DePaul University
Biosafety Manual
Institutional Biosafety Committee
&
Office of Risk Management & Environmental Health & Safety
Page 1 of 30
TABLE OF CONTENTS
1.0 Preface ---------------------------------------------------------------------------------------4
2.0 Introduction ---------------------------------------------------------------------------------4
3.0 Responsibilities------------------------------------------------------------------------------4
3.1 Department Chairperson------------------------------------------------------------4
3.2 Principal Investigator----------------------------------------------------------------5
3.3 Research Laboratory Personnel----------------------------------------------------5
3.4 Office of Risk Management & Environmental Health & Safety--------------5
3.5 Institutional Biological Safety Committee---------------------------------------5
4.0 DePaul University Biological Safety Requirements ---------------------------------6
4.1 Registration with the Institutional Biosafety Committee-----------------------6
4.2 Training--------------------------------------------------------------------------------7
4.3 Risk Group Determination---------------------------------------------------------7
4.4 Prudent Practices for Infectious Agents in the Laboratory---------------------8
4.5 Recombinant DNA Experiments---------------------------------------------------9
4.6 Infectious Agents in the Laboratory------------------------------------------------9
4.6.1 Human Blood, Body Fluids, Tissue, and Other Potentially Infectious
Materials-----------------------------------------------------------------9
4.6.2 Research Animals---------------------------------------------------------10
4.6.3 Care and Handling of Infected Research Animals--------------------10
5.0 Immunizations --------------------------------------------------------------------------- 11
6.0 Medical Restrictions --------------------------------------------------------------------- 11
6.1 Pregnancy----------------------------------------------------------------------------11
6.2 Reproductive Biological Hazards-----------------------------------------------------------12
6.3 Other Restrictions--------------------------------------------------------------------------------- 12
7.0 Emergency and Accident Procedures ------------------------------------------------ 12
7.1 Laboratory Emergency Contacts--------------------------------------------------12
7.2 Emergency Equipment-------------------------------------------------------------12
7.3 Evacuation Routes------------------------------------------------------------------12
7.4 Spills and Emergencies-------------------------------------------------------------13
7.4.1 Spill Procedures for the Research Laboratory -----------------------13
7.4.2 Spill in a Biological Safety Cabinet------------------------------------14
7.4.3 Personal Contamination -------------------------------------------------14
7.4.4 Emergency Procedures for Exposure Incidents----------------------14
7.4.5 Reporting Incident-------------------------------------------------------15
7.4.6 Medical Assistance------------------------------------------------------15
7.4.7 Investigation of Laboratory Accidents--------------------------------15
8.0 Risk Assessment and Risk Management----- ----------------------------------- --- 15
8.1 Routes of Exposure------------------------------------------------------------------16
9.0 Laboratory Practices -------------------------------------------------------------------- 16
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9.1 Human Factors and Attitudes in Relation to Laboratory Accidents----------16
9.1.1 Biosafety Levels 1 and 2 -----------------------------------------------17
9.2 Labeling of Equipment sent out for repair, surplus or disposal---------------17
9.3 Cell and Tissue Culture-------------------------------------------------------------17
9.4 Basic Microbiological Practices---------------------------------------------------18
9.5 Maintaining a Clean and Orderly Laboratory-----------------------------------18
9.6. Transport Shipping Biological Specimens -----------------------------------------18
9.6.1 Transport of Biological Materials on/off campus--------------------19
10.0 Personal Protective Equipment (PPE) -------------------------------------------- -19
10.1 Laboratory Clothing---------------------------------------------------------------20
10.2 Gloves-------------------------------------------------------------------------------20
10.3 Face and Eye Protection ------------------- --------------------------------------21
10.4 Best Procedure for Removing PPE ---------------------------------------------21
11.0 Laboratory Equipment ---------------------------------------------------------------- 21
11.1 Biological Safety Cabinets-----------------------------------------------------21
11.2 Centrifuges-----------------------------------------------------------------------21
11.3 Syringes and Needles-----------------------------------------------------------22
11.4 Pipettes----------------------------------------------------------------------------22
11.5 Blenders, Mixers, Sonicators and Cell Disruption Equipment------------23
11.6 Miscellaneous Equipment (Waterbaths, Cold Storage, Shakers)---------23
12.0 Decontamination and Disposal of Bio-hazardous Waste ----------------------- 24
12.1 Definitions------------------------------------------------------------------------24
12.2 Waste Procedures----------------------------------------------------------------25
12.3 Decontamination of Laboratory Surfaces-------------------------------------25
13.0 Opening and Closing a Research Laboratory ------------------------------------ 25
13.1 Radioactive Materials-----------------------------------------------------------26
13.2 Hazardous Materials------------------------------------------------------------26
13.3 Infectious Agents----------------------------------------------------------------26
13.4 Suggestions for Setup of a New Laboratory---------------------------------26
14.0 APPENDIX ------------------------------------------------------------------------------- 28
14.1 APPENDIX A: Biosafety Levels----------------------------------------------28
14.2 APPENDIX B: Routes of Exposure-------------------------------------------29
14.3 APPENDIX C: Transport-------------------------------------------------------30
15.0 References -------------------------------------------------------------------------------- 31
16.0 Contacts ----------------------------------------------------------------------------------- 31
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1.0 Preface
The policy of DePaul University is to provide a safe and healthful work environment for employees,
students, and visitors. It is the intent of DePaul University to minimize, to the extent practical, all
recognizable hazards and to comply with all Federal and State laws and regulations. The implementation
of this policy is the responsibility of all employees of the University. Supervisors at all levels shall be
accountable for the health and safety of employees engaged in activities under their supervision.
Supervisors shall insist that employees comply with health and safety rules and work in a safe manner.
2.0 Introduction
Microbiological, molecular biology, and biomedical research laboratories pose a number of special
circumstances that set them apart from other types of laboratories. Most of these circumstances exist
because the work conducted within them often involves organisms that are potentially harmful to humans.
The purpose of this manual is to define the appropriate kinds of facilities and work practices for
microbiological, molecular biology, and biomedical research laboratories to reduce the probability of
infection to laboratory personnel. All principal investigators, faculty, staff, and students working with
infectious agents or recombinant DNA are expected to abide by the provisions of this manual.
The Institutional Biosafety Committee (IBC) has adopted the Center for Disease Control (CDC)
www.cdc.gov and National Institute of Health (NIH) Biosafety in Microbiological and Biomedical
Laboratories (BMBL) http://www.cdc.gov/od/ohs/biosfty/bmbl5/BMBL_5th_Edition.pdf as the University's
official biosafety guidelines for the use of potentially infectious agents. Recombinant DNA research is
regulated by NIH guidelines (Guidelines for Research Involving Recombinant DNA Molecules)
http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm.
Additional
information
regarding safe practice and procedures is available through the Office of Risk Management and
Environmental Health & Safety (RM & EHS) http://rmehs.depaul.edu
Research involving infectious agents or rDNA shall be reviewed by the IBC. All proposals, regardless of
funding source, are subject to this review. The IBC determines the proper hazard classifications, ensures
that proper safety precautions and procedures have been met, and recommends approval or disapproval
of the protocol.
3.0 Responsibilities
3.1 Department Chairperson
The Department Chairperson is responsible for making faculty aware of the institutional research
protection committees and resources for the safe conduction of experiments in the teaching and
research laboratory. The Chairperson shall encourage compliance among researchers and lab
personnel with safe practices and procedures in the laboratories.
3.2 Principal Investigator
The key person in the laboratory is the Principal Investigator (PI) who is in charge of the
facility. This is the individual who has been assigned the responsibility and discretionary
authority to set work practices. The attitude of this person will be reflected by others working
in the facility. It is the policy of the University that the Principal Investigator is responsible for
complying with this Biosafety Manual, the procedures recommended in the CDC/NIH
handbook for infectious agents, and the NIH guidelines for rDNA research (where applicable).
The Principal Investigator is also responsible for compliance with CDC Select Agent and
Occupational Safety and Health Administration (OSHA) regulations.
Specifically, the Principal Investigator has the primary responsibility for:
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







Determining the real and potential biohazards of the proposed research.
Performing risk assessments for procedures to be performed in the lab.
Determining the appropriate level of biological and physical containment.
Selecting the microbiological practices and laboratory techniques for handling
potentially infectious agents and recombinant DNA materials.
Preparing procedures for dealing with accidental spills on personnel and
environmental contamination of personnel.
Determining the applicability of various precautionary medical practices, serological
monitoring, and immunization.
Securing approval by the appropriate University research compliance committees
(e.g., IRB, IACUC) of the proposed research prior to initiation of work.
Providing appropriate training and personal protective equipment for laboratory
personnel.
3.3 Research Laboratory Personnel
Research laboratory personnel are ultimately responsible for working safely in the laboratory
or research environment. Personnel shall ensure that all work is conducted in compliance
with NIH, CDC, OSHA and other applicable guidelines and regulations. Personnel should
follow the DePaul University Biosafety Manual and Bloodborne Pathogens requirements
(where applicable). Personnel need to know specific laboratory practices, potential hazards
of infectious agents in use, and emergency and spill procedures. Research laboratory
personnel shall complete all required training and help to maintain laboratory safety through
compliance with laboratory procedures and communication with the PI.
3.4 Office of Risk Management and Environmental Health and Safety
The Office of Risk Management and Environmental Health and Safety provide consultation
on the operation of the laboratory to ensure compliance with CDC, NIH, OSHA, federal, and
state criteria. This department also provides information on regulations that apply to the
laboratory, advice on safe methods for new procedures, and provides advice in the event of a
biohazardous material spill and or chemical spill.
3.5 Institutional Biological Safety Committee
The Institutional Biological Safety Committee (IBC) performs duties for the University as
defined in the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH
Guidelines). As such, the Committee shall review applications for research involving rDNA to
determine whether the facilities, procedures, and practices meet the standards required by
the University and the NIH. It shall, in addition, have the responsibility to certify annually to
the NIH that such facilities, procedures, and practices, and the training and expertise of
personnel meet NIH standards. Meetings called for the purpose of such review and
certification may be open to the public. Minutes of these meetings shall be kept and made
available for public inspection. DePaul University also requires the IBC to review all research
involving infectious agents and other potentially infectious materials to ensure compliance
with the OSHA Bloodborne Pathogens Standard, 29CFR 1910.1030.
Some of the specific biological safety services provided by the IBC include:
 Evaluation and inspection of laboratory facilities for work with infectious agents
and other hazardous biological agents.
 Maintenance of training records for compliance with federal, state and university
requirements.
 An online Biosafety Training Slide Presentation located on the IBC website:
http://research.depaul.edu/.
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4.0 DePaul University Biological Safety Requirements
The following information describes the requirements for DePaul University researchers as defined by the
Institutional Biosafety Committee. It is the responsibility of each Principal Investigator to ensure the
laboratory is in compliance.
4.1 Registration with the Institutional Biosafety Committee
Each Principal Investigator (PI) is responsible for the preparation of a registration form for all
research involving biohazardous materials or agents including the assignment of the required
Biosafety Level to the proposed research (See below). This includes research involving:




Recombinant DNA, including experiments that are specifically exempt under the NIH
Guidelines.
Agents that are potentially infectious to humans such as bacteria, viruses, fungi, etc.
Live viruses.
Human blood, tissue, and cell lines.
DePaul University researchers can download and complete submission forms for research
involving both rDNA and biohazardous materials (http://research.depaul.edu). Before submitting
an application, please follow the instructions below:

Determine if IBC review is necessary: All research involving rDNA and other
biohazardous agents must register with DePaul University's IBC. Research that uses
biohazardous agents that require full IBC review must be submitted to the IBC before
receiving materials or initiating research. Information on determining the necessity of IBC
review
can
be
found
in
the
IBC's
Policies
and
Procedures
http://policies.depaul.edu/policy/policy.aspx?pid=183 . Final determination will be made
by the IBC, and PIs will be notified if their work does not require IBC review.

Determine Biosafety Level (BSL). Currently, only research falling into either BSL 1 or 2
can be conducted at DePaul University. Information on BSL and Risk Group Levels can
be found on page 7 of this document and in the IBC Polices and Procedures.
The IBC will review all submitted registration forms; confirm, where applicable, that exempt status
is appropriate for certain rDNA work; and consider approval for those registration forms that are
complete and which provide for safe handling of potentially bio-hazardous materials under the
appropriate Biosafety Level. Two registration forms are available:



rDNA Registration Form http://research.depaul.edu/ – for work involving rDNA
Infectious Agents Registration Form http://research.depaul.edu/ – for work involving all
other biohazards
In some cases, both forms will need to be completed
Copies of the registration forms can be downloaded from the IBC website
http://research.depaul.edu or can be obtained by contacting Diana Alfaro at dalfaro@depaul.edu
or 312-362-7592.
4.2 Training
Successful completion of a range of Biosafety training programs is required for individuals
conducting BSL2 at DePaul University. The first is an online Biosafety Training presentation for
laboratory procedures, the second a CD for the proper receiving and shipping of BSL2 materials.
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You can access training information at the Office of Research Protections website at:
http://research.depaul.edu/
4.3 Risk Group Determination
The investigator must make an initial risk assessment based on the Risk Group (RG) of an agent.
There are four Risk Groups, according to their relative pathogencity for healthy adult humans.
DePaul University currently can only support research with Risk Groups 1 or 2.
Risk Group 1: Agents not associated with disease in healthy human adults. A complete list of
these agents can be found in the NIH Guidelines under Appendix B. Examples include
asporogenic Bacillus subtilis or Bacillius lichenformis. A strain of E. coli is considered RG1 if it
does not possess a complete lipopolysaccharide (lacks the O antigen) and does not carry any
active virulence factor or colonization factors and does not carry any genes encoding these
factors.
Risk Group 2: Agents associated with human disease which is rarely serious and for which
preventative or therapeutic interventions are often available.
Risk Group 3: Agents are associated with serious or lethal human disease for which preventive
or therapeutic interventions may be available.
Risk Group 4: Agents are likely to cause serious or lethal human disease for which preventive or
therapeutic interventions are not usually available.
Typically, once a risk group is determined, the agent is assigned a comparable biosafety level
and treated using the guidelines assigned to the BSL below: At this time, DePaul University can
only support work done at BSL1 and BSL2.
Biosafety
Level
1
2
Agents
Not known to
cause disease
in healthy
adults.
Associated with
human
disease,
hazards are
autoinoculation,
ingestion,
mucous
membrane
exposure.
Practices
Standard
Microbiological
Practices.
BSL-1 practice plus:
Limited access;
Biohazard warning
signs; "Sharps"
precautions;
Biosafety manual
defining any needed
waste
decontamination or
medical surveillance
policies.
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Safety Equipment
(Primary Barriers)
None required.
Facilities
(Secondary Barriers)
Open bench top, sink
required.
Class I or II BSCs or
other physical
containment devices
used for all
manipulations of agents
that cause splashes or
aerosols of infectious
materials; PPE:
laboratory coats; gloves;
face protection as
needed.
BSL-1 plus an
autoclave is available.
3
Indigenous or
exotic agents
with potential
for aerosol
transmission;
disease may
have serious or
lethal
consequences.
Dangerous or
exotic agents
which pose
high risk of lifethreatening
disease,
aerosoltransmitted lab
infections; or
related agents
with unknown
risk of
transmission.
4
BSL-2 practice plus:
Controlled access;
Decontamination of
all waste;
Decontamination of
lab clothing before
laundering; Baseline
serum.
BSL-3 practices plus:
Clothing change
before entering;
Shower on exit.
All material
decontaminated on
exit from facility.
Class I or II BSCs or
other physical
containment devices
used for all
manipulations of agents;
PPE: protective lab
clothing; gloves;
respiratory protection as
needed.
All procedures
conducted in Class III
BSCs or Class I or II
BSCs in combination
with full-body, airsupplied, positive
pressure personnel suit.
BSL-2 plus physical
separation from access
corridors, self-closing,
double- door access,
exhausted air not
recirculated, negative
airflow into laboratory.
BSL-3 plus separate
building or isolated
zone, dedicated
supply/exhaust,
vacuum, and decon
systems, other
requirements outlined
in BMBL.
4.4 Prudent Practices for Infectious Agents in the Laboratory
The control of biological hazards in the laboratory/ research environment shall be maintained by:
 Limiting access to the laboratory in general.
 Limiting access to the material in question.
 Limiting the handling of material to the minimum compatible with efficient usage.
 Insuring proper disposal of material after usage.
 Insuring proper usage of appropriate safety equipment, precautions, and procedures
when handling the materials.
 Maintaining appropriate levels of identification, warning, and security in storage of the
material. The universal biohazard warning label (see below) shall be posted on the
outside door of each laboratory and on equipment used for storage or manipulation of
biohazardous material which is potentially infectious to humans.




Submitting an Infectious Agent Registration Form for each infectious agent received by
the laboratory.
Maintaining proper air supply to the laboratory so that air in the laboratory always
remains negative in relationship to the hallways and surrounding areas.
Keeping laboratory doors closed during operations involving the material.
Additional standard and special practices for Biosafety Levels 1 through 4 are described
in the Biosafety in Microbiological and Biomedical Laboratories, 5th Ed, which can be
found
at
the
Centers
for
Disease
Control
and
Prevention:
http://www.cdc.gov/od/ohs/biosfty/bmbl5/BMBL_5th_Edition.pdf
Page 8 of 30
The following is an outline of safety procedures that should be applied for all those working in a
DePaul University lab:
 Access to the laboratory is limited or restricted at the discretion of the laboratory director
when experiments or work with cultures and specimens are in progress.
 Persons wash their hands after they handle viable materials, after removing gloves, and
before leaving the laboratory.
 Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food
for human use are not permitted in the work areas. Persons who wear contact lenses in
laboratories should also wear goggles or a face shield. Food is stored outside the work
area in cabinets or refrigerators designated and used for this purpose only.
 Mouth pipetting is prohibited; mechanical pipetting devices are used.
 Policies for the safe handling of sharps are instituted.
 All procedures are performed carefully to minimize the creation of splashes or aerosols.
 Work surfaces are decontaminated at least once a day and after any spill of viable
material.
 All cultures, stocks, and other regulated wastes are decontaminated before disposal by
an approved decontamination method such as autoclaving. Materials to be
decontaminated outside of the immediate laboratory are packaged in accordance with
applicable local, state, and federal regulations before removal from the facility.
 A biohazard sign must be posted at the entrance to the laboratory whenever infectious
agents are present. The sign must include the biosafety level and the name and phone
number of the investigator.
4.5 Recombinant DNA Experiments
Institutional Biosafety Committee (IBC) approval is required prior to the initiation of most nonexempt recombinant DNA experiments. A brief description of non-exempt and exempt
recombinant DNA experiments can be found in the NIH Guidelines. Principal Investigators must
contact the IBC to register non-exempt recombinant DNA work, update current registration if the
scope of the work has changed, or ask any questions regarding recombinant DNA work. NIH
Guidelines
(Guidelines
for
Research
Involving
Recombinant
DNA
Molecules)
http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm are available on-line.
4.6 Infectious Agents in the Laboratory
The term infectious agent is used broadly to apply to agents that could possibly cause harm to
humans, animals, plants, or insects. The following are examples of Specific Infectious Agents.
4.6.1 Human Blood, Body Fluids, Tissue and Other Potentially Infectious Materials
The Occupational Safety and Health Administration (OSHA) created the Occupational
Exposure to Bloodborne Pathogens Standard, 29 CFR Part 1910.1030 (Bloodborne
Pathogens Standard)
http://osha.gov/SLTC/bloodbornepathogens/index.html to minimize or eliminate exposure
to infectious agents that may be present in human blood, tissues, or certain body fluids
(Bloodborne Pathogens). The Bloodborne Pathogens Standard applies to all employers
having employees that are “occupationally exposed” to human blood or other potentially
infectious materials. An employee is considered occupationally exposed if there is
“reasonably anticipated skin, eye, mucous membrane, or potential contact with human
blood, or other potentially infectious materials in the performance of an employee’s
duties”.
Other potentially infectious materials (OPIM) include:
o Human cell or tissue cultures
o Organ cultures
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o
o
o
o
Any unfixed tissue or organ, from a human being (living or dead)
HIV- or HBV- containing culture media or other solutions
Human body fluids such as, urine, feces, saliva, or tears unless visibly
contaminated with blood
Blood, organs or other tissues from experimental animals infected with HIV, HBV,
or other Bloodborne pathogens
An individual is also considered occupationally exposed if they do not have direct contact
with blood or other potentially infectious material, if the employee uses equipment that is
used to process or store blood, other potentially infectious materials or Bloodborne
Pathogens. All occupationally exposed employees are required by OSHA to attend a
Bloodborne Pathogens training session prior to beginning work and annually thereafter.
Bloodborne Pathogens training is available through RM & EHS, contact Jenny Graham at
jgraham5@depaul.edu. Additionally, individuals are required to be immunized against
Hepatitis-B or sign a waiver.
For more information contact: Diana Alfaro at
dalfaro@depaul.edu or Jenny Graham at jgraham5@depaul.edu.
OSHA has determined that occupational exposure to human blood, tissues, and body
fluids poses a significant health risk because these may contain the following Bloodborne
Pathogens.
4.6.2 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 and those which involve biohazards will also require submission of an IBC protocol. The
IBC shall review work with human pathogens or recombinant DNA in animals (including
transgenic animals) prior to initiation.
4.6.3 Care and Handling of Infected Research Animals
Comprehensive reviews indicate that animals infected with a wide range of etiologic agents are
capable of shedding infectious microorganisms in the saliva, urine, or feces. In the absence of
specific information to the contrary, all infected animals should be regarded as potential
shedders.
Procedures appropriate for the handling of infected animals are given below:
o
o
o
o
Careful handling procedures should be employed to minimize the dissemination of
dust from animal and cage refuse.
Cages should be sterilized by autoclaving. Refuse, bowls, 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.
Page 10 of 30
o Double gloves should be worn or use forceps when feeding, watering, handling, or
o
o
o
o
5.0
removing infected animals. Bare hands should never be placed in the cage to move
any object therein.
When animals are to be injected with infectious agents, the animal caretaker should
wear protective gloves and the laboratory workers should wear double gloves.
Animals should be properly restrained (e.g., use of squeeze cage for primate
inoculation) or tranquilized to avoid accidents that might result in disseminating
infectious agents, as well as to prevent injury to the animal and to personnel.
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 static micro
isolation cage or in rooms designed for protection of personnel by use of biological
safety cabinets.
Additional standard and special practices for Animal Biosafety Levels 1 through 4 are
described in the Biosafety in Microbiological and Biomedical Laboratories, 5 th Ed.
which can be found at the Centers for Disease Control and Prevention:
http://www.cdc.gov/od/ohs/biosfty/bmbl5/BMBL_5th_Edition.pdf
Immunizations
If required by the type of research conducted by DePaul University personnel, a consultation with a
Health Advisor i.e. Illinois Masonic Hospital or Sage Medical, may be necessary. For a particular
employee, the recommendations from a Health Advisor might call for any of a number of precautionary
measures, including immunizations or a periodic physical examination. In these instances, the principal
investigator would provide a Health Advisor with guidelines and descriptions of conditions that might have
significance for personnel assigned to the laboratory. For more information about these programs contact
the RM & EHS at 773-325-4201.
Individuals that work with Bloodborne Pathogens as defined as OSHA are required to be immunized
against Hepatitis-B. For more information contact: Diana Alfaro at dalfaro@depaul.edu or Jenny Graham
at jgraham5@depaul.edu
6.0
Medical Restrictions
A Health Advisor is available to discuss medical conditions that can affect employee safety in the
laboratory. Services can be scheduled by contacting the RM & EHS at 773-325-4201. Payment for
services must be coordinated through the individual employee’s department.
6.1 Pregnancy
It is recognized that exposure to certain infectious agents may adversely affect a fetus during
pregnancy if the mother is infected with the agent. Therefore, if pregnancy is possible while you
are working in an infectious disease laboratory or laboratory engaged in work with infectious
agents you should consult your principal investigator. A Health Advisor is also available for
questions regarding the potential harm from the biological agents present within your laboratory.
Women that are pregnant or become pregnant are encouraged to inform their supervisors or
principal investigators. Employees are urged to discuss exposure issues related to pregnancy
with their supervisors or principal investigators.
Please consult the following list of reproductive hazards
http://www.oehha.ca.gov/prop65/prop65_list/Newlist.html designated by the state of California.
6.2 Reproductive Biological Hazards
Page 11 of 30
Consultations with a Health Advisor can be coordinated through the IBC for any woman or man of
childbearing age working with reproductive pathogens or other potentially infectious materials.
Reproductive biological hazards include, but are not limited to the following:
 Cytomegalovirus (CMV)
 Hepatitis B virus (HBV)
 Hepatitis E virus
 Human Immunodeficiency virus (HIV)
 Human parvovirus B19
 Rubella (German Measles)
 Lymphocytic Choriomeningitis virus
 Toxoplasma gondii (Toxoplasmosis)
 Listeria monocytogenes
 Varicella-zoster virus (chicken pox)
Whenever necessary, the IBC will offer an opportunity to review work procedures in the lab to
ensure that potential exposure is minimized. Consideration for reassignment to other tasks that
don't involve exposure to the reproductive hazard (generally with actual pathogens, not
necessarily for only other potentially infectious materials such as blood or body fluids) should be
given. Also, investigators actively working with reproductive hazards should explain the risk
assessment at time of hire.
6.3 Other Restrictions
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. Therefore, if you are suffering from any of the above
conditions, you should inform your physician about the biohazards you are exposed to in the
laboratory to assess the risk involved.
7.0
Emergency and Accident Procedures
7.1 Laboratory Emergency Contacts
Phone numbers of responsible individuals to be contacted in case of emergencies should be
posted on the outside door of each laboratory. Also, a list of the significant hazard classes (e.g.,
biohazard level, oxidizing agents, toxic chemicals, carcinogens) found within the laboratory is to
be posted for use by emergency personnel.
7.2 Emergency Equipment
All personnel should be familiar with the location and use of all the emergency equipment in their
laboratory. This includes fire extinguishers, eyewash stations, emergency deluge showers,
chemical spill kits, absorbent materials, and fire alarm pull stations.
7.3 Evacuation Routes
All personnel should be familiar with the primary and secondary evacuation routes from their area
to the nearest building exit. RM & EHS can assist in developing these routes. The secondary
route should be used when the primary route is blocked for some reason. All personnel should
be told what method is used to signal a building evacuation (e.g. a building alarm or manually
operated air horn).
7.4 Spills and Emergencies
Page 12 of 30
Principal investigators are responsible for developing spill and emergency guidelines suitable for
biological operations in their specific areas. These guidelines shall contain proper procedures for
containing and decontaminating any spills that may occur and any emergency actions to be
taken. Personnel working in the areas of operation should be trained on these emergency
guidelines. Guidelines should be posted in the laboratory. The guidelines below are those
developed by the RM&EHS and are recommended for use in the research laboratory.
7.4.1 Spill and Clean –up Procedures for the Research Laboratory
If potentially hazardous biological material is spilled in the laboratory, the first essential
practice is to avoid inhaling any airborne material by holding the breath and leaving the
laboratory. Warn others in the area and go directly to a wash or change room area. If
clothing is known or suspected to be contaminated, remove the clothing with care, folding
the contaminated area inward. Discard the clothing into a bag or place the clothing
directly in an autoclave. Wash all potentially contaminated areas as well as the arms,
face, and hands. Shower, if facilities are available. Reentry into the laboratory should be
delayed for a period of 30 minutes to allow reduction of the aerosol generated by the spill.
Advance preparation for management of a spill is essential. A "spill kit” including leakproof containers, forceps, paper towels, sponges, disinfectant, eye protection, and rubber
gloves should be readily available.
If the emergency involves personal injury or biological contamination, call Public Safety at
773-325-7777. Be sure to state the type of contaminant on the victim. The caller should
remain available to brief emergency responders on the type of contamination and proper
procedures for handling the material.
Clean-up
o
o
o
o
o
o
o
o
o
o
o
o
Block off the spill area and alert personnel in the area to the presence of the spill.
Put on appropriate personal protective equipment (PPE). This may include
gloves, gown, additional eye protection, or face mask depending on the size of
the spill, and the type of material.
Cover the spill with paper towels or other absorbent materials to contain spill.
Carefully pour a freshly prepared 1:10 dilution of household bleach around the
edges of the spill and into the spill. Avoid splashing.
Allow 20 minute contact period.
Pick-up all absorbent material and place carefully in a biohazard bag for
autoclaving and subsequent disposal. Do not seal the bag at this point.
Use forceps, plastic scoop, or other mechanical means to remove any broken
glass or other sharp objects from the spill area. Take care not to create aerosols.
Place these items into a small cardboard box, thick walled plastic bag, or other
container that will prevent them from puncturing the biohazard bag (or your
hand). Place the sharp items into the biohazard bag for disposal. Do not seal
the bag at this point.
Clean spill area with fresh paper towels soaked in disinfectant. Place used paper
towels in biohazard bag for disposal. Do not seal the bag at this point.
Once spill is completely cleaned, place all used spill control equipment in
biohazard bag for disposal. Do not seal the bag at this point.
Remove PPE and decontaminate or place in biohazard bag for disposal.
Do not remove PPE from face with soiled gloves. Remove soiled gloves first and
place them in the biohazard bag for disposal.
Once all used PPE, spill control equipment, and other potentially contaminated
items are in the biohazard bag loosely seal the bag, place in a secondary
container, and transport to autoclave facility.
Page 13 of 30
o
Wash your hands with soap and water.
7.4.2 Spill in a Biological Safety Cabinet
A spill that is confined to the interior of the biological safety cabinet should present little or
no hazard to personnel in the area. However, chemical disinfection procedures should be
initiated at once and while the cabinet ventilation system continues to operate, in order to
prevent escape of contaminants from the cabinet. Spray or wipe walls, work surfaces,
and equipment with a disinfectant. A disinfectant with a detergent has the advantage of
detergent activity, which will help clean the surfaces by removing both dirt and
microorganisms. Use appropriate commercially-available chemical germicides known to
be effective against the organism in use or 1 part household bleach (e.g. Clorox) to 9
parts of tap water with 0.7% nonionic detergent, prepared fresh daily. The operator
should wear gloves and eye protection during this procedure. Use sufficient disinfectant
solution to ensure that the drain pans and catch basins below the work surface contain
the disinfectant. Lift the front exhaust grill and tray and wipe all surfaces. Wipe the catch
basin and drain the disinfectant into a container. This disinfectant, gloves, wiping cloth,
and sponges should be discarded into an autoclave pan and autoclaved. Note: This
procedure will not disinfect the filters, blower, air ducts or other interior parts of the
cabinet. Decontamination of these parts shall be performed by a contractor, and the
biosafety cabinet will have to be recertified.
7.4.3 Personal Contamination
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Remove any contaminated clothing or personal protective equipment.
If skin has been contaminated, wash with soap and warm water.
If eyes have been splashed, rinse under tepid running water (eyewash or faucet) for
at least 15 minutes.
Contact Public Safety at 773-325-7777.
Contact your PI or supervisor and inform them of the situation.
Visit a Health Advisor; Public Safety will provide transportation.
7.4.4 Emergency Procedures for Exposure Incidents
An "exposure incident" is specific contact (e.g. contact with the eye, mouth, other mucous
membrane, respiratory tract via inhalation, non-intact skin, or parenteral) with potentially
infectious materials that results from the performance of an employee's duties. An
employee who sustains a known or potential exposure incident shall remove gloves and
treat the affected area immediately by following the appropriate exposure incident
response below.


Percutaneous Injury
o Wash the affected area with antiseptic soap and warm water for 15 minutes.
Splash to Face
o Flush affected area in eyewash for 15 minutes.
 Aerosol Exposure
o Hold breath and immediately leave room.
o Remove Personal Protective Equipment (PPE) carefully. When
removing PPE make sure to turn the exposed areas inward.
o Wash hands well with soap and water.
o Post spill sign on lab entry; lab should be evacuated for at least
30 minutes.
o The researcher must clear lab for re-entry.
Page 14 of 30
7.4.5 Reporting Incident
The employee must report the incident to his/her supervisor. The supervisor shall
complete an incident report documenting the route of exposure and the circumstances
under which the incident occurred. Contact Public Safety, 773-325-7777 to fill out a
report and transported to a Health Advisor.
7.4.6 Medical Assistance
Employees are urged to call Public Safety at 773-325-7777 after they have received
proper first aid available at site of exposure. For certain exposures, such as non-human
primate bites or scratches, tick or insect bites, or exposure to infectious agents; the
employee is advised to see a Health Advisor.
A Health Advisor will provide the post-exposure evaluation and follow-up at no cost to
employees who experience "exposure incidents". The post-exposure monitoring periods
are dependent on the type of exposure. This time period is related to the various
incubation periods of the infectious agents.
7.4.7 Investigation of Laboratory Accidents
RM & EHS and Public Safety, in cooperation with principal investigators and their staff,
will conduct the necessary investigation of laboratory accidents. The goal of the
investigation is to prevent similar accidents and to obtain information concerning the
circumstances and number of employees who have been exposed to the agent in
question. In addition, RM & EHS, in consultation with a Health Advisor, might institute
further steps to monitor the health of those who may have been exposed to the agent in
question.
It should be emphasized that the reporting of accidents to the principal investigator or
laboratory supervisor is the responsibility of the employee who has the accident. The
principal investigator or the laboratory supervisor should then report the incident to Public
Safety, 773-325-7777. Please also report incidents that did not result in an exposure
(near miss) to Public Safety.
Whenever an injury involves a sharp device and exposure to human material (e.g. body
fluid, tissue, cell line) the IBC shall perform an investigation to determine if a safe sharps
device is available to prevent future occurrences of the injury.
8.0
Risk Assessment and Risk Management
Responsibility for biosafety exists at all levels and is shared throughout the University. The President
acknowledges the institution’s role in providing a safe workplace and has given the Institutional Biosafety
Committee (IBC) the authority to administer the campus biosafety program. IBC establishes policies for
the safe use of biohazards and for compliance with all applicable regulations.
The researchers, clinicians, and technicians who perform work with biohazards are perhaps the most
important component of the biosafety program, as they shall incorporate the biosafety requirements and
safety precautions into all facets of their work.
The principal investigator is ultimately responsible for safety within the laboratory. An integral part of this
responsibility is to conduct a review of proposed work to identify potential hazards (risk assessment) and
to adopt appropriate safety procedures before initiation of the experiments (risk management). Certain
experiments require advanced registration and IBC approval.
Page 15 of 30
The five P’s of risk assessment and risk management are:
 Pathogen – hazardous biological agent.
 Procedures – proposed experimental manipulations and safe work practices.
 Personnel – appropriate training and skills.
 Personal Protective Equipment (PPE) – protective clothing and safety equipment.
 Place – laboratory design.
Consider the five P’s in each facet of laboratory work. Properly conducted, risk assessment can help
prevent exposure to biohazards and minimize the potential for laboratory acquired infection. Remember
that prior planning prevents poor performance.
8.1 Routes of Exposure
In order for biological agents to cause disease, they must first enter or invade the body in
sufficient numbers. Routes of entry include oral, respiratory, parenteral, mucous membrane and
animal contacts (e.g. bites, scratches). Once inside the body, biohazards must meet other
requirements to cause disease; they shall colonize and establish in body cells, tissues and/or
organs, overcome the body’s natural defense mechanisms and mutate or adapt to body changes.
Other factors contribute to an individual’s susceptibility to the disease process. These include
age, immunological state, occupation, physical and geographic environment and predisposing
conditions (such as alcoholism and other drug abuse, pregnancy and diseases such as diabetes).
It is difficult to determine a minimum infectious dose when discussing biohazards. The same dose
of a pathogen may produce no disease symptoms in one individual but may cause serious or
even fatal disease in another. There are microorganisms for which it is thought one organism
entering the body is sufficient to invade and promote the disease process; the bacterium that
causes tuberculosis is an example. For many pathogens, 10 to 100 or more organisms may enter
the body to cause infection leading to disease. See Appendix B for additional information on
routes of exposure.
9.0
Laboratory Practices
In this section, an attempt has been made to provide information regarding hazards involved with certain
laboratory practices and methods for preventing them. Prevention is an important element to biohazard
control, and it is recommended that anyone working in a laboratory read this section carefully.
9.1 Human Factors and Attitudes in Relation to Laboratory Accidents
For the purpose of safety, an attitude can be defined as an accumulation of information and
experience that predisposes an individual to certain behavior. Human factors and attitudes result
in tendencies on the part of the individual to react in a positive or negative fashion to a situation, a
person or an objective. Laboratory supervisors and principal investigators should understand the
importance of attitudes and human factors in their own efforts to control biohazards in their
laboratory. Each employee working with biohazardous agents shall be consistently aware of the
importance of the proper attitude in preventing accidents in the laboratory.
Some observations that may be of help to supervisors are listed below:
 The lack of accident perception ability is often a significant factor in laboratory accidents.
Inflexibility of work habits, that tend to preclude last minute modification when an accident
situation is recognized, plays a part in the causation of some laboratory accidents.
 Working at an abnormal rate of speed is a significant causal factor.
 Intentional violations of regulations are a frequent cause of accidents. This is termed
excessive risk taking.
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


The performance of routine procedures such as diluting and plating cultures is the most
frequent task being performed at the time of laboratory accidents.
Working when one is very tired is more likely to create a higher potential for accidents.
Working at a well-organized and uncrowded laboratory bench will help in the prevention of
lab accidents.
9.1.1 Biosafety Levels 1 and 2
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Allow only persons informed of the research to enter BL2 areas.
Avoid using hypodermic needles.
Control insect and rodent infestations.
Decontaminate all biological wastes before discard. Decontaminate other
contaminated materials before washing, reuse, or discard.
Do not mouth pipette, use mechanical pipetting devices.
Do not smoke, eat, drink, store food or apply cosmetics in BL1 and BL2 areas.
Ensure that all biomedical waste containers are labeled with the biohazard symbol.
For off-site decontamination, package contaminated materials in closed, durable,
leak-proof containers.
Keep animals not used in BL2 experiment out of the laboratory.
Keep laboratory door closed.
Maintain a biological spill kit within the laboratory.
Post a biohazard sign on the door of the laboratory indicating the agent being used,
specific precautions for exit and entry, PI’s name and contact info, Emergency
Contact Info and the IBC protocol number.
Report spills, accidents, near misses, and disease symptoms related to laboratory
acquired infection to the PI.
Substitute plastic for glass where feasible.
Use biological safety cabinets to contain aerosol-producing equipment.
Wash hands after completing experimental procedures and before leaving laboratory.
Wear laboratory gowns or coats when appropriate. Change PPE when soiled or
compromised.
9.2 Labeling of Equipment Sent Out for Repair, Surplus, or Disposal
Contaminated and potentially contaminated equipment sent out for repair, surplus, or disposal
shall be decontaminated as thoroughly as possible. Affix a tag to the equipment indicating when
the equipment was decontaminated, what disinfectant was used, and the name of the person who
performed the decontamination. Thorough decontamination of highly technical or sensitive
equipment or equipment with limited access to contaminated areas may not be possible.
Decontaminate the equipment to the degree possible (flushing lines or wiping down the exterior)
and affix a label to the equipment before sending it out for repair.
9.3 Cell and Tissue Culture



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


Wear long sleeved gowns with knit cuffs and long gloves when working in the biosafety
cabinet.
Glassware and other contaminated items should be disinfected or autoclaved before
washing, reuse, or disposal.
Maintain a clean lab gown reserved solely for cell culture work.
Place pipettes on a rack to avoid disrupting airflow when removed.
Keep open tubes parallel to the airflow.
After transferring inoculums always recap vials.
Do not place tubes on work surface.
Page 17 of 30




Discard empty tubes immediately.
Work with one specimen at a time; recap before going to the next.
Cell culture wastes shall be decontaminated.
Autoclave verification should be performed routinely.
9.4 Basic Microbiological Practices
In the absence of definite accidents or obvious spillage, it is not certain that the opening of plates,
tubes and bottles of other microorganisms has caused laboratory infection. However, it is
probable that among the highly infective agents some infections have occurred by this means.
Particular care is required when opening plates, tubes, or bottles containing fungi, for this
operation may release a large number of spores. Such cultures should be manipulated in a
biological safety cabinet.
Ensure that all hazardous fluid cultures or viable powdered infectious materials in glass vessels
are transported, incubated, and stored in easily handled, non-breakable leak-proof secondary
containers that are large enough to contain all the fluid or powder in case of leakage or breakage
of the glass vessel. The secondary container shall be labeled with a biohazard label.
9.5 Maintaining a Clean and Orderly Laboratory
Well-defined cleaning and organizational procedures are essential in reducing the risks
associated with working with pathogenic agents. This is particularly true in the laboratory
operating under less than total containment concepts and in all areas used for the housing of
animals.
A well-conceived and well-executed cleaning and organizational program limits
physical clutter that could distract the attention and interfere with the activities of laboratory
personnel at a critical moment in a potentially hazardous procedure, provides a work area that will
not in itself be a source of physical injury or contamination, and provides an area that promotes
the efficient use of decontaminants in the event of inadvertent release of an etiologic agent.
The objectives of cleaning and organizational procedures in the laboratory are to:

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Provide an orderly work area conducive to the accomplishment of the research
program.
Provide work areas devoid of physical hazards.
Provide a clean work area with background contamination ideally held to a zero level
but more realistically to a level such that extraordinary measures in sterile techniques
are not required to maintain integrity of the biological systems under study.
Prevent the accumulation of materials from current and past experiments that
constitute a hazard to laboratory personnel.
Prevent the creation of aerosols of hazardous materials as a result of the
housekeeping procedures used.
9.6 Transport Shipping Biological Specimens
Any laboratory wishing to ship biohazards must have an individual who has been properly trained
by the University in the approved DOT/ IATA Shipping Training. This training is mandatory for
individuals shipping and receiving BSL2 agents and is recommended to be completed by at least
one individual in the laboratory. This individual shall be responsible for all package preparation
and shipment of biological specimens. This training is available in the Biology Department – 773325-7595.
Any laboratory wishing to import biological samples shall complete the CDC Importation Permit
Application and fax this document to the CDC. Please contact the IBC for assistance in
completing the importation permit.
This document may be found on-line at
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http://www.cdc.gov/od/eaipp/. Once the document has been completed fax to the number
indicated on the form.
9.6.1. Transport of Biological Materials on/off Campus
Biological specimens transported between laboratories and medical facilities should be
properly contained in a sealed, leak-proof, shatter-proof secondary container. This
container should be sealed in the laboratory and the outside should be disinfected. (See
Figure 1) This will allow for safe transport of the specimen without gloves. An example of
proper transportation would be a sealed tissue culture flask placed into a sealed plastic
bag and then placed into a small cooler with a tight-fitting lid. Consult the Environmental
Health & Safety office for assistance in choosing an appropriate secondary container.
See Appendix C on how to contain a specimen.
10.0 Personal Protective Equipment (PPE)
Research conducted in DePaul University laboratories requires that personal protective equipment
(protective clothing and safety apparatus/equipment) be used to protect the researcher from contact with
infectious, toxic and corrosive agents, excessive heat, cold, fire, and other physical hazards. Good
laboratory techniques, procedures, and appropriate laboratory equipment are the primary barriers against
potential exposure to hazardous agents. PPE serves as a second line of defense.
General Guidelines for the Use of PPE in the Laboratory:
 Overt exposure to agents at all level of risk should be followed by immediate decontamination
of the PPE and change into clean PPE to protect the worker, the experiments and the
environment.

personnel, if applicable.

cafeteria, or other places accessible to the public.
 Personnel should be encouraged to use disposable facial tissues instead of personal
handkerchiefs.

washing, decontamination or disposal.
 PPE with extensive contamination m
 Do not take PPE home to launder; a laundry service is currently available through the
Biology Department. Contact 773-325-7595.


ar appropriate sizes and keep an adequate supply of PPE available in the laboratory.
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Wear closed-toe shoes and long pants to guard against skin contamination or chemical
exposure. Do not wear sandals or shorts in the laboratory.
10.1 Laboratory Clothing
A commonly used PPE item within the laboratory is special clothing. Both reusable and
disposable clothing is available. Whichever is used, it must be durable, designed to provide
protection and prevent exposure of the skin to harmful agents, as well as be compatible with the
methods of decontamination employed. Laboratory clothing serves to protect the wearer, the
experiment, and the environment against contamination. Laboratory clothing includes lab coats,
lab gowns, dedicated clothing or scrubs, shoe covers, and dedicated shoes and socks. The
Page 19 of 30
necessary laboratory clothing needed should be determined by a lab-specific risk assessment of
procedures with the infectious agent in use.
10.2 Gloves
Gloves should be comfortable and of sufficient length to prevent exposure of the wrist.
Depending upon intended use, the composition and design of the glove may vary to provide the
desired level of flexibility, strength, impermeability, and resistance to penetration by sharp
objects, as well as protection against heat and cold. No glove can be expected to be satisfactory
for all intended uses. Some gloves are pre-tested for viral penetration, chemical resistance, and
puncture resistance. Obtain pre-tested, if necessary, ask glove manufacturers for documentation
regarding any pre-testing of gloves.
Disposable (single use) gloves provide a barrier between infectious agents and the skin. Glove
use is a basic precept of preventing infectious agent transmission. Breaks in the skin barrier of
the hand (damaged cuticles, scrapes, micro-cuts, dermatitis, etc.) are common and the use of
hand-lotions is recommended to prevent this. Latex gloves can be degraded by common hand
lotions which contain petroleum based products, therefore it is important to choose a water-based
hand lotion for laboratory applications.
Gloves should be removed and hands washed before exiting the laboratory. Use the one glove
method or an appropriate secondary container when transporting materials through common use
areas.
General Guidelines for the Use of Gloves in the Laboratory:
 Change gloves periodically, when gloves become soiled, and always wash hands after
removing gloves or other PPE.
 Gloves will not prevent needle sticks or other puncture injuries.
 Check gloves for visible tears before use.
 Do not reuse disposable latex or nitrile gloves.
 Discard contaminated gloves in a biohazard bag immediately after use.
 Double glove when cleaning spills.
10.3 Face and Eye Protection
Protection of the face and eyes is of prime importance in laboratories due to the potential for foreign
material, both liquid and solid, to splash on the head, face and eyes, or contact lenses. A variety of
face shields, head covers/hoods, protective goggles, and lenses are available from safety supply
houses. The selection is dependent upon materials of construction, fit, comfort, and compatibility
with the work and the overall facial area requiring protection. Face shields and goggles can provide
splash protection. Safety glasses provide impact protection from projectiles in the laboratory.
Contact lenses do not provide eye protection. It is recommended that contact lenses not be worn
when working around chemicals, fumes, and other hazardous material and dust particles since
these items may become trapped in the space between the contact lens and the cornea. When
contact lenses are worn, eye protection, such as tight fitting goggles, shall be worn. Contact lenses
should never be handled in the laboratory.
10.4 Best Procedure for Removing PPE
When removing gloves, grip the outside of one glove at wrist with the other gloved hand, pull glove
off inside-out and gather in palm of gloved hand. Place index or middle finger of the ungloved hand
on wrist of gloved hand, slide finger under the glove opening and pull glove off inside out.
Page 20 of 30
When removing PPE, remove lab coat or solid front gown first, then remove gloves (aseptically),
remove face protection last to avoid touching your face with contaminated hands. If wearing double
gloves, remove outer gloves before removing lab coat or solid front gown.
11.0 Laboratory Equipment
11.1 Biological Safety Cabinets
Biological safety cabinets (BSCs), when used properly, provide a clean work environment for
research activities. Biological safety cabinets offer personnel, product, and environmental
protection. The BSC provides primary containment for infectious materials. The efficacy of BSCs
depends upon the behavior of the operator and the orientation of the unit in the facility.
BSCs isolate biohazards from personnel by confining the biohazardous material to the unit and
remove aerosolized biohazardous material by moving air through high efficiency particulate air
(HEPA) filters. The intake air is filtered through a HEPA filter before entering the BSC work area.
Exhaust air also passes through a HEPA filter. Aerosols generated in the work area of the BSC are
contained within the BSC, as long as the efficacy of the BSC has not been compromised by the
actions of the personnel.
The efficacy of BSCs depends upon the behavior of the operator, the unit’s orientation in the facility,
and the movement of personnel in the laboratory. All BSCs at DePaul University shall be certified
at least annually. Any BSCs not certified will be reported to the IBC and PI as a serious lab
violation. It is your responsibility to participate in the required annual certification process.
11.2 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.
The following procedures for centrifugation are recommended:
 Examine tubes and bottles for cracks or stress marks before using them.
 Fill and decant all centrifuge tubes and bottles within the biological safety cabinet for BSL2.
 Wipe outside of tubes with disinfectant before placing in safety cups or rotors.
 Never overfill centrifuge tubes as leakage may occur when tubes are filled to capacity. The
maximum for centrifuge tubes is 3/4 full.
 Always cap tubes before spinning. Use screw cap tubes.
 Place all tubes in safety buckets or sealed rotors when centrifuging infectious materials.
Inspect the "O" ring seal of the safety bucket and the inside of safety buckets or rotors.
Open safety buckets or rotors in a biological safety cabinet.
 Never exceed safe rotor speed.
 Stop the centrifuge immediately if an unusual condition (noise or vibration) begins.
11.3 Syringes and Needles
The hypodermic needle can be a dangerous instrument if not properly used. To reduce the chance
of accidental injection, aerosol generation, or spills, the use of syringes should be avoided when
alternate methods are available. Needles used to transfer and inject biological and non-biological
solutions should be avoided as this is an unnecessary use of sharps in the lab. Replace sharp
needles with blunt needles of the same gauge whenever possible.
Page 21 of 30
The following practices are recommended for hypodermic needles and syringes:
 Use the syringe and needle in a biological safety cabinet only and avoid quick and
unnecessary movements of the hand holding the syringe.
 Examine glass syringes for chips and cracks and needles for barbs and plugs prior to use.
This should be done prior to use. Use needle-locking syringes only, and be sure that the
needle is locked securely into the barrel. Replace glass syringes with plastic disposable
syringes whenever possible.
 Expel excess air, liquid, and bubbles from a syringe vertically into cotton square moistened
with an appropriate disinfectant.
 Do not use the syringe to forcefully expel a stream of infectious fluid into an open vial for
the purpose of mixing.
 If syringes are filled from test tubes, take care not to contaminate the hub of the needle, as
this may result in the transfer of infectious material to the fingers.
 When removing a syringe and needle from a rubber-stopper bottle, wrap the needle and
stopper in cotton square moistened with an appropriate disinfectant. If there is concern of
the disinfectant contaminating sensitive experimental materials, sterile cotton square may
be used and immediately discarded into a biohazard bag.
 When inoculating animals, position the hand that is holding the animal “behind” the needle
in order to avoid puncture wounds.
 Be sure the animal is properly restrained or for BSL-2 anesthetized or sedated prior to the
inoculation.
 Discard syringes into an appropriate sharps container.
 Do not bend, shear, recap, or otherwise manipulate the needle.
11.4 Pipettes
The following is excerpted from Laboratory Safety, Principles and Practices 3 rd Ed., ASM Press.

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Never suction or pipette by mouth; always use some type of pipetting aid when pipetting
infectious materials. All activities should be confined to a biosafety cabinet for BSL-2.
Pipetting of toxic chemicals should be performed in a chemical fume hood.
Infectious or toxic materials should never be forcefully expelled from a pipette.
Infectious or toxic fluids should never be mixed by bubbling air from a pipette through the
fluid.
Infectious or toxic fluids should never be mixed by alternate suction and expulsion through
a pipette.
Discharge from a pipette should be as close as possible to the fluid or agar level, and the
contents should be allowed to run down the wall of the tube or bottle whenever possible,
not dropped from a height.
Pipettes used for transferring infectious or toxic materials should always be plugged with
cotton, even when safety pipetting aids are used.
Avoid accidentally dropping infectious or toxic material from the pipette onto the work
surface. Place absorbent material on the work surface and autoclave before discard.
Plastic backed bench paper is suitable for this purpose.
Contaminated pipettes that will be reused should be completely immersed in a suitable disinfectant,
such as hypochlorite, to allow complete immersion of the pipettes. All other contaminated pipettes
should be disposed of in a biohazard bag and autoclaved.
11.5 Blenders, Mixers, Sonicators, and Cell Disruption Equipment
Hazardous aerosols are created by most laboratory operations involving blending, mixing, and
stirring, grinding, or disrupting biohazardous materials. Even the use of a mortar and pestle can be
Page 22 of 30
a hazardous operation. Other devices that may produce aerosols are ball mills, colloid mills, jet
mills, tissue grinders, magnetic mixers, stirrers, sonic cleaning devices, ultrasonic cell
disintegrators, French presses, and shakers.
The laboratory practices generally required when using equipment that may generate aerosols with
biohazardous materials are as follows:
 Operate blending, cell disruption, and grinding equipment in a biological safety cabinet for
BSL-2.
 Use safety blenders designed to prevent leakage from the rotor bearing at the bottom of the
bowl. In the absence of a leak-proof rotor, inspect the rotor for leakage prior to operation.
A preliminary test run with sterile water, saline, or methylene blue solution is recommended
prior to use.
 If the blender is used with infectious material place absorbent material moistened with an
appropriate disinfectant over the top of the blender. Sterilize the device and residual
contents promptly after use.
 Glass blender bowls are undesirable for use with infectious material because of the
potential for glass bowls to break.
 Before opening the safety blender bowl permit the blender to rest for at least one minute to
allow settling of the aerosol cloud.
 Homogenization of tissue containing infectious agents should be performed in a BSC,
double bagged in a Stomacher, or in a sealed homogenizer.
 Grinding of infected tissues or materials with any open device is best done within a
biological safety cabinet.
11.6 Miscellaneous Equipment (Waterbaths, Cold Storage, Shakers)
Water baths should be treated with chemicals to prevent growth of microorganisms and should be
regularly cleaned and decontaminated. Deep freezers, liquid nitrogen containers, refrigerators, and
dry ice chests should be checked and cleaned out periodically to remove any broken ampoules or
tubes containing infectious material and decontaminated. All infectious or toxic material stored in
refrigerators or deep freezers should be properly labeled. Security measures should be
commensurate with the hazards.
The degree of hazard represented by contaminated liquid nitrogen reservoirs will be largely
dependent upon the infectious potential of the stored microorganisms, their stability in liquid
nitrogen, and their ability to survive in the airborne state. Investigations suggest that storing tissue
culture cell lines in containers other than sealed glass ampoules might result in potential intercontamination among cell lines stored in a common liquid nitrogen repository.
Shaking machines should be examined carefully for potential breakage of flasks or other containers
being shaken. Screw-capped durable plastic or heavy walled glass flasks/vials should be used.
These should be securely fastened to the shaker platform. An additional precaution would be to line
the shaker with absorbent material.
12.0 Decontamination and Disposal of Biohazardous Waste
The following guidelines describe the safe and appropriate handling and disposal of infectious waste. These
wastes include human blood and body fluids, sharps, infectious microbiological materials, pathological
specimens, and blood, body fluids, and tissues from infected animals.
12.1 Definitions
Autoclave - a device utilized for exposure of instruments, liquids, and potentially infectious waste to
steam at a high pressure in order to decontaminate the materials or render them sterile.
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Biohazardous Waste - human or animal tissue or fluids that are contaminated or may be
contaminated with pathogenic organisms or recombinant DNA which may be hazardous to humans,
animals, plants, or the environment.
Blood and Blood Products - human blood, blood products (such as serum, plasma, and other
blood components), and body fluids.
Decontamination - use of physical or chemical means to remove, inactivate, or destroy agents on
a surface or item to the point where they are no longer infectious particles and the surface or item is
rendered safe for handling, use, or disposal. Decontamination is generally considered to be log 6
reductions, not sterilization.
Disinfectant - an agent that destroys harmful bacteria and/or viruses on inanimate surfaces.
Common types include household bleach, quaternary ammonium compounds, phenolic
compounds, and iodophors. Products making disinfectant claims must be registered with the
Environmental Protection Agency (EPA), and state it on the label with a registered EPA number.
Infectious Waste - hazardous waste which is capable of causing infections in humans, including
but not limited to, contaminated animal waste human blood and blood products, pathological waste,
and discarded sharps.
Medical Waste - any solid waste that is generated in the diagnosis, treatment, or immunization of
human beings, in research pertaining thereto, or in the production or testing of biological, including
but not limited to: blood-soaked bandages, discarded surgical gloves - after surgery, used sharps,
cultures, stocks, and swabs used to inoculate cultures, removed body organs.
Microbiological - wastes including all cultures and stocks of infectious agents. These should be
collected in appropriate containers, autoclaved, and discarded with regular trash (solids) or into the
sanitary sewer via sink (liquids).
Pathological - wastes including tissues, organs, and body parts discarded from surgical,
obstetrical, autopsy, and laboratory procedures.
Sharps - includes needles, syringes, scalpels, and glass vials. These should be placed in sharps
containers.
12.2 Waste Procedures
Separation and labeling of infectious waste (which may include red bagging, universal biohazard
symbol, etc.) shall be done at the point of generation. During collection, storage and transportation,
all waste shall be managed such that the integrity of the packaging is preserved and that rapid
microbial growth and putrefaction is inhibited. Sharps containers should be rigid, impervious and
puncture-resistant; plastic bags should be tear-resistant, leak-resistant, and sturdy enough to
withstand handling. Whenever possible, infectious waste should be treated to render it noninfectious and non-recognizable as to its former character.
Infectious waste - Should be properly treated to render it non-infectious. Autoclaving and chemical
treatment are the most common methods. This material shall be placed in autoclave bags or other
clearly identifiable containers and properly labeled with autoclave tape or other means that show
that the waste is no longer hazardous. Human blood and blood products SHALL NOT be disposed
into the sanitary sewer.
Autoclaving of Biohazardous Waste - Autoclave bags labeled with Biohazard symbol, containing
biohazardous waste should be decontaminated in an autoclave designated for this purpose
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Autoclave bags should not be overfilled and should be placed in a secondary container prior to
autoclaving. If you do not have access to a designated biohazardous waste decontamination
autoclave, please contact the IBC or EH&S.
Medical waste – Red bags with the universal biohazard warning label are for waste which has not
been treated to make it non-infectious. Material that must be incinerated includes all forms of
medical waste. The use of a red bag says that “this bag and its contents must be incinerated”.
Designate a point in the laboratory for storage and pick up of medical waste bags, sharps
containers and other infectious waste.
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 from
infected animals should be considered infectious. When preserved animal carcasses or tissue are
ready for disposal, the researcher should contact RM & EHS. Animal waste will be placed in
plastic-lined 55-gallon drums. The RM & EHS will then be contacted to arrange for the disposal of
the waste. Do not place animal carcasses in with other biohazardous waste to be autoclaved.
12.3 Decontamination of Laboratory Surfaces
Laboratory surfaces should be decontaminated after work with biohazardous materials and at the
end of the day. Disinfectants should be examined for efficacy against the infectious agent in use.
Disinfectants should be prepared according to the manufacturer directions for dilution and shelf-life.
Storage of disinfectants should be in properly labeled containers.
13.0 Opening and Closing a Research Laboratory
It is the responsibility of all laboratory directors, faculty members conducting laboratory research, and
principal investigators conducting such work to ensure that the appropriate units below receive advance
notification when they:
 move into new laboratories,
 move into different laboratories,
 expand into new laboratory space,
 leave the University,
 cease laboratory research, or
 change, initiate, or cease their use of radioactive materials, hazardous materials, or infectious
agents within their laboratories.
They will also be responsible for prompt notification of the appropriate director or department chair
concerning any such changes and will provide the director or chair with copies of all correspondence taking
place between them and the units concerned. Directors and chairs should send new faculty a copy of the
University's safety requirements for accepting or transferring hazardous, radioactive or infectious materials
well in advance of their moving to DePaul University. Directors and chairs are responsible for
developing their own internal procedures to ensure that labs are not vacated and left in an unsafe
condition.
Labs being vacated will be inspected for contamination by the appropriate department or individuals
depending on hazard type and any hazardous or radioactive waste will be removed; unneeded chemicals
and radioactive materials will also be removed. New lab spaces will be inspected for compatibility with the
intended research operations and any safety deficiencies will be identified. Failure to give advance
notification of laboratory moves could result in delays in start up of new labs, additional cleanup and
disposal costs
13.1 Radioactive Materials
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Exiting existing labs and set up of new labs for radioactive materials use shall be approved by Dr.
John Dean, the Radiation Safety Officer at (773) 325-2187. Authorized users of radioactive
materials shall contact the Radiation Safety officer for an inspection one week before the
anticipated move or closure date. Authorized users are responsible for correcting any problems
identified during the inspection.
13.2 Hazardous Materials
Lab directors, faculty members and principal investigators are responsible for knowing the identity
of and labeling all chemicals and other hazardous materials in areas under their control and
cleaning areas where chemical contamination has occurred prior to leaving and turning the space
over to someone else. RM & EHS can consult on the clean up process. Unusable chemicals shall
be labeled as hazardous waste.
13.3 Infectious Agents
An inventory of infectious agents (i.e., cell cultures, parasites, microorganisms, vectors, etc.) shall
be submitted to the IBC two weeks in advance of the lab move or closure date. Lab directors,
faculty members and principal investigators are responsible for appropriately disposing of infectious
material and decontaminating their laboratories and equipment prior to departure.
13.4 Suggestions for Setup of a New Laboratory
The following items are suggested:
 A folding stepstool is a much safer choice than a rolling stepstool.
 A complete, contained first aid kit.
 A spill kit including leak-proof containers, forceps, paper towels, absorbent material
(Bentonite clay/cat litter), disinfectant, eye protection, and rubber gloves.
 All personal protective equipment for procedures to be performed should be in stock in
sufficient quantities.
 Hand care items including antimicrobial soap or regular hand soap and non-petroleum based
hand lotion should be available.
 Sufficient training of laboratory personnel should be completed prior to the commencement of
research.
 Submission of a research protocol to the IBC, shall be done prior to the initiation of any
laboratory experiments involving biohazardous agents.
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14.0 APPENDIX
14.1 APPENDIX A: Biosafety Levels
Below is a summary of practices, equipment and facility requirements for agents assigned to
biosafety levels 1–4 (BL 1–4). Additional information on biosafety levels may be found in the
BMBL,
which
is
available
on-line
at
update
link
http://www.cdc.gov/od/ohs/biosfty/bmbl5/BMBL_5th_Edition.pdf.. Only work at biosafety levels 1-2
is permitted at DePaul University. No work at biosafety level 3 and 4 is allowed at DePaul
University.
Biosafety
Level
1
2
3
4
Agents
Not known to
cause disease
in healthy
adults.
Associated with
human
disease,
hazards are
autoinoculation,
ingestion,
mucous
membrane
exposure.
Indigenous or
exotic agents
with potential
for aerosol
transmission;
disease may
have serious or
lethal
consequences.
Dangerous or
exotic agents
which pose
high risk of lifethreatening
disease,
aerosoltransmitted lab
infections; or
related agents
with unknown
risk of
transmission.
Practices
Standard
Microbiological
Practices.
BSL-1 practice plus:
Limited access;
Biohazard warning
signs; "Sharps"
precautions;
Biosafety manual
defining any needed
waste
decontamination or
medical surveillance
policies.
BSL-2 practice plus:
Controlled access;
Decontamination of
all waste;
Decontamination of
lab clothing before
laundering; Baseline
serum.
BSL-3 practices plus:
Clothing change
before entering;
Shower on exit.
All material
decontaminated on
exit from facility.
Safety Equipment
(Primary Barriers)
None required.
Facilities
(Secondary Barriers)
Open bench top, sink
required.
Class I or II BSCs or
other physical
containment devices
used for all
manipulations of agents
that cause splashes or
aerosols of infectious
materials; PPE:
laboratory coats; gloves;
face protection as
needed.
Class I or II BSCs or
other physical
containment devices
used for all
manipulations of agents;
PPE: protective lab
clothing; gloves;
respiratory protection as
needed.
All procedures
conducted in Class III
Basks or Class I or II
Basks in combination
with full-body, airsupplied, positive
pressure personnel suit.
BSL-1 plus an
autoclave is available.
BSL-2 plus physical
separation from access
corridors, self-closing,
double- door access,
exhausted air not
recalculated, negative
airflow into laboratory.
BSL-3 plus separate
building or isolated
zone, dedicated
supply/exhaust,
vacuum, and deacon
systems, other
requirements outlined
in BMBL.
Adapted from the Office of Health and Safety, Centers for Disease Control and Prevention
Page 27 of 30
14.2 APPENDIX B: Routes of Exposure
Page 28 of 30
14.3 APPENDIX C: Transport
Page 29 of 30
15.0 References:
Biosafety in Microbiological and Biomedical Laboratories
http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm
NIH Guidelines for Research Involving Recombinant DNA Molecules
http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm
Rutgers Environmental Health and Safety Website
http://rehs.rutgers.edu/
University of Kentucky Environmental Health & Safety Website
http://ehs.uky.edu/welcome.html
World Health Organization Laboratory Biosafety Manual
http://www.who.int/csr/delibepidemics/WHO_CDS_CSR_LYO_2004_11/en/
Yale University Biological Safety Manual
http://www.yale.edu/ehs/Documents/Bio/Biosafety%20Manual.pdf
16.0 CONTACTS FOR IBC
o
Joanna Brooke, Ph.D., IBC Chair
 jbrooke@depaul.edu or (773) 325-1161
o
Jenny Graham, RM & EHS Analyst
 jgraham5@depaul.edu or (773) 325-4201
o
Diana Alfaro, Research Protections Assistant
 dalfaro@depaul.edu or (312) 362-7592
o
Dr. John Dean, Radiation Safety Officer
 jdean@depaul.edu or (773) 325-2187
o
Facilities Operations
 (773) 325-7377
o
Public Safety
 (773) 325-7777
o
Emergencies
 911
Page 30 of 30