1. Select appropriate containers/bags for autoclaving. b. Ensure that contaminated materials are free of sharp objects which may puncture bag.

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1 Requirements for Decontamination by Autoclaving Summary: Steam sterilization (autoclaving) is one of the most effective methods for decontaminating biohazardous material. Autoclaves use saturated steam under high pressure to sterilize infectious materials (cultures, cells, contaminated glassware, pipettes, etc.), media, lab ware and other items. Biohazardous material and sharps containers generated within research labs can be handled in one of two ways: unsterilized and picked up for handling by an outside vendor (submit an on-line request) or sterilized using departmental autoclaves. When using lab or departmental autoclaves, the Principal Investigator (or Department Head) is responsible for meeting the following requirements: 1. Maintain a log book in accordance with Section 4A below. 2. Autoclave for a MINIMUM of 60 minutes (see Section 4B) unless validation data demonstrates that les time is sufficient for decontamination. 3. Document proper validation techniques when decontaminating material (see Section 8). The recommendations below serve as guidelines to help autoclave operators ensure a safe and effective process. For questions concerning autoclave requirements or guidelines or safety, contact the Office of Regulatory Compliance & Safety (ORCs) at What To Do 1. Select appropriate containers/bags for autoclaving. For biohazardous DRY materials: a. Use polypropylene bags or reusable containers. Make sure your plastic container is suitable for autoclaving. Some plastics such as polyethylene or HDPE are not. b. Ensure that contaminated materials are free of sharp objects which may puncture bag. c. Fill bags ~2/3 full. Ensure adequate steam penetration by closing bags loosely leaving an ~ one inch opening. Close with autoclave indicator tape. d. Red sharps containers may be autoclaved, labeled as TREATED if run is successful (see Section 8) and placed in a large broken glassware box for subsequent removal to dumpster. For biohazardous liquids: e. Fill liquid containers ~1/2 full. Loosen caps or use vented closures. f. Never put sealed containers in an autoclave. They can explode. Large bottles with narrow necks may also explode if filled too full. For non-biohazardous labware: g. Cap pyrex bottles loosely whether empty or filled to prevent explosions due to expansion. h. Use aluminum foil to cover or wrap labware.

2 2. Place autoclave bag or other container into a secondary container. a. Make sure your plastic secondary container is suitable for autoclaving (not polystyrene or HDPE). b. Polypropylene, polycarbonate or stainless steel tubs are typically used for secondary containment. c. Select a container with the lowest sides and widest diameter. This will promote steam penetration and will collect nay leakage of fluid overflow. d. Leave space between items/bags to allow steam circulation. Water can also be added to plastic pans at a depth of one inch or into bags with solid materials. WRONG WAY! 3. Select the correct autoclave cycle for your materials and check the drain strainer. a. Review the manufacturer s operating manual and any lab SOPs for the autoclave unit. Training should be provided for any new autoclave operators. b. Slow Exhaust is the recommended cycle for biohazardous dry waste and liquids. c. Check the drain strainer at the bottom of the chamber before using the autoclave. Clean out any debris. d. For efficient heat transfer, steam must flush the air out of the autoclave chamber. If the drain screen is blocked with debris, a layer of air may form at the bottom of the autoclave and prevent proper operation. e. Autoclave the load immediately after preparation. Do not leave unprocessed items in the autoclave overnight.

3 4. Fill out the autoclave logbook and be aware of required cycle times. PLEASE NOTE THE FOLLOWING REQUIREMENTS (a,b) a. Each unit used to sterilize biohazardous waste must have an autoclave log where the operator records the date, name, cycle time and indicator check results. For both dry and liquid biohazardous materials: b. Cycle time must be set for a minimum of C, 15 psi 1 unless decontamination in less time can be demonstrated. See section 8 for information on verification procedures. c. Liquids and large loads containing many bags may require more time. For sterilization of non-biohazardous labware or media: 5. Follow these precautions when the autoclave cycle is finished. d. Cycle time for sterilization will vary and does not require verification. For larger volumes, allow extra time and for dry goods allow ~ 20 minutes of drying time. Dry time may need to be increased for enclosed items such as pipette tips or bottles with lids. a. Wear personal protective equipment: Lab coat, eye protection, closed toe shoes, heat resistant gloves to remove hot items. b. Never open an autoclave set for slow exhaust until the cycle is complete. Superheated liquids can boil over and damage the autoclave and injure the operator. c. Open the door cautiously. Stand behind the door and open slowly. d. Let liquids stand another minutes after autoclave is opened to avoid any movement that can cause boiling over. e. Do not override the autoclave s built-in safety control features unless authorized by autoclave repair vendor.

4 6. Properly dispose of materials. a. Biohazardous material NOT autoclaved (either it cannot be autoclaved or the lab chooses not to do it themselves) should be placed into an infectious waste box supplied by ORCS. The box should not weigh more than 40 pounds and should not contain more than small amounts of liquid. TREATED WASTE b. Biohazardous waste that has been autoclaved should be labeled as TREATED WASTE, placed into a black plastic trash bag and then taken out to the nearest dumpster by laboratory personnel. Custodians should never handle lab waste. 7. Materials NOT appropriate for autoclaving! a. Never put materials containing solvents, corrosives or radioactive materials in the autoclave (i.e. phenol, chloroform, pyridine or bleach 2 ). b. Pathological lab materials such as whole human cadavers, recognizable human body parts must not be autoclaved. Animal carcasses or tissues may be evaluated on a case by case basis. Contact the Biosafety Office ( ) for disposal instructions.

5 8. Perform autoclave verification tests For each load: Chemical indicator a. Place a chemical indicator (CI) strip in one of the red bags being autoclaved. b. After the run is completed, retrieve the strip and record the result in the log book. If the strip has demonstrated adequate sterilization (as per the manufacturer), the biohazardous waste may be discarded as treated waste. If the strip fails, repeat the run. If it fails again, contact the service vendor for the autoclave. Monthly: Biological indicator c. Place a biological indicator (BI) in one of the red bags being autoclaved. BIs are the gold standard for autoclave verification because they directly determine whether the most resistant microorganisms (Geobacillus or Bacillus spores) are killed as opposed to determining whether the physical and chemical conditions necessary for sterilization are met 3. d. Upon completion of the cycle and following the manufacturer s instructions, the BI is incubated (24 or 48 hours) and observed for signs of growth (turbidity, color change). Record the result in the log book. If growth does occur, the autoclave is not sterilizing properly. Contact the service vendor immediately. 9. Perform preventative maintenance a. Autoclaves sterilizing biohazardous waste should be maintained on a regular service contract. b. Calibration should be performed annually 4 and can be performed by the service vendor as part of the annual maintenance contract. c. If you suspect a problem with your autoclave, contact your service vendor for assistance. References 1 Le, R.N., et al (2005) Autoclave Testing in a University Setting. Applied Biosafety, 10(4), Trace amounts of bleach used to decontaminate laboratory materials during procedures (e.g. decontaminating pipette tips or other lab ware) may be autoclaved. 3 Centers for Disease Control and Prevention, Oral Health Resources Sterilization-Monitoring FAQs, April ASME Boiler and Pressure Vessel Code and Public Law PL