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BSL 2 Laboratory
Biosafety Manual
Version 1.0
Idaho State University, Office for Research
Institutional Biosafety Committee (IBC)
1651 Alvin Ricken Drive, Pocatello, ID 83201-8046
Phone: 208-282-2179 Fax: 208-282-4723
Principal Investigator:
Laboratory Location:
TABLE OF CONTENTS
SECTION 1.0 RESPONSIBILITIES
1.1 Principal Investigator
1.2 Research Personnel
SECTION 2.0 AGENTS AND PROJECTS COVERED UNDER THIS MANUAL
2.1 Infectious Agents
2.2 Recombinant DNA (rDNA)
2.3 Human or Nonhuman Primate Materials
2.4 Biological Toxins
2.5 Select Agents
SECTION 3.0 MEDICAL SURVEILLANCE PROGRAM
3.1 General
3.2 Bloodborne and Airborne Pathogens
3.3 rDNA
3.4 Agent Specific
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SECTION 4.0 PROCEDURES FOR BSL-2
4.1 General Signage
4.2 Entry and Exit Procedures
4.3 Procedures for Working in Biosafety Cabinet (BSC)
4.4 Handling of Sharps
4.5 Decontamination of Liquid Waste
4.6 Decontamination of Solid Waste
4.7 Decontamination of Work Surfaces
4.8 Spill Clean Up
4.9 Transport of BSL-2 Agents
4.10 Shipping and Receiving
4.11 Protocol Specific Procedures
4.12 Procedure for Visitors
4.13 Handling of Animals
4.14 The Safe Use of Autoclaves
SECTION 5.0 CERTIFICATIONS
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SECTION 1.0- RESPONSIBILITIES
1.1- Principal Investigator
1. Will assure that all research and support personnel obtain required training on the
potential hazards associated with the work involved, the necessary precautions to
prevent exposures, the exposure control/medical surveillance plan, and the incident
reporting procedures.
2. Will assure that all research personnel are proficient in standard and special
microbiological practices before working with BSL2 agents.
3. Will assure that documentation of training is maintained in the laboratory and available
for inspection.
4. Will assure that biosafety procedures are incorporated into standard operating
procedures for the laboratory and that the laboratory maintains written policies and
procedures for handling of biohazardous agents.
5. Will assure that personal protective equipment and necessary safety equipment is
provided and used.
6. Will assure that all laboratory personnel and support personnel are compliant with the
relevant regulations, guidelines, and policies.
7. Will submit an incident report form to the IBC concerning reportable incidents as
outlined in the ISU IBC Policy.
8. Will review and update the Laboratory-Specific Biosafety Manual at least annually and
more frequently if procedures and practices change.
9. Additional responsibilities in this laboratory:
1.2- Research Personnel
1. Will participate in and complete all required training.
2. Will follow biosafety procedures and practices outlined in this manual and the ISU
Biosafety Manual.
3. Will report incidents of exposure or accidents as outlined in the ISU IBC Policy to the
Principal Investigator/Laboratory Supervisor.
4. Will comply with all aspects of the exposure control/medical surveillance plan for the
agents covered by this biosafety manual.
5. Will review this Biosafety Manual at
least annually and more frequently if procedures
and practices change.
6. Additional responsibilities in this laboratory:
SECTION 2.0- AGENTS AND PROJECTS COVERED UNDER THIS MANUAL
2.1 Infectious Agents-Applicable Not Applicable
List all infectious agents including viral vectors (name and strain designation) used in
the laboratory that will be covered by this biosafety manual:
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2.2 rDNA or synthetic DNA Materials- Applicable Not Applicable
List all rDNA or synthetic DNA materials other than viral vectors used in the laboratory
that will be covered by this biosafety manual:
2.3 Human or Nonhuman Primate Materials-Applicable Not Applicable
List all human/nonhuman primate derived materials (e.g., primary tissues, blood/serum,
bodily fluids, cell lines) that are used in the laboratory that will be covered by this
biosafety manual:
2.4 Biological Toxins-Applicable Not Applicable
List all biological toxins that are used in the laboratory that will be covered by this
biosafety manual:
2.5 Select Agents-Appli
cable Not Applicable
List all select agents that are used in the laboratory that will be covered by this biosafety
manual:
Section 3.0 MEDICAL SURVEILLANCE PROGRAM
3.1 General
All personal are to be instructed that their health status may have an impact on their
susceptibility to infection and, if required, their ability to receive immunizations or prophylactic
interventions. Therefore, all laboratory personnel and particularly women of childbearing age
will be provided with information regarding immune competence and conditions that may
predispose them to infection. Personnel that have conditions that would render them more
susceptible to infection or who are pregnant, will be encouraged to self-identify to these issues
to the Principal Investigator and their personal physician
such that appropriate counseling and
guidance can be provided.
3.2 Bloodborne and Airborne Pathogens
Bloodborne pathogens are defined as pathogenic microorganisms that are present in human
blood and that can cause disease in humans. These pathogens include but are not limited to
hepatitis B (HBV) and human immunodeficiency virus (HIV). Other potentially infectious
material (OPIM) includes the following: (1) human body fluids: cerebrospinal, synovial, pleural,
pericardial, peritoneal, amniotic, semen, vaginal secretions saliva in dental procedures; all body
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fluids, and secretions; all body fluids in situations when it is difficult to differentiate between body
fluids; (2) Any unfixed tissue or organ (other than intact skin) from a human living or dead; (3)
HIV-containing cell or tissue culture, organ culture, and HIV or HBV-containing culture medium
or other solutions; and (4) blood, organs or other tissues from experimental animals infected
with HIV or HBV. For the purposes of this manual, all human cell lines, as well as nonhuman
primate blood, unfixed tissues, body fluids, cells, and cell lines, will also be considered to have
the potential of carrying bloodborne pathogens.
Airborne pathogens are disease-causing microorganisms that spread from person to person in
the form of droplet nuclei in the air. Airborne pathogens can be viral, bacterial or fungal in
nature. Meningitis, influenza, pneumonia, and tuberculosis are examples of diseases
transmitted through the air. Personnel receiving bloodborne pathogens training will also receive
training on airborne pathogens.
All personnel working with any of the potentially infectious materials listed above must
complete Bloodborne pathogen training on an annual basis. Documentation of this
training must be maintained with this manual and provided to the IBC or TSO upon
request. Bloodborne pathogen training certifications are attached to this manual.
Applicable Not Applicable
All personnel will be provided information on the Hepatitis B vaccine that will include: efficacy of
the vaccine, its safety, method of administration, benefit of administration, benefits associated
with vaccination, and encouraged to obtain the vaccine from their personal physician.
An exposure incident is defined as a specific eye, mouth, other mucous membrane, non-intact
skin, or parenteral contact with blood or other potentially infectious materials that resulted from
the performance of an employee's duties. If exposed to blood or other potentially infectious
materials, first determine if it meets the definition of an exposure incident. Blood or fluids
splashed onto intact skin are not exposure incidents, but require skin to be washed immediately.
Exposure to saliva that is not visibly contaminated with blood does not constitute an exposure
incident. If it is determined that an exposure incident has occurred, the exposed employee will
immediately report the incident to his/her supervisor. The Supervisor/PI must report the
exposure to the ISU Technical Safety Officer. The employee will report to their personal
physician or an emergency department for post exposure evaluation and follow-up. Employees
must familiarize themselves with ISU’s policies on exposure control for bloodborne pathogens.
3.3 rDNA
In general, the risk of exposure and illness from working with rDNA is very low; however, risks
do exist and all personnel working with these reagents must be aware of these potential risks.
The viral supernatants produced by transfecting packaging cell lines with recombinant viral DNA
could, depending on the DNA insert, contain potentially hazardous recombinant virus. Due
caution must be exercised in the production and handling of recombinant viruses even if they
are replication defective. The potential risks include exposure to unknown pathogens from the
cell culture systems used to propagate/package the vectors, anticipated or unknown effects
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from expression of a foreign gene or overexpression of an endogenous gene, anticipated or
unknown effects from suppression of expression of an endogenous gene, or insertional
mutagenesis. These issues are especially of concern when viral vectors are used to express a
known or suspected oncogene or cell cycle regulatory proteins, or when they are used to
suppress the expression of endogenous tumor suppressors.
If an exposure incident as defined under section 3.2 occurs, the same procedure for reporting
and follow-up is to be followed. In addition, the principal investigator must complete an IBC
incident report form and submit it to the Institutional Biosafety Committee. The report must be
submitted within one week of the incident.
3.4 Agent Specific Medical Surveillance Plans
For all BSL-2 pathogens used in the laboratory and covered by this manual, list the signs and
symptoms of illness from these pathogens, the usual sequelae of the disease, the natural, as
well as, laboratory routes of transmission, and indicate the actions that employees should take if
personnel display these signs and symptoms.
If risk assessment by IBC determines it is necessary, a medical surveillance plan may need to
be developed in coordination with the IBC and the Technical Safety Officer.
If an exposure incident as defined under section 3.2 occurs, the same procedure for reporting
and follow-up is to be followed. In addition, the principal investigator must complete an IBC
incident report form and submit it to the IBC. The report must be submitted within one week of
the incident.
Section 4.0 PROCEDURES FOR BSL-2
4.1 General Signage
1. A Universal biohazard symbol will be posted at the entrance to the laboratory when
infectious agents or materials that are handled at BSL-2 (e.g., recombinant DNA,
human cells, and tissues, biological toxins) are present. The biohazard label will read
“Biohazard” “Low Risk.”
2. A Laboratory Door Sign will be posted at the laboratory entrance. The sign must
include the following information:
a. Room, Building, Department, and Date.
b. Room Use/Description such as Research Laboratory.
c. The types of potential hazards present in the laboratory will be checked off on
the card. For Biological Hazards the terminology “Low Risk” will be checked off.
The specific type of agent in use will not be listed.
d. Emergency Con
tact information for the following personnel will be posted on the
card:
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4.2 Entry and Exit Procedures
1. Access to this laboratory is restricted to those personnel approved by the principal
investigator, , when work with BSL-2 agents is in progress.
2. Entry into this laboratory requires participation in medical surveillance plan.
Applicable Not Applicable
3. Entry into this laboratory requires vaccination against:
Applicable Not Applicable
4. Good Laboratory Practices must be followed at all times.
a. Eating, drinking, chewing gum, smoking, handling contact lenses, or applying
cosmetics is prohibited in this laboratory.
b. All food for human consumption must be stored outside the laboratory area in
cabinets or refrigerators designated for this purpose.
c. Mouth pipetting is prohibited. Mechanical pipetting devices must be used.
d. Personal items such as coats, boots, bags and books should not be stored in
the laboratory.
e. All procedures will be conducted such that the creation of splashes and
aerosols are minimized. Whenever possible, all procedures that generate
aerosols will take place in a biosafety cabinet.
5. No animals or plants may enter this laboratory unless used specifically for the
research being performed and approved by the IACUC. If animals are used in the
context of an IBC protocol, use in the laboratory must be approved by the IBC.
6. Appropriate PPE must be worn when handling BSL-2 agents.
a. Inside biosafety cabinet, the following PPE must be used.
i. Gloves must be worn when handling or working with BSL-2 agents in
the biosafety cabinet.
ii. Gloves must be changed when contaminated, integrity has been
compromised, or when otherwise necessary. Two pair of gloves may be
required for some procedures.
iii. Laboratory coat, gown, smock, sleeve protection, or uniform designated
for laboratory use must be worn while working with BSL-2 agents.
b. Outside biosafety cabinet on open bench
i. Gloves must be worn when handling or working with BSL-2 agents
outside the biosafety cabinet.
ii. Gloves must be changed when contaminated, integrity has been
compromised, or when otherwise necessary. Two pair of gloves may be
required for some procedures.
iii. Laboratory coat, gown, smock, or uniform designated for laboratory use
must be worn while working with BSL-2 agents.
iv. Eye Protection and Face protection (goggles, mask, face shield, or other
splatter guard) must be worn if procedure may produce aerosols
(splashes or sprays).
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v. Respiratory Protection (N95 or PAPR) must be worn if risk analysis
determines that procedure/agent requires this protection.
7. Upon completion of work with BSL-2 agents, the following procedures must be done.
i. Remove and discard gloves in biohazard waste. Disposal gloves may
not be washed or reused.
ii. Wash hands.
iii. Remove Laboratory coat, gown, smock, or uniform before leaving
laboratory for non-laboratory areas such as cafeteria, library, or
administrative offices. For disposable protective clothing, place in
biohazardous waste. For reusable protective clothing, hang in
designated area in laboratory for reuse, or place in designated area for
laundering by the institution. Protective clothing should not be taken
home.
iv. Eye and face protection must be disposed of with contaminated waste
or decontaminated after use.
v. Wash hands before exiting.
4.3 Procedures for Working in Biosafety Cabinet in PI’s Laboratory.
Make
Model
Location (Bldg/Room)
Cabinet Last Certified
1. Biosafety cabinet is operated: Continuously As Needed
If as needed is checked, please indicate the minimum time cabinet blower is on to prior
beginning work: After work in completed.
2. Other activities (e.g., rapid movement, open/closing room doors, etc.) in the room will
be minimized when operations are being conducted in the biosafety cabinet to avoid
disrupting the cabinet air barrier.
3. PPE as outlined in section 4.2 will be worn when working in the biosafety cabinet.
4. Before beginning work, stool height will be adjusted such that personnel’s face is
above the front opening. The sash should be set at the recommended height in order
for proper cabinet operation and user protection. The cabinet user should adjust their
shoulder height to be level with the lower edge of the sash.
5. Closure of
the drain valve under the work surface will be done prior to beginning work
so that all contaminated materials are contained within the cabinet should a large spill
occur.
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6. Wipe down the interior of the cabinet with an appropriate surface disinfectant(e.g.,10%
commercial bleach solution, 70% alcohol, or similar non-corrosive antimicrobial agent)
7. Materials needed for work in the biosafety cabinet will be placed in the cabinet prior to
beginning work to avoid disruption of airflow. Materials will be placed as far back in the
cabinet as is practical.
8. All operations within the cabinet will be performed on the work surface at least four (4)
inches from the inside edge of the front grille.
9. If plastic-backed absorbent toweling is placed on work surface it will be placed such
that it does not cover front or rear grille openings.
10. The front grille will not be blocked with research notes, discarded plastic wrappers,
pipetting devices, etc.
11. The number of arm-movement disruptions across the air barrier of the cabinet will be
minimized.
12. If aspirator suction flasks are used, two flasks will be connected in series, and they
will be pre-filled with appropriate disinfectant such that the final concentration is
sufficient to kill the microorganisms. A filter (either 0.3 µM or HEPA) will be placed in-
line along with a second flask to prevent overflow into building vacuum system.
List disinfectant and final concentration:
10% final concentration of household bleach
Other – List agent and concentration:
Not Applicable
Contact time must be at least 30 minutes and then liquid waste should be
handled as described in 4.5. NOTE: Disposal must be done on at least a daily
basis.
13. Horizontal pipette discard trays containing an autoclave bag or an appropriate
chemical disinfectant will be used within the cabinet. Upright pipette collection
containers placed on the floor outside the cabinet or autoclavable biohazard collection
bags taped to the outside of the cabinet should not be used. The frequent
inward/outward movement needed to place objects in these containers is disruptive to
the integrity of the cabinet air barrier and can compromise both personnel and product
protection.
14. Active work should flow from the clean to contaminated area across the work surface.
Bulky items such as biohazard bags, discard pipette trays and suction collection flasks
must be placed to one side of the interior of the cabinet.
15. Use of glass Pasteur pipettes is discouraged. Glass pipettes should be replaced with
safer alternatives (i.e., plastic) as recommended by the World Health Organization
Biosafety Manual (WHO, 2003). Contact the Technical Safety Office for more
information on safer alternatives.
16. Indicate if open flame will be used in BSC and describe use or indicate N/A:
17. Indicate if UV light will be used in BSC and describe use or indicate N/A:
18. Upon completion of work, the interior surfaces of the cabinet will be wiped down with
the following disinfectant:
10% final concentration of household bleach made daily followed by 70% ethanol
to remove bleach residue.
Chlorine Dioxide. Final concentration:
Other – List agent and concentration:
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hands will be washed.
1. Sharps, such as needles, scalpels, contaminated glass pipettes, and broken
contaminated glassware will be handled in the following manner:
a. Whenever possible, use of sharps with potentially hazardous material will be
avoided. Plasticware will be substituted for glassware whenever possible.
b. The handling of sharps will be minimized. Needles will not be bent, sheared,
broken, recapped, removed from disposable syringes, or otherwise manipulated
by hand before disposal.
c. Used disposable needles and syringes will be carefully placed in a puncture-
resistant containers used for sharps disposal. Sharps containers will not be
beyond ¾ full.
d. Broken glassware will not be handled directly. It will be removed using a brush
and dustpan, tongs, or forceps and properly disposed of glassware waste.
2. Disposal of biohazardous sharps. When sharps containers are ¾ full, contact
at extension for disposal.
3. Non-disposable sharps that are contaminated with infectious material will be placed in
a hard walled container for transport to a processing area for decontamination.
Not Applicable or Applicable
List Non-disposable sharps used:
1. The following method will be used for decontamination of liquid biohazardous waste:
10% final concentration of household bleach
Other – List agent and concentration:
Not Applicable
Contact time must be at least 30 minutes
2. Following decontamination, liquid may be disposed of down the sink and the sink
rinsed with water.
1. Solid waste that has been in contact with potentially infectious materials will be disposed
of in the following manner:
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lines and tissues, and containing replication incompetent viral vectors).
NOTE: ALL waste generated by the laboratory within a specific location must qualify as low risk.
IF not, ALL waste generated in that location much be treated as moderate risk.
Autoclaved and disposed – All biohazardous waste is placed in biohazard bag, placed in
autoclavable container (i.e., polypropylene tray), and transported to autoclave on a cart. Waste
is autoclaved in the tray and then placed in black plastic bag and discarded in standard waste.
Commercial. All biohazardous waste is placed in biohazard bag inside solid biohazard
container. TSO approved commercial biohazard disposal company retrieves and disposes
biohazard waste in compliance with State and Federal regulation.
Moderate Risk Waste (BSL-2 waste containing any replication competent infectious agent
or BSL-1 waste generated in the same location as moderate risk waste).
Autoclaved and disposed – All biohazardous waste is placed in biohazard bag, biohazard
bag is placed in autoclavable container (i.e., polypropylene tray), and transported to autoclave
on a cart. Waste is autoclaved in the tray and then placed in black plastic bag and discarded in
standard waste. (This is only for those laboratories that do not have commercial waste disposal).
Autoclaved and then commercial disposal. All biohazardous waste is placed in biohazard
bag, biohazard bag is placed in autoclavable container (i.e., polypropylene tray), and
transported to autoclave on a cart. Waste is autoclaved in the tray and then placed inside solid
biohazard container. TSO approved commercial biohazard disposal company retrieves and
disposes biohazard waste in compliance with State and Federal regulation.
1. Work surfaces will be decontaminated after completion of work and immediately
cleaned after any spill or splash of potentially infectious material. The following
method will be used for routine decontamination of work surfaces:
10% final concentration of household bleach made daily followed by 70% ethanol to
remove bleach residue.
Chlorine Dioxide. Final concentration:
Other – List agent and concentration:
2. All equipment coming into contact with biohazardous material must be decontaminated
before repair, maintenance, or removal from the laboratory.
1.
Spills will be handled in accordance with ISU policies described in the ISU Hazardous
Waste Policy Manual. http://www.physics.isu.edu/health-physics/tso/
hazman2008complete.pdf All personnel will be familiar with these procedures.
Spills > 1 lit
er are considered large spills and must be immediately reported to
Technical Safety Office (208-282-2310 or pfarina@isu.edu).
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3. Spills that result in potential exposure to BSL2 agents or that occur outside of the
biosafety cabinet must be reported to the principal investigator. The principal
investigator must complete an IBC incident report form and submit to the Institutional
Biosafety Committee. The report must be submitted within one week of the incident.
1. The following potentially infectious materials will be transported from to
.
2. The materials/agents will be contained in the following primary containment .
3. The primary containment will be placed in a secondary containment, which is non-
breakable and sealed.
1. If potential infectious materials will be shipped from the facility, the technical safety
office must be contacted for guidance, x2310. All appropriate local, state and federal
(U.S. Department of Transportation) regulations must be followed. For air or
international shipments, International Air Transportation Association (IATA) rules must
be followed.
2. Personnel responsible for shipping will complete the appropriate training for packaging,
labeling and shipping of all infectious materials. Contact the Technical Safety Office
for training on shipping dangerous goods. Documentation of this training must be
maintained with this manual and provided to the IBC or TSO upon request.
3. For receiving only, the ISU policies on receiving potentially infectious material must be
followed. Please contact the Technical Safety Office for guidance prior to receiving
any potentially infectious materials.
Describe any laboratory specific procedures required when handling biohazardous material.
Examples of procedures needed include, but are not limited to, centrifugation, sonication,
shakers, etc.
1. Centrifuge use:
Not Applicable Applicable
Describe the containment including if sealed rotors are used:
2. Shaker use:
Not Applicable Applicable Describe the containment:
3. Sonicator use:
Applicable
Not Applicable Describe the containment:
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4. Other: Not Applicable Applicable Describe the containment:
ISU BSL2 Laboratory Biosafety Manual/IBC
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4.12 Procedures for Visitor
1.
All visitors to the laboratory must comply with the ISU’s VISITORS TO CAMPUS
LABORATORIES PROCEDURES POLICY to ensure that they are informed of the
potential hazards and receive the appropriate training.
2.
All volunteers must sign an Acceptance of Risk, Waiver and Release.
4.13 Procedures for Handling Animals
Disregard this section if it is not applicable. Procedures described apply to rodents and must be
modified for non-rodent species.
Unless specifically described in approved IACUC and IBC protocols all BSL-2 work performed in
rodents will be performed in designated BSL-2 rooms in core animal facilities. Animals
infected/inoculated with replication incompetent/defective adeno-associated, adenoviral,
retroviral, or lentiviral vectors must be housed at BSL-2 for 7 days post-infection due to the
potential for shedding of the viral vectors (see section 3.3). Animals infected with replication
competent viral vectors or infectious agents must be housed at BSL-2 for the entire duration of
the study post-inoculation. To gain access to these rooms, personnel must first be listed on an
approved IACUC and IBC protocol that covers the BSL-2 work to be performed. If work with the
agent requires vaccination, this is administered by the employee’s personal physician.
Personnel must meet with an approved veterinarian to receive training in animal BSL-2
procedures before access to the room will be granted. In addition, annual training is required for
all personnel with access to BSL2- rodent rooms.
1.
Access: All BSL-2 rodent rooms are locked at all times. Access is restricted to only
individuals that have completed BSL-2 training with a veterinarian.
2.
Signage: For each agent being used in the BSL-2 room, a BSL-2 Laboratory sign is
listed on the outside of the door. The sign indicates: agent, entry requirements,
required PPE and emergency contact information.
3.
PPE: Personal Protective Equipment (PPE) is provided in BSL-2 rodent rooms.
Required PPE includes gown or tyvek jumpsuit, hair bonnet or cap, N95 respirator
mask, nitrile gloves and shoe covers.
4.
Handling of sharps: Sharps containers are readily available in the room. All needles
and other sharps are disposed of in the sharps container. Needles should not be
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5. Storage: Any items to be stored in the room must be contained in containers that are
readily disinfected. No paper bags, cardboard boxes or Styrofoam are allowed for long-
term storage in the room.
6. Handling of infectious agent/ inoculum: Investigators only bring enough infectious
agent to the room to complete the study for a given day. Infectious materials should
transported to and from the room in a spill and leak proof secondary container. No
storage of infectious agent is allowed in the animal room. Containers or syringes with
infectious agent must be under the direct supervision of the research personnel at all
times in the animal room.
7. Work Within the Biosafety Cabinet: All BSL-2 rodent rooms are equipped with a
biosafety cabinet (BSC). All work with animals, cages or handling of infectious
materials is performed within the BSC according to the guidelines outlined in section
4.3:
a. The work surface of the hood is thoroughly disinfected with clidox.
b. Required materials are disinfected and placed in the hood.
c. Personnel ensure that the sash is placed at the proper level as indicated by a
line on the frame of the sash.
d. The fluorescent light and blower fan is turned on. Personnel check to be sure
that the magnehelic gauge reads between and .5 +/-.1 psi. If the gauge reads
below this range, contact the area supervisor or veterinarian. The blower fan on
the hood must run for at least 5 minutes prior to beginning work.
e. If the hood alarms determine and correct the cause of the alarm (e.g. lower the
sash to the appropriate level). If a cause cannot be determined, personnel are
required to immediately stop work and contact the area supervisor or
veterinarian.
f. When work is completed, the hood is thoroughly disinfected with clidox, and
allowed to run for at least 5 minutes prior to turning off.
8. Disinfection: Chlorine dioxide disinfectant is readily available in the room and is used
to disinfect all work surfaces in the room. If warranted by the infectious agent,
alternative disinfectants will be considered. Standard operating procedures for
handling mice SPF mice are used when working with mice in the BSC.
9. Euthanasia: CO2 is available in all BSL-2 rodent rooms for euthanasia. Rodents
infected with a BSL2 agent should not be removed from the BSL-2 rodent room, for
any reason, unless approved in both corresponding IBC and IACUC protocols.
10. Disposal of animal carcasses and waste: Following the completion of work,
personnel dispose of all waste, thoroughly disinfect the work surface of the BSC and
any other surfaces in the room that may be contaminated.
a. Trash is disposed of in autoclavable biohazard trash bags within red foot
operated trash cans.
b. Animal carcasses are placed in clear plastic bags. A red biohazard sticker
labeled with the agent is placed on the outside of the bag, which is then placed
in refrigerator designated for carcasses within the room.
c. Soiled cages are reassembled and bagged in autoclavable biohazard trash
bags within the BSC. Up to three cages may be placed in a single bag. The bag
is closed and secured with a rubber band. A red biohazard sticker labeled with
the agent is then placed on the outside of the bag. The bag is thoroughly
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sprayed with disinfectant and placed in a designated location within the room.
11. Decontamination of waste: All waste and soiled cages is removed regularly by the
BRL staff to be decontaminated by autoclaving for 250
0
F for 40 minutes unless another
method is indicated by the agent and approved by the IBC. Autoclaved, soiled cages
are then transported to the BRL cage wash area for sanitization. Autoclaved trash from
the room is transported to BRL room 11 to be removed by Stericycle.
12. Exit procedure: Any items to be removed from the room are contained in a closed
impermeable container and the outside of the container is thoroughly sprayed with
clidox disinfectant. Under no circumstances should live animals be removed from the
room unless an exception has been granted by the IBC and IACUC.
13. Removal of PPE: To exit the room, personnel thoroughly spray the floor delineated
with yellow and black tape near the door, the inside of the door (including handle), and
red trash can with chlorine dioxide. Wearing clean gloves, PPE is removed in the
following order: hair cover, mask, gown or tyvek jumpsuit and shoe cover. As the shoe
covers are removed, personnel step into the delineated clean area near the door. PPE
is then placed in the foot-operated red trash can. Personnel then exit the room.
14. Spill Clean-up: Spills of infectious agent are handled according section 4.8 of the
investigator’s BSL2 Biosafety Manual. In addition, spills of infectious agent that occur
within the animal facility are reported to the animal facility supervisor or veterinarian. If
cages are spilled, personnel immediately collect any loose animals and put them in a
new clean cage. The spill contents are thoroughly sprayed with chlorine dioxide and
allowed to soak for 15 minutes. The contents of the spill are swept up and placed in a
clear autoclavable bag, labeled with a biohazard sticker, and placed in the designated
location for dirty cages. The floor where the spill occurred is thoroughly sprayed with
chlorine dioxide and mopped up. Upon completion of work, personnel report the spill
to the animal facility supervisor or veterinarian.
15. Exposure procedure: If an individual experiences an injury, such as a needle stick or
bite from a rodent, they immediately stop working and thoroughly wash the site with
soap and water. The individual informs the animal facility supervisor or veterinarian of
the incident. The individual notifies the PI of the incident who then completes an injury
report form. In addition, the PI submits an incident form to the IBC. During and after
normal working hours, the individual goes to the nearest Emergency Room.
4.14 The Safe Use of Autoclaves
1. There are many different types of autoclaves used on the ISU campus. It is the
responsibility of the supervisor to ensure that all authorized individuals are properly
trained on the use of the autoclave(s) used by laboratory personnel.
2. The autoclaves used by this laboratory are listed in the table below:
Location (Room and Building) Department or PI responsible for Autoclave
Page 15 of 17
ISU BSL2 Laboratory Biosafety Manual/IBC
PI:
Version 1.0
3. Training on the use of autoclaves will consist of the following and documentation of
training will be maintained in the laboratory and will be available for review by TSO and
IBC upon request.
a. Appropriate PPE requirements such as the use of heat resistant gloves, lab
coats, and safety eye and face protection.
b. A discussion of the types of items that can and cannot be autoclaved.
c. Proper packaging of biohazardous wastes for autoclaving.
d. Methods for loading materials into an autoclave and unloading procedures.
e. The use of test strips and biological indicators for quality control.
f. Autoclave operational procedures including emergency shutdown precautions.
g. How to dispose of autoclaved waste.
h. Record keeping
4. Maintenance and Testing of Autoclaves
a. Department or PI responsible for autoclave must properly maintain and service.
If autoclave is owned by PI, list service provider and frequency of preventative
maintenance on autoclave:
b. Department or PI responsible for autoclave must occasionally quality tested to
ensure proper sterilization procedures are met and decontamination of
biohazardous waste is complete. If autoclave is owned by PI, list method of
testing autoclave and frequency of testing on autoclave:
Page 16 of 17
Principal Investigator Certification
I hereby certify that I have reviewed these practices and procedures and they represent the
current operating practices in my laboratory.
Signature: Date: Annual Review
(
Personnel Certification
We, the undersigned, have reviewed these practices and procedures, have been trained in the
appropriate methods and practices for handling potential infectious material and agree to follow
the stated practices and procedures. We understand that we must review and document
compliance with these practices and procedures on an annual basis.
Name
Signature
Date
Page 17 of 17
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