Marywood University Biological Waste Disposal Standard Operating Procedure
Biological Waste Disposal
Overview Biological waste must be sterilized before disposal. Sterilization serves to destroy all microorganisms that may contaminate materials used in the laboratory. The waste sterilization procedure used autoclaving. An autoclave creates moist heat at temperatures high enough to kill bacterial endospores. Steam under pressure at 15 lb/in2 creates the needed temperature of 121◦C. Processing time necessary to achieve decontamination of biological material depends on several loading factors. Load size, type of container, and moisture content all impact decontamination time. An extended autoclave cycle (excluding exhaust time) is recommended for autoclaving waste to allow enough time for the center of the load to come up to temperature and have sufficient steam penetration. This will insure that the temperature in all parts of the load has reached 121ºC for at least 20 minutes.
Types of Biological Waste (See Appendix A) Autoclave waste bags - Autoclave waste bags are used for bulk dry disposable materials such as paper towels, contaminated gloves, plastic centrifuge tubes, plastic Pasteur transfer pipettes and plastic petri dishes (with or without agar-based culture media). Autoclave bags used for biological waste at MU are made of translucent, colorless, steam resistant plastic film with no markings. Autoclave bags should be loosely taped or tied during decontamination to allow steam to penetrate into the bag. Air pockets in tightly sealed bags may cause localized sterilization failure. Bottles – Glass bottles or glass tubes with screw or push tops are used for autoclaving liquids such as water, some buffers and culture media. Always loosen the screw or push top so that it barely engages the thread or neck of the bottle or tube. If using plastic bottles and or caps, always verify that the plastic is heat-resistant before using them in an autoclave. Miscellaneous – Steam and heat-resistant pouches are commonly used to sterilize surgical instruments. Metal cans with snug-fitting metal lids may be used to sterilize glass serological pipettes (although this is less common nowadays because disposable pipettes are widely used). Hard-walled plastic sharps containers should not be autoclaved. These should be disposed of through the sharps waste stream. Report full sharps containers to the Lab Manager so they may be transported to the medical waste accumulation site. Medical waste bags are red or orange with a preprinted universal biohazard symbol. They contain medical or pathological waste such as cell lines, tissues and body fluids or consumables contaminated with body fluids. Red bag waste should not be autoclaved. Report full red bag waste to the Lab manager so they may be transported to the medical waste/pathological waste accumulation site. Burn-Up Bins contain medical waste bags that hold plastic disposable serological pipettes that are contaminated with medical or pathological waste such as cell lines, tissues and body fluids. Report full burn-up bins to the Lab Manager so the medical waste bags inside may be transported to the medical waste/ pathological waste accumulation site.
Applicability This procedure applies to all University Faculty, Staff, and Students and any other university employee who is involved with the disposal of laboratory waste.
Requirements Petri plates, disposable plastic pipettes, culture media and gloves must be placed in the “Waste to be Autoclaved” red or white trash cans that are lined with clear autoclave bags. Test tubes with media or other non-disposable waste to be autoclaved are to be placed on the waste cart located beside the autoclaves.
Page 1 2/18/2015 Marywood University Biological Waste Disposal Standard Operating Procedure
Autoclave Procedures for Biological Waste Disposal of waste in clear autoclave bags 1. Laboratory coat, gloves and goggles must be worn. Autoclave gloves must be worn when handling hot materials. 2. Remove the autoclave bag from the red waste can and refill with a new clear autoclave bag. 3. Loosely seal the bag with autoclave tape, removing as much air as possible before sealing. o Visually inspect bag for tears or holes. It is occasionally advisable to double bag the waste. 4. Place the bag into the autoclave inside a secondary tray (to catch any leaks). 5. Close and secure autoclave door. Please refer to Use of Autoclaves SOP for details on autoclave operation. 6. Set autoclave timer for 60 minutes and temperature to 121◦C. 7. Start the autoclave. 8. Allow the autoclave to reach zero pressure and a temperature below 121◦C before opening door. 9. While wearing autoclave gloves, remove the secondary tray containing the bag of waste and place on the waste cart. 10. Make sure the autoclave tape has turned black. 11. Allow the bag to cool slightly and then place into the garbage can. o If the bag appears to have any holes, double bag it before placing it in the trash.
***Note that 60 minutes of sterilizing averages 2 hours for full cycle which includes an average 30 minute heat time, and 30 minute cool down time. When up to temperature the autoclave will read 121°C, the timer begin count down from 60 minutes and the psi will average 15. ***
Disposal of waste from glass test tubes 1. Laboratory coat, gloves and goggles must be worn. Autoclave gloves must be worn when handling hot materials. 2. Organize test tubes in separate racks according to tube type and size, loosen lids, and remove labels (Labels must be removed before autoclaving). 3. Label the test tube racks as “Waste” with autoclave tape. 4. Place organized racks of tubes into a secondary container and then in the autoclave. 5. Close and secure autoclave door. Please refer to Use of Autoclaves SOP for details on autoclave operation. 6. Set autoclave timer for 30 minutes and temperature to 121◦C. 7. Start the autoclave. 8. While autoclave is processing waste, proceed to number 17 if applicable (if tubes are soaking by sink) otherwise proceed with number 9. 9. Allow the autoclave to reach zero pressure and a temperature below 121◦C. 10. While wearing autoclave gloves, remove the tray containing the racks of tubes and place by the sink. Allow tubes to cool enough so that they are cool enough to handle with nitrile gloves. 11. Make sure the autoclave tape has turned black. 12. Obtain two black containers found below the sink. Place one in sink for the tubes and one on the countertop for lids. 13. While wearing nitrile gloves remove each test tube from the rack take, off its lid. Run lid under warm water and place in “lid” bucket. Pour tube contents down the drain with warm running water, make sure label is removed and place in “tube” bucket. 14. All tubes should be placed in bucket in the same direction with a separate bucket for each tube type and size. A separate bucket should also be used for lid type and size. 15. When tubes have all been emptied, measure 60mL of Versa Clean Liquid concentrate(soap) into each black container and fill with warm water. 16. Place all black containers on countertop and allow tubes and lids to soak for at least one hour. 17. After tubes and lids have soaked, drain water from black containers. 18. Rinse and scrub each test tube and lid as necessary to remove any residual waste. 19. Place test tubes face down into a metal wire container, found on shelves, and place in the dishwasher. All tubes in each container should be of same type and size. 20. Place lids in white basket with lid. All lids in each basket should be of same type and size. Place in dishwasher. Make sure lid is on basket before placing in dishwasher.
Page 2 2/18/2015 Marywood University Biological Waste Disposal Standard Operating Procedure
21. Fill dishwasher with dishwasher detergent and run the dishwasher on normal cycle to clean all test tubes and lids. 22. When dishwashing is complete, return test tubes to proper storage bins on shelves. 23. Take the white baskets containing clean lids and flip it over to drain water and let lids dry. Once dry add lids to stock. 24. Keep tube and lid stock organized. The key to organized stock key to follow the steps in this procedure.
***Note that 30 minutes of sterilizing averages an hour and half for full cycle which includes an average 30 minute heat time, and 30 minute cool down time. When up to temperature the autoclave will read 121°C, the timer begin count down from 30 minutes and the psi will average 15. ***
First Aid
In the event of skin contact with a hot liquid, seek medical attention. Report any incident to the laboratory manager.
Disposal Requirements
Spills can be cleaned up with a paper towel and disposed of in the trash. Use caution with hot media.
References:
1. Techniques in Microbiology: A Student Handbook, Lammert, John M., Pearson Education, Inc., 2007 2. Biological Safety Principles and Practices (4th edition) edited by Diane Flemming & Debra Hunt, ASM Press, Washington D.C., 2006
Appendix A: Disposal of Laboratory Waste
Disposal of Sharps and Burn-Up Bins Sharps which include used microscope slides, syringes/needles, swabs, scalpel blades and lancets are disposed of in a red sharps container located on the counter top. Contaminated plastic disposable serological pipettes are collected in red biohazard bags inside of burn-up bin boxes. The Lab Manager is notified when sharps and burn-up bin containers are full. Sharps containers and biohazard waste bags from burn up bins will be stored in the Medical Waste Accumulation Area and disposed of properly as medical waste.
Page 3 2/18/2015 Marywood University Biological Waste Disposal Standard Operating Procedure
Contaminated Consumables (Autoclave Waste Bags) Contaminated consumables which include disposable plastic transfer pipets, gloves, paper towels (used to clean lab benches), exudates, body fluids and cultured Petri dishes are to be placed in the red receptacles marked waste to be autoclaved. These receptacles are lined with clear autoclave bags. This waste will be autoclaved and disposed of in the regular trash.
Dissection Tissues (Medical/Pathological Waste Bags) Used dissection organs/tissues and contaminated gloves are collected in the red biohazard bags located on the counter top near the sink. To prevent leakage, each bag should be placed in another red bag essentially double bagging it. At the end of each lab period or when a bag is full, it is placed either by the laboratory instructor or laboratory assistant in the -20°C freezer located in CNHS 95 marked Medical/Pathological Waste Accumulation Area. These bags are stored and disposed of as pathological waste.
Media/Liquid Waste Cultured bottle/tube media including all slant, deep, and broth agars are placed on the waste cart in CNHS 107 located next to the autoclaves. When full, plastic biohazard liquid waste bottles from biological hoods are moved to the waste cart in CNHS 107. This waste will be autoclaved and disposed of in the regular trash. The glass tubes and plastic bottles will be washed and reused.
Chemical Hazardous Waste Hazardous waste which includes stains, dyes and their wash liquids are collected as indicated in each course lab needs outline. Once collected they are transferred to a properly labeled hazardous waste container. When full or at the conclusion of the lab experiment, the waste container should immediately be transferred to the Fume Hood in CNHS 304 by the lab assistant or lab instructor. The Chemical Hygiene Officer will examine the waste and it will be disposed of properly as hazardous waste.
Broken Glassware Broken glassware is disposed of in the white glass container located in each lab. This does not include contaminated broken slides which should be disposed of in a red sharps container.
Regular Trash Non contaminated paper towel and non contaminated gloves may be disposed of in the regular trash
Page 4 2/18/2015