THE WISTAR INSTITUTE

The Wistar Institute

3601 Spruce Street

Philadelphia, PA 19104-4268

Biosafety Level 3 Laboratory

Code of Practice

(Revised August 1999)

Table of Contents

Subject Page

Introduction 3

Authorized Personnel 3

Permitted Work 3

Access 4

Precautions and Safety Concerns 4

Eating, Drinking, Smoking 4

Sharps and Glass 4

Personal Protective Equipment 4

Serological Surveillance 5

Re-evaluation and Safety Meetings 5

Equipment and Supplies 5

Biological Safety Cabinets 6

Centrifuges 7

Non-hermetically Sealed 7

Hermetically Sealed 7

Spills in a Centrifuge 7

Microscopes, Cameras, and Video Equipment 7

Computer Equipment and Note Taking 8

Waste Handling, Decontamination, Disinfection and Disposal 8

The Disinfectant 9

Decontamination of Infectious Materials for Removal 9

General, Uncontaminated Waste 9

Contaminated Waste 10

Plastic Ware 10

Sealable Items 10

Large Non-Sealable Items 10

Small Non-Sealable Items 10

Containers for Glass Patient Blood Sample Tubes 10

Philadelphia FIGHT Samples 11

Radioactive Waste Handling, Decontamination, Disinfection and Disposal 11

Liquid Radioactive Waste 11

Solid Radioactive Waste 12

Accidents 12

General Guidelines 12

Laboratory Fumigation 13

Storage and Transport of Infected Material 13

Storage of Infectious Materials 14

Transport of Infectious Materials 14

Preparing Equipment to be Serviced by Outside Personnel 15

Shipping Equipment 15

In-House Service 15

BSL-3 Authorization Checklist 16

Serum Banking Consent Form 17

THE WISTAR INSTITUTE

3601 Spruce Street

Philadelphia, PA 19104-4268

Biosafety Level 3 (BSL-3) Laboratory

Code of Practice

Revised August 1999

This Code of Practice and all updates will be issued to each person intending to work in the BSL-3 isolation facility. BSL-3 users must read, understand, accept, and adhere to all the rules herein. A signed form from each authorized user noting receipt, understanding, and acceptance of the Code of Practice will be maintained in Wistar’s Safety Office. Failure to follow the rules of the Code of Practice may result in serious accident. Anyone failing to follow this Code will be ineligible to use the BSL-3 facility further.

1. Authorized Personnel

The names of those currently authorized to work in the BSL-3 are on file at the Security Office. Only authorized users are permitted to work in the BSL-3. In order to become authorized, potential BSL-3 users must:

Have attended a bloodborne pathogen training session in the past year.
Be vaccinated against Hepatitis B.
Receive University of Penn RSO radiation safety training.
Provide a baseline serum sample drawn by the Wistar Phlebotomist.
Attend a BSL-3 Code of Practice training session with the Wistar Safety Officer.
Attend a hands on training session with the BSL-3 Laboratory Manager.
Read, understand, accept, and adhere to all rules in this BSL-3 laboratory Code of Practice.

2. Permitted Work

The BSL-3 pathogens that may currently be used in the BSL-3 facility are posted at the entrance to the BSL-3 facility. Work that does not require level 3 containment (for example, the manipulation of fixed or non-infectious materials, cultivation of uninfected cell lines, storage of chemicals, and the making of buffers and media) should be conducted elsewhere in external labs whenever possible.

Work involving radioactive isotopes will be permitted within the BSL-3 facility for authorized, licensed users and only in designated areas. A radioactive logbook will be maintained, recording the date, user, isotope, and waste of each experiment. Each radioactive user will be responsible for monitoring and cleaning the designated area and sinks after each use.

3. Access

The external entry and exit doors to the BSL-3 facility are controlled by a card-key access system. The door lock is deactivated when an authorized user’s identification badge is "swiped" through the door-lock control. A light on the door-lock control will change from red to green to indicate that the door may be opened. In the event that two users are entering or exiting the BSL-3 facility at the same time, both users are required to swipe their identification badges through the lock control.

The internal entry door to the BSL-3 facility is controlled by negative pressure and will not open until the external door is fully closed. The entryway between the two doors is supplied with personal protective clothing and storage lockers (see Section 4C, "Protective Clothing"). Authorized users may enter the BSL-3 facility on a 24-hour basis

4. Precautions and Safety Concerns

A. Do not eat, drink, mouth-pipette, or smoke in the BSL-3 facility!

B. Sharps and Glass

The major causes of infection of laboratory personnel by human retroviruses are scratches, cuts, and needle-stick injuries. Because of this, the following are not allowed in the BSL-3 facility without proper safety training and precautions and prior authorization from the Laboratory Manager: hypodermic needles; razors or scalpel blades; glass pipettes; pasteur pipettes; and laboratory glassware. All medium and solutions used inside the facility are to be contained ONLY in plastic bottles. Plastic and/or disposable alternatives must be used for all glassware items which have such suitable alternatives. Any other items which might pose some danger (e.g., damaged apparatus) must be disposed of immediately.

HIV patient samples arrive in glass tubes. Specific handling procedures for these glass tubes are covered in sections 11.C.3 and 12 of this Code of Practice, but a good rule of thumb when handling them is "the less amount of handling, the less chance of accident".

C. Personal Protective Equipment

Before entering the BSL-3 facility, users must "gown up" in the entryway. BSL-3 users must wear the provided gloves and back-fastening lab coats at all times while in the facility. Users must also wear conventional shoes, socks, and long trousers or the provided disposable shoe cover and pant alternatives. Disposable yellow sleeves must be used working at the hoods or if working with radioactive materials. Face masks and eye shields are provided both within the entryway and inside the BSL-3 facility. Other disposable personal protective equipment, such as hairnets, is also provided for users inside the entryway.

The lab coats provided are reusable. There are double-sided lockers within the entry and exit ways of the facility for lab coat storage between uses. The lab coats should be thrown away after 5 uses or if they have become contaminated.

Double gloves are mandatory when working in the BSL-3 facility. The first pair of gloves, ones that should be long enough to tuck gown sleeves under the latex, is provided in the entryway. The second pair of gloves is provided inside the facility and should be changed frequently, particularly when the user changes stations (e.g., when the user is moving from a biosafety cabinet to the microscope or the computer; or when the user is answering the phone). While inside the facility, users should not touch their faces, eyes, hair, or exposed skin.

Minor skin abrasions should be protected by applying bandages under the first pair of gloves. Users should not work in the BSL-3 laboratory if they have skin abrasions, cuts, or conditions that seriously impair the integrity of the skin. BSL-3 users should also not use petroleum jelly or other agents that weaken glove latex.

D. Serological Surveillance

A study concerning the serological monitoring of HIV workers reported by the Division of Safety, National Institutes of Health USA, and published in Morbidity and Mortality Weekly, Vol. 37 No. S-4, pp. 19-22, recommends:

"serum samples should be obtained at least once a year and analyzed for sero-conversion. Results should be reported to individual workers in a timely manner. Counseling services should be available for workers who have positive serologic results. Procedures that maintain strict confidentiality should be adopted."

These recommended guidelines have been adopted for Wistar Institute’s BSL-3 facility, except in the timing of the sample drawing and the serum testing. All personnel working in the BSL-3 facility are required to sign a consent form to have a serum sample drawn prior to working in the BSL-3 facility and then re-drawn every 6 months thereafter. Confidential testing for HIV status and for seroconversion of the drawn samples is at the discretion of the user, that is, the samples may be immediately tested or they may be stored, untested, after being drawn for testing at a later time, if necessary.

E. Re-evaluation and Safety Meetings

Meetings for the re-evaluation of the Wistar Institute’s Biological Safety Level-3 Code of Practice will be called on occasion. All authorized BSL-3 facility users must attend these meetings in order to maintain their authorization. In addition, it will also be required of all authorized BSL-3 users to attend regular safety meetings and to review laboratory procedures. Notification of such meetings will be at least ten (10) working days.

5. Equipment and Supplies

The BSL-3 facility is equipped with a multitude of items necessary for autonomous work inside the laboratory. There are many tabletop and micro- centrifuges, biological safety cabinets, tissue culture incubators, microscopes, computers, refrigerators, -80⁰C freezers, and a liquid nitrogen storage tank that can be found in various areas around the BSL-3 laboratory. Some of this equipment is explained in more detail in sections to follow. All common use equipment inside the facility should be treated carefully and used with regard to all other persons authorized to work within the BSL-3 facility.

Supplies for tissue culture and for other required work inside the BSL-3 facility can be found in various designated areas around the laboratory and on the supply carts near each of the biosafety cabinets. General supplies will be kept stocked by the BSL-3 Laboratory Manager. The supply cart near each biosafety cabinet must be stocked with all necessary items by the current user. All personal supplies must be removed from the cart when work in the biosafety cabinet is finished. These supplies should be stored in the users drawer space.

The Laboratory Manager should be notified if stocks within the BSL-3 itself are running low or are out, if a BSL-3 user needs a specific item which they can not find within the facility, or if equipment within the facility needs service or repair.

Each BSL-3 facility user must contact the Laboratory Manager to review equipment and supply charges in order to determine a method of payment or repayment for use of the facility.

6. Biological Safety Cabinets

The BSL-3 facility is equipped with 3 working biological safety cabinets that are rigorously tested semi-annually by the Wistar Institute Safety Office. All aseptic procedures, tissue culture work, blood processing, and operations in which infectious liquids or cell cultures are transferred from one container to another is to be done inside one of the three biosafety cabinets provided within the BSL-3 facility. In addition, all work that may result in the production of aerosols must be conducted inside a biosafety cabinet as well. This includes filtration of infectious liquids, any work on patient blood sample tubes, or the opening of potentially contaminated containers such as centrifuge rotors and tubes.

Before doing any work in the biosafety cabinets, BSL-3 users should make sure to remove any unnecessary items that may impede the flow of air inside the cabinet. This is facilitated by the supply carts that are provided beside each biosafety cabinet for the stocking of necessary tissue culture supplies and other equipment required for the work.

BSL-3 users should prepare the biosafety cabinet and make sure to have at-hand items for disposal and decontamination of the waste produced (see Section 10 on waste handling and decontamination). Before doing any work in the biosafety cabinets, BSL-3 users must make sure they have sufficient amounts of active Virkon solution and 70% ethanol (both are provided in trigger-operated wash bottles on the carts near each biosafety cabinet. See Section 10A for an explanation of what Virkon is and how to use it effectively). Users should also line the plastic pipette cans found in each of the biosafety cabinets with a new small red plastic bag. Large screw-top plastic 2 liter bottles are provided near the sink and should be filled partially with a freshly made active Virkon solution and placed inside the biosafety cabinet. Finally, users should also make sure that the large autoclavable biohazardous waste bucket found near each biosafety cabinet is accessible, is fitted with a large red autoclave bag, and that it’s not overly full. If the bucket is close to full, it should be closed, fitted with it’s lid, and placed in the area designated by the Lab Manager for waste decontamination (currently, the area directly in front of the autoclave). A new biohazardous waste bucket should then be fitted with a large red autoclave bag and placed near the biosafety cabinet. Empty biohazardous waste buckets are located beside the autoclave.

At the end of each working session, users must remove all extra materials and all waste from the biosafety cabinet. The internal surfaces of the biosafety cabinet should then be decontaminated and cleaned by first spraying and wiping it with active Virkon solution and then by spraying and wiping it with 70% ethanol.

7. Centrifuges (All centrifuge buckets must be covered with bucket covers while centrifuging and may only be opened in the biosafety cabinet.)

A. Non-hermetically sealed centrifugation

Any centrifuge operation in the BSL-3 involving non-hermetically sealed containers, carriers, rotors, or tubes must be carried out inside a biosafety cabinet.

B. Hermetically sealed centrifugation

Centrifuges that have carriers, rotors, or tubes that can be hermetically sealed can be used outside of the biosafety cabinets, provided that all potentially aerosol-producing manipulations are carried out within the biosafety cabinets prior to centrifugation. Carriers, rotors, and tubes must be loaded, balanced, sealed, and reopened only in the biosafety cabinet. Tissue culture plate carriers have no hermetically sealable lids. However, plates can be spun in the benchtop centrifuges at low speeds in their dedicated carriers. These plates must first be well sealed with tape and tightly enclosed in small biohazardous waste bags before being placed into the carrier, and that they are not opened at any time outside of the working environment of the biosafety cabinets.

C. Spills in a centrifuge

If a user suspects that there has been a spillage inside a centrifuge within the BSL-3 facility, the centrifuge should not be opened! The centrifuge should be marked visibly with some sort of sign or some tape placed over the still closed handle that warns other users: "suspected contamination: DO NOT OPEN!" The incident must be reported to the BSL-3 Laboratory Manager or the Safety Officer immediately. They will do a risk assessment of the situation and decide on a course of action for decontamination.

8. Microscopes, Cameras, and Video Equipment

All BSL-3 users interested in using the inverted microscope, cameras, and video equipment must arrange time with the BSL-3 Laboratory Manager in order to be trained before handling them. Proper training is required since the equipment is easily misadjusted by improper manipulations.

Users must change their outer gloves before moving to the microscope area from any other work area inside the BSL-3 facility.

It should be noted that there is no photographic developing facility within the BSL-3 laboratory, and in order to be able remove exposed film from the facility without destroying it by disinfection, users must show extreme caution and be careful that the film does not become contaminated with infectious agents. Therefore, plastic hemacytometers containing exposed infected cell cultures to be counted should be transported to and from the microscopes inside a closed container (e.g., a large plastic petri dish). Users should exercise caution when manipulating infected cell and tissue cultures at the microscope area, and users should address "film work" as a separate from "data collection". That is to say, users must change gloves between loading/manipulations of the film and any manipulations of infected materials they are working with or photographing.

9. Computer Equipment and Note Taking

A Wistar Institute-networked common-use Power Macintosh 8500 that is equipped with a scanner and video-linkage to a Nikon inverted microscope is set up inside the BSL-3 facility in order to allow users external access to any data collected and/or experiments performed inside the BSL-3 facility. Users should contact the BSL-3 Laboratory Manager in order to obtain authorized access to the BSL-3 facility’s computer equipment.

Although permanent notebooks are permitted inside the BSL-3 facility, users are encouraged to use the computer to record experiments and data. Data and notes should be kept inside the individual personal folders for each authorized BSL-3 user and NOT on the desktop of the computer. Unknown-author documents will be periodically removed from the system if not inside a clearly labeled personal folder. All BSL-3 users are responsible for back-up of their own files.

Users must change their outer layer of gloves before moving into the computer area from any other work area inside the BSL-3 facility. Also, users may NOT place any infectious or other experimental material within the computer area. No transit of paper will be permitted back and forth from the biosafety cabinets to the writing benches, as these two areas should be addressed as separate working environments. NO paper may be left in work areas (outside of the writing bench) after a user has left the facility. It is recommended that rough notes on paper scraps be transcribed into the computer or into permanent lab notebooks and that the scrap paper be discarded into the general waste autoclave buckets before the user leaves the BSL-3 facility.

10. Waste Handling, Decontamination/Disinfection, and Disposal

This section provides for the safe storage, decontamination, and disposal of waste from the BSL-3 laboratory. All disinfection and decontamination procedures carried out are designed to eliminate all possibility that users will accidentally contaminate themselves with infectious material while in the facility, or that waste will remain infectious once leaving the BSL-3 facility.

Liquid waste must be immediately decontaminated with disinfectant and contaminated solids must be either rinsed in disinfectant or sealed in spill-proof containers. For the safety of all BSL-3 users, NO contaminated or infectious material should remain exposed to the air or be left in an easily accessible location. The procedures listed below are for decontamination in common situations and are sufficient to inactivate most infectious agents. Other methods of disinfection should be tested under fair conditions first before using in the BSL-3. If a BSL-3 user would like to use alternate methods to decontaminate infectious waste, they must get approval from the BSL-3 Laboratory Manager and the Safety Officer first.

A. The Disinfectant

All or most BSL-3 decontamination procedures involve Virkon. A surfactant is present in Virkon to disrupt viral envelopes and cellular membranes and to allow the penetration of its disinfectants (oxidizing and alkylating agents). Virkon is a powerful disinfectant when used at the recommended dilution (1% w/v) and it is know to inactivate most viruses within 5 to 20 minutes of contact. It does not give rise to formaldehyde vapor nor does it corrode stainless steel. Virkon is a red solution when freshly dissolved and active. Since this red coloration fades when the solution is no longer active, solutions of Virkon should not be used unless they are red in color. Virkon must be used for all decontamination purposes except on items that will be placed in an incubator (for these items, exterior swabbing with 70% ethanol is acceptable). BSL-3 users must disinfect any used work surface with Virkon before leaving the facility. Any area of suspected spillage should be sprinkled with Virkon powder, then thoroughly wiped up with paper towels after at least 20 minutes of incubation.

Decontamination of Infectious Materials for Removal from the BSL-3 Facility

Any materials that are removed from the BSL-3 Facility must be decontaminated before removal to a room that does not operate under BSL-3 containment procedures. Materials that are decontaminated by heat inactivation (65° C for at least 30 minutes) or fixed by an approved method may be removed from the facility. Infectious material may be removed from the BSL-3 without using decontamination procedures only if it is to be received in a laboratory that is also operating Biosafety Level-3 containment procedures (see Section 14B). If you must remove materials to a room that is not operating under BSL-3 containment procedures contact the BSL-3 Laboratory Manager.

C. General, Uncontaminated Waste

Items such as paper, paper towels, gloves, etc., should be placed directly into the large red-bag-lined autoclavable waste buckets marked by a large biohazard symbol. These are positioned at various places around the BSL-3 facility-- they can be found near each biosafety cabinet, in the writing area, in the entryway and the exit. Empty buckets are positioned in the rear of the BSL-3 facility. When a bucket is near full capacity, the current user should twist the red autoclave bag closed, close the lid, and place the entire bucket in the zone set aside for the purpose of disposal by the BSL-3 Laboratory Manager (currently, this is the area directly in front of the BSL-3 facility autoclave). Users should make sure to replace the bucket with an empty one containing a fresh autoclave red bag. Because these buckets and their contents are handled personally by the Laboratory Manager during autoclaving and disposal, users must make sure that any waste considered potentially "sharp" (such as unprotected pipette tips, broken plastic) not be put into the general waste.

D. Contaminated Waste

D1 Liquid Waste

Liquid waste can be decontaminated by a) pouring it into screw-top 2 liter bottles containing an active Virkon solution; b) adding an equal volume of 2% Virkon to the vessel containing the contaminated liquid; or, c) adding dry Virkon powder to the contaminated liquid to give a final concentration of 1%. After 20 minutes (or preferably overnight), the liquid can then be disposed of down the sink along with plenty of water.

Larger volumes of liquid waste may require autoclaving for total decontamination. The BSL-3 Laboratory Manager or Safety Officer should be notified when large volumes of contaminated liquid needing decontamination is being produced.

D2 Plastic Ware

i. Sealable Items. (tissue culture flasks, centrifuge tubes, etc.)

Items such as these that have contained infectious material should be emptied of all liquid (see Section 11C1), closed tightly, and then placed inside the large red-bag-lined autoclavable biohazardous waste buckets found next to the biosafety cabinet.

ii. Large Non-sealable Items. (pipettes, multi well tissue culture plates, etc.)

Pipettes should be rinsed with the active Virkon solution in the screw-top 2 liter bottles before being placed in the red-bag-lined plastic pipette can inside the biosafety cabinet. Multi well plates should be aspirated of liquid waste by using a pipette or vacuum aspirator, sprayed with active Virkon solution, closed, and then placed in the red-bag-lined plastic pipette can inside the biosafety cabinet. These bags are then to be sealed at the end of the work session and placed into the large red-bag-lined autoclavable biohazardous waste buckets.

iii. Small Non-sealable Items. (pipette tips, cryovials, microfuge tubes)

These are to be placed directly into the active Virkon solution inside the 2 litre bottles. The 2 litre bottles are then sealed by the user at the end of the work session, removed from the biosafety cabinet, and placed in the sink area for at least 20 minutes. The liquid inside the bottles should then be disposed of down the sink with plenty of water and the small items can be tossed into the large red-bag-lined autoclavable biohazardous waste buckets.

D3 Containers for glass patient blood sample tubes

Containers holding potentially infectious glass vacutainer tubes from HIV patient blood samples (see Section 11) must stay inside the biosafety cabinets so as not to expose any BSL-3 facility user to the potentially infectious material it contains. When they are filled, the current user must close the lid tightly before removing the container from the biosafety cabinet. Then place it into the zone set aside for the purpose of disposal by the BSL-3 Laboratory Manager (currently, this is the area directly in front of the BSL-3 facility autoclave).

11. Philadelphia FIGHT Samples

HIV patient blood samples come into The Wistar Institute through a variety of sources, the most common through a collaboration with the HIV/AIDS education/outreach group known as Philadelphia FIGHT. After the samples have been logged into the books, the jars containing the glass tube samples may be transferred and opened inside the BSL-3 facility.

The patient samples are in glass vacutainer tubes and should be handled with the utmost care. A good rule of thumb when handling these tubes is "the less amount of handling, the less chance of accident". All patient blood sample tubes, due to the vacuum inside, have the potential of creating an aerosol of their contents upon opening. Therefore, vacutainer blood tubes may only be opened in the biosafety cabinet. To open vacutainer tubes first spray a paper towel with Virkon solution and then place the paper towel over the top of the tube. Open the tube slowly and carefully with the tube at eye level. Screw-top plastic 2 litre buckets are available at the sink area. These should be partially filled with an active solution of Virkon and placed into the biosafety cabinet. Vacutainer caps and all infectious wastes from the patient samples should be deposited into these Virkon-filled 2 litre containers during processing of the blood. Emptied vacutainer tubes should be carefully and gently placed into the red "sharps" containers that are located in each of the biosafety cabinets (see Section 10.D.3). In the case that sample vacutainer tubes arrive broken, the sample tubes are packaged in such a way that any spill or breakage of a vacutainer sample tube will be contained by a larger biohazardous container. This larger vial containing the broken or leaked vacutainer sample tubes should be opened only inside a biosafety cabinet and filled to the point where all it’s original contents are covered by active Virkon solution. After a period of at least 20 minutes, the Virkon solution should be disposed of down the sink (see Section D1) and all vacutainer sample tubes found inside the larger container should be discarded carefully into the red "sharps" containers (see Section 10D3).

12. Radioactive Waste Handling, Decontamination/Disinfection, and Disposal

Work involving radioactive isotopes is permitted in the BSL-3 facility for authorized, licensed users and only in designated areas. All BSL-3 facility users who work with radioactive materials must record all radioactive usage and disposal or decay in logbooks and must adhere to University of penn Radiation Safety and NRC guidelines. All work surfaces, gloves, and equipment as well as the user must be monitored during and after use of radioactivity. Paper towels, gloves, etc., used during the process must be placed inside a radioactive waste bag for disposal.

Any contamination must be cleaned up with a mixture of 2% Virkon. Large spills must be reported to the BSL-3 Laboratory Manager or the Wistar Safety Officer.

A. Liquid Radioactive Waste

If the radioactive liquid waste is contaminated with infectious materials, it must be treated with an equal volume of active 2% Virkon disinfectant for at least 20 minutes (overnight incubation is preferable) before it is disposed of.

Non-infectious or decontaminated radioactive waste can be gently poured down the radioactive sink, without splashing and with copious amounts of running water.

All liquid radioactive waste is disposed of down the sink and therefore must be recorded in the sewer disposal logs.

Note that current limits for sewer disposal are £ 200mCi of ³H OR £ 50mCi of ³²P or ¹²⁵I. The addition of 1% Virkon before disposal helps reduce radioactive contamination to the sink and collection containers.

B. Solid Radioactive Waste

Solid radioactive waste should be placed into a sealable plastic bag, labeled clearly and correctly, and stored in the waste container designated for the particular isotope used. Disposal and decay of solid radioactive waste must be logged in the correct logbook. All solid radioactive waste in the BSL-3 facility is treated as infectious and therefore will undergo a period of decontamination -- this usually consists of an incubation of at least one hour in a 65° C oven-- before disposal or decay.

B1 ³H and ¹²⁵I (disposed outside of the BSL-3 facility)

Any waste bag produced must be decontaminated before being removed from the BSL-3 facility and disposed of properly in the radioactive disposal areas of the Wistar Institute (currently, the Fourth Floor disposal area).

B2 ³²P (decayed inside the BSL-3 facility)

Any waste bags produced must be placed inside the containers designated for decay. Waste bags, once decayed, will then be decontaminated before being disposed of in the large red-bag-lined autoclavable biohazardous waste buckets used for "general waste".

13. Accidents

A. General Guidelines

All dangerous spills, injuries, or other accidents must be immediately reported to the Principal Investigator in charge of the BSL-3 facility, to the BSL-3 Facility Lab Manager, and/or to the Wistar Institute’s Safety Officer.

Some guidelines for accidents are as follows:

If any infected material comes into contact with a BSL-3 user’s outer pair of gloves, the gloves should be discarded immediately.

If any infectious material comes into contact with a BSL-3 user’s exposed skin, the affected area should be washed immediately and quite thoroughly. The incident must be reported immediately.

If any penetrating or "stick" injury occurs, such as from the breakage of a glass vacutainer blood sample tube, the wound should be encouraged to bleed immediately and the area washed quite thoroughly before bandaging. The incident must be reported immediately.

As covered in section 7C, if a user suspects that there has been a spill inside a centrifuge, the centrifuge should not be opened! It should be marked visibly with some sort of sign or some tape placed over the still closed handle that warns other BSL-3 facility users: "suspected contamination: DO NOT OPEN!" The incident must be reported immediately.

If there has been a major spill in the BSL-3 facility which is too widespread for superficial disinfection, the incident must be reported immediately, all work must stop, and the laboratory must be sealed shut with warning tape. The Laboratory Manager or Wistar’s Safety Officer will do a risk assessment of the situation and decide on a course of action for decontamination.

B. Laboratory Fumigation

If there has been a spill of infectious materials that is so widespread, to the extent that standard clean up can not contain it or decontaminate it, the BSL-3 facility must be entirely fumigated for decontamination. The following procedure will be performed by the BSL-3 Laboratory Manager or the Wistar Institute Safety Officer. Materials stored in vials at -80⁰C and over liquid nitrogen should be safe from the effects of this fumigation. However, no similar guarantees can be made about the viability of materials stored in incubators or elsewhere within the BSL-3 facility.

Formaldehyde Fumigation Procedure:

Prepare BSL-3: Remove all lab personnel. Close all incubator and refrigerator doors.

Set air conditioning to 28° C (heat room).

Turn off biosafety cabinets (timed period, for 13 hours only).

Fumigation: Make formalin mixture in fume hood (75 ml 37% formaldehyde,

150 ml H₂O). Transport mixture into BSL-3 in a suitable carrier.

Add mixture to a metal fumigation beaker on hot plate that has

been hooked up to a timer set for two hours and that has been

placed in view of the window.

Leave BSL-3 immediately after turning on timer to the hotplate.

Seal outer door and window filters, put biohazard tape visibly

around door, and lock securely.

Incubation: Allow beaker to boil dry and leave room "as is" overnight.

The formaldehyde is removed from the facility by the Class II

cabinets, which turn on automatically after 13 hours.

Once the room is at negative pressure, unseal tape from around

the window filters.

After 24 hours, the formaldehyde should have been evacuated. Unseal the door and

return air conditioning to normal setting. Make sure formalin has dispersed before

allowing any BSL-3 personnel in.

14. Storage and Transport of Infected Material

The BSL-3 facility is equipped with 4° C/-20° C, -80° C, and liquid nitrogen storage freezers. Space for individual users is available (please see Laboratory Manager). All material stored in these freezers should be in securely closed, clearly labeled (i.e. name, date, and description of the sample) containers that cannot be opened accidentally. A few other guidelines for storage and transportation of infected materials are as follows.

A. Storage of Infectious Materials

A1 Room Temperature, 4⁰C, and -20⁰C

It is inadvisable to store any infectious material under these conditions for long term. If infectious material needs to be stored temporarily before analysis, it should optimally be stored in the -20⁰C freezer portion of the yellow "infectious refrigerator".

Samples stored for longer than three months under these conditions and left untouched will be subject to disposal without notice.

A2 -80⁰C

Storage racks are provided in the 80⁰C freezers. BSL-3 users should contact the Laboratory Manager for space. Material should not be stored outside the racks. Any clutter is subject to removal and destruction without notice.

A3 Liquid Nitrogen

When infected cells are to be frozen for storage, they must be suspended in the freezing medium and transferred into freezing vials within a safety cabinet. These vials should then be slowly cooled (at one degree/minute) in the -80⁰C freezers before they are transported to the liquid nitrogen freezer. When anything is retrieved from the liquid nitrogen freezer, users must wear a protective visor and facemask in case the material explodes on contact with the warmer surrounding air of the BSL-3 facility. Visor is located on top of the liquid nitrogen freezer. Contact the Laboratory Manager for space.

B. Transport of Infectious Materials

All materials that are handled within the BSL-3 facility, with the exception of photographic film (see Section 8), are automatically considered to be infectious unless specifically disinfected by one of the approved methods (see Section 10) and should NOT be removed from the facility. Infectious material may be removed from the BSL-3 without using decontamination procedures only if it is to be received in a laboratory that is also operating Biosafety Level-3 containment procedures. Before transport, storage vials containing contaminated materials need to be securely closed and then surface decontaminated, usually by a swabbing of the outside of the container with 70% ethanol. The vial should then be placed inside a larger container (i.e., a sealable or taped-shut bag or a screw-top vessel) that carries enough absorbent material inside it to soak up any spill that may occur, and securely closed. This larger container should then be placed inside a third container that is to be used as the transportation carrier and which is clearly marked with a biohazard symbol and the words "biohazardous material inside".

The containers used for the transport must be sufficient to prevent accidental contamination and must be approved by the Laboratory Manager and Wistar’s Safety Officer.

15. Preparing Equipment to be Serviced By Outside Personnel

Some equipment in the BSL-3 facility will need to be serviced regularly or might need maintenance. The Laboratory Manager should be notified that equipment within the facility needs service or repair. The equipment will either have to be shipped out to the contractor or be serviced in-house.

A. Shipping Equipment

The equipment to be serviced must be completely disinfected before removal from the facility. This procedure is usually no more extensive than a thorough swabbing of the equipment with 70% ethanol.

B. In-House Service

In such circumstances, the BSL-3 facility must be made safe for the outside personnel before service can be carried out. The Laboratory Manager will be given written notice by Physical Plant of the appointed time of service, and will then inform all the BSL-3 facility users of the expected disruption with as much time in advance as possible. One day before the work is due to start, the Laboratory Manager will ensure that the equipment to be serviced and all surfaces with which the service personnel are likely to come in contact with have been thoroughly disinfected. The Laboratory Manager will also bar access to all other areas of the BSL-3 facility. Physical Plant or the service personnel will then inform the Laboratory Manager when work has been completed.

In addition, all work by BSL-3 users must stop. Under no circumstances may any manipulations of infectious material be carried out by any BSL-3 user during the period of in-house servicing by outside personnel.

If superficial disinfection procedures are inadequate to protect the service personnel, fumigation of the laboratory will be necessary (see Section 16). In such cases, all

authorized personnel will be contacted before fumigation and given at least one week’s notice, whenever possible.

THE WISTAR INSTITUTE

3601 Spruce Street

Philadelphia, PA 19104-4268

Biosafety Level 3 (BSL-3) Laboratory Authorization Checklist

Revised August 1999

In order to become an authorized BSL-3 user you must:

Have attended a bloodborne pathogen training session in the past year. When did you attend ______________ . Where: ________________________________________________.
Have received the hepatitis B vaccination. When did you receive it: _________________.
Have received University of Penn radiation safety training. When ___________________.
Have provided a baseline serum sample. Complete the attached consent form and present the form to the Wistar Phlebotomist in room G-1. The Phlebotomist must verify by signing:________________________________________ Date: ________________.
Have attended a BSL-3 Code of Practice training session with the Wistar Safety Officer (L84B). Safety Officer’s signature: ________________________ Date: ___________.
Have attended a training session with the BSL-3 Laboratory Manager (room 480).

BSL-3 Laboratory Manager signature: _______________________ Date: __________.

Read, understand, and adhere to all rules in this BSL-3 laboratory Code of Practice.
Contact Security Office (L84) to have your Wistar ID card activated for BSL-3 entrance.

I have read and understood this Code of Practice for the Biosafety level 3 facility at the Wistar Institute. I agree to abide by the BSL-3 Code of Practice of The Wistar Institute and understand that I may be prevented from using the laboratory if I fail to do so. I will keep a copy of the Code of Practice and will remain familiar with its contents.

Name (printed): ________________________________________________________

Signature: ________________________________________ Date: ________________

Principal Investigator: _______________________________ Date: _________________

Return the completed form to the Wistar Institute Safety Office, room L84B.

The Wistar Institute Serum Banking Program Consent Form

In the course of conducting biomedical research, laboratory personnel use biological agents that may be harmful to human health. The Institute requires personnel to take extensive safety precautions when using these agents; however, it is impossible to completely eliminate the risks associated with their use.

Recognition of this fact has led the Institute to institute a serum collection and banking program for BSL-3 users. Serum samples are commonly used to help determine potential origins of pathogenic exposure or infection. Under this program, employees provide a serum sample at the time of employment, and at the time of termination. Those individuals working with certain biological agents (e.g. HIV) will provide serum samples periodically during the course of employment, consistent with recommendations of the Centers for Disease Control or National Institutes of Health. Each serum sample that is collected is given an identifying number, and is placed in storage. Samples are not analyzed, reviewed or tested unless the donor and the Institute subsequently determine that testing is warranted, and the donor gives written consent to the testing.

By signing this form, you consent to give the sample of blood obtained from you. You agree that the blood sample you provide will belong to The Wistar Institute, but will not be analyzed without your written consent. You have been informed that your participation in this program is voluntary, and not a condition of your employment. You have been given the opportunity to ask any questions you may have about drawing blood or about how your blood sample will be stored and/or used. You acknowledge that you may obtain further information from the Associate Director for Science Administration, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 (telephone: 215-898-3898). You have the right to obtain a copy of this consent form. After having completely read and understood the above, you hereby agree to have blood drawn by a representative of The Wistar Institute trained for this purpose.

__________________________ _____________________________

Full Printed Name Signature

________________ _____________________________

Date Witness

Aug 1999