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Autoclaving Guidelines for Sterilization of Lab Equipment

Operating an autoclave requires caution and adherence to strict regulatory requirements. Follow these autoclaving guidelines for sterilizing equipment and research materials.

by Lab Manager
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Operating an autoclave requires caution and adherence to strict regulatory requirements. Follow these autoclaving guidelines for sterilizing equipment and research materials.

1. Check the autoclave's drain screen.

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. Check the drain screen at the bottom of the chamber before using the autoclave. Clean out any debris.

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2. Prepare research materials for sterilization.

  • Glassware:
    • Cap Pyrex bottles loosely–whether empty or filled–to prevent explosions due to expansion.
    • Cover bottles that are not made of safety glass (e.g. not Pyrex) with aluminum foil.
  • Liquids: Fill containers only half full
  • Combination loads: Do not combine strong oxidizing material (such as dry hypochlorites) with organic materials (such as paper, cloth or oil).

3. Use secondary containment for all items being autoclaved.

  • Polypropylene or stainless steel tubs are typically used for secondary containment.
  • Make sure your plastic container is suitable for autoclaving. Not all plastics can be autoclaved. Plastic types can be identified by looking for initials imprinted on the container bottom.
    • Use:
      • Polypropylene (PP, recycle #5)
      • Polycarbonate (PC, no recycle number assigned)
    • Do not use:
      • Polyethylene (PE, recycle #1)
      • High-density polyethylene (HDPE, recycle #2)
    • If you're unsure about a new container, place it in an autoclave-safe container the first time. 
  • Select a container with the lowest sides and widest diameter possible for the autoclave.
  • Leave space between items to allow steam circulation.

4. Close the autoclave door securely.

Be sure the door is sealed before selecting the cycle.

5. Choose the correct cycle.

  • For liquids, always choose the liquid or "slow exhaust" cycle.
  • Ask your lab manager which cycle is recommended for sterilizing dry goods or equipment.
  • For descriptions of the 2 basic autoclave cycles, read Autoclave Overview.

6. Set the appropriate time for sterilization and drying.

These guidelines contain recommended sterilization times. Always follow your lab's written operating procedures.

  • Nonhazardous dry goods: 30 minutes of sterilization plus 20 minutes of drying time. Dry time may need to be increased for enclosed items such as pipette tips or bottles with lids.
  • Liquids (add 10–20 minutes for crowded items):
    • Less than 500 milliliters (ml): 30 minutes
    • 500 ml – 1 liter: 40 minutes
    • 2–4 liters: 55 minutes
    • More than 4 liters: 60 minutes
  • Let liquids stand for at least 10 minutes after the cycle is complete before opening the door.

Note: Autoclaving new glassware for 90 minutes will partially temper it, increasing its strength.

7. Start the autoclave.

Push the "start" button on the control panel to initiate the cycle.

8. Fill out the autoclave log.

Each machine must have an autoclave log where the operator records the date and other details. Fill out the log while the autoclave is "charging" or starting.

9. Follow these precautions when the autoclave cycle is finished.

  • Wear personal protection equipment:
    • Lab coat
    • Eye protection
    • Closed-toe shoes
    • Heat-resistant gloves to remove items, especially hot glassware
  • Wait for the pressure gauge to drop to zero with zero time remaining before opening the door.
  • Never open an autoclave set for "slow exhaust" until the cycle is complete. Super-heated liquids can boil over and damage the autoclave and the operator.
  • Open the door cautiously. Stand behind the door and slowly open it. Allow all steam to escape before reaching inside.
  • Let liquids stand another 10-20 minutes after the autoclave is opened to avoid any movement that could cause them to boil. Remove items carefully.