4.10 Biological Waste Management
4.10.1 Contaminated Waste Items Generated In A Biosafety Cabinet
Items may be removed from the BSC after they are decontaminated with an appropriate disinfectant, such as being sprayed with 70% ethanol, or they can be placed within a bag or sealed container in the BSC, which is sprayed with disinfectant before removal from the BSC.
4.10.2 Liquid Biohazardous Waste
Preferred Method For Liquid Biowaste Containing No Disinfectant Or Other Chemical Components:
- Collect the waste in a well-labeled autoclavable container (containing no bleach), then autoclave the waste on a liquid cycle at 121˚C, with the sterilization time determined by the liquid volume. After cooling, the waste may be poured down the lab drain.
- If using the above method is not possible, liquid biowaste can be discarded into a container containing a sufficient quantity of bleach (e.g., pure bleach to yield a 1:5 dilution. (Example: 100 mls. household bleach added to 400 mls. tissue culture media).
- After the required exposure time in the BSC, the liquid waste plus bleach may be disposed of down the lab drain, followed by a water flush. If a different disinfectant is used, it must be disposed of as chemical waste.
- Decontamination by bleach a less reliable decontamination method because of the opportunities for a failed result if the bleach used is 1) expired, 2) present in the wrong proportion to liquid waste volume, and 3) not given sufficient contact time before disposal.
Liquid Biowaste Containing Bleach, Another Chemical Disinfectant, Or Other Chemical Constituents Such As Sodium Azide:
- Dispose of as liquid chemical waste. DO NOT AUTOCLAVE THIS WASTE.
Liquid Biowaste Containing Antibiotics:
Media wastes containing the following antibiotics can be autoclaved, cooled and flushed down the lab sink drain because the antibiotics are broken down by heat, and are then environmentally safe to go into the domestic sewer.
Media wastes containing the following heat-stable antibiotics cannot be autoclaved and discarded into the domestic sewer because of their relatively long half-life which persists in the environment; add bleach in a 1:5 vol/vol concentration to deactivate biologicals, then dispose of as liquid chemical waste.
- Hygromycin B
- Nalidixic acid
If antibiotics are used that are not on these lists, contact EHS for disposal consultation, or simply submit for disposal as liquid chemical waste.
Media with additives such as growth factors, metals or other chemicals must be disposed of as liquid chemical waste.
Liquid decontamination method(s) should be verified and documented.
Liquid biowaste containers must be appropriately labeled as such, with biohazard signage; liquid chemical waste containers must be appropriately labeled and well-identified as a different waste stream.
Liquid Wastes Containing Extracted DNA/ Cellular Components/ Lysates, etc.
- Biological material treated with extraction kit chemicals or other chemical treatments that lyse cell membranes renders bioagents non-viable and no longer hazardous. This material must be disposed of as chemical waste.
4.10.3 Solid Biohazardous Wastes
BSL-1: Collect in a non-colored autoclave bag with no Biohazard symbol.
BSL-2: Collect in an ORANGE autoclave bag with a Biohazard symbol.
Autoclave bags must be kept inside appropriately labeled biowaste containers, equipped with a closeable lid.
Solid biowaste containers must remain closed except when in use.
At end of a work session or when bag is 2/3 full, securely close the bag, place it in a secondary container (Nalgene or stainless steel pan) reserved for this purpose, and spray it with appropriate disinfectant prior to transport on a cart to the autoclave room.
Proceed with autoclaving the waste according to standard procedures provided in Section 4.14. Bags of solid biowaste must be autoclaved on a gravity or pre-vacuum cycle at 121˚C; the length of cycle used must be determined by the size and density of the autoclave load. Each load must be checked with a verification device to confirm that kill conditions were met.
Bags must be autoclaved daily when possible, or as soon as an autoclave is available. No full bags should be left in the laboratory or the autoclave/glassware room OVER WEEKENDS OR BREAKS.
Bags must remain closed until they are ready to be autoclaved, at which time their closures should be loosened to at least a 1-inch opening to allow steam penetration.
All BSL-2 solid waste is to be decontaminated by autoclaving and disposed of as Regulated Medical Waste.
Decontaminated BSL-1 waste can be discarded into regular trash, or disposed of in Regulate Medical Waste (see Section 4.8.3).
Agar Plates With Antibiotics In The Medium:
- BSL-1 – It is acceptable to autoclave BSL-1 waste containing heat-stable or heat-labile antibiotics, and once decontaminated, dispose of waste in dumpster trash bound for landfill. Bagged waste is well contained, allowing adequate time for breakdown of antibiotics when disposed of in a landfill.
- BSL-2 – It is acceptable to autoclave BSL-2 waste containing heat-stable or heat-labile antibiotics, and once decontaminated, this waste must be discarded in Regulated Medical Waste – the same disposal method as BSL-2 plates containing no antibiotic.
Solid Biowaste/Lab Debris Mixed with Chemical Waste: Contact EHS to determine the best disposal method.
4.10.4 Sharps Biohazardous Wastes
Sharps contaminated with biohazardous materials include anything that could puncture an autoclave bag. Examples include:
- Pipette tips
- Wood applicator sticks/swabs
- Syringes and needles
- Glass slides/cover slips
- Serological pipettes
- Glass Pasteur pipettes
- Disposable plastic pipettes
- Broken glass
- Blood tubes/capillary tubes
Discard all sharps used with biologicals into lidded, rigid, labeled Bio-sharps containers. EHS supplies containers that meet the requirements of being closeable, puncture-resistant and leakproof; they can be requested through the EHS online Safety Management System.
Sharps waste containers:
- Must be located for ease of accessibility; i.e., at or near point-of-use.
- Must be replaced routinely
- Must be securely closed before removal from point-of-use
- Must have NO chemical or liquid waste placed within (with the exception of specimen tubes containing blood)
Never re-cap needles or scalpel blades before disposal into a sharps container.
Avoid forcing sharps waste into a full container; this can cause puncture/ cut injuries.
All sharps containers are to be autoclaved using the same cycle as used for biological solid waste and then disposed of in the same manner as BSL-2 waste; i.e., in Regulated Medical waste.
In situations when collection of Sharps waste is intermittent or of smaller volume, avoid collecting them in large sharps containers. Use smaller Sharps containers whenever possible, as these will fill more quickly and thus can be decontaminated more expeditiously.
Rigid plastic laundry detergent bottles with lids can be used but must be well labeled.
4.10.5 Regulated Medical Waste (RMW)
RMW collection boxes are used for end-point disposal of DECONTAMINATED (i.e., autoclaved) biological Sharps and ALL BSL-2 solid waste.
Decontaminated BSL-1 waste can be disposed of in regular trash cans lined with black bags, or in RMW.
RMW must not contain any liquids in containers, or free liquids.
RMW Box Assembly and Use:
- Tape bottom center seams 2-3X, and tape open side seams one time with wide cellotape.
- Line each RMW box with 1-2 red RMW plastic liner bags. If using 2 bags, place one inside the other. Remember, these liners cannot substitute as autoclave bags – they disintegrate in high heat.
- Do not overfill RMW boxes, or pack them so that they are too heavy; limit is 50 lb.
RMW Final Packaging:
- When a RMW box is full, twist the top of the inner bag enough times so that you can loop it over on itself in a “gooseneck.”
- Seal the twisted top by wrapping with tape. Seal a second, outer bag by the same method.
- Close the box’s top and seal X3 on center seam with wide cellotape, then tape the open side seams 1X.
- Fill out the white label for the box that identifies the generators of the waste, and the date the box is closed and sealed. Be sure to list all labs that contributed waste to the box. Apply the label in the designated area on the box.
- Be sure you write the date of closure on the box, not the date the box was assembled and began to be used.
- Notify EHS when you have RMW waste for pickup using the EHS online request system.
4.10.6 Animal and Animal-Related Wastes
Lab workers who produce a minimal volume of biological material with no chemical present (e.g., tiny pieces of dissected unfixed tissue) can dispose of that material in solid biowaste which will be autoclaved.
Lab workers who produce larger volumes of this waste or other types of animal/animal-related wastes should refer to Virginia Tech’s Animal and Animal-Related Waste Procedures charts to determine proper waste handling and disposal. Categories of wastes included in the charts are:
- Animal tissue with/ without fixative or other chemicals present
- Animal tissue or carcasses with/ without hazardous biological agents, rDNA, etc.
- Companion animal carcasses
- Related wastes: bedding, disposable containers, fecal material, dressings, etc.
- Associated sharps and liquids
- Blood collection tubes
- Radioactive wastes
4.10.7 Plant and Plant-Related Wastes
BSL-1P, BSL2-P Greenhouse Plant Waste. Examples include transgenic, exotic, or infected plant material; soil; pots; etc.
Collect in clear autoclave bags or other bags/containers appropriate for the method of biological deactivation to be used.
Heat inactivation can be accomplished by:
- Autoclaving (for smaller volumes) at 121˚ C, 15-30 psi, for 15-180 minutes, depending on type and state of material.
- Treatment in greenhouse steam box/soil sterilization box (for larger volumes, for fungal, viral or nematode plant pathogens under permit) at ≥ 104˚ C for 3 hours. NOTE: Permission to use greenhouse steam box must be obtained from the greenhouse manager.
BSL-1P material can be composed or desiccated, according to IBC approved protocols.
Deactivation of plant/seed material must be confirmed before disposal. Acceptable methods of confirmation include:
- Recording time/temperature criteria of heat treatment used.
- If autoclaving, use verification devices.
- Following successful deactivation, material can be discarded in regular trash cans with black liners
Plain Plant Waste
Non-transgenic, non-exotic, non-infective laboratory plant waste is still laboratory-generated waste, and can appear identical to other more hazardous plant wastes.
According to your lab-specific situation, this type of waste can be handled in several ways, at the PI’s discretion:
- Collect, deactivate and discard this waste as if it is BSL-1P or BSL2-P waste.
- Collect material in opaque trash bags; when full, close securely and discard bags in dumpster.