaning with a hospital
grade disinfectant-detergent registered with the EPA should be carried out on all
accessible surfaces, including legs and wheels.
3. Disinfection may be performed in surgical and laboratory areas with diluted
chlorine bleach solution (a 1 to 10 dilution of liquid household bleach with tap
water), a fresh 5% solution of hydrogen peroxide, or a 1:100 dilution of LysolTm.
4. Some disinfectants, such as ethyl alcohol, 1% phenol, 1% formalin and quaternary
ammonium compounds, are not effective.
5. Dr. Frank Gohr, University of California, in 1979, found the most effective,
practical and economical germicide for cleaning potential Coxiella burnetti
contaminated areas and equipment relatively free of organic deposits was standard
bleach diluted 1:100 with water (1 ml bleach, 99 ml water) to which was added 1
gram of a neutral detergent such as AllTm laundry detergent.
6. If heavy organic material is present (sheep manure, shavings, heavy blood or tissue
contamination), use chlorine bleach diluted 1:10 with water (1 ml bleach, 9 ml
water) with 1% vol/vol added detergent. Contact time should be at least 20 minutes.
The germicidal solution should be mixed daily and checked to assure a pH of 7.
Good scrubbing may be needed to assure proper germicide contact.
Section II -- Safety Compliance
32
BIOSAFETY LEVEL 1 GUIDELINES
Biosafety Level 1 (BSL 1) is suitable for work involving agents of no known or of minimal
potential hazard to laboratory personnel and the environment. The laboratory is not
separated from the general traffic patterns in the building. Work is generally conducted on
open bench tops. Special containment equipment is not required or generally used.
Laboratory personnel have specific training in the procedures conducted in the laboratory
and are supervised by a scientist with general training in microbiology or a related science.
Refer to Section III -- Recombinant DNA Guidelines.
Section II -- Safety Compliance
33
BIOSAFETY LEVEL 2 GUIDELINES
Biosafety Level 2 (BSL 2) is similar to Biosafety Level 1 and is suitable for work in
clinical, diagnostic, teaching, research or production facilities involving agents of moderate
potential hazard to personnel and the environment.
It differs in that; (1) laboratory personnel have specific training in handling pathogenic
agents and are directed by competent scientists, (2) access to the laboratory is limited when
work is being conducted, and (3) certain procedures in which infectious aerosols are created
are conducted in biological safety cabinets or other physical containment equipment. Refer
to Section III -- Recombinant DNA Guidelines.
Section II -- Safety Compliance
34
BIOSAFETY LEVEL 3 GUIDELINES
Biosafety Level 3 (BSL 3) is applicable to clinical, diagnostic, teaching, research, or
production facilities in which work is done with indigenous or exotic agents which may
cause serious or potentially lethal disease as a result of exposure by the aerosol route.
Laboratory personnel have specific training in handling pathogenic and potentially lethal
agents and are supervised by competent scientists who are experienced in working with
these agents. All procedures involving the manipulation of infectious material are
conducted within biological safety cabinets or other physical containment devices or by
personnel wearing appropriate personal protective clothing and devices. No children are
permitted in the laboratory.
The laboratory has special engineering and design features. It is recognized, however, that
many existing facilities may not have all the facility safeguards recommended for Biosafety
Level 3 (e.g., access zone, sealed penetrations, and directional air flow, etc.). In these
circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g.,
diagnostic procedures involving the propagation of an agent for identification, typing, and
susceptibility testing) in laboratories where facility features satisfy Biosafety Level 2
recommendations provided the recommended "Standard Microbiological Practices,"
"Special Practices", and "Containment Equipment" for Biosafety Level 3 are rigorously
followed. The decision to implement this modification of Biosafety Level 3
recommendations should be made by the laboratory director on advice of the Biosafety
Officer. A specific facility operations manual is prepared, reviewed by the Biosafety
Officer, and adopted. Refer to Section III -- Recombinant DNA Guidelines.
Section II -- Safety Compliance
35
ANIMAL BIOSAFETY LEVELS
Laboratory animal facilities, operational practices, and quality of animal care must meet
applicable standards and regulations. Also, appropriate species must be selected for animal
experiments (Refer to Guide for the Care and Use of Laboratory Animals, HHS Publication
No. 86-23, Rev. 1985, and Laboratory Animal Welfare Regulations, 9 CFR, Subchapter A,
Parts 1, 2, and 3). Refer to Section III -- Recombinant DNA Guidelines.
Section II -- Safety Compliance
36
BIOSAFETY REFERENCES
1. Classification of Etiologic Agents on the Basis of Hazard. (4th Edition, July, 1974).
U.S. Department of Health, Education, and Welfare. Public Health Service.
Centers for Disease Control, Office of Biosafety, Atlanta, Georgia 30333.
2. Biosafety in Microbiological and Biomedical Laboratories, Centers for Disease
Control, Atlanta, Georgia, National Institutes of Health, Bethesda, Maryland, U.S.
Department of Health and Human Services, Public Health Service, HHS Publication
No. (CDC) 93-8395, 3rd Edit., May 1993.
3. USDA permit, required for import and interstate transport of pathogens, may be
obtained from the Animal and Plant Health Inspection Service, USDA, Federal
Building, Hyattsville, MD 20782.
4. National Cancer Institute Safety Standards for Research Involving Oncogenic
Viruses. (October, 1974), U.S. Department of Health, Education, and Welfare
Publication No. (NIH) 75-790.
5. Guidelines for Protecting the Safety and Health of Health Care Workers. U.S.
Department of Health and Human Services, Public Health Service, Centers for
Disease Control, and National Institute for Occupational Safety and Health
{Division of Standards Development & Technology Transfer, 4676 Columbia
Parkway, Cincinnati, OH, Telephone (513) 533-8287}; DHHS (NIOSH)
Publication No. 88-119, September, 1988.
6. Radiation Safety Manual, The Johns Hopkins Medical Institutions, Revised July,
1990.
7. Safety & Emergency Procedure Manual, The Johns Hopkins Hospital, Revised
August, 1992.
8. Use of Experimental Animals at the Johns Hopkins University, The Johns Hopkins
University School of Medicine, Division of Comparative Medicine, Revised
September, 1992.
9. Laboratory Biosafety Guidelines, Medical Research Council of Canada and
Laboratory Centre for Disease Control, Health Protection Branch, Health and
Welfare Canada, Cat. No. MR 21-1/1990E, 1990.
10. Johns Hopkins University Safety Policy and Procedure Manual, Johns Hopkins
University, Revised August 1994.
Section II -- Safety Compliance
37
11. Biohazards Management Handbook, Second Edit., D.F. Liberman, Marcell Dekker,
Inc., New York, 1995.
12. Laboratory Safety Principles and Practices, Second Edit., D. Fleming, J.
Richardson, J. Tulis, D. Vesley, ASM Press, Washington, DC, 1995.
Section II -- Safety Compliance
38
BIOSAFETY LEVEL 3 FACILITY SPECIFICATIONS
A. PERFORMANCE
Laboratory must be effectively air tight and liquid tight.
B. LOCATION
1. Lab separated from unrestricted traffic.
2. Access to lab through two sets of doors.
3. Access to laboratory from an anteroom or change room.
C. FLOORS
1. Integral cove preferred.
2. Seamless or welded vinyl coving sealed to the floor with silicone.
D. WALLS
1. Washable and resistant to detergents and disinfectants, ie., formaldehyde.
2. Durable paint such as epoxy or formica or tile surface.
E. CEILING
1. Monolithic construction (ie., gypsum board, not removable tiles).
2. Same finish as walls.
3. Any access to HVAC, plumbing, or electrical above ceiling must be through access
doors that are closed and sealed.
F. ACCESS ZONE
Passage through two sets of doors (anteroom, change room) is mandatory.
G. DOORS
1. Self-closing.
2. Inward opening.
Section II -- Safety Compliance
39
3. No gaskets around door jams.
H. WINDOWS
Closed and sealed.
I. PLUMBING
1. All penetrations are horizontal.
2. Penetrations are caulked with fire retardant seal.
3. No drainpipes or other plumbing penetrates the floor.
J. VACUUM SYSTEM
1. Independent of building central vacuum system.
2. HEPA filtered exhaust.
K. ELECTRICAL
1. All outlets surface-mounted.
2. No junction boxes set into wall.
3. Surface conduit preferred.
4. Wall or ceiling penetrations of electrical cable are to be kept to a minimum and
sealed with fire retardant material (ie., Fireseal).
5. Each biological safety cabinet shall have independent circuits.
L. LIGHT FIXTURES
Electrical connections to fixture made with either sealed penetrations or
surface mounted conduit.
M. LAB FURNITURE
1. Sturdy.
2. Spaces between benches and equipment for cleaning access.
Section II -- Safety Compliance
40
3. Bench tops impervious to water.
4 Bench tops resistant to acids, alkalis, organic solvents.
5 Bench tops resistant to moderate heat.
N. HAND WASHING SINK
1. In laboratory, near exit door.
2. Wrist, elbow, foot, automatically operated.
O. EYE, FACE AND BODY SPRAY FIXTURE
1. Located in laboratory near exit door.
2. Near or on hand washing sink.
P. AUTOCLAVE
1. Must be available on same floor as the laboratory.
2. Preferred within lab or with pass-through to anteroom or glassware processing area
with bioseal at wall.
3. Autoclave drain must be inside the laboratory.
4. Penetration of drain through the floor, if essential, must be sealed.
Q. VENTILATION
1. Ducted exhaust must be discharged directly outside the building with dedicated
primary and secondary backup fans.
2. Exhaust is not to be recirculated to any other part of building.
3. Exhaust discharge outside must be away from occupied areas and building air
intakes.
4. Exhaust discharge should be HEPA filtered with a bag-in-bag-out prefilter and bag-
in-bag-out HEPA filter housings.
Section II -- Safety Compliance
41
5. Ventilation system (supply & exhaust) must contain 100% shut off dampers to
isolate the laboratory from other areas of the building.
6. Supply and exhaust systems must be interlocked so that supply shuts off when
exhaust is off.
R. BIOLOGICAL SAFETY CABINETS
1. Class II, Type B3 recommended.
2. HEPA-filtered exhaust through thimble connection.
3. Thimble exhaust shall not interfere with the air balance of the room.
4. Ceiling around thimble should allow opening of the thimble door(s) for certification
tests.
5. Exhaust air flow balance in thimble connection shall be 150% of the biological
safety cabinet manufacturer's exhaust specification.
6. All utility lines to the BSC shall be installed behind the cabinet.
7. The BSC shall be set six inches out from the real wall.
8. Gas line to the BSC shall have access to a shutoff valve with handle on either the
left or right-hand side of the cabinet, depending on which side the internal gas valve
is located.
9. Gas supply line shall have a pipe union between the shutoff valve and the
connection to the BSC.
S. CHEMICAL FUME HOOD
1. The sash opening shall be set to 18 inches and the exhaust air shall be balanced to
provide an airflow of 100 linear feet per minute at the work opening.
2. Flammable storage cabinet and/or acid-base cabinet shall be vented into the
chemical fume hood with pipe extending from a fire arrestor screen at the rear of the
flammable storage cabinet and extending behind and four inches above the bottom
of the rear baffle at the rear of the fume hood.
T. AIR PRESSURE DIFFERENTIAL
1. Anteroom shall be at least 50 CFM negative with respect to corridor.
Section II -- Safety Compliance
42
2. Laboratory shall be at least 50 CFM negative with respect to anteroom.
U. DIRECTIONAL AIRFLOW
Ducts to be installed so air flows from area of least hazard to area of greatest hazard
potential.
V. AIR SUPPLY VENTS
1. Vents shall be located away from the face of biological safety cabinets and CO2
incubators.
2. The vents shall be four way, three way, two way, or one way depending on the
locations of areas of greatest hazard potential.
3. Low velocity, large surface area supply vents are preferred to directional vents.
W. FIRE PROTECTION
1. If required, laboratory may have a sprinkler system.
2. Sprinkler escutcheon plates must be sealed.
Section II -- Safety Compliance
43
SUMMARY OF BIOSAFETY LEVELS
_________________________________________________________________________________
Facility Biosafety Practices & Safety Facilities
Level Techniques Equipment
_________________________________________________________________________________
1 Standard microbiological Containment provided by None
practices. adherence to standard
laboratory practices
during open bench operations.
2 Level 1 practices plus: Partial containment Basic
Laboratory coats; decon- equipment,(i.e., Class
tamination of all II biological
infectious wastes; safety cabinets) used
limited access; protec- to conduct mechanical
tive gloves and biohazard and manipulative pro-
warning signs as indicated. cedures that have high
aerosol potential that
may increase the risk
of exposure to personnel.
3 Level 2 practices plus: Partial containment Containment
special laboratory equipment used for all
clothing; controlled manipulations of infec-
access. tious material.
4 Level 3 practices plus: Maximum containment Maximum
entrance through equipment (Class III Containment
changeroom where street biological safety
clothing is removed cabinet or partial
and laboratory clothing containment equipment
is put on; shower on exit; in combination with
all wastes are decon- full-body, air-supplied,
taminated on exit from positive-pressure
the facility. personnel suit used for
all activities.
Section II -- Safety Compliance
44
CLASSIFICATION OF ETIOLOGIC AGENTS ON THE BASIS OF HAZARD
The CDC originally prepared a document which provided a standard for
evaluating the hazards associated with the various bacterial, fungal, parasitic, and
viral etiologic agents.1 The list of agents was revised by the NIH for the
Recombinant DNA guidelines. A more recent document describes biosafety
precautions for use of agents which have resulted in known laboratory infections.2
In the CDC documents, human etiologic agents were placed in classes of increasing
hazard. The classification is included here as a general reference. Specific examples
of agents are listed under each class.
A. Class 1 Agents of no or minimal hazard under ordinary conditions of handling at
Biosafety Level 1.
B. Class 2 Agents of ordinary potential hazard. This class includes agents which may
produce disease of varying degrees of severity from accidental inoculation or
injection or other means of cutaneous penetration but which are contained by the
ordinary laboratory techniques described in Biosafety Level 2.
C. Class 3 Agents involving special hazard or agents derived from outside the United
States which require a federal permit for importation unless they are specified for
higher classification. This class includes pathogens which require special
conditions for containment, usually at Biosafety Level 3.
D. Class 4 Agents that require the most stringent conditions for their containment
because they are extremely hazardous to laboratory personnel or may cause
serious epidemic disease. This class includes Class 3 agents from outside the
United States when they are employed in entomological experiments or when
other entomological experiments are conducted in the same laboratory area.
Biosafety Level 4 facilities are limited to places such as the CDC, NIH, etc. No
Biosafety Level 4 work is permitted on campus. Collaboration may be arranged
with United States Army Medical Research Institute of Infectious Diseases or the
Frederick Cancer Research Center in nearby Frederick, MD.
E. Class 5 Foreign animal pathogens that are excluded from the United States by law
or whose entry is restricted by USDA administrative policy. Note: Federally
licensed vaccines containing live bacteria or viruses are not subject to these
classifications. These classifications are applicable, however, to cultures of the
strains used for the vaccine production, or further passages of the vaccine strains.
This classification does not include strictly animal pathogens. A PHS permit is
Section II -- Safety Compliance
45
required to import any agent or to transfer within the United States any agent
imported under permit. Containment conditions are established by the USDA on a
case-by-case basis. No Biosafety Level 4 work is permitted on campus.