Collecting waste
Autoclaving your waste
Autoclave validation program
Steps for autoclave validation
Autoclave validation for BL2+ and BL3 labs
Steps to take if an autoclave fails validation
Supplies
Autoclave log
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Collecting waste

Items that should not be placed in the autoclave waste bag include:

• Biologically contaminated sharps: Commonly referred as biosharps, these are disposed in leak-proof, puncture-proof containers. To request containers or to schedule a biosharps pick up, email biosharps (at) mit.edu
  
• Hazardous chemicals: To schedule a chemical waste pick-up or to request red tags, satellite accumulation tags or secondary containers, please fill out the Chemical Waste Collection form at http://web.mit.edu/environment/ehs/waste.html#2
  
• Radioactive materials: Contact the EHS Radiation Protection Program, x2-3477, for information on proper disposal of radioactive materials. To schedule a radioactive waste pickup please fill out the Radioactive Waste Collection form at https://web.mit.edu/environment/ehs/topic/mitonly/
  radiowastepickup.html
  
• Mixed wastes. If your biological waste also contains hazardous chemicals or radioactive materials, contact the Biosafety Program (x2-3477 or bsp(at)mit.edu) for assistance.

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Autoclaving your waste

If you have never operated the autoclave you will be using, contact an experienced user in your lab, your EHS Rep or your EHS Coordinator to be shown how to operate it. The following are general instructions for autoclave use but do not replace hands-on training.

1. When a bag is 75% full, it’s time to autoclave it. Put the bag in a shallow tray and place it in the autoclave. If the bag must be stored before autoclaving, place it in a tray or bin marked with the universal biohazard symbol.
   
2. Fill out a numbered autoclave tag for the bag using a permanent marker. Tape the tag to the bag. Do not seal the bag; leave it open so that steam can get in.
   
3. Fill out a line in the autoclave log, noting the autoclave tag number, date, time, planned temperature and length of run, your name and your Principal Investigator or lab name.
   
4. Close and seal the autoclave door.
   
5. Select the run time and temperature, or the “waste” cycle. Generally recommended parameters are 60 minutes and 2500°F/ 121°C at 15psi, but your autoclave may require different settings.
   
6. Run the autoclave cycle.
   
7. At the end of the autoclave cycle, check the chamber pressure and temperature gauges to verify that it is safe to open the autoclave. The chamber pressure should be zero. To avoid the hazards of escaping steam, wait several additional minutes before opening the door.
   The greatest risk of personal injury caused by steam occurs during the process of unloading the autoclave. Wear safety glasses, long sleeves and heat-resistant gloves for the following steps.
   
8. Open the door slowly and only slightly, while standing behind the door and leaning away. Allow steam to escape.
   
9. Allow items to cool in the autoclave for at least 10 minutes after opening the door. Approach liquid contents with care as they will still be very hot.
   
10. Check the autoclave recorder chart to verify that the minimum time and temperature were reached. If possible, make a note of the tag number(s) on the chart. (If the minimum time and temperature were not attained, contact the person responsible for maintenance of the unit to initiate repairs. Leave a note on the autoclave describing the problem and that it cannot be used until the problem has been resolved. Autoclave your waste again in another autoclave.)
    
11. Remove the bag from the autoclave.
    
12. If any liquid has collected in the bottom of the bag, carefully drain it into the sink.
    
13. Use the autoclave tag to seal the bag, and then place it in the regular trash.

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Autoclave validation program

Autoclave validation should be performed monthly using vials containing spores of the heat-resistant bacterium Geobacillus stearothermophilus, supplied by the Biosafety Program. Vials are autoclaved by the lab groups in a representative bag of waste and then sent to the EHS Office for incubation and recording of results in a database.

For shared autoclaves, labs using the autoclave often create a schedule showing which lab is responsible for validation each month. Special schedules may be approved by the Biosafety Program for autoclaves which are rarely used.

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Steps for autoclave validation

1. Pick up spore vial(s), label(s), string and pre-printed labels in 46-6062, VWR Office 56-022, 68-204 or from the coordinator of your autoclave facility. Check that the vial hasn’t expired.
   
2. Place the vial in the bag of waste near the center of the load. One way to do this without contaminating your hands/arms is to tie a string around the vial and then loop the string around the end of a plastic serological pipette, which can be used to insert the vial into the center of the waste. Then pull the pipette out and place it in a bio-sharps container. If the string is left hanging out of the bag, you can easily remove the vial after autoclaving.
   
3. Autoclave waste as usual, and allow it to cool.
   
4. Remove the bag and retrieve the vial from the bag of waste.
   
5. Allow the vial to cool for 10 minutes. Caution: The vial is hot and under pressure. Failure to allow sufficient cooling time may result in an injury if the vial bursts.
6. Put the vial in a conical tube.
   
7. Enter the relevant information on a pre-printed label and attach it to the conical tube.
   
8. Leave autoclaved vials at a drop off location: 46-6062, VWR Office 56-022, 68-204 or E19-105 (near the loading dock), or mail to “EHS Autoclave Validation” at N52-496.
   
9. Results of your test will be sent via email within about a week.

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Autoclave validation for BL2+ and BL3 labs

BL2+ and BL3 labs should have specific written policies and procedures covering use and validation of autoclaves. Due to the higher potential hazard of the materials used in these areas, special autoclave use or validation procedures may be warranted. The Biosafety Program will work with these groups on development of policies and procedures.

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Steps to take if an autoclave fails validation

If validation test results indicate that the autoclave failed to kill the spores, first check the recorder chart to make sure the correct temperature and cycle time were obtained. If not, adjust settings to the correct time/temperature, or call for service if the autoclave is not reaching the desired temperature.

If the correct time and temperature were achieved in your initial test, and the results were positive, you will need to change one or more of these parameters when you do a re-test:

Time: Increase the cycle time by 15 minutes and insert another vial to do a retest. If your results are positive again, increase the cycle time by 15 minutes increments until the results are negative. Reminder: The cycle time required will vary with the composition of the load.

Density: Load the bag to 75% of the holding capacity or less. Don’t compress waste to fit more in the bag, because steam cannot penetrate completely through densely packed waste bags.

Steam: Add approximately one half cup of water to your dry waste loads to facilitate steam generation.

Loading techniques: You may wish to try changing how you load the autoclave to allow steam to better move from the top of the chamber to the bottom and penetrate the load:

• Place bags in the back of the autoclave rather than in the front of the autoclave, which is the coldest section.
  
• Avoid crowding or stacking.
  
• Check that the bags are not touching the top or sides of the autoclave as you push the tray inside.
  
• Try using shallower trays (but make sure they will still contain any spills).

Record what parameters you changed for the re-test on the vial label. They should become the new parameters for autoclaving waste in your autoclave, so be sure to make whatever changes are necessary to protocols and instructions for autoclave use, and to inform others who use the autoclave.

If none of the above gives negative test results, ask your EHS Coordinator or the person responsible for maintenance of the unit to initiate repairs.

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Supplies

Autoclave Bags:
Bags used by most labs should be clear with no biohazard symbol.

In the VWR Stockroom: #14232-184, size is 24x36 inches

On-line ordering via ECAT: In the VWR section, enter item #14233-764, size is 38x48 inches.

Note: These bags should be used as a liner in a marked biological waste container

Orange, red or clear bags with the biohazard symbol:
These bags are only used by the Medical Department, Clinical Research Center, Broad Institute and Division of Comparative Medicine. These groups ship their waste out for incineration, so these bags are not ending up in the regular trash. If another lab finds this type of bag, please contact the EHS Office Biosafety Program, ext 2-3477.

Autoclave Tags (sequentially numbered autoclave tags)
Autoclave tags are available from:
• The EHS Coordinator in Biology, HST, and BCS/PCLM
• Administrative Officer in some departments
• EHS Office Biosafety Program, ext 2-3477 or email bsp@mit.edu
These tags are assigned to each lab. Tags are used to identify who generated the waste if the waste hauler has a concern.

Gloves rated for hot items:
These are available in the VWR stockroom.
online ordering via Ecat: In the VWR section, enter item # WLS 40254-10

Temperature charts (circular or strip type):
Change these charts regularly. It is recommended that BL1 & 2 labs retain these charts. BL2+ and BL3 labs must keep temperature charts for 10 years.

The chart is different for each type of autoclave and there are many types at MIT. They may be ordered from your autoclave vendor or service representative.

Temperature -Sensitive Tape (aka Autoclave Tape):
This is available in the VWR stockroom.
On-line ordering via ECAT: In the VWR section, enter item # 58752-704

When this tape has been exposed to high temperatures, lines will appear. While the use of temperature -sensitive tape can be helpful to indicate that materials have been autoclaved, it is not proof that the autoclave cycle was successful at decontaminating or sterilizing the contents. A biological indicator or other means must be used to validate sterilization.

If the temperature -sensitive tape fails to change color, or the word “autoclaved” fails to appear after autoclaving, this could indicate that there is a problem with your autoclave. You may wish to autoclave the bag again in a different machine. Label the autoclave so others know that it did not function properly and call the autoclave repair vendor.
Follow the steps to take if an autoclave fails validation.

Trays:
Use a tray to contain leaks that could contaminate the autoclave. The tray also prevents having to scrape off melted plastic.

Features of trays:
Sides should be high enough to contain liquid that leaks out of the bag, but not excessively high. Metal trays will transfer heat to bags better than plastic trays and this compensates for the restricted airflow in the autoclave caused by the trays. Restricted airflow becomes much more a factor with deeper trays.

Autoclavable plastic trays
Polypropylene trays can withstand autoclaving without melting. (Polyethylene and polystyrene will melt.) Old plastic trays can become brittle or soft when heated. Regularly inspect trays and discard when they are compromised.

Polypropylene trays are available in the VWR stockroom.
On-line ordering via ECAT: In the VWR section, enter item # 62662-048

Metal trays
(not available in the VWR stockroom)
On-line ordering via ECAT: In the VWR section, enter item # 19758-058

Waste Containers:
These containers should have the following features:

1. The foot pedal opens the container cover. Hands-free operation reduces the risk of glove contamination.
   
2. Lid closes automatically to control odors and pests.
   
3. Enclosed sides to be rodent proof and fly tight
   
4. Biohazard symbol is prominent (request from the Biosafety Program rep)
   
5. Leak proof (in case there is a hole in the bag)
   
6. Can be cleaned with bleach or can be put in the autoclave
   
7. Holds the autoclave bag securely inside the container

Note that the open sided, scissors-frame type of bag holder does not meet Mass. Dept. of Public Health requirement that waste be contained and stored in leak proof, rodent proof, fly tight containers at all times.

Some acceptable containers include:

• plastic models by Eagle -- VWR #56610-177, 56610-226, and 56610-181
  
• metal models by Justrite – VWR #56617-832
  
• step-can type trash receptacles available at local retailers

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Autoclave Log:
Fill in the autoclave log for each run, noting the autoclave tag number, date, time, temperature and length of run, your name and your Principal Investigator or lab name. Keep logs next to autoclaves or in an area of the lab that is accessible to all autoclave users. Current MIT records retention procedures require that autoclave logs be retained for 10 years. EHS staff and Coordinators will check the logs during Level 2 inspections. It is preferable to attach the temperature chart or the tape of the run to the log.

Link to Blank autoclave log sheet.

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