Laboratory Biosafety Manual
(BSL Level)
Room (Number)
Building (Name)
Department (Name)
PI/Lab Director (Name)
Revised by OSEH 2/14/2013
1
Validation and History for Biosafety Manual
Director’s Certification:
I hereby certify that I have reviewed the contents of this manual and that it reflects my
current operating policy for the laboratories (room numbers) located in (building).
(Principal Investigators Name – please print on line
)
(Signature)
(Date)
(List all Co-Principal Investigators Name– please print)
(Annual Review Dates)
2
Chapter 1– Introduction .................................................................................................. 6
Acknowledgments ..................................................................................................................................... 7
Definitions ................................................................................................................................................. 7
Risk Group Classification ........................................................................................................................ 9
Laboratory Biosafety Level ..................................................................................................................... 9
Chapter 2 - General Information and Structure ......................................................... 10
Commitment to Safety.............................................................................................................................10
Management Structure and Responsibility ...........................................................................................11
Oversight ..................................................................................................................................................11
Chemical Hygiene Plan (CHP) ...............................................................................................................11
Research Agents.......................................................................................................................................11
Laboratory Access ...................................................................................................................................13
Laboratory Security ................................................................................................................................13
Select Agents ........................................................................................................................................13
Personnel Training ..................................................................................................................................14
Facilities ....................................................................................................................................................15
Negative pressure tissue culture rooms .............................................................................................15
Bench Tops ...........................................................................................................................................15
Laboratory Furniture .........................................................................................................................15
Workplace Practice Controls..............................................................................................................15
Vaccinations (if applicable) .....................................................................................................................16
Posting and Labeling Requirements ......................................................................................................16
Signs ..........................................................................................................................................................16
Labels and Tags .......................................................................................................................................16
Audit Management ..................................................................................................................................17
Storage of Biohazardous Materials ........................................................................................................17
Transport of Hazardous Materials to Storage Areas ...........................................................................18
Chapter 3 - Laboratory Practices .................................................................................. 18
Standard Microbiological Practices .......................................................................................................18
Biosafety Procedures ...............................................................................................................................18
Personal Protective Equipment ..........................................................................................................18
Laundry and Decontamination of Protective Clothing ........................................................................19
Standard Microbiological Practices BSL2+ ..........................................................................................20
Good Microbiological Techniques..........................................................................................................21
Use of pipetting aids ............................................................................................................................21
Minimization of aerosol production .......................................................................................................22
Protection of vacuum line .......................................................................................................................22
Syringes and Needles ...............................................................................................................................22
Biological Safely Cabinet Use .................................................................................................................23
Biohazardous Waste Disposal Methods .................................................................................................24
Biohazardous Glassware, Plastics and Other Solid Waste ..............................................................24
Biohazardous Liquids– Sanitary Drain .............................................................................................25
OSEH Hazardous Materials Management (HMM) - Collection of Waste .........................................25
Biohazardous Glassware, Plastics and Other Solid Waste ..............................................................25
Biohazardous Regulated Sharps - Note Sharps cannot be treated by the laboratory ...................25
Biohazardous Liquids .........................................................................................................................26
Chapter 4 - Research Animals ....................................................................................... 27
Use of Sharps with Animals ....................................................................................................................28
Animal waste ............................................................................................................................................28
Chapter 5 – Specific Standard Operating Procedures ................................................ 29
Biological Safety Data Sheets ..................................................................................................................29
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Chapter 6 – Equipment and Facility Management ..................................................... 30
Equipment Care and Use ........................................................................................................................30
Proper Use of Autoclave......................................................................................................................30
Proper Use of Centrifuges ...................................................................................................................32
Spills and Leaks .......................................................................................................................................33
Blenders, Ultrasonic Disrupters, Grinders and Lyophilizers ..........................................................34
Miscellaneous equipment ........................................................................................................................34
Microscopes ..........................................................................................................................................34
Microtomes...........................................................................................................................................34
Cryostats...............................................................................................................................................34
Water baths ..........................................................................................................................................35
Loop Sterilizers and Bunsen Burners ................................................................................................35
Open Flames in Biosafety Cabinets....................................................................................................35
Other Physical and Chemical Hazards ..................................................................................................36
Dry Ice ..................................................................................................................................................36
Liquid Nitrogen ...................................................................................................................................37
Ultraviolet light ....................................................................................................................................39
Electrical Hazards ...............................................................................................................................40
Gel Electrophoresis .............................................................................................................................40
Ethidium Bromide ...............................................................................................................................40
Acrylamide ...........................................................................................................................................40
Additional Chemical Usage.....................................................................................................................41
Phenol ...................................................................................................................................................41
Chloroform...........................................................................................................................................41
Equipment Maintenance .........................................................................................................................42
Cleaning and Decontamination ..............................................................................................................42
Chapter 7 – Radiation Management ............................................................................. 43
Use of Radioactive Isotopes ....................................................................................................................43
Disposal of Isotopes after Usage .............................................................................................................44
Chapter 8 - Emergency Management ........................................................................... 45
Emergency Procedures............................................................................................................................45
Accidental Exposure to an Infectious Agent .........................................................................................45
Spill Response Procedures for Infectious agents and Recombinant DNA ..........................................46
Spill in the Laboratory ............................................................................................................................47
Skin, Mucous Membrane, or Injury Exposure .....................................................................................48
For exposures to recombinant DNA, perform the following: ..........................................................48
Fire Fighting Procedures ........................................................................................................................49
Reporting and Recordkeeping ................................................................................................................49
Chapter 9 - Shipment and Receipt of Infectious Materials......................................... 50
Receipt of Infectious Materials ...............................................................................................................50
Transportation of Biohazardous Materials ...........................................................................................50
On-Campus Transportation ...................................................................................................................51
Hand Carry Between University Labs or Buildings .............................................................................51
Transport by University Vehicle ............................................................................................................51
Off-site Transportation by Courier .......................................................................................................52
Chapter 10 – Appendices................................................................................................ 53
Appendix 1 ....................................................................................................................... 54
Emergency Call List ................................................................................................................................54
Appendix 2 ....................................................................................................................... 55
Schematic of Laboratory.........................................................................................................................55
Appendix 3 ....................................................................................................................... 56
Roster of Approved Personnel and Training Records .........................................................................56
Appendix 4 ....................................................................................................................... 57
Training Completion Form.....................................................................................................................57
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Training Checklist ...................................................................................................................................58
Appendix 5 ....................................................................................................................... 61
Housekeeping Inspections .......................................................................................................................61
Appendix 6 ....................................................................................................................... 62
Pathogen Safety Data Sheets for Infectious Agents ..............................................................................62
Appendix 7 ....................................................................................................................... 63
Accident Report Forms ...........................................................................................................................63
Appendix 8 ....................................................................................................................... 65
Standard Operating Procedure ..............................................................................................................65
Appendix 9 ....................................................................................................................... 68
Autoclave Use Log ...................................................................................................................................68
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Chapter 1– Introduction
While this manual is not intended to cover every aspect of biosafety, essential basic
procedures, precautions and guidelines are discussed. If basic (BSL level) laboratory
practices are performed carefully, along with the appropriate experimental design and
common sense, workers should have few problems working safely in the (BSL level)
environment.
It is essential that laboratory personnel maintain good, sound laboratory work practices
and precautions. An awareness and respect for the dangerous nature of certain pathogens
should always be maintained. Extensive guidelines regarding biosafety containment
levels and various agent summary statements can be obtained from the U.S. Department
of Health and Human Services, Centers for Disease Control and Prevention and National
Institutes of Health publication entitled Biosafety in Microbiological and Biomedical
Laboratories 5th Edition. This document is available from the Department of
Occupational Safety Environmental Health (OSEH) website at
http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm
The guidelines should be read before work in the (BSL Level) laboratory is initiated. In
addition, those who plan to initiate work in the lab must be familiar with the standard
practices that apply as well.
_____________________________
Lab Director
___________________
Date
6
Acknowledgments
This Biosafety Manual is the result of assessing the safety-management needs of a variety of
laboratories that use biological materials at the University of Michigan. We would like to
acknowledge and express our appreciation to all the researchers who contributed to this
effort.
Definitions
Biohazardous Material - any material known to harbor organisms or agents capable of
infecting or infesting human or animal hosts or causing environmental harm if released.
Biosafety - Biosafety is a specialized practice for proper handling and working with
biohazardous organisms or biological material, which may harbor biohazardous
organisms.
Biosafety fits neatly into the traditional programmed approach to safety:
 Administrative controls to standardize methods to reduce exposure risk
 Mechanical engineering controls for containment of hazardous materials
 Medical surveillance and PPE for potentially exposed employee
 Workplace monitoring to determine exposure levels
Biosafety Levels aka Biological Safety Level- (BSLs) describe the combination of safety
practices, safety equipment and facility design used to contain the hazards associated
with specific risk groups of microorganisms and is based on risk assessment. Biosafety
levels are different from risk groups however risk group information is critical in
determining the correct biosafety containment level. See below
Blood borne Pathogen - an agent known to be transmissible through contact with human
blood, such as the human immunodeficiency virus (HIV) or the hepatitis B virus (HBV).
Containment:
Primary Containment – The protection of personnel and the immediate laboratory
environment from exposure to infectious agents is provided by both good microbiological
technique and use of appropriate safety equipment.
Secondary Containment – The protection of the environment external to the laboratory
from exposure to infectious materials, is provided by a combination of facility design and
operational practices
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Infectious Substance - a viable micro-organism, or its toxin, which causes or may cause
disease in humans or animals, and includes those agents listed in 42 CFR 72.3 or any other
agent that causes or may cause severe, disabling, or fatal disease.
IBC - Institutional Biosafety Committee
Occupational Exposure – an exposure that may place personnel at risk of injury or infection
is defined as percutaneous (e.g., a needle stick or cut with a sharp object), contact of mucous
membranes, or contact of skin (especially when the exposed skin is chapped, abraded, or
afflicted with dermatitis or the contact is prolonged or involving an extensive area) with
blood, tissues, or other bodily fluids to which universal precautions apply. For the purpose of
this manual, the occupational exposure must be to fluids or aerosols known to be infectious.
OSEH - University of Michigan, Department of Occupational Safety and Environmental
Health
OVPR – Office of the Vice President for Research
Potentially Infectious Material - any material, which may or is known to contain an etiologic
agent of human or animal disease.
Principal Investigator/Lab Director - the University of Michigan faculty member responsible
for the research underway in the laboratory. For the purposes of this protocol, the Principal
Investigator is (List PI, address and phone number)
Recombinant DNA Molecules - molecules that are constructed outside living cells by
joining natural or synthetic DNA segments to DNA molecules that can replicate in a
living cell; or molecules that result from the replication of those described above.
UCUCA - University Committee on Use and Care of Animals
Risk Groups- are the classification of infectious microorganisms based on principle
characteristics such as; route of transmission and severity of disease. See below
Standard Microbiological Practices (SMP) - basic safe laboratory work protocols for
working with biological materials based on containment level.
-
The main objective of SMP is to provide safety controls needed to protect
workers and the environment from contamination in the event that the agents are
accidentally released from their primary container.
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Risk Group Classification
Risk Group 1 (RG1) agents are defined and characterized strains of viable
microorganisms not known to consistently cause disease in healthy adult humans or
animals. Laboratory personnel may become infected through high doses or unusual
routes of exposure that are not commonly encountered in a natural setting. Opportunistic
RG1 pathogens may cause serious disease in elderly persons and infants, and persons
with compromised immune systems. A risk assessment should be used for vaccine
strains, as multiple passages in vivo do not ensure avirulence.
Risk Group 2 (RG2) agents are associated with human or animal disease which is rarely
serious and for which preventive or therapeutic interventions are often available. RG2
organisms have the capability to cause serious disease based on dose, route of exposure,
and immune status. Laboratory exposures may cause serious infection, but the risk of
spread of infection is limited. The risk assessment should give special attention to those
RG2 organisms for which preventative or therapeutic interventions are not available.
Risk Group 3 (RG3) agents are associated with serious or lethal human or animal
disease and have the potential for respiratory transmission, and for which preventive or
therapeutic interventions may be available. Preventative or therapeutic interventions may
not be available for some RG3 agents, or are less available than for RG2 organisms.
Risk Group 4 (RG4) agents are associated with serious or lethal human or animal
disease. RG4 agents may be transmitted via the aerosol route, can be readily transmitted
from one individual to another, directly or indirectly, and for which there is no available
vaccine or therapy. RG4 agents are NOT permitted at the University of Michigan.
Laboratory Biosafety Level
BSL1 practices, safety equipment, and facility design and construction include
appropriate laboratories in which work is done with defined and characterized strains of
viable microorganisms not known to consistently cause disease in healthy adult humans,
and are of minimal potential hazard to laboratory personnel and the environment. The
laboratory is not necessarily separated from the general traffic patterns in the building.
Work is generally conducted on open bench tops using standard microbiological
practices. Special containment equipment or facility design is neither required nor
generally used. Laboratory personnel have specific training in the procedures conducted
in the laboratory and are supervised by a scientist with general training in microbiology
or a related science.
BSL2 practices, equipment, and facility design and construction are applicable to
teaching and research laboratories in which work is done with the broad spectrum of
indigenous moderate-risk agents that are present in the community and associated with
human disease of varying severity. U of M requires all activities involving infectious
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materials be conducted in biological safety cabinets. No work with infectious materials
in open vessels will be conducted outside the cabinets.
 RG2 agents infectious to humans or vertebrate animals are stored or
manipulated;
 Human blood, blood products, or other potentially infectious materials,
including commercial cell lines;
 animal tissues or fluids, especially those collected from nonhuman
primates, that are contaminated with an agent infectious to humans or
vertebrate animals, or materials that were collected from an animal known
to be sick are manipulated, including commercial cell lines;
 Certain types of research utilizing toxins or venom.
BSL3 practices, safety equipment, and facility design and construction are
applicable to clinical, diagnostic, teaching, research, or production facilities in which
work is done with indigenous or exotic agents with a potential for respiratory
transmission, and which may cause serious and potentially lethal infection.
Primary hazards to personnel working with these agents relate to autoinoculation,
ingestion, and exposure to infectious aerosols. All laboratory manipulations should be
performed in a BSC or other enclosed equipment, such as a gas-tight aerosol generation
chamber. Secondary barriers for this level include controlled access to the laboratory and
ventilation requirements that minimize the release of infectious aerosols from the
laboratory.
 RG3 agents such as; Mycobacterium tuberculosis, St. Louis
encephalitis virus, and Coxiella burnetii are representative of the
microorganisms assigned to this level.
BSL-4 No work at BSL4 is conducted at the University of Michigan
Animal Biosafety Levels
A similar set of four biosafety levels are provided for work with vertebrate animals
infected with agents which may infect humans. These Animal Biosafety Levels, ABSL 1
thru 4, provide for practices, equipment, and facilities that are comparable to the
laboratory biosafety levels described above. However, there are unique hazards
associated with infected animals that must be understood by those personnel with animal
contact and addressed in the animal facility. Animal activity can create aerosols and bites
and scratches can occur.
Chapter 2 - General Information and Structure
Commitment to Safety
It is the objective of this laboratory, and its management to practice safety in science and
to exercise all reasonable and prudent precautions generally accepted as research industry
standards. Guidelines recommended by the CDC and NIH for biosafety will be strictly
10
observed and enforced by the laboratory director and management. At the conclusion of
initial personnel training, each lab employee will sign a Training Completion Form (See
Appendix 4) to be retained with the training records.
Management Structure and Responsibility
Overall supervision of the (BSL level) laboratory is the responsibility of (Name of
Principal Investigator). Management of daily operations in the lab is the responsibility
of (Name of Lab Manager or Responsible Person). Access to the laboratory must be
obtained from one or both of the above.
Oversight
This plan meets the quality standards for laboratory safety management as required by
UM-OSEH and OVPR.
Chemical Hygiene Plan (CHP)
The Laboratory Safety Standard requires employers to write and implement laboratoryspecific Chemical Hygiene Plans (CHP). According to this regulation, a Chemical
Hygiene Plan applies to all employers engaged in the laboratory use of hazardous
chemicals. A “laboratory” is defined as a facility where the laboratory use of hazardous
chemicals occurs. A “hazardous chemical” is defined as a chemical for which there is
evidence that acute or chronic health effects may occur in exposed employees. The CHP
should include specific work practices, procedures, and policies to ensure that employees
are protected from all potentially hazardous chemicals used. Recommendations for
completing each section of the Chemical Hygiene Plan are provided on OSEH’s Internet
web site. The web site contains a generic CHP that can be down loaded and modified for
use in your Lab (http://www.oseh.umich.edu/research/chem-hygiene.shtml ). If you have any
questions regarding the CHP, please contact OSEH at 76 3-6973.
Research Agents
Until otherwise specified, infectious agents approved for use in the laboratory include:
(please use genus species naming format and list the risk group (1-3))
Name
Name
11
Name
Name
Pathogen Safety Data sheets (PSDS) if available.
http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php
Printed copies for the organisms listed above can be found in Appendix 6.
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Laboratory Access
1)
The Principal Investigator or his/her designee authorizes access to the laboratory.
Persons requesting to use the laboratory or equipment shall be advised of the
potential hazards involved and shall follow all biosafety guidelines as presented in
this manual.
2)
Access to the (BSL level) laboratory is restricted when work with infectious
agents is in progress, after hours, or when laboratory personnel are not available.
3)
Restrictions or recommendations will be made on an individual basis. Examples
of conditions that might warrant special precautions are HIV infection,
immunosuppressive conditions, and drug therapies that suppress the immune
system. Additionally, it is recognized that exposure to certain infectious agents
may adversely affect a fetus during pregnancy if the mother is infected with the
agent. Employees, who fall into any of the above conditions, should inform their
personnel physician and the Principal Investigator about the situation.
Laboratory Security
Certain biohazardous microorganisms and toxins may be of interest to persons or groups
involved in terrorism or other illegal activities. Therefore infectious agents that could
pose a serious threat to humans, agriculture, or the livestock industry should be kept
under secure conditions within the laboratory.
Select Agents
In compliance with the “Public Health Security and Bioterrorism Preparedness Response
Act of 2002” (Public Law 107-188) and the Select Agents regulations from the Centers
for Disease Control 42 CFR 73 (http://www.cdc.gov/od/sap/42_cfr_73_final_rule.pdf)
and USDA 9CFR 121, 7 CFR 331 (http://www.aphis.usda.gov/programs/ag_selectagent
/), any research project which will utilize Select Agents and High Consequence Livestock
Pathogens and Toxins on campus requires special registration. Please contact OSEH if
you plan to use any of the listed agents or toxins in your research (List of Agents and
Toxins, http://www.cdc.gov/od/sap/docs/salist.pdf).
The highest level of security is reserved for materials referred to as Select Agents, and
these require a Biosecurity Plan to be implemented. The Institutional Biosecurity Plan is
available through UM-OSEH and is administered in conjunction with Department of
Public Safety. The Plan defines which viable agents and quantities are regulated.
If a request is received from another institution or corporate entity for a dangerous
organism for academic purposes, the Principal Investigator is responsible for ensuring that
the receiving entity is a valid research organization and that the transfer has administrative
13
approval from both institutions. When a request is received, the Principal Investigator must
notify the Biological Safety Officer for approval to send the agent.
Personnel Training
1)
Once a year all personnel working in the (BSL level) laboratory will be instructed
by the Principal Investigator or Laboratory Manager on the special biological
safety procedures to be used. Attendance at this session is mandatory.
2)
OSEH conducts the Comprehensive Laboratory Safety training course several
times a month and upon request. All new lab employees and anyone who has not
yet attended must attend this course. OSEH recommends that employees be
trained at least every three years thereafter. See OSEH’s web page
http://www.oseh.umich.edu/training/mylinc.shtml for training dates, times and
sign-up procedures.
3)
Personnel must have prior experience with the agent in use or must be provided
with suitable and sufficient information, instruction and training on working with
the agent prior to initiating work. A training course entitled “Working Safely
with Viral Vectors is available for new or entry-level lab personnel who plan to
use viral vectors in vitro or in vivo, and who cannot demonstrate significant
previous experience and expertise in the necessary aspects of biosafety and
regulatory compliance. The course is provided by the University of Michigan
Vector Core Laboratory and is sanctioned by the Institutional Biosafety
Committee (IBC) and the Department of Occupational Safety and Environmental
Health (OSEH). Registration for the course is available on the OSEH website at
http://www.oseh.umich.edu/training/mylinc.shtml
4)
New employees must read a copy of the Biosafety Manual and associated
documents as listed in the Training Completion Form (Appendix 4) prior to
starting work in the laboratory.
5)
New employees must exhibit competency in good microbiological techniques as
listed in the Recombinant DNA/ Infectious Agent Training Checklist
(appendix 4) prior to starting work in the laboratory.
6)
It is the direct responsibility of the Principal Investigator or Lab Manager, to
initially instruct new employees of the safety procedures used in the laboratory.
This includes a thorough review of the appropriate operating procedures of the
laboratory. See Recombinant DNA/ Infectious Agent Training Checklist
(appendix 4)
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Facilities
Negative pressure tissue culture rooms
In general, a separate tissue culture room provides a higher level of containment for
working with potentially airborne recombinant DNA vectors (ex. adenoviral vectors) or
infectious agents than the general laboratory. Mechanical ventilation should provide an
inward flow of air (negative pressure) without recirculation to spaces beyond the
laboratory. This is sometimes referred to as a one-pass ventilation system. Laboratory
personnel should verify that the direction of airflow is into the laboratory. UM-OSEH
provides this check as a service during inspections.
Bench Tops

Bench tops should be impervious to water and resistant to acids, alkalis, organic
solvents, and moderate heat.

The work areas should be kept clean and dust free as to prevent contamination of
samples and laboratory acquired infections.

Bench tops should be disinfected with 1:10 dilution of bleach solution or other
appropriate disinfectant daily, following any spill, and at least once a month when
bench top is used infrequently.
Laboratory Furniture
Laboratory furniture should be capable of withstanding anticipated loading and uses.
Spaces between benches, cabinets, and equipment should be accessible for cleaning.
Chairs and other furniture used in laboratory work should be covered with a non-fabric
material that can be easily decontaminated.
Workplace Practice Controls
All personnel must routinely use appropriate controls when handling biohazards or materials
that may harbor biohazards. These include:
Engineering Controls
-
Primary Barriers (e.g., biological safety cabinets and their respective
ventilation systems) are the preferable method for containment of biohazards.
Aerosol-generating procedures with biohazardous materials that pose an
inhalation risk should always be handled in a biosafety cabinet. A biosafety
cabinet is designed to contain microorganisms, which are released during
work within the cabinet. Selection of the type of primary barrier should be
based on the risk assessment.
-
Secondary Barriers (e.g., building design features include floor to ceiling
walls, operating areas under negative pressure and using closed doors). Many
15
laboratories have monitoring systems built-in to indicate any system failures
that could affect secondary containment. Laboratory personnel should be
familiar with these devices if they are available.
Vaccinations (if applicable)
Lab personnel must be provided with information regarding vaccines that may be
available to protect them against laboratory acquired infection. Under some
circumstances, the Laboratory Director may require immunization as a condition of
employment within the biohazard laboratory. Information to be provided to employees
includes: efficacy, side effects, booster schedule, etc. Vaccinations should be provided
to employees free of charge and during working hours. Vaccinations are provided
through a certified Occupational Medical service, such as Occupational Health Services
(OHS). Personal health status may impact an individual’s susceptibility to infection,
ability to receive immunizations or prophylactic interventions. Therefore, all laboratory
personnel and particularly women of childbearing age should be provided with
information regarding immune competence and conditions that may predispose them to
infection.
Posting and Labeling Requirements
The necessity for establishing policies and procedures for proper identification of
hazardous biological agents within the University's laboratories is to alert support and
emergency personnel who may enter the area to take precautionary measures and to
restrict traffic to potentially hazardous areas.
Signs
All areas and laboratories that contain biohazardous agents must be posted with a
“Caution” sign. A biohazard label, incorporating the universal biohazard symbol, should
be placed on the face of these signs. These signs shall:



Indicate the biosafety level of the laboratory.
List the name and telephone number for the Principal Investigator to facilitate
contact in case of emergency.
Required procedures for entering and exiting the laboratory.
Contact OSEH at 647-1143 to request a customized door sign.
Labels and Tags
Biological hazard warning labels must be used to identify containers of infectious materials,
infectious waste, refrigerators, incubators and/or freezers where biohazards are stored,
infectious waste containers, equipment which may be contaminated through normal use of
16
biohazards, laboratory animals (cages) which are potentially infectious or combinations thereof
which are contaminated with biohazardous materials. The label must consist of the universal
"Biohazard" Symbol). These labels should be affixed as close as safely possible to the
container, refrigerator/freezer, equipment, animal cage or other containers.
Audit Management
General
Maintaining accurate records and documentation is a critical part of any
Biosafety Program. In order to prove that specific requirements of the Biosafety
Program have been accomplished, appropriate documentation must be filed.
Documentation is required for the following:

Biosafety training: Documentation should be available to prove that employees have
been trained in the proper use of the specific biohazards with which they work.
Documentation should be provided for new personnel and to document retraining (See
Appendix 4)

Accident Investigation and Injury Illness Recordkeeping.
http://www.umich.edu/~connect/pdf/iirf.pdf

Inspection/Audit Reports: Self-inspection and follow-up reports should be maintained
for at least one year. http://www.oseh.umich.edu/pdf/BL2checklist.pdf

In the case of highly hazardous organisms (Select Agents), electronic entry logs for the
laboratory must be maintained.
Storage of Biohazardous Materials
All infectious materials to be stored must be clearly labeled. The storage space (e.g.,
freezer, refrigerator) must also be labeled with the universal biohazard symbol.
Additional information including contact name and emergency numbers should be visible
in case of emergency, i.e., freezer breakdown.
Expired and other unwanted material must be decontaminated properly. Materials for
long-term storage must be annually inspected and each container must be checked for
cracks and other damages and properly disposed or replaced.
In the event of a freezer melt-down, all materials that are unable to be salvaged must be
properly treated by autoclaving or chemical disinfection.
17
Transport of Hazardous Materials to Storage Areas
Live infectious materials, which are removed from the facility for storage in liquid
nitrogen or –80 º freezers, should be stored in non-breakable, cryovials. The vials must
be surface decontaminated with 70% ethanol after sealing and then transported to the
freezers in non-breakable, impermeable, closed containers (ex. Biotransport Carrier).
The container used for this purpose must be labeled with a biohazard label.
Chapter 3 - Laboratory Practices
All Biosafety Containment level practices and facilities encompass all the requirements
described below as well as containment level specifics outlined in this manual.
Standard Microbiological Practices








Persons working in the laboratory must be fully aware of the potential hazards to
themselves and their co-workers. Personnel must meet the specific entry/exit
requirements for this space.
Persons must wash their hands after working with potentially infectious materials
and before leaving the laboratory.
Eating, drinking, smoking, handling contact lenses, applying cosmetics, and
storing food for human consumption are not permitted in the laboratory
Food must be stored outside the laboratory area in cabinets or refrigerators
designated and used for this purpose.
Mouth pipetting is strictly prohibited; mechanical pipetting devices must be used
Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and
broken glassware must be developed and implemented. Whenever practical,
laboratory supervisors should adopt improved engineering and work practice
controls that reduce risk of sharps injuries.
Laboratory coats, gloves, and protective eyewear must be worn when working in
the laboratory
Non-experimental animals and plants are not permitted in the laboratory.
Biosafety Procedures
Personal Protective Equipment
Personal Protective Equipment (PPE). PPE should be selected in accordance with the
hazards identified. All laboratory personnel are required to wear appropriate PPE
corresponding to the chemical, biological and radiological substances being used.
Appropriate PPE may include: gloves, safety glasses, safety goggles, face shields and
laboratory coats. The minimum level of protective equipment in a lab should include lab
coats, safety glasses and appropriate gloves. Persons who wear contact lenses should
18
also wear eye protection. To determine if the laboratory personnel are wearing the
appropriate type of glove, you can refer to a glove selection chart. Glove selection charts
are available through various laboratory supply catalogs. Two pairs of gloves should be
worn when appropriate. Alternatives to latex gloves should be available. Dispose of
protective clothing appropriately, or deposit it for laundering. Do not launder lab coats at
home.
OSEH can assist in the correct selection of PPE. Laboratory dress code should strongly
discourage the wearing of shorts and open toe shoes. The Personal Protective Equipment,
General Guideline has been developed to provide the University community with the
necessary information to identify work situations that require the use of PPE, the proper
selection and use of PPE, and documentation of this information. This information is
important to help ensure the safety and health of all UM employees. The Personal
Protective Equipment, General Guideline can be found on OSEH’s web site at
http://www.oseh.umich.edu/pdf/guideline/ppe_guideline.pdf .
Special handling practices include:
 The frequent changing of PPE; changing gloves when the gloves are
contaminated or the integrity has been compromised, or when otherwise
necessary; do not wash or reuse disposable gloves.
 Eye, face and respiratory protection should be used in rooms containing infected
animals as determined by the risk assessment.
 The removal of PPE when work has been completed with infectious materials or
when leaving the lab followed by hand washing with an appropriate
decontaminating soap
 Disposing of PPE with other contaminated laboratory waste or decontaminated it
before reuse.
 Ensuring eye and face protection (goggles, mask, face shield or other splatter
guard) is used for anticipated splashes or sprays of infectious or other hazardous
materials when the microorganisms must be handled outside the biosafety cabinet
or containment device.
 Prohibition of eating, drinking, smoking, chewing gum, removing contact lenses,
or applying cosmetics either while in the biohazard area or while wearing
potentially contaminated PPE.
Laundry and Decontamination of Protective Clothing
Potentially contaminated lab coats or protective outer garments are to be handled as little as
possible with a minimum of agitation to prevent contamination of the air or of persons
handling them. The risk of actual disease transmission from contaminated laundry is low,
however care should be taken when handling such clothing. Lab coats should not be taken
home for laundering. Professional laundering service is available. Disposable lab coats are
available as an option.
19
Standard Microbiological Practices BSL2+
All of the above plus
1) All procedures are performed carefully to minimize the creation of splashes or
aerosols. All infectious agents are handled in a biosafety cabinet.
2) Work surfaces should be decontaminated at least once a day and after any spill or
splash of potentially infectious material with appropriate disinfectant
.
3) All cultures, stocks, and other regulated wastes are decontaminated before disposal by
an approved decontamination method, such as autoclaving immediately after use.
Materials to be decontaminated outside of the immediate laboratory are to be placed
in a durable, leak-proof container and secured for transport from the laboratory.
4) Laboratory personnel must be provided medical surveillance, as appropriate, and
offered available immunizations for the agents handled or potentially present in the
laboratory.
5) Careful management of needles and other sharps are of primary importance. Used
disposable needles must not be bent, sheared, broken, recapped, removed from
disposable syringes, or otherwise manipulated by hand before disposal. Used
disposable needles must be carefully placed in conveniently located punctureresistant containers used for sharps disposal.
6) Do not handle broken glassware directly by hand. The glassware should be removed
by mechanical means such as a brush and dustpan, tongs, or forceps. Plastic ware
should be substituted for glassware whenever possible.
7) Place all cultures, tissues, or specimens of body fluids in a durable leak-proof
containers that prevents leakage during collection, handling, processing, storage,
transport, or shipping.
8) PPE requirements; dedicated lab coat that remains in lab, gloves and eye protection.
Laboratory personnel must wash their hands after working with potentially hazardous
materials and before leaving the laboratory. Fabric lab coats autoclaved before
laundering.
9) Spills and accidents should be immediately reported to the Principal Investigator and
the University Biological Safety Officer (647-1143).
20
Good Microbiological Techniques
Use of pipetting aids
Pipettes are used for volumetric measurements and transfer of fluids that may contain
infectious, toxic, corrosive or radioactive agents. Laboratory associated infections have
occurred from oral aspiration of infectious materials, mouth transfer via a contaminated
finger and inhalation of aerosols. Exposure to aerosols may occur when liquid from a
pipette is dropped onto the work surface, when pipetting liquid cultures, or when the last
drop of an inoculum is blown out. A pipette may become a hazardous piece of equipment
if improperly used. A variety of pipetting aids are available, but caution must be
exercised with all of them as the use of excessive force when inserting a pipette can
fracture the pipette and cause serious injury to the hands. The safe pipetting techniques,
which follow, are required to minimize the potential for exposure to hazardous materials.












Mouth-pipetting any substance, however innocuous, is prohibited
Replace glass pipettes with disposable options whenever possible.
If working with biohazardous or toxic fluid, confine pipetting operations to a
biosafety cabinet.
Always use cotton-plugged pipettes when pipetting biohazardous or toxic
materials, even when safety pipetting aids are used.
Check your pipette at the beginning of the working day for dust and dirt on the
outside. If needed, wipe with 70% ethanol.
Do not prepare biohazardous materials by bubbling expiratory air through a liquid
with a pipette.
Do not forcibly expel biohazardous material out of a pipette.
Never mix biohazardous or toxic material by suction and expulsion through a
pipette.
Do not discharge material from a pipette at a height. Whenever possible allow the
discharge to run down the container wall.
Prevent cross contamination by: using sterilized tips or sterilized filter tips and
autoclave the pipette. Avoid contamination to or from fingers by using the tip
ejector. Keep the pipette vertical when pipetting in order to prevent the liquid
from running into the pipette body
Place contaminated, reusable pipettes horizontally in a pan containing enough
liquid disinfectant to completely cover them. Do not place pipettes vertically into
a cylinder. Autoclave the pan and pipettes as a unit before processing them for
reuse.
Pans or collection containers for contaminated pipettes should be placed inside
the biosafety cabinet.
21
Minimization of aerosol production


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



Procedures with a high potential for creating aerosols, such as vigorous shaking
and vortexing, should be performed in the back third of the biological safety
cabinet.
Never pour or decant virus suspensions.
Do not forcibly expel the last drop of infectious agent suspension from a pipette.
Discharge pipetted material near the surface of fluid or down the wall of a tube.
When opening culture tubes, bottles and flasks, manipulate them slowly.
When resuspending liquid cultures, use a swirling action to create a homogeneous
suspension with a minimum of aerosolization. Once cultures are resuspended,
wait a few minutes to reduce aerosols before opening the container.
Do not spray disinfectant directly onto liquid spills, as this will create aerosols.
Instead cover spill with paper towels and pour disinfectant, around and over the
spill. Allow suitable contact time (20 minutes).
Protection of vacuum line
Vacuum lines should be protected by a disinfectant trap. Use a plastic aspirator suction
flask with a second vacuum flask connected to it as a backup. To the second vacuum
flask attach a high efficiency particulate air (HEPA) vacuum filter (ex. VacuShield
product). These filters can isolate and confine infectious materials, preventing fluid and
aerosol contamination of vacuum pumps or aspiration suction systems. Filters are
available through laboratory supply catalogs. Contact OSEH (647-1143) if you need
more information regarding HEPA filters for your vacuum system. The vacuum
collection flasks should be attached in series from the vacuum source in the biosafety
cabinet or a vacuum pump. If this set up is placed outside of the biosafety cabinet, it
should be contained in a tray or pan to prevent accidental spills.
Syringes and Needles
Syringes and hypodermic needles are dangerous instruments. The use of needles and
syringes should be restricted to procedures for which there is no alternative. Blunt
cannulas should be used as alternatives to needles wherever possible (i.e., procedures
such as oral or intranasal animal inoculations). Needles and syringes should never be
used as a substitute for pipettes. When needles and syringes must be used, the following
procedures are recommended:


Use disposable needle locking syringe units whenever possible.
When using syringes and needles with biohazardous or potentially infectious
agents:
a. Work in a biosafety cabinet whenever possible.
b. Wear gloves.
c. Fill the syringe carefully to minimize air bubbles.
22
d. Expel air, liquid and bubbles from the syringe vertically into a cotton
pledget moistened with disinfectant.
e. Do not use a syringe to mix infectious fluid forcefully.
f. Do not contaminate the needle hub when filling the syringe in order to
avoid transfer of infectious material to fingers.
g. Wrap the needle and stopper in a cotton pledget moistened with
disinfectant when removing a needle from a rubber-stoppered bottle. If
there is concern of the disinfectant contaminating sensitive experimental
materials, sterile cotton square may be used and immediately discarded
into an autoclave bag.



Bending, recapping, clipping or removal of needles from syringes is prohibited. If
it is essential that a contaminated needle be recapped or removed from a syringe,
the use of a mechanical device or the one handed scoop method must be used. The
use of needle clipping devices is prohibited and the devices must be discarded.
Use a separate pan of disinfectant for reusable syringes and needles. Do not place
them in pans containing pipettes or other glassware in order to eliminate sorting
later.
Used disposable needles and syringes must be placed in conveniently located
appropriate sharps disposal containers and discarded as infectious waste.
Biological Safely Cabinet Use
The biological safety cabinet (bsc) is the principal device used to provide containment of
infectious splashes or aerosols generated by many microbiological procedures.
To assure sterility inside the cabinet and establish proper air flow for containment, the
blower should be turned on at least 10 minutes before infectious materials are to be put in
the biosafety cabinet. Check to ensure that the airflow markers fall within the posted safe
ranges before working in the hood. Airflow alarms are present on all cabinets.

If airflow is incorrect, discontinue work and contact OSEH at 647-1143. Make
sure that all biohazard materials are properly secured, and notify the Principal
Investigator or Laboratory Manager.
Before using the biosafety cabinet, wipe down the interior so as to avoid accidental
exposure to potentially infectious agents and to avoid contamination of cultures. Use
10% bleach, followed in 5 to 10 minutes with 70% ethanol to prevent pitting of the
stainless steel, and allows to dry. Always keep a bottle of disinfectant (e.g., bleach, 70%
ethanol, etc.) in the hood for decontaminating, or in case of a spill.
23
All activities involving infectious materials must be conducted in biological safety
cabinets. No work with infectious materials in open vessels will be conducted outside the
cabinets.
Work should be performed as deeply into the biological safety cabinets as possible.
NEVER place anything over the front grille of a cabinet. Disrupting the airflow in this
manner allows contaminated air from inside the cabinet to blow out of the cabinet into
the lab or directly at the person sitting at the cabinet. It also allows non-sterile air from
the room to blow into the cabinet over your experiments.
Materials should be placed in the bsc so as to not block air flow into the rear grille.
Leave a few inches for air to flow around things. Any disruption of the airflow in the
cabinet decreases its effectiveness.
Before manipulating infectious materials, try to make sure that you have everything you
need in the bsc. The fewer times you pull your hands out of the bsc, the less disruption of
the airflow.
Any infectious agents that are centrifuged must be contained in screw cap tubes and the
aerosol-barrier rotor caps used. The centrifuge container must be loaded and unloaded in
the biological safety cabinet.
Any waste generated in the biological safety cabinet must be decontaminated or
autoclaved for disposal.
The biological safety cabinet must be disinfected with the appropriate disinfectant after
each use.
Biohazardous Waste Disposal Methods
The following are various methods of disposal/treatment for different types of
biohazardous waste.
Biohazardous Glassware, Plastics and Other Solid Waste
Autoclave (Steam Sterilization)
Laboratories with access to an autoclave should treat their own solid biohazardous waste
and verify the sterilization of waste monthly by using a biological indicator (Appendix
9). Check that there are no standing liquids or hazardous chemicals. Place waste in a
university standard autoclave bag – a clear bag with a biohazard symbol and a printed-on
sterilization indicator badge (example Fisher Scientific part number 01-826A).
Autoclave with bag open or loosely tied. After sterilization cycle, close bag. The
sterilized waste can be transported directly to the dumpster by Building Services or
laboratory staff, without boxing or labeling.
24
Chemically Disinfect
Soak contaminated materials in 1:10 dilution (10% concentration) of household bleach
(contains 5% sodium or calcium hypochlorite) to water or other suitable commercial
disinfectant. Note: if contaminated materials contain a high organic load use 1:5
dilution (20% concentration) of bleach, 1% sodium or calcium hypochlorite. Following
treatment, place the waste in an impervious container such as a cardboard box and label
as uncontaminated glass or plastic waste. Building Services or laboratory staff can
transport this waste to the dumpster.
Biohazardous Liquids– Sanitary Drain
Ensure there are no chemicals in this waste. Treat liquid biohazardous waste with a 1:10
dilution of household bleach (contains 5% sodium or calcium hypochlorite) to water for
at least 30 minutes or use another suitable commercial disinfectant. Note: if liquid
materials contain a high organic load use 1:5 dilution (20% concentration) of bleach.
Follow manufacturer’s label instructions for appropriate concentration and contact time.
Rinse the treated material down the drain with copious amounts of water
OSEH Hazardous Materials Management (HMM) - Collection of Waste
Biohazardous Glassware, Plastics and Other Solid Waste
***If a laboratory does not have access to an autoclave, HMM (763-4568) must be
contacted for collection of the waste.
Solid biohazardous waste should be collected in a properly labeled biohazard pail or
biohazard box. Various containers are available through Hazardous Materials
Management (HMM). Containers can be ordered by calling HMM at 763-4568. All
containers of solid biohazardous waste must have a “biohazard label” attached. Labels
are available through HMM for identifying biohazardous waste. Labels can be ordered
by calling HMM at 763-4568. When waste accumulation begins the accumulation start
date must be written on the label. The accumulation start date is the date the waste is first
generated. All biohazardous waste must be treated or sent for disposal within 60 days.
Biohazardous Regulated Sharps - Note Sharps cannot be treated by the laboratory
All sharp objects such as scalpel blades, hypodermic needles, Pasteur pipettes and,
contaminated broken glass etc. must always be disposed of in approved "sharps collection
bins" (available from HazMat). A fluorescent orange or orange-red label that has the
biohazardous symbol in a contrasting color must be affixed to the container.
25
Biohazardous Liquids
***If a laboratory cannot dispose of the material in the sanitary drain, HMM can be
contacted for collection of the waste.
Various containers are available through Hazardous Materials Management (HMM) for
the collection of Liquid biohazardous waste. Containers can be ordered by calling HMM
at 763-4568. Package the container in a cardboard box so that the material can safely be
moved by HMM staff. All containers of solid biohazardous waste must have a “biohazard
label” attached. Labels are available through HMM for identifying biohazardous waste.
Labels can be ordered by calling HMM at 763-4568. When waste accumulation begins
the accumulation start date must be written on the label. The accumulation start date is
the date the waste is first generated. All biohazardous waste must be treated or sent for
disposal within 60 days.
Pathological Waste- Note all pathological waste must be sent for proper disposal
through HMM
Place pathological waste in a 5-gallon bucket and securely seal the lid. A fluorescent
orange or orange-red label that has the biohazardous symbol in a contrasting color must
be affixed to the container. Call HazMat at 763-4568 for waste pick-up.
For further information see the Laboratory Refuse Collection Poster
http://www.oseh.umich.edu/labcoll.shtml and/or Biohazardous Medical Waste
Guideline http://www.oseh.umich.edu/pdf/guideline/guidbmwd.pdf .
26
Chapter 4 - Research Animals
All research experiments involving animals shall be conducted in accordance with the
associated Institutional Animal Care and Use Committee (IACUC) approved protocol.
Animal research that involves a hazard (biological, radiological, or chemical) shall be
reflected in the approved IACUC protocol. Contact the IACUC office for additional
information. The Department of Biological Safety shall approve work with human
pathogens or recombinant DNA in animals (including transgenic animals) prior to
initiation. Contact the Department of Biological Safety to initiate the approval process.
Care and Handling of Research Animals
Comprehensive reviews indicate that animals infected with a wide range of biological
agents are capable of shedding infectious micro-organisms in the saliva, urine or feces. In
the absence of specific information to the contrary, all infected animals should be
regarded as potential shedding infectious micro-organism.
Procedures appropriate for the handling of infected animals are given below:
Careful handling procedures should be employed to minimize the dissemination of dust
from the animal, bedding and the cage.
Cages should be sterilized by autoclaving. Feeding and watering devices should remain
in the cage during sterilization. All water devices should be of the "non-drip" type.
Cages should be examined each morning and at each feeding time so that dead animals
can be removed. Dead animals should be placed in leak-proof containers (plastic bags,
covered metal trays, canisters, or fiber cartons) that are appropriately marked with date,
experiment, biohazard label, cage number, etc., and stored in designated refrigerators or
cold rooms prior to necropsy or disposal.
Heavy gloves should be worn when feeding, watering, handling, or removing infected
animals. Bare hands should never be placed in the cage to move any object.
When animals are to be injected with infectious agents, the animal handler should wear
appropriate PPE including protective gloves should be worn according to procedures
posted. All disposable PPE is single use and should be disposed of in the designated
waste container upon exiting the space. Protective eyewear including safety glasses or
goggles should be decontaminated and remain in the room.
Restraint devices and practices should be used to reduce the risk of exposure during
animal manipulation and administration should take place inside a certified biosafety
cabinet. All lab staff administering biohazardous material to animals or staff who will be
handling animals following administration must complete the Unit for Laboratory Animal
Management (ULAM) Hazard Containment Lecture and Workshop. Sign-up for these
courses is available on the ULAM website www.ulam.umich.edu
27
Infected animals to be transferred between buildings should be placed in ventilated cages
or other aerosol-proof containers.
Animals exposed to infectious agents in aerosols should be housed in individually
ventilated cages in gas-tight cabinet systems, or in rooms designed for protection of
personnel by use of ventilated suits or biological safety cabinets.
Additional standard and special practices for Animal Biosafety Levels 1 through 4 are
described in Biosafety in Microbiological and Biomedical Laboratories. No work for
animal biosafety level 4 is permitted at U of M.
Use of Sharps with Animals



When inoculating animals, position the hand that is holding the animal “behind”
the needle or use a pair of forceps to hold the animal in order to avoid puncture
wounds.
Be sure the animal is properly restrained prior to the inoculation and be on the
alert for any unexpected movements of the animal.
Before and after injection of an animal swab the injection site with an appropriate
antiseptic.
Animal waste
Animal waste shall be considered infectious if it is derived from animals infected with
zoonotic diseases (transmissible from animals to human) or purposely infected with
agents infectious to humans. Carcasses, body parts, tissue, body fluids, excreta, and
bedding should be considered infectious. Animal waste can be properly disposed of in
the DLAR facility. Animal carcasses that have not been intentionally infected should be
returned to DLAR for disposal. Do not place animal carcasses in with other
biohazardous waste to be autoclaved.
Infected animal carcasses or tissue that is also contaminated with hazardous chemicals or
radioactive materials is a type of mixed waste. This type of waste poses special safety and
regulatory problems and should not be generated if at all possible. The Office of
Environmental Management should be consulted before generating this type of waste.
28
Chapter 5 – Specific Standard Operating Procedures
Standard operating procedures for research involving biohazardous and rDNA
materials must be developed. Laboratory staff must be trained on SOPs annually
and/or whenever new SOPs are added.
Required elements of an SOP include: risk identification, exposure controls, and
emergency response procedures for handling spills and injuries, waste disposal,
shipment and/or receipt of materials. Additional procedures should be written to
protect laboratory and animal care staff from animals that have been exposed to
biological agents.
Examples of specific procedures for individual operations can be found in Appendix 8.
Safety precautions should be noted for each procedure. A Standard Operating Procedure
template is available in Appendix 8.
Biological Safety Data Sheets
In addition to the inventories of Safety Data Sheets (SDSs) compiled for hazardous
chemicals, all laboratories performing research with known or potential bio-hazardous
agents must maintain a comprehensive collection of Pathogen Safety Data Sheets(PSDSs)
for each bio-hazardous agent. Questions or concerns regarding acquisition of PSDSs of
biohazardous materials should be directed to OSEH at 647-3133.
29
Chapter 6 – Equipment and Facility Management
Equipment Care and Use
Proper Use of Autoclave
1)
Steam Sterilization is defined as 121C for at least 15 minutes peak temperature.
A conventional autoclave used in laboratories may take as long as 15 minutes to
reach peak temperature, and may take 15 minutes or more to be safe to open.
Therefore, a standard autoclave “cycle” is at least 45 minutes total, under ideal
conditions. Any interfering factors would necessarily increase this treatment
time.
2)
An autoclave must be available for the (BSL Level) laboratory and must only be
operated by personnel who have been properly trained in its use. Autoclave Safety
Training is available through OSEH. See the OSEH website for further
information and registration http://www.osehtraining.umich.edu/osehtraining/ .
Improper sterilization could result in laboratory personnel, other personnel
involved in disposal of laboratory waste, or the community at large being exposed
to potentially infectious agents. Chemical integrators are recommended for every
load; place on the middle, largest, or densest of the waste bag. A subsequent color
change indicates that the load has been exposed to the required conditions for a
sufficient length of time. Record autoclave waste runs in the log form appendix 9.
3)
Biohazardous materials must not be placed in autoclaves overnight in anticipation
of autoclaving the next day.
4)
Wrap packages to allow for steam penetration; aluminum foil does not allow
steam penetration, and should not be used for wrapping.
5)
Do not overload the chamber.
6)
Avoid over packing of autoclave bags.
7)
Do not seal bags or close bottles and other containers tightly.
8)
Do not stack containers.
9)
Label waste by writing lab name and date on autoclave tape
The changes that are seen on autoclave indicator tapes following an autoclave cycle do
not guarantee that the contents of containers are sterile: they indicate only that the tape on
the outside of the packages has been exposed to a certain amount of heat or steam. Proper
autoclave performance is essential for sterilization. The time required for effective
30
sterilization depends on the size of the load, volumes of liquid and density of materials to
be autoclaved. Assessing autoclave performance regularly (at least once per month) is
critical, the use of a heat-resistant biological indicator (BI) such as Bacillus
stearothermophilus, should be used to ensure that the cycle in use really achieves
sterilization conditions. (See Autoclave Performance Program)
Safe work practices when using an autoclave include the following:



Read the operating manual and post proper work procedures near the autoclave.
Never autoclave hazardous chemicals.
Dry hypochlorites, or any other strong oxidizing material, must not be autoclaved
with organic materials such as paper, cloth or oil:
OXIDIZER + ORGANIC MATERIAL + HEAT = MAY PRODUCE AN
EXPLOSION.

Special precautions should be taken to prevent accidental removal of material
from an autoclave before it has been sterilized or simultaneous opening of both
doors on a double door autoclave.


Open the door slightly to allow escape of steam before unloading.
Wear insulated gloves or mitts when unloading.
Autoclave Performance Program Purpose: To ensure autoclaves are performing
correctly. All general use autoclaves used for the sterilization of biological and
recombinant DNA-containing waste must be validated at least once per month. A heatresistant biological indicator (BI) such as Bacillus stearothermophilus, must be used to
test the microbial killing power of the sterilization process; it is important to use a
positive control (an indicator not autoclaved). Ensuring that sterilization is occurring in
the autoclave is of the utmost importance as it relates to human safety; to facilitate this
process a log sheet to document monthly performance verification of sterilization is
provided below for printing. Log sheet should be kept in biosafety manual page 67.
Guidance on basic autoclave procedure:
Estimate the total volume of solids and add time to the sterilization cycle for larger
amounts of material.
• 2 liters = 30 minutes (e.g. 2 sleeves of LB plates made from 2 liters of media)
• 4 liters = 60 minutes
• 6 liters = 90 minutes
Add water to bags of waste containing mostly dry materials (paper towels, gloves, tips,
etc.) to help generate steam for sterilization.
Put bags of waste in a heat resistant autoclave bin or tray to prevent fluid from leaking
out of bag into the autoclave. (See example image below)
31
Do not twist the bags closed and tape shut them when preparing them for autoclaving as
this prevents the steam from being able to penetrate the waste and prohibits the
sterilization process. (See example image below)
Do put lab name and date on autoclave tape in case there are questions regarding the
waste the appropriate lab can address concerns. (See example image below)
Failure Procedure: If the autoclave has failed to pass criteria that indicate sterilization
has occurred. The tests should be repeated once more and if the failure is confirmed, the
appropriate contact person for the autoclave must be notified of the autoclave failure. The
autoclave contact person is expected to follow up the maintenance service provider for
the autoclave. Notice of failure must be provided to all users by placing an “Out of Order,
Do Not Use!” sign conspicuously on the autoclave concurrent with a request for repair.
Proper Use of Centrifuges
All centrifugation shall be done using centrifuge safety buckets or sealed centrifuge tubes
in sealed rotors. Each person operating a centrifuge should be trained on the proper
operating procedures. Improperly used or maintained centrifuges can present significant
hazards to users. Failed mechanical parts can result in release of flying objects, hazardous
chemicals and biohazardous aerosols. The high-speed spins generated by centrifuges can
create large amounts of aerosol if a spill, leak or tube breakage occurs.
The following procedures for centrifugation are recommended:
1)
2)
Check glass and plastic centrifuge tubes for stress lines; hairline cracks and
chipped rims before use. Use unbreakable tubes whenever possible.
Fill and decant all centrifuge tubes and bottles within the biological safety
cabinet. Avoid filling tubes to the rim. The maximum for centrifuge tubes is ¾
32
3)
4)
5)
6)
7)
8)
9)
10)
11)
full. Wipe outside of tubes with disinfectant before placing in safety cups or
rotors.
Use screw top caps or stoppers on centrifuge tubes. Avoid using lightweight
materials such as aluminum foil as caps.
Once samples to be centrifuged are prepared, load tubes into buckets inside
the biological safety cabinet and seal carefully before moving to centrifuge.
Aerosol-free (sealed) centrifuge buckets or rotors are required for all
centrifuging of infectious substances. Only the correct size tubes should be
used in any centrifuge bucket.
Buckets should be kept clean and free of broken glass and plastic.
Follow manufactures recommendations for tube and rotor maximum safe
operation speeds.
Ensure the rotor has stopped before opening the centrifuge lid when the run is
complete.
After centrifugation, buckets should be opened in a biological safety cabinet
to prevent exposure from aerosolized particles. Always visually inspect rotor
for signs of tube leakage prior to opening buckets.
Decontaminate the outside of the cups or buckets before and after
centrifugation. Clean and disinfect rotors, and centrifuge interiors. Inspect Orings regularly and replace if cracked or dry. Never leave a centrifuge in a
contaminated state.
Ensure that the centrifuge is properly balanced.
When using high-speed or ultra-centrifuges, additional practices should include:

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
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
Connect the vacuum pump exhaust to a disinfectant trap.
Record each run in a log book: keep a record of speed and run time for each rotor.
Install a HEPA filter between the centrifuge and the vacuum pump.
Never exceed the specified speed limitations of the rotor.
Regularly inspect the rotor for contamination, corrosion, or cracks.
Spills and Leaks
Report all spills of infectious agents to OSEH Biosafety.
In the event a sample may spill or leak inside the centrifuge due to failure of the rotor or
associated centrifuge parts the following steps should be followed:


Close the centrifuge lid immediately with the samples remaining inside and turn
the centrifuge off.
It may be necessary to vacate the lab depending on the nature of the spilled
material and its ability to generate an aerosol. When the lab must be vacated,
33



secure the room such that others cannot gain access. Post signage indicating
restricted entry.
All personnel should decontaminate themselves and seek medical attention as
necessary.
Notify principal investigators, lab supervisors, and coworkers as soon as possible.
Wait at least 30 minutes before entering the lab to allow aerosols to settle and
decontaminate. Note this step is based on risk assessment lab should have SOP based on
agents used in lab.
Blenders, Ultrasonic Disrupters, Grinders and Lyophilizers
The use of any of these devices results in considerable aerosol production.
Blending, cell-disrupting and grinding equipment must be used in a biological
safety cabinet when working with biohazardous materials.
Miscellaneous equipment
Microscopes
Tighten caps on flasks of infectious culture before transporting to the microscope.
Infectious cultures in plates or other containers without tight fitting lids must be carried to
the microscope in a tray. When using the hemacytometer to count cells, enclose the
hemacytometer in a 70% ethanol-disinfected petri dish for transport to the microscope.
Disinfect the viewing platform of the microscope after each use.
Microtomes
Microtome blades are extremely sharp and must be handled with great care and stored
safely when not in use. If the knife projects beyond the sectioning area, a suitable guard
must be fitted. Handling and changing of microtome blades causes many injuries, and
great care must be exercised when performing these operations. Always carry the knife,
in its case, to the microtome. Never leave the knife on a microtome. After use, always
return the knife to its case or dispose of immediately. Disinfect the microtome by wiping
with bleach or sodium hydroxide solution. Slide the ''back'' on to the knife before
removing it.
Cryostats




Frozen sections of unfixed human tissue or animal tissue infected with an
infectious agent pose a risk of infection. Freezing tissue does not inactivate
infectious agents.
Freezing propellants under pressure should not be used for frozen sections as they
may cause spattering of droplets of infectious material.
Gloves should be worn during preparation of frozen sections.
Consider the contents of the cryostat to be contaminated and decontaminate it
frequently with 70% ethanol.
34




Consider trimmings and sections of tissue that accumulate in the cryostat to be
potentially infectious and remove them during decontamination.
Defrost and decontaminate the cryostat with a tuberculocidal hospital disinfectant
once a week and immediately after tissue known to contain infectious substances
is cut.
Handle knives with extreme care. Stainless steel mesh gloves should be worn
when changing knife blades.
Consider solutions for staining potentially infected frozen sections to be
contaminated
Water baths
Pathogenic or nonpathogenic agents may contaminate water baths. It is recommended
that either 1 oz. of bleach or 1 oz. of phenolic detergent be added to each gallon of water
used in a bath. Phenolic disinfectants are preferred over bleach, but phenolics must be
replenished regularly. Propylene glycol has been used effectively as an alternative in
cold-water baths. Raise the temperature to 90ºC or higher for 30 minutes once a week for
decontamination purposes. Avoid using sodium azide to prevent growth of
microorganisms (sodium azide forms explosive compounds with some metals).
Thimerosol should also be avoided as a bacteriostat or fungistat as it contains mercury.
All forms of mercury are poisonous if absorbed. Treated water must be disposed of as
hazardous waste. To prevent electrical shocks, unplug the unit before filling or emptying.
Loop Sterilizers and Bunsen Burners
Sterilization of inoculating loops or needles in an open flame generates small-particle
aerosols, which may contain viable microorganisms. A shielded electric incinerator or hot
bead sterilizer should be used to minimize aerosol production. Disposable plastic loops
and needles are good alternatives. Alternatively, disposable plastic loops and needles
may be used for culture work where electric incinerators are not available. The loops are
semi quantitative and can be used for counting bacteria.
Open Flames in Biosafety Cabinets
Open flames inside a biosafety cabinet create airflow turbulence that may compromise
sterility and worker protection, and heat buildup may damage the HEPA filters. Open
flames are extremely dangerous around flammable materials, such as ethanol, which is
often found in biological safety cabinets. Follow these tips for avoiding fires in your
biological safety cabinet:

Use disposable pre-sterilized loops and spreaders.

Replace Bunsen burners with alternative technology such as electric loop
sterilizers
35

Use only Bunsen burners without pilot flame, excess-temperature protection,
flame monitor, and regulated timer.

Ensure that the gas supply is clearly labeled inside the cabinet. Inspect your gas
lines inside the cabinet before use for kinks, tears, holes, and loose connections
and replace worn/damaged lines.

Stabilize the alcohol container so that it cannot be tipped over.

Reduce the amount of flammable chemicals, equipment and supplies in the
cabinet. Use only enough alcohol for one day’s work.

Have a “snuffing” lid available in case the alcohol in the container catches fire.
Water is not a good choice for putting out fires in biological safety cabinets.

If you smell gas, turn off the exterior gas valve and wait until the gas has fully
dissipated before lighting any flames. Remember that a biological safety cabinet
recirculates air and vapors may build up inside the cabinet.
Other Physical and Chemical Hazards
Dry Ice
Dry ice is the solid form of carbon dioxide, non-combustible, available in flakes, pellets
or block form. Dry ice will sublime (vaporizes directly to the gas state) at a temperature
of –78.5C (-109.3 F) or higher. Dry ice is commonly purchased from a commercial
manufacturer. Dry ice is commonly used to cool reactions or to ship biological
specimens.
All University lab personnel must follow the safe storage, usage, and handling of dry ice.
University personnel responsible for shipping packages containing dry ice, must be
properly trained according to Department of Transportation (DOT) and International Air
Transport Association (IATA) Regulations.
Storage


Dry ice is to be stored in a well-ventilated location and placed in a Styrofoam
chest, insulated cooler, or a special cooler designed for the storage of dry ice.
Because of the thermal expansion of dry ice (one pound of dry ice produces about
250 liters of gaseous carbon dioxide), sufficient gaseous carbon dioxide can be
released in a sealed container to cause an explosion. Dry ice is NEVER to be
stored in any type of tightly sealed devices such as an ultra-low freezer or
plastic/glass container.
36


Dry ice will sublimate about five to ten pounds every 24 hours (blocks last longer)
in a typical storage cooler. Plan on purchasing dry ice as close as possible to the
time needed.
Normal air is composed of 78% nitrogen, 21% oxygen, and only 0.04% carbon
dioxide. Concentrations greater than 0.5% (5000 ppm) can become dangerous.
Therefore, handle dry ice in well-ventilated locations.
Hazards/Precautions:

Burns/frostbite: Dry ice can cause burns to the skin in short periods of times.
Thermal gloves are to be used if it is necessary to handle dry ice.
 Suffocation: carbon dioxide is a simple asphyxiant. Always store dry ice in a
well-ventilated area to minimize the buildup of carbon dioxide. Personnel must
use caution should dry ice be stored in a deep cooler. Personnel must never stick
one’s head into the chest to obtain the dry ice.
 Explosions: Placing dry ice into a tightly sealed container can permit sufficient
gas build up to cause an explosion. Never place dry ice inside an ultra-low freezer
or other enclosed space.
 Placement of dry ice in rooms with little or no ventilation can result in a build-up
of the carbon dioxide in the area. Do not store dry ice in a confined area such as
walk-in coolers, refrigerators, freezers, closets, or cars/vans.
 When using dry ice to ship materials, the shipper must abide to all applicable
shipping regulations.
Disposal




Dispose unneeded dry ice by letting the unused portion sublimate (recommended
for well-ventilated locations because it will occur over a period of several days
and the ventilation will take care of the gas liberated);
NEVER dispose of dry ice in a sink, toilet or other drain (such action can destroy
the structure because of the temperature difference);
NEVER dispose of dry ice in the trash or garbage; and
NEVER place unneeded dry ice in corridors (some corridors may not be well
ventilated and the oxygen level can be reduced to low levels).
Liquid Nitrogen
Liquid nitrogen is frequently used in chemical research laboratories for the purpose of
cooling. There are two major risks associated with the use and handling of liquid nitrogen
which present potential hazards:
-
Liquid nitrogen is extremely cold. At atmospheric pressure, liquid
nitrogen boils at -196°C.
-
Liquid nitrogen produces a large amount of nitrogen gas.
37
Do not allow objects cooled by liquid nitrogen to touch your bare skin.
Contact with the skin may cause serious frostbite. Because it is extremely cold, it can
freeze human flesh almost instantaneously. Use forceps or tongs to remove straws or
canes from the storage container.
Protective clothing can reduce the hazards of handling liquid nitrogen.
Insulated or heavy leather gloves should always be worn when handling any object that has been
in contact with liquid nitrogen. Loose fitting gloves are recommended so that they may be
discarded quickly in the event that any liquid nitrogen splashes into them.
Special containers are required.
Cryobiological storage containers are specifically designed and constructed to withstand the
extreme temperature variances involved in handling liquid nitrogen. These special containers
should be filled slowly to avoid the expansion stress that occurs as a result of the rapid cooling.
Too much stress can damage the container.
Do not seal the containers.
Cryobiological storage containers are designed to function with little or no internal
pressure. The use of any tight-fitting stopper or plug that prevents the adequate venting of
gas builds up pressure that could severely damage or even burst the container. Even icing
or accumulated frost can interfere with proper venting and containers should be checked
for such obstructions.
Transfer liquid nitrogen with care.
The primary hazards of transferring liquid nitrogen from one container to another are
spilling and splashing. Special funnels (with the top partially covered) will reduce
splashing. For cryobiological storage containers a self-pressurizing discharge device is
available that allows controlled liquid nitrogen, withdrawal up to two liters per minute.
Always follow carefully the instructions on containers or accessories when transferring
liquid nitrogen. NEVER overfill the containers. Filling above the specified level is likely
to produce spillage when the neck tube core is replaced.
Use solid metal or wooden dipsticks.
Because of the extremely low temperature of liquid nitrogen, plastic measuring devices tend to
become very brittle or even shatter. NEVER use hollow rods or tubes; the gasification and
expansion of the rapidly cooling liquid inside the tube will force liquid to spurt from the top of
the tube. Always wear insulated or heavy gloves when measuring.
Nitrogen gas is colorless, odorless, tasteless ... and deadly!
It reduces the concentration of oxygen and can cause suffocation. Since it cannot be detected by
sight, taste or smell, it may be inhaled as if it were air. That is why liquid nitrogen must always
be stored and used ONLY in areas that are fully ventilated. As liquid nitrogen evaporates, the
resulting nitrogen gas displaces the normal air-and breathing air that is less than 18% oxygen
may cause dizziness, unconsciousness and even death.
Nitrogen gas is extremely cold.
The eyes can be damaged by exposure to this gas even when the contact is too brief to affect the
skin.
38
Handle containers with care.
Containers should always be stored in an upright position. Tipping the container or letting it lie
on its side can result in spillage and may damage the container or the materials stored in it.
Walking or dragging containers could result in a partial or complete vacuum loss. For containers
that cannot be easily and safely carried, a roller base can provide safe and easy movement of
containers.
Container Contents.
The extremely low temperature of the liquid nitrogen provides the protection of the
materials stored in cryobiological storage containers. When all of the liquid nitrogen has
evaporated, the temperature inside the container will rise slowly. The rate of evaporation
depends upon the age, condition and use pattern of the container. Opening and closing the
container or moving it about will reduce its cooling efficiency. You should check the
liquid nitrogen level in your containers at least weekly; make sure there is enough liquid
nitrogen in the container to maintain the required temperature to avoid damage to the
ampules, canes, straws or vials stored in the container. If the liquid has evaporated faster
than usual or if the container is covered with frost or condensation, the vacuum system
may be damaged. In such instances, transfer the contents to another container and remove
the damaged one from service.
Ultraviolet light
UV lamps in biological safety cabinets are no longer recommended or supported by UMOSEH. When the lights are working properly, they emit radiation at 254 nanometers,
which is can kill infectious agents on the interior surfaces of the cabinets. This
wavelength also happens to induce tumors in laboratory rodents and presents an
occupational hazard to laboratory workers.
Visualization of DNA often involves the use of UV light. Ethidium bromide fluoresces
under UV light. The lamp used to generate the UV light is usually a mercury arc lamp.
The primary emission of the light is 354 nm. There are other emissions from the visible
range (400 nm+) down to and below 254 nm. The most hazardous region for human skin
is 270-310 nm. Some mercury arc lamps put out a significant portion (20-30%) of their
power in this range. To reduce exposure employees should not use the lamp facing up.
While using the lamps, wear a protective face shield and cover exposed skin. The effects
of UV are erythema (red skin), photokeratitis (small lacerations of the cornea, or
“welder’s flash) and skin cancer.
When using a UV microscope personnel must wear protective goggles or glasses. In
addition, anyone else in the room during such use should also wear similar protective
equipment.
39
Electrical Hazards
Electrical hazards can be present in electrophoresis because electricity is fundamental to
the process. New electrophoresis machines come with UL and CE designations. These
pieces of equipment have past stringent tests for electric shock protection. If the
electrophoresis machines do not have these approvals, the operator must ensure that no
exposed live wires or contact are exposed. A ground fault interrupter (GFI) can be added
to automatically shut off the electricity in the event of an electrical fault. The voltages
used for electrophoresis are sufficient to cause electrocution. Cover the buffer reservoirs
during electrophoresis. Always turn off the power supply and unplug the leads before
removing a gel.
Gel Electrophoresis
The major hazards of gel electrophoresis are related to the chemical and physical agents.
DNA is separated using gels (mainly agarose), a buffer solution, and an electric field (80110 volts).
Ethidium Bromide
Ethidium bromide is a moderately toxic chemical. It has been shown to be mutagenic.
Most suggest handling ethidium bromide as a carcinogen. No skin contact is permitted.
When working with ethidium bromide, try to minimize the potential for spills. Where
practical, purchase ready-made stock solutions from chemical manufacturers in lieu of
mixing your own solutions. If you prefer to mix your own solutions of ethidium bromide,
protect yourself by doing this process in a fume hood. Perform all processes that generate
ethidium bromide dusts or mists inside the fume hood to minimize inhalation exposures.
Prevent accidents by transporting small quantities of ethidium bromide in a secondary
container instead of carrying large quantities.
Acrylamide
Acrylamide is a common research laboratory chemical. Widely used as a cross linking
agent for electrophoresis separation procedures, acrylamide is a basic requirement for
various biochemical techniques. This familiarity may cause some lab personnel to
overlook the hazardous nature of this toxic substance. Acrylamide is a powerful central
and peripheral nervous system toxicant. Acute (short-term) exposures to low levels of the
monomer can damage nerves and cause effects such as drowsiness, lack of coordination,
hallucinations, and confusion. Chronic (long-term) exposures can cause severe nerve
damage and result in sensory and motor impairment marked by numbness and weakness
in the hands and legs, and difficulty walking and speaking. Based upon a number of
laboratory and epidemiological studies, the U.S. EPA has classified acrylamide as a
probable human carcinogen.
All measuring, mixing, and handling of the acrylamide monomer should take place in a
chemical fume hood while wearing latex gloves, which extend over the cuffs of the lab
40
coat. Once the monomer has polymerized it is no longer hazardous, however, since there
is never 100% polymerization, there will always be toxic monomer contamination. For
this reason polymerized gels should be treated with the same caution as the monomer.
Elimination of the hazardous powder is one of the best methods to decrease the risk of
acrylamide exposure in the lab. Where practical, purchase pre-mixed acrylamide
solutions available from various vendors. These solutions have the added advantage of
being specifically designed for each application and can provide a high level of purity
and reproducibility.
Additional Chemical Usage
Phenol
Phenol is a very caustic organic solvent that is used to extract protein from DNA preps.
Phenol can be readily absorbed through the skin, whereupon it can affect the central
nervous system and cause damage to the liver and kidneys. It is also a mutagen, and there
is some evidence that phenol may be a reproductive hazard. When heated, phenol will
produce flammable vapors that are highly toxic and explosive. Whenever possible, work
with phenol in a chemical fume hood, especially when heating it. Never heat or melt
phenol in an incubator, microwave, drying oven, or similar appliance. Prevent phenol
from contacting skin by wearing neoprene gloves and a laboratory coat. Change gloves
frequently. Wear chemical goggles to protect the eyes. Always wash hands thoroughly
after handling phenol, even if gloves are used.
Chloroform
Chloroform is widely used in molecular biology as a solvent in organic extraction. It has
been shown that generation of phosgene from chloroform has occurred with or without
the exposure to flames, electrical arcs, intense sunlight and hot surfaces. Recently it has
been suggested that over time chloroform can break down and form phosgene in older,
particularly unstabilized, chloroform containers. Researchers should purchase stabilized
chloroform whenever possible. Although amylene is used as a stabilizer, there is evidence
that it may not prevent phosgene generation.
If unstabilized chloroform is necessary for your work, you must treat it like peroxide
forming compounds; dated when received, used quickly, and discarded after a year. If
you have opened unstabilized chloroform that has been in the laboratory for more than
one year, you should discard it as hazardous waste. Storing chloroform in a dark place
(cabinet) in an amber bottle can reduce the rate of chloroform decomposition.
41
Equipment Maintenance
1)
Autoclaves, centrifuges, biological safety cabinets, and fume hoods should
undergo regular preventative maintenance by qualified personnel.
2)
The airflow must be regularly checked on the biological safety cabinets and filters
change by qualified personnel. If cabinets are not functioning correctly, contact
OSEH (647-1143) to perform maintenance operations.
3)
Preventative maintenance records should be kept on all equipment.
Cleaning and Decontamination
Housekeeping
1)
General laboratory Procedures
 All areas of the (BSL Level) laboratory must be kept clean and orderly.
 Dirt, dust and clutter are safety hazards and are not consistent with acceptable
biological research.
 Stock solutions of disinfectants should be maintained at each bench top and
biological safety cabinet work area:
1. Bleach, 10% sodium hypochlorite
2. Ethanol, 70% solution
3. Other appropriate disinfectants





Vacuum lines should be protected by a disinfectant trap (an aspirator suction
flask containing bleach) and a HEPA filter between the vacuum port and the
aspiration flask to prevent pathogens from entering the vacuum system.
All infectious materials, contaminated plastic ware/glassware, and
contaminated waste will be disinfected prior to washing or disposal.
Contaminated materials are to be placed in sealed clear biohazard bags with
red or orange lettering indicating biohazard and printed-on sterilization
indicator badge prior to autoclaving.
Surfaces are to be decontaminated after each use.
The Lab Manager or other laboratory personnel should conduct periodic
inspections of the (BSL Level) Laboratory. A copy of a BL2 Recombinant
/Infectious Agent Laboratory Audit Checklist can be found on OSEH’s
homepage at http://www.umich.edu/~oseh/BL2checklist.pdf for the
laboratory’s use. OSEH recommends that inspections be performed and
recorded every six months. Records of these inspections should be kept in
appendix 5.
42
Chapter 7 – Radiation Management
Use of Radioactive Isotopes
All personnel working with radioactive materials should be instructed in the use of
necessary safeguards and procedures and all visitors should be informed of pertinent
precautions to be taken.

All personnel and visitors should be supplied with such auxiliary devices as may be
necessary for protection. This includes devices such as:
1. Lead or Plexiglas©
2. Radiation badges, rings, gloves, and
3. Radioactive spill cleaning materials

The radiation safety coordinator should ensure that every visitor has proper
authorization and should recommend that no unnecessary visit to areas involved with
radioactive use be made.

Radioactive material should be prevented from leaving the jurisdiction of the
radiation safety coordinator under circumstances that may subject other personnel to
radiation in excess of the limits prescribed by the OSEH Radiation Safety Service.

All areas that are to be used for radioactive work must be clearly labeled with the
appropriate radiation hazard signs. Contact OSEH, Radiation Safety Service at7644420 for further information.

Measures should be taken to ensure that no modification of equipment or installations
that might lead to unforeseen radiation hazards is made without provision of the
appropriate safeguards.

Measures should be taken to ensure that no radioactive material is used by
unauthorized personnel who do not have the required training to use the
radioisotopes.

The radiation safety coordinator must keep concise and up-to-date records of usage,
inventory, and disposal of radioisotopes.

Any accident, unusual incident, or personal injury, must be reported to the radiation
safety coordinator, OSEH Radiation Safety Service, and the Principal Investigator or
Lab Manager.

Areas where radioactive isotopes are used should be routinely monitored by
systematic swipe sampling and sample analysis via a scintillation counter.

Shielding
43
1. Radiation emitted from isotopes must be shielded away from the user.
2. When working with 32P, the radiation may be shielded behind a ½ to ¾
inch Plexiglas © shield.
3. When working with 125I, lead must be used to shield the worker from
radiation.
4. Contact OSEH, Radiation Safety Service at 764-4420 for any questions
regarding shielding.
Disposal of Isotopes after Usage
When working with potentially infectious agents, any radioactive reagents coming in
contact with the biohazard must first be immersed in bleach prior to being discarded into
a radioactive waste bag or container. Note: Do Not Use bleach with 125I as it produces
a volatile form of the isotope.
It is imperative that all potentially infectious waste be disinfected with bleach prior
to disposal.
44
Chapter 8 - Emergency Management
Emergency Procedures
The fundamental rule in dealing with a biological spill is to be prepared. Preparation
involves identification of the biohazard risks, both actual and potential, that are involved on
the site and determining the types of potential spills or emergencies which can occur. In order
to prepare for a biohazard spill, you must first:

Know the ventilation system serving the laboratory or room, the corridors and the
building in order to enable you to know how aerosols or airborne particles would
move;

Know where fume hoods and biological safety cabinet exhaust ducting goes after
leaving the lab area;

Know where biohazard areas are and where biohazardous materials are stored and
assessed and what hazard could result in the event of a fire, flood, or explosion;

Establish evacuation routes and procedures to be used in the event of an
emergency with biohazardous materials;

Establish rules for safe handling, storage and disposal of biohazardous materials
to minimize accidental release and set standards for use to avoid conditions which
might lead to an accidental spill;

Establish a standard operating procedure (SOP) to be followed if a spill should
occur. This SOP would consist of a step-by-step procedure to follow if a spill
occurs. Spill kit materials should be present in proximity to the area where
biohazardous materials are handled;

Know the occupational medical services' procedures for reporting and dealing
with exposure to biohazardous materials.
Accidental Exposure to an Infectious Agent
Laboratory personnel who are accidentally exposed to a potentially infectious agent must
report the incident as soon as possible to the Principal Investigator and the OSEH
Biological Safety Officer (763-3133). The Principal Investigator will see that necessary
treatment or health monitoring is obtained without delay. University of Michigan
Occupational Health Services (C380 Med Inn Building 734-764-8021) will provide
follow-up and counseling on risk of infection and its consequences. The
Work~Connection Employee Illness or Injury Report form must be completed for all
workplace injuries and illnesses (see Appendix 7).
45
Spill Response Procedures for Infectious agents and Recombinant DNA
Spills and accidents should be immediately reported to the Principal Investigator and
the Department of Occupational Safety and Environmental Health (OSEH) (6471143).
Large Spills Outside of the Biosafety Cabinet
OSEH will respond to large spills (greater than 200 ml) of BSL-2 (or above) containment
level material. Contact OSEH (647-1143) for help with large spill cleanup.
Small Spills Outside of the Biosafety Cabinet
1. Don double gloves and eye protection if not already wearing them.
2. Cover spilled material with an absorbent paper towel or Kimwipe. Once the absorbent
material is in place over the spill, wet the material with a 10% solution of bleach
(1:10 dilution of Clorox containing sodium hypochlorite) or other appropriate
disinfectant.
3. Let stand 15-20 minutes, wipe up and wash surface with appropriate disinfectant.
4. Wipe down all equipment and surfaces which may have been splashed.
Spill in a Biosafety Cabinet
Note: Leave the cabinet turned on.
1. Don double gloves, a lab coat, and eye protection if not already wearing them.
2. Cover spilled material with an absorbent paper towel or Kimwipe. Once the absorbent
material is in place, wet material with 10% solution of bleach (1:10 dilution of Clorox
containing sodium hypochlorite) or other appropriate disinfectant. Let stand 15-20
minutes, wipe up and wash surface with appropriate disinfectant.
3. If personnel are contaminated, remove potentially contaminated garments at the BSC
and decontaminate garments by saturation with 70% ethanol or place in autoclave bag for
autoclaving. Wash hands and other potentially exposed skin surfaces thoroughly with
soap and water. Don fresh PPE, return to worksite, and spray walls, liners, and equipment
with an appropriate disinfectant.
4. Spray or wipe container walls, other work surfaces and equipment with the appropriate
disinfectant
46
5. If necessary, flood the work surface, drain pan and catch basin below the work surface
with disinfectant. Allow at least 15-20 minutes contact time.
6. Soak up the disinfectant and drain the catch basin into a container. Lift the front
exhaust grille and tray and wipe all surfaces. Ensure that no foreign materials are blown
into the area below the grille.
7. If a 10% bleach solution is used on metal surfaces, rinse with water or 70% ethanol
after decontamination is complete.
8. If the spill overflows into the interior of the cabinet, more extensive decontamination
of the cabinet may be necessary. Contact OSEH (763-6973) for decontamination of the
cabinet.
Spill in the Laboratory
1. If an infectious aerosol may exist outside of a biosafety cabinet, leave the room
immediately, lock the door, post a warning sign and inform your supervisor. If clothing
is contaminated, remove and turn the exposed side of fabric in on itself and place in
autoclave bag or biohazard container. Wait at least 30 minutes before reentering the lab
to allow dissipation of aerosol created by the spill. During this time, review clean-up
procedures, assemble decontamination materials, PPE and contact OSEH (647-1143).
2. Don fresh gloves, a lab coat or tyvek suit, and eye protection.
3. Carefully lay disinfectant-soaked towels over the spill and pour more around the spill.
Use more concentrated disinfectant if the volume of material will significantly dilute the
disinfectant.
4. Allow 15-20 minutes contact time.
5. Use forceps to place sharp objects into a sharps container. Using a dustpan and
dustbroom, tongs, etc., transfer all contaminated materials (paper towels, gloves, labware,
etc.) biohazard waste containers and contact OSEH HazMat (763-4568) for removal.
6. Wipe surrounding surfaces with disinfectant to cover all splash areas. Wipe flat
surfaces to remove any material that may have splashed out and settled on those surfaces.
7. Place all contaminated materials, including protective clothing, into an autoclave bag
or biohazard waste container.
8. Wash hands with soap and warm water.
9. If a personnel exposure occurred, complete an Illness or Injury Report form
(https://www.workconnections.umich.edu/illnessorinjury.php) summit to
47
Work~Connections and report incident to OSEH (763-6973). See additional procedures
noted below.
Skin, Mucous Membrane, or Injury Exposure
1. If there is an agent-specific protocol for exposures, follow that (e.g., HIV, Herpes B).
2. In the case of skin contact or injury with a contaminated instrument:
a. Thoroughly wash area with soap and water. Do not squeeze the wound to induce
bleeding.
b. Avoid use of abrasive chemical soaps or disinfectant washes as they can cause skin
abrasions and a possible additional route of entry for the agent.
c. For mucous membranes (e.g., eyes, mouth), flush for a minimum of 15 minutes.
3. Notify UM-Occupational Health Services (OHS) for evaluation of exposure following:
a. Contact with mucous membranes;
b. Contact with non-intact skin;
c. Percutaneous exposure; or
d. Any type of exposure that involves concentrated virus.
U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am – 4:30 pm
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021
begin_of_the_skype_highlighting
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
5. Complete an Accident-Illness Report Form
(https://www.workconnections.umich.edu/illnessorinjury.php)
submit to
Work~Connections and report incident to OSEH (647-1143).
For exposures to recombinant DNA, perform the following:
1.
Any situation involving recombinant DNA that poses a threat to an individual's health,
safety, or welfare should be handled with the appropriate care including emergency
response (911) if necessary.
2. Wash the area thoroughly with soap and water.
48
3. Cover the wound with a sterile dressing.
4.
Following this, reporting must occur to:



Principal Investigator or Director of Lab (immediately)
Biosafety Officer (647-3133) (immediately)
Complete an Accident-Illness Report Form
(https://www.workconnections.umich.edu/illnessorinjury.php)


submit to
Work~Connections (within 24 hours)
IBC (may be reported through the Biosafety Officer)
NIH/OBA (report will be coordinated by the IBC)
Fire Fighting Procedures
Personal safety is each worker’s primary concern in the event of fire
1)
Upon learning of the threat of fire within the building, laboratory personnel will,
to the extent possible:



2)
Turn of all gas burners, biological safety cabinets, electric motors, and other
electrical equipment.
Place containers of infectious materials into autoclaves, incubators,
refrigerators, freezers or other storage areas.
Leave the laboratory as quickly as possible using designated fire evacuation
routes.
Personnel should be trained by OSEH Fire Safety Services in the operation of fire
extinguishers (615-6764).
Reporting and Recordkeeping
Accidents and spills occurring outside the biological safety cabinets will be reported and
accident report forms will be filed under Appendix 7.
49
Chapter 9 - Shipment and Receipt of Infectious Materials
General
Procedures for receiving and unpacking infectious materials must be established by the labs
receiving these materials. Employees who have responsibilities that include the receipt of
packages must be given specific instructions regarding the receipt of infectious materials and
should be trained and qualified to recognize the hazardous nature of the material being received
and recognize whether or not the material has been packaged, labeled and manifested or
documented appropriately. This constitutes an audit function for regulatory compliance. Any
given package may receive several such audits and visual inspections along its shipping route.
Receipt of Infectious Materials
Shipments of hazardous materials must be received (generally) by those to whom it is
addressed. This is most easily accomplished via a certified carrier such as Federal
Express. University Mail Services is certified to accept and transfer hazmat packages
from the U.S. Postal Service (USPS).
Employees should receive shipments in a designated and secure area of the laboratory.
This person should have at-hand and utilize all appropriate personal protective equipment
and containment devices (biological safety cabinet or chemical fume hood). Before
accepting, the parcel should be carefully inspected for leakage indicated by broken or
improperly sealed containers. If the package is rejected (not accepted) due to leakage or
other damage, the carrier will work with the shipper to resolve the problem. If the
shipment is critical and must be accepted, OSEH should be contacted at 763-6973, and
further activities should be conducted with care in a containment device. All
contaminated materials should be collected for proper disposal through consultation with
OSEH. Employees must review and sign the Training Sheet for the Receipt of Biologics.
http://www.oseh.umich.edu/Transportation_of_Biologics_Appendix%20I.pdf . Signed sheets should
be faxed back to OSEH (763-1185).
Transportation of Biohazardous Materials
General
All shipments of infectious or diagnostic specimens must be packaged, labeled, and
transported in conformance with all applicable local, federal, and international
transportation and shipping regulations, including U.S. Department of Transportation
(DOT) regulations. Materials that are transported by airline carrier will also comply with
packaging and shipping regulations set by the International Air Transport Association
(IATA). Personnel who are involved in the preparation, packaging or shipment of
infectious substances, biological materials, human/ animal specimens, genetically
50
modified microorganism or any shipment involving the application or use of dry ice must
be trained and certified by OSEH. Classes are scheduled monthly and held at the OSEH
office. Registration is available at http://www.osehtraining.umich.edu/osehtraining/ .
NOTE – this training is required every two years.
On-Campus Transportation
Packaging
The materials must be properly packaged for transport (similar to how you would package them
if they were going by courier). For liquids, the inner packaging must be leak proof, and the outer
packaging must contain sufficient absorbent material to absorb the entire contents of the inner
packaging. For solids and liquids, the outer packaging must be a strong, tight packaging securely
closed and secured against movement. The outside of the secondary container must be free of
any biohazardous material so that the package can be carried safely between buildings without
wearing gloves or lab coats outside. The package must be taken directly to its intended location.
If a spill occurs during transport, do not attempt to clean it up without appropriate spill response
material and PPE. Keep other persons clear of the spill. Contact OSEH (647-1143) for additional
assistance.
Hand Carry Between University Labs or Buildings
Infectious substances must be transported or moved between laboratories in way as to
prevent spills and accidental exposure or release including:



Placing material in a primary (specimen) container that is leak-proof and secured
with a tight-fitting cap, parafilm, or lab tape.
Placing absorbent material (diapers, absorbent towels, pads) around the primary
containers for transport of liquids.
Placing the primary containers in a secondary transport container that is also
sealed and labeled with a biohazard symbol. These materials may be moved on a
cart or other device between rooms or buildings.
Transport by University Vehicle
Unregulated or exempted biological materials may be transported either on campus or to
off-site research locations for university business in a university vehicle as long as they
meet all packaging, labeling, and training requirements. OSEH does not recommend use
of a personal vehicle for transporting materials off campus. Insurance companies may
not cover an accident where hazardous materials were transported in a personal
vehicle. A university or city of Ann Arbor bus or shuttle cannot be used for the transport
of infectious substances or other biohazardous materials.
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For materials that qualify for transport in university vehicles, the following precautions
should be followed:








Must have a valid driver’s license
Must have authorization to use a university vehicle
All materials must be packaged according to a triple packaging system or more
restrictive packaging requirements.
Hazardous materials should be transported in the trunk, or as far away from
passengers as possible.
All containers should be clearly labeled with content information.
A complete inventory of the material must accompany the package.
The vehicle must be driven directly from the point of origin to the intended
destination without stopping at other locations on the way.
Materials needed to contain or clean-up a spill, such as sorbent pads, gloves, and
eye protection, should be available in the vehicle.
Off-site Transportation by Courier
Biohazardous materials which are to be transported off-site from a facility by a courier must be
shipped in accordance with the appropriate DOT or International Regulations. The following
information must be known prior to shipment:



The infectious agent name;
The amount of material to be transported (milliliters or ounces);
The name and address of the shipper and the consignee.
The material must then:




Be packaged in accordance with the appropriate regulation(s);
Be labeled in accordance with the appropriate regulation(s);
Be accompanied by the appropriate documentation as required;
Be transported by an authorized carrier.
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Chapter 10 – Appendices
The following forms and diagrams are referenced in the text of the Laboratory Biosafety
Manual. Included in the Appendices are:
1)
2)
3)
4)
5)
6)
7)
8)
9)
Emergency Call List
Schematic of laboratory
Roster of approved laboratory personnel and training records
Training Completion Form
Housekeeping Logs
PSDS Sheets for Infectious Agents Located in Laboratory
Accident Report Forms
Recombinant DNA Registration Form
Standard Operating Procedures Template
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Appendix 1
Emergency Call List
Name
Work Phone
Home Phone
Lab personnel
OSEH
Janet Follo
UM Biological Safety Officer, OSEH
647-3133
Terry Alexander
Director UM OSEH
647-1143
Mark Driscoll
UM Radiation Safety Officer, OSEH
764-4420
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Appendix 2
Schematic of Laboratory
55
Appendix 3
Roster of Approved Personnel and Training Records
Personnel
Type of Training
Date Completed
Signature of Trainee
Detailed list of training materials
1)
2)
3)
4)
5)
UM OSEH, Radiation Safety Training Course
UM OSEH, Biological and Laboratory Safety New-Hire Training Course
DOT shipping of Infectious and Diagnostic Specimens (if applicable – contact
OSEH at 3-6973))
ULAM Training (if applicable – contact ULAM at 4-4420))
Reading List
 Laboratory Biosafety Manual
 CDC/NIH Biosafety in Microbiological and Biomedical Laboratories
 UM, OSEH Biohazardous (Medical) Waste Disposal Guideline
 Laboratory Safety Practices for Progress
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Appendix 4
Training Completion Form
RECOMBINANT DNA/INFECTIOUS AGENT TRAINING
Name _____________________________Department__________________
Campus Location ___________________ Campus Phone________________
Employee Classification _____________ Supervisor ___________________
All employees working with recombinant DNA or infectious agents are to be provided
with biosafety training at the initial assignment to tasks where occupational exposure may
take place and annually thereafter. Lab specific training appropriate to the skill level
required for the employee to perform their job safely will be conducted for lab personnel
by the lab supervisor or other qualified personnel. Prior to initiating research, the
Principal Investigator shall:
1. Make available to all laboratory staff the protocols that describe
the potential biohazards and the precautions to be taken.
2. Instruct and train laboratory staff in the practice and techniques
required to ensure safety and the procedures for handling spills.
3. Inform laboratory staff of the reasons and provisions for any
precautionary medical practices advised or requested (e.g.,
vaccinations or serum collection)
In addition, each lab is required to develop a laboratory biosafety manual, which includes
laboratory specific work practices and procedures to ensure that employees are protected
from all potentially infectious materials or recombinant DNA. New employees must
review this document and associated training materials. Training should be documented.
By your signature below, you acknowledge that you have read and understood the
contents the biosafety manual and standard operating procedures and know its location
within the laboratory.
________________________________________________________________________
Employee Signature
Date
Document specific employee training below:
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Description of Training
Date
Provided By
A copy of this form should be maintained in the laboratory's Biosafety Manual.
Training Checklist
This checklist may be used to assist employers with the laboratory specific training
requirements for work with infectious agents or recombinant DNA.
Introduction to laboratory specific Biosafety Manual:
Location and contents
Introduction to laboratory specific Standard Operating
Procedures (SOP’s)
Review description of the Principles of Biosafety, Principles of
Good Microbiological Practice and Universal Precaution.
Review modes of transmission, epidemiology and symptoms of
pathogenic microbes
Review emergency information: Spills, Personal Injury, Fire, and
Power Failure
Accident reporting and investigation procedures
Spill control and clean-up
Fire extinguisher
First Aid Supplies
Evacuation Plans
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Basic Safety Rules
Note rules with special importance for your laboratory.
Identify specific areas for food consumption.
Review procedures for working after hours.
Review the inventory of biohazardous materials for your
laboratory
Review the UM (OSEH) Waste Handling Procedures
Autoclaving (bags, periodic monitoring, preventative maintenance, log)
Chemical Disinfection
Sharps Disposal
Review procedures for procurement, distribution, and storage
Review Standard Operation Procedures for use of biohazardous
materials
Storage (freezer, refrigerator, etc.)
Personal Protective Equipment (PPE)
Location of procedure (biological safety cabinet, laboratory bench)
Waste Disposal (aqueous, solid, biohazardous, radioactive)
Review procedures for use of compressed gas cylinders
Protective Apparel and Equipment (PPE)
Discuss when safety glasses, goggles, or face shields are required.
Discuss any need for other protective equipment (aprons, mask, shoe
covering)
Discuss selection of gloves (latex, cold/hot environments)
Discuss disposal procedures for PPE
Housekeeping, Maintenance, and Inspections
Discuss materials stored or frequently present on the floor.
Discuss maintenance items for scientific equipment.
Discuss formal and internal inspection programs.
Discuss recordkeeping requirements for the lab.
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Environmental Monitoring
Discuss building ventilation.
Discuss use of fume hoods, biological safety cabinets or other mechanical
ventilation systems.
Review SOP’s for working with biologically hazardous materials
Review Exposure Control Plan if working with human blood or other
potentially infectious materials.
Review Hepatitis B Vaccination Program
Working with Radioisotopes
Review radiological safety practices
Review Dosimetry Program
Review waste disposal procedures (including waste that may be both
radioactive and infectious)
Medical Program
Discuss need for any medical surveillance (Respirator use, Potentially
Hazardous Materials, Animal Handler, Tb, etc…)
Discuss vaccinations, if applicable (Hepatitis B,
Training Program
Discuss laboratory-specific training and OSEH training sessions
Additional Safety Session Topics
Review recent incidents/accidents/injuries and how to prevent
reoccurrence.
Review new equipment at least annually.
Review new procedures at least annually
Review results of recent inspections and how to correct problem areas.
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Appendix 5
Housekeeping Inspections
This section should contain information on laboratory inspections (including the date and
recommendations). Inspections may be conducted by the laboratory, OSEH, MIOSHA,
NRC, and possibly other organizations. This section should also contain records
associated with corrective actions.
Laboratories conducting recombinant DNA work at Biosafety Level 2 (BL2) or
Biosafety Level 3 (BL3) must be inspected by UM OSEH prior to the initiation of
the work and on a regular basis thereafter. The items that OSEH will review
during their inspection, as a minimum, are contained on their BL2 Recombinant
Laboratory checklist. Key items are proper employee training, availability of a
completed Biosafety Manual, and appropriate written procedures.
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Appendix 6
Pathogen Safety Data Sheets for Infectious Agents
PSDS sheets on infectious agents (Office of Laboratory Security) can be found at the
following address:
http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php
Additional PSDS Sheets for the following materials can be obtained by request (6471143).
Adenovirus Type C Strain 5 and Recombinant Vectors
Moloney Murine Leukemia Virus (MoMuLV) Recombinant Vectors
Adeno-Associated Virus (AAV) Serotype 2 Recombinant Vectors
Respiratory Syncytial Virus (RSV)
Adenovirus Fact Sheet
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Appendix 7
Accident Report Forms
Keep Copies Here
Forms available at: http://www.workconnections.umich.edu/forms.html
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Appendix 8
Standard Operating Procedure
EXAMPLE ONLY!!!
Laboratory Standard Operating Procedure
(For the use of biohazardous materials or equipment)
Name of Procedure: Working with biohazardous materials in general (Insert agent
name(s) here)
Prepared By: John Doe
Revision Date: Insert Date
APPROVALS - Experiments using recombinant DNA require approval from the
UM- Institutional Biosafety Committee (IBC) before initiation of experiments. The
use of infectious agents is reviewed by OSEH.
CONTAINMENT LEVEL – Biological agents may be contained at varying
biosafety levels. The IBC will assign the appropriate containment level for the lab
and animal work involving recombinant DNA projects. OSEH will assign the
appropriate lab and animal containment level for the use of infectious agents.
LOCATION - This procedure may be performed at the following location(s):
Procedures are performed in Lab (insert lab locations, include animal procedure rooms, surgery rooms
or housing areas if applicable) in the biological safety cabinet. The biohazardous materials are stored in a
(freezer, refrigerator, incubator) located in room (insert room location)
HAZARDS - The following materials and equipment associated with this procedure
presents exposure or physical health hazards. Safety precautions are prudent and
mandatory:
Eyewash/Safety shower is located (insert room location). Remove contaminated clothing and vigorously
wash exposed area with soap and water for 10 minutes. Obtain immediate medical attention. Report the
incident to the Laboratory Director. See Chapter 7 of the Biosafety Manual for additional information on
Emergency Management.
ENGINEERING CONTROLS - Prior to performing this procedure, the following
safety equipment must be accessible and ready for use: (ex. biological safety cabinet,
laminar flow hood, biological disinfectant)
Biological safety cabinet is located in (insert room location). Perform all work that may create an aerosol
in the biological safety cabinet. The concentrated stock of biological disinfectant is located in the cabinet
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labeled disinfectant in room 00. Dilute stock solutions are located in the (tissue culture room, biological
safety cabinet, in cabinet)
PERSONAL PROTECTIVE EQUIPMENT - Prior to performing this procedure,
the following personal protective equipment must be obtained and ready for use:
(ex. Disposable gloves, safety eyewear, lab coat, apron)
Wear gowns, lab coats, aprons or similar protective clothing. Wrap around outer clothing when
introducing biological agents into animals or performing necropsies. Lab coats are adequate for tissue
culture manipulations. Wear fluid-resistant clothing if there is a potential for splashing or spraying of
blood. Wear gloves for all blood and tissue sample collection.. Wear disposable (single use) latex or nitrile
gloves. Gloves are located in (insert room location). Replace gloves as soon as possible when visibly
soiled, torn or punctured. Wash your hands or any other contaminated skin with soap and water
immediately or as soon as possible after removal of gloves and after visible contact with blood or other
potentially infectious materials. Use facial barrier protection whenever splashes, spray, droplets, or aerosols
may be generated (NOTE: Opening containers creates aerosols). Face protection may include the
following: hood sashes, shields, masks and safety glasses, or chin-length face shields. Perform work in a
biosafety cabinet when working with aerosols. Remove all PPE immediately upon leaving the work area
and as soon as possible if overtly contaminated. Contaminated PPE will be DISPOSED of as biohazardous
waste or decontaminated.
Animal Use - Approvals are needed from the University Committee on the Use and
Care of Animals (UCUCA) and the Institutional Biosafety Committee (IBC) ( if
involving rDNA)
Perform inoculations carefully to reduce the possibility of creating splashes or aerosols. Ensure appropriate
PPE is worn according to the procedures posted in the animal room. All PPE is single use and should be
disposed of in the designated waste containers upon exiting the ULAM space. Used needles must be
disposed of in an approved sharps container immediately after use. Used needles should not be set on the
bench, sheared, bent or re-capped prior to disposal. Whenever feasible, self-sheathing needles should be
used to avoid the potential for accidental needle stick injuries. Restraint devices and practices should be
used to reduce the risk of exposure during animal manipulation and administration should take place inside
a certified biosafety cabinet. All lab staff who will be administering biological materials to animals must
receive appropriate training. OSEH will review the protocol details and recommend the appropriate
housing. Employees may be required to complete the ULAM Hazard Containment and Hands-On Training
if Animal Containment Housing is recommended by OSEH. Employees must follow ULAM Containment
Policies and Procedures SOP if Containment Housing is indicated. Arrangements must be made with
OSEH for proper disposal of animal carcasses.
WASTE DISPOSAL - This procedure will result in the following regulated waste,
which must be disposed of in compliance with environmental regulations:
Sharps- Dispose of sharps such as needles, broken glass, scalpels in labeled, hard walled containers such as
the five gallon pails available from Stores. Label with an “infectious substance” marking. Call for pick-up
by HazMat (3-4568).
Solids- Place solid waste such as laboratory coats in special biohazardous waste boxes available from
HazMat or 55 gallon fiber drums labeled as infectious substance.” Call for pick-up by HazMat.
Liquids- If disinfected with bleach, blood and blood products can be poured down the drain. If the
biohazardous liquid waste contains other chemicals besides bleach, manifest as chemical waste and call
HazMat for pick-up.
66
Autoclaved Waste- Place waste that will be autoclaved in clear autoclave bags that have an indicator badge
that turns black once the waste is autoclaved. Do not put sharps or standing liquids in autoclave bags.
ACCIDENTAL SPILL - In the event that a hazardous material spills during this
procedure, be prepared to execute the following emergency procedure:
If a small spill, alert people in the immediate area of the spill. Put on protective equipment. Cover the spill
with paper towels or other absorbent materials. Carefully pour a freshly prepared 1 in 10 dilution of
household bleach around the edges of the spill and then into the spill. Avoid splashing. Allow a 20-minute
contact period. Use paper towels to wipe up the spill, working from the edges into the center. Clean the
spill area with fresh towels soaked in disinfectant. Place towels in a sealed container and put an “infectious
substance” marking on the container. Report the spill to the Laboratory Director. If a large spill, evacuate
the lab and call HazMat at 3-4568 for proper clean-up.
PRIOR APPROVAL - This procedure is considered hazardous enough to warrant
prior approval from the laboratory director. YES / NO
ADDITIONAL PRECAUTIONS & REFERENCES –
CERTIFICATION - I have read and understand the above SOP. I agree to contact
my Supervisor or Lab manager if I plan to modify this procedure.
Signature
Name (Print)
Date
Room #
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Appendix 9
Autoclave Use Log
68
Autoclave Use Log Form
Record daily waste runs or monthly performance runs in log
Bldg location of
autoclave & Rm#
Lab
Bldg/Rm#
Autoclave operator
(name & lab ph #)
Run date/Cycle
time: e.g. 01-12-13
45min cycle
Load description:
# of bags, agar plates
Cycle Used
Gravity/Fast ex
Liq/Slow ex
Autoclave contact
Name - number
Chemical
Integrator
Pass/Fail
Biological
Indicator
Pass/Fail
Additional comments (please include: date of concern/comment, operator name, and date person contacted regarding concern)
69
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