Biohazardous and infectious (or potentially infectious) agents are routinely handled and manipulated in biomedical and animal laboratories. Biohazards include human samples and tissue cultures, bacteria, viruses, prions, parasites, toxins, recombinant DNA (rDNA) - which is used in expression vectors such as plasmids, infected or transfected/transduced cells, infected human or animal tissues, transgenic animals, and any material contaminated with a biohazardous agent. To prevent the escape of biohazards into the environment, waste from biohazardous operations must be treated and/or disposed of in a special way. The following section reviews how to process and discard biohazardous waste appropriately, as well as other types of waste frequently generated in biomedical and animal laboratories.
The Department of Environmental Conservation (DEC) of the State of Vermont regulates the disposal of Solid Waste (SW). An important part of solid waste generated in the medical industry requires special handling and treatment prior to disposal in order to protect public health, safety, and the environment. This particular type of SW is known as Regulated Medical Waste (RMW), biohazardous waste, or infectious medical waste. RMW is heavily generated, not only by industries in the healthcare sector, but also by research institutions across the world.
Procedures addressing definitions, handling, and treatment of RMW were published by Vermont's DEC in 2001 in the following document: www.anr.state.vt.us/dec/wastediv/solid/pubs/MedWaste.pdf
An updated list of the types of solid waste that are considered Regulated Medical Waste was published in the Solid Waste Management Rules (SWMR) of Vermont's DEC in 2006: www.anr.state.vt.us/dec/wastediv/solid/pubs/SWMRules.pdf
The following types of solid waste (some examples pictured below), which you may encounter during your research or clinical work, fall under the category of Regulated Medical Waste:
1. Pathological Waste: Human tissues, organs and body parts.
2. Human blood, blood products, and other body fluids including:
- Liquid waste human blood, blood products and components
- Other potentially infectious liquid body fluids: amniotic, cerebrospinal, peritoneal, pleural, and synovial fluid
- Items saturated or dripping with blood or potentially infectious body fluids and those caked with dried blood or potentially infectious dried body fluids
3. Cultures and stocks of infectious agents, including:
- Cultures and stocks of infectious agents from research, industrial and educational laboratories
- Discarded live and attenuated vaccines
- Culture dishes and devices used to transfer, inoculate and mix cultures
4. Sharps - objects capable of cutting or penetrating the skin and inducing subdermal inoculation of an infectious agent, including:
- Pasteur pipettes
- Scalpels and blades
- Discarded unused sharps
5. Animal waste, including animal carcasses, body parts, bedding and other items from animals known or suspected by either the Department of Health or the Department of Agriculture of being contaminated with organisms that can produce disease in humans, and for which disposal by burial or other ordinarily acceptable means would not sufficiently reduce the risk of disease transmission to humans or other animals.
6. Chemotherapy waste: Any material containing cytotoxic and/or antineoplastic agents during the preparation, handling or administration of the agent, including:
- PPE: masks, gloves, gowns...
- Empty IV tubing bags and vials
- Other contaminated materials
7. Infectious isolation waste: biological waste and discarded materials contaminated with blood, body fluids, excretion, exudates or secretion from isolated humans with dangerous communicable diseases.
8. Biotechnological by-product effluents: any discarded preparation made from genetically altered living organisms and their products.
All biohazardous waste containers must be labeled with a biohazard label (red or red/orange label with the universal biohazard symbol and the word BIOHAZARD underneath). Please contact us at: Safety@uvm.edu if you need biohazard labels.
In addition, red or red/orange biohazard bags should be used for the disposal and/or autoclaving of biohazardous waste. Clear biohazardous waste bags are not allowed on our campus any longer, since they can be easily mistaken with trash bags. Red biowaste bags are usually available in the HSRF biowaste shed, Terrill 109 and the biowaste room in the Colchester Research Facility.
Liquid biohazardous waste can be disposed of in different ways depending on the type of biological agent present, containing vessel, etc. The most frequently used method to discard liquid biohazardous waste is:
- Chemical inactivation and pouring down the sink drain: Growth medium used in mammalian tissue culture, and bacteria or yeast culture, should be collected and inactivated for 20 minutes in a recipient containing a fresh bleach solution before being discarded down the drain. To collect such waste, aspirate it into a flask containing bleach (A) connected to a second overflow collection flask (B) also with bleach, and separated from the vacuum system (D) by an in-line filter (C). The final concentration of bleach in the flasks should be 10 to 20%, depending on the amount of organic load present. Bleach solutions maintain their activity for approximately a week, after which a new solution should be prepared. To ensure that all the biohazardous waste is neutralized before disposal, mix the contents of the flasks thoroughly with the bleach and allow a minimum contact time of 20 minutes (see picture below; source: Biosafety in Microbiological and Biomedical Laboratories, 5th Edition). Note: If using a disinfectant other than bleach, please contact Environmental Safety at: Safety@uvm.edu to confirm whether the disinfectant can be safely and legally disposed of down the drain.
A large portion of the waste generated in microbiological and biomedical laboratories belongs to the category of Regulated Medical Waste and is treated as such. Before reaching its final destination, a cardboard biowaste box, the waste is discarded either in a properly labeled biohazard container lined with a red biohazard bag or a sharps container (or equivalent), as described below.
- Collection in biowaste containers lined with red biohazard bags: Biologically contaminated solid waste should be deposited into biohazard containers (red biohazard bag in biohazard container). Some examples of biohazardous waste commonly found in the lab include: flasks, plates, and tubes used for cell culture; ELISA plates; bacterial plates; PCR, DNA, RNA, and protein-containing tubes and plates; flow cytometry tubes; parafin blocks with tissue; contaminated liners, paper tissue, and Kim wipes; blood and tissue samples... All biohazardous waste containers should be covered when not in use.
- Collection in sharps containers: Objects that can penetrate the skin such as scissors, scalpels, needles, razor blades, histology slides and coverslips, pasteur pipettes, serological pipettes, pipette tips and broken glass are considered sharps. Needles, razor blades, pasteur pipettes and broken glass contaminated with biological agents should only be collected in biohazard sharps containers. Similarly, all histology slides and coverslips used with biohazardous materials, whether fixed or not, should be discarded into a biohazard sharps container that will ultimately be deposited into a biowaste box. Biologically contaminated serological pipettes should be collected in a sharps container, or an equivalent receptacle (e.g., an empty serological pipette cardboard box with a biohazard sign), before being deposited into a Clean Harbors biowaste box. The picture below illustrates how to discard biologically contaminated sharps correctly.
NOTE: Non-contaminated serological pipettes and pipette tips can be discarded in a broken glass box
- Temporary collection in puncture-proof containers: Pipette tips can be collected in a puncture-proof container on the open bench and discarded into a biowaste box when full.
- Autoclaving and collection in biowaste containers lined with biohazard bags: Based on a risk assessment of your work, you may need to autoclave the biowaste generated. For questions regarding whether you should autoclave your biowaste, please contact the Biosafety Officer, Jeff LaBossiere, at: firstname.lastname@example.org
We recommend the autoclaving of highly infectious materials, and disposable labware that has been contaminated with it, prior to its disposal as Regulated Medical Waste. Autoclaving is used to achieve the sterilization or complete destruction of the microorganisms present in the autoclaved materials,
Both solid and liquid materials can be autoclaved using different programs. Special autoclave biohazard bags should be used for items that must be bagged before autoclaving, and a chemical indicator such as heat-sensitive tape (autoclaving tape) should be placed on every load. This tape changes color (from white to black) after being exposed to a specific temperature, verifying that the correct temperature was reached. However, the change in color does not confirm whether the appropriate contact time or sterilization was achieved.
If autoclaving biohazardous waste, autoclave effectiveness should be verified and documented, at least monthly, using a biological indicator. The spores of Geobacillus stearotermophilus are used as a biological indicator because they are among the most resistant living forms to steam autoclaving. To determine the effectiveness of the autoclaving process, the biological indicator must be placed in a typical test load (solid or liquid) and exposed to standard cycle conditions. Follow these procedures to use biological indicators:
1. Read and follow the supplier's instructions.
2. Place G. stearotheromophilus in the center of a representative test load.
3. Process load in normal fashion.
4. Extract and incubate the G.stearothermophilus sample as instructed by the manufacturer. Incubate another ampoule (from the same lot number) that has not been autoclaved (positive control).
5. Check for color changes in the culture media at regular intervals during the incubation period (8, 12, 24, and 48 hours). If the media is yellow and turbid, the autoclave process has FAILED. Immediately re-run all samples with new biological indicators.
6. If failure continues to be noted, either increase the exposure time or initiate repairs to the autoclave. Note: The autoclave cannot be used again until validation procedures indicate that the autoclave is properly sterilizing the contaminated materials.
7. Record all results (positive and negative) in the autoclave notebook.
Note: If you need autoclaving tape or biological indicators, please contact Environmental Safety at: Safety@uvm.edu
Biowaste Box Marking Procedure
Biowaste boxes, bags, and tape are available in the HSRF biowaste shed, CRF biowaste storage room, and Terrill 109.
Biowaste boxes must clearly indicate the contact information of the lab where they originated. Please clearly write with a BLACK marker the following information in your biowaste box:
- Lab Building and Room Number
- PI or Lab Group Name, and
- Lab Phone Number.
Properly packed and marked boxes can be deposited in the HSRF biowaste shed, CRF biowaste storage room, or Terrill 109 depending on where your lab is located. Please contact Sonia at email@example.com if you need your CATcard activated for any of these locations.
Note: The Hazardous Waste Team (Brian Hodge and Brian Medor) will pick up biowaste boxes from the BioResearch Center, Cook, Dewey, Perkins, Terrill 109 and Votey. Please make sure that you pack your biowaste boxes correctly.
Biowaste boxes should not contain any free liquid. If boxes are leaking, they will be left behind and you will be asked to repack them for the next pick-up. No chemicals should ever be packed in biowaste boxes.
Frozen biohazardous material, such as boxes containing frozen carcasses from the Animal Care Facility, should be brough to the HSRF Biowaste shed no earlier than Monday evenings. This will reduce damage to the boxes caused by thawing of the material before the Tuesday morning Clean Harbors biowaste pick-up.
Box Weight Limit
One individual biowaste box should never weigh more than 55 lbs. Less weight is better than more. If you think you are nearing the 55 lb limit, start a second biowaste box.
Double (Red) Bag
2 red bags are required inside each biowaste box. Tie both bags before taping all sides of the carton with clear tape.
Animal carcasses must be disposed through the Office of Animal Care Management (OACM). This type of waste is placed into sturdier biowaste boxes closed with a lid and labeled with Animal Care generator labels (instead of HSRF labels). Please direct your questions about animal disposal to the OACM at 656-1006.
Improper handling of biohazardous waste can result in accidental exposure to infectious agents. Thus, all researchers who work with biohazardous agents are responsible for the proper treatment and disposal of biohazardous waste. They must keep in mind that other persons, such as custodians who clean the lab and the contractor who handles biowaste for its final transportation and disposal, are at increased risk for exposure. The pictures below show some incorrect ways of disposing of biowaste in the lab and packaging it. Let's review what's wrong with each of them.
Picture 1: A serological pipette has been discarded in the regular trash. Even if the pipette was not contaminated with a biohazardous agent, it is considered a sharp because it can puncture the skin. Non-contaminated pipettes should only be discarded in the regular trash if properly packaged inside a puncture-resistant cardboard box.
Picture 2: Serological pipettes have carelessly been thrown into a biowaste box sticking out in different directions. Biologically contaminated pipettes should always be discarded in a sharps container or a puncture-resistant carboard box before being deposited into a biowaste box. Non-contaminated plastic sleeves that could go in the regular trash have also been discarded in the biowaste box. In addition, biowaste boxes and containers should be closed when not in use.
Picture 3: An overpacked box is bulging and exposing the inner biohazard lining bag. Biowaste boxes should be properly closed in a way that the inner contents are not exposed to the outside. In addition, they should not be overpacked and cannot exceed 55 lb. The biohazardous waste carrier may decline pick up of heavy and incorrectly packed boxes like this one, in which case the responsible researchers will be asked to open the box and repack the contents again into two different boxes.
Picture 4: The biowaste box is grossly contaminated near the bottom. Biowaste boxes should be free of exterior contamination. The biohazardous waste carrier may decline pick up of contaminated boxes like this one, in which case the responsible researchers will be asked to open the box and repack the contents again.
Biowaste box covers and dollies can be purchased through the ChemSource program. Please consider using them if you have Clean Harbors biowaste boxes inside your lab.
Please, help us keep yourself and the others safe by handling your biowaste correctly. If you have any question regarding biowaste management, feel free to contact us at: Safety@uvm.edu
Laboratory equipment and appliances that have been used with biohazards should be thoroughly decontaminated, and have the biohazard labels removed, before being disposed of through the UVM Surplus Disposal Program. Please follow this link with instructions on how to get biologically contaminated equipment and appliances ready for disposal:
The picture below illustrates both the correct way of disposing of an incubator (Incubator 1: decontaminated, without biohazard labels and with Surplus Form attached) and the wrong way (Incubator 2: not decontaminated, with biohazard label and without Surplus Form).
If you have any question or concern, please do not hesitate to contact Environmental Safety at 656-5400 or: Safety@uvm.edu
Last updated: January 31, 2014