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 Since this red coloration fades when the solution is no longer active, solutions of Virkon should not be used unless they are red in color.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. B. 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

1. Liquid Waste

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.

2. 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.

3. 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).

The Wistar Institute
Biosaftey Level 3 Laboratory Code of Practice