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University of Virginia Biosafety Manual
And Standard Operating Procedures
For Biosafety Level 2 Agents
Dr. J. Thomas Parsons
University of Virginia Microbiology Department
Rooms 223, 224, 2216B Jordan Hall
Charlottesville, VA
March 26, 2008
University of Virginia
Biosafety Manual
Principal Investigator Dr. J. Thomas Parsons
Lab Building/Rooms Jordan Hall/ Rooms 223, 224, 2216B
Agents used:
BSL-2 MicroorganismsRooms 2216B & 223 / Human-derived materials
Room 224 / Biotoxins/room
Adenovirus / A549 / IGROV
Lentivirus / Asp-Pc1 / Ishikawa / None
BxPc3 / L3.6pl
CaOV-3 / LNCap
Colo357 / LP-9
DU145 / MCF-7
ES-2 / MD-MBA-231
FG / MD-MBA-468
H23 / MeT5A
H460 / MPanc 96
H1650 / OvCar-3
H1975 / Panc1
HEC-1A / PC3
HEC-50co / PC12
HEK293 / RL-95
HeLa / SKOV-3
SK-UT
Principal Investigator’s Certification
I hereby certify that I have reviewed the contents of this manual and that it reflects my current operating practices.
Signature______Date______
Annual Review:
Signature______Date______
Signature______Date______
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Contact Information
Office of Environmental Health and Safety 982-4911
Institutional Biosafety Committee (IBC) 982-1597
UVA-WorkMed (formerly Occupational Health Services) 243-0075
UVA Employee Health 924-2013
Student Health Center 924-5362
Principal Investigator’s Home Phone 540-248-1225
SIGNATURE and ACKNOWLEDGEMENT PAGE
Authorization
All members of the J.T. Parsons Lab who have signed the list below are approved for entry into Rooms 2-23, 2-24, and 2-2216B while work with BSL 2 agents is in progress. HOWEVER, only those persons who have attended the OSHA Bloodborne Pathogens lecture and are otherwise specifically trained (e.g., the University Biosafety Officer provides a short training program entitled, “Fundamentals of Biosafety”) may perform work with samples or cell cultures in Rooms 2-23, 2-24, 2-2216B.
Anyone (including any workers not in the P.I.’s Lab) who uses Rooms 2-23, 2-24, and 2-216B must sign the disclaimer below.
Disclaimer
We, the undersigned, understand that adenovirus and lentivirus are in use in the Tissue Culture Room (2-23), Room 2-24, and Room 2216B on the second floor of Jordan Hall, and that these agents are infectious to humans. We agree to follow BL2 containment when using adenoviruses, lentiviruses, or virus-infected cells, and to follow appropriate precautions to prevent inadvertent exposures to these agents.
Further, we have read and understood this manual and agree to attend required Bloodborne Pathogens lectures prior to handling samples in Rooms 2-23, 2-24, and 2216B, Jordan Hall.
Name / Signature / Date / HBVYes/No/ Declined? /
Todd Bauer
Cheryl Borgman
Catherine Cowan
Pablo Grigera
Christine Harrer
Dan Hershey
Linda Patchel
Amy Koski
Marcin Iwanicki
Tom Parsons
Jill Slack-Davis
Jayme Stokes
Rob Tilghman
Authorization and Disclaimer for Weber Laboratory
We, the undersigned, understand that adenovirus and lentivirus (low titers) are in use in the Tissue Culture Room (2-23), and Room 2-24, on the second floor of Jordan Hall. We have been informed that the Biosafety Manual for the JT Parsons laboratory is available for review and is located in room 225 A and on the JT Parsons lab web page (http://faculty.virginia.edu/tparsons/). We agree to follow BL2 containment when using adenoviruses, lentiviruses, or virus-infected cells and to follow appropriate precautions.
Name / Signature / Date /
Rooms 223 and 232
Rooms 224 & 225
Table of Contents
Principal Investigator Certification and Agent Inventory 2
Signatures and Acknowledgement of Risk 4, 5
Biosafety Manual (must be completed by all Investigators working with BSL2 microorganisms and/or human-derived materials and/or toxins of biological) origin)
1. Purpose 11
2. Responsibilities 11
3. Background 12
4. Work Practices 14
A. Standard Practices for BSL-2 agents 14
B. Safety Equipment 16
C. PPE 16
D. Lab Standard Operating Procedures 17
5. Waste Disposal 17
A. Liquids 18
B. Sharps 18
C. Solid Wastes 18
D. Research Animal Carcasses 19
6. Spill Clean Up Procedures 19
A. Spills Outside of a Containment Device 19
B. Spills inside a Biosafety Cabinet 20
C. Spills in a Centrifuge 20
D .Biological/Radioactive Emergencies/Spills 21
7. Proper Use of a Biosafety Cabinet 21
8. Emergency Phone Numbers and Procedures 22
A. Fire 23
B. Injury 23
C. Exposure to Biohazardous Materials 23
D .Security Incidents 23
9. Shipping Infectious Substances 24
Appendix A. Exposure Control Plan (must be completed by investigators working with human-derived materials including human cell lines) 25
Appendix B. Toxin Safety Plan (must be completed by investigators working with toxins of biological origin) 37
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1. Purpose
This Biosafety Manual outlines procedures for using and disposing of Biosafety Level 2 (BSL-2) agents. All University laboratories using BSL-2 agents must comply with the procedures in this manual. Principal Investigators or laboratory supervisors must contact the Office of Environmental Health & Safety if they are uncertain how to categorize, handle, store, treat or discard any biohazardous material.
2. Responsibilities
The Principal Investigator:
1. Ensures that laboratory and support personnel receive appropriate training for the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures.
2. Ensures biosafety procedures are incorporated into standard operating procedures for the laboratory.
3. Ensures personal protective equipment and necessary safety equipment is provided and used.
4. Ensures compliance by laboratory personnel with the relevant regulations, guidelines, and policies
5. Reviews and updates the Biosafety Manual annually.
6. Submits a written report to the Institutional Biosafety Committee concerning:
· Any accident that results in inoculation, ingestion, and inhalation of biohazardous materials or recombinant DNA
· Any incident causing exposure of personnel or danger of environmental contamination.
· Any problems pertaining to operation and implementation of biological and physical containment safety procedures or equipment or facility failure.
Laboratory Personnel
1. Participates in appropriate training and instruction
2. Complies with biosafety procedures.
3. Reports all accidents, major spills, or exposure incidents to supervisor
4. Reviews the Biosafety Manual annually.
Additional Responsibilities for this lab -
3. Background
The focus of the laboratory of Dr. J. Thomas Parsons is to study adhesion signaling as it relates to cell growth, migration and metastasis. The potentially infectious biological materials used in this work include viral vectors (adenovirus and retrovirus), and human cells and tissues which will be handled at the BSL2 level. Also, BSL 1 level agents are used such as recombinant DNA, E.coli K12, and baculovirus. Specific details regarding the use of BSL2 level agents is described below.
Avian retroviruses are commonly used to transfer new genetic constructs into cultured chick embryo fibroblast cells (CEF). The constructs used in the Parsons laboratory use a vector system, RCAS-A and RCAS-B, designed by Hughes, et al (J. Virol. 61:3004-3012) in which the gene of interest is cloned into a replication-competent plasmid and transfected into CEF cells. This results in the expression of the introduced gene in CEF cells and also the production of recombinant infectious virus, containing the viral recombinant RNA genome and the viral structural proteins. Virus generated by this procedure is used in further infections of CEF cells. These avian retroviruses are restricted to avian cell lines and are NOT capable of infecting cells of other species. Nevertheless, it is prudent that these viruses and infected cells be handled as a potential hazard if the gene introduced is biologically active (e.g. an oncogene). Examples of such oncogenes used in the laboratory include the tyrosine kinase Src and the small GTPases Ras and Cdc42. Thus, RCAS virus stocks, infected and transfected CEF cells will be handled at the BL2 level of containment, as described below.
Human Adenovirus Type 5 (Ad5) will be employed to transfer genetic constructs into mammalian cancer cell lines and primary rodent cells. The Ad5 genome (100 map units, mu) containing a 4.3 kB insert which renders the virus defective in packaging will be co-transfected into human HEK293 cells with a shuttle plasmid containing the CMV promoter and the coding sequence of the gene of interest flanked by 0-1 mu and 9-16 mu of the Ad5 genome. Following recombination, the cassette will replace the 4.3 kB insert and the E1 region of the genome, making the virus capable of packaging but replication defective. The HEK293 cell line is Ad5 transformed and expresses the E1 region (1-11.3 mu) in trans which allows for replication of the recombinant adenovirus. Virus is collected from cells and purified by cesium-chloride gradient. Virus is then incubated with the cells of interest at 20-100 plaque forming units/cell. The Ad5 virus stocks, infected HEK293 packaging cells and target cells will be handled at the BSL2 level of containment, as described below.
Retroviruses will be used to infect rodent and human cells lines to allow for over expression of defined proteins or expression of small interfering RNAs to selectively suppress the expression of targeted proteins. Appropriate virus stocks are generated using transfection of HEK293 cells with three separate plasmids, each containing a different component of the virus. The transducing vector is pLKO.1 puro (Sigma); it contains a hairpin insert corresponding to the gene to be targeted, an RRE rev response element, self-3' inactivation long terminal repeat and RNA packaging signal. The packaging plasmid is CMVdeltaR8.2 (obtained from David Rekosh, UVa. It has HIV-1 structural proteins, NEF and Rev downstram of CMV promotor. The envelope vector, MDg (David Rekosh) encodes VSV-G under beta globin promotor. These plasmids are transfected into HEK293 T cells and supernatants are collected 48 hrs later. Transfection of HEK293 cells with the three plasmids generates replication defective virus particles that can be used to infect human or rodent cells to effect the knockdown of selected genes in the target cells. The tripartite vector system ensures that there is no infectious virus produced. Generation of the virus and subsequent infection of target cells is carried out using BSL2 containment.
Human cell lines and archival human tumor samples will also be handled at the BSL 2 level because of the possibility that they contain unidentified etiologic agents.
The Parsons laboratory does not use any biotoxins regulated by the Antiterrorism and Effective Death Penalty Act of 1996.
4. Work Practices
Many laboratory procedures using BSL-2 agents can be safely done on the open laboratory bench utilizing good microbiological techniques and appropriate Personal Protective Equipment (PPE) provided the potential for producing splashes or aerosols is low. Work on the open laboratory bench is permitted, except when performing activities with BSL-2 agents that may produce aerosols or splashes.
Procedures with a potential for creating infectious aerosols or splashes must be conducted in a certified Biosafety Cabinet (BSC). These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals or embryonate eggs.
A. Standard Microbiological Practices for Biosafety Level 2 agents
1. Access to the laboratory is restricted when work with BSL-2 agents is in progress. Access may be restricted by locking doors, posting warning signs or using other suitable methods as determined by the Principal Investigator.
2. A biohazard sign must be posted on the entrance to the laboratory when BSL-2 agents are in use. Information to be posted includes the agent(s) in use, the Biosafety level, the investigator's name and telephone number, and any personal protective equipment that must be worn in the laboratory.
3. Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.
4. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the laboratory. Food is stored outside the laboratory.
5. Mouth pipeting is prohibited; mechanical pipeting devices are used.
6. All procedures are performed carefully to minimize the creation of splashes or aerosols.
7. Work surfaces are decontaminated with disinfectants that are effective against the infectious agents present upon completion of work or at the end of the day and after any spill or splash of viable material
8. Chairs and other furniture are covered with a non-fabric material that can be easily decontaminated. Carpets and rugs in laboratories are inappropriate.
9. All cultures, stocks, contaminated wastes and other Regulated Medical Waste (RMW) are disposed of in accordance with University of Virginia Infectious Waste Disposal Procedures (see page 15).
10. A sharps management program is in place including:
a. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative.
b. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in a conveniently located Sharps Container. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
c. Syringes which re-sheathe the needle, needleless systems, and other safety devices are used when appropriate.
d. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps.
11. Cultures, tissues, specimens of body fluids, or potentially infectious wastes are placed in a container with a cover that prevents leakage during handling, processing and storage.
12. Transport of BSL-2 agents to sites within the grounds of UVA, must be placed in a secondary leak proof carrier that can contain the contents if the primary container were to leak or break. Carriers must have the biohazard label affixed to the outer surface of the transport container
13. Contaminated equipment must be decontaminated before it is sent for repair or maintenance or before removal from the laboratory.
14. Spills and accidents that result in overt exposures to infectious materials are immediately reported to the Principal Investigator and UVA-WorkMed.
15. Animals not involved in the work being performed are not permitted in the lab.
16. Additional Special Practices for this Laboratory
· Spills and accidents which result in overt exposure to virus containing oncogenic recombinant DNA molecules will be immediately reported to J.T. Parsons.
· Medical evaluation, surveillance and treatment will be provided as outlined in the CDC-NIH Biosafety Manual for personnel at risk.