Autoclave Safety and Operation

Autoclaves use high pressure and high temperature steam to kill microorganisms and render biohazardous material inactive. For effective sterilization, the materials/load must be saturated with steam. Air pockets or insufficient steam supply will prevent effective sterilization. Proper cycle parameters for effective decontamination of infectious waste are done using autoclave indicators and performing autoclave validations. For more information see Autoclave - Waste and Validation.

Potential risks of using an autoclave are heat and steam burns, hot fluid scalds, injuries to hands and arms from the door, and bodily injury in the event of an explosion. Exposure to biohazardous material may occur if biohazardous waste is improperly packaged or manipulated. Onsite training on how to use the autoclave properly and safely is essential for all new employees to prevent injury. The use of heat-insulating gloves, lab coat, and closed-toe shoes help prevent burns and scalds during loading and unloading the autoclave.

If you have never operated the autoclave you will be using, contact an experienced user in your laboratory for instruction on safe operation. For autoclaves in your building, you may need to contact the facilities manager or department safety contact. For additional assistance; contact the Division of Research Safety at 217-333-2755 or via e-mail.

Autoclave Safety

Prevent injuries by:

  • Wearing appropriate Personal Protective Equipment (PPE) including a lab coat, heat resistant gloves, and eye protection, especially when unloading the autoclave.
  • Never sealing containers; under pressure they pose an explosion risk.
  • Never opening the door to the autoclave if there is water running out the bottom. Clogged steam lines, equipment malfunction, or plugged drains may cause a buildup of scalding water.
  • Waiting for the pressure to reach zero and the temperature is at or below 121°C before opening the door at the end of a cycle to avoid steam burns and shattered glassware. Do not stand directly in front of the door.
  • Never superheating liquids. Superheating is a condition that occurs when liquids are at a temperature above their normal boiling point but do not appear to be boiling. Any disturbance of the liquid could cause some of it to violently flash to steam and spraying. In situations where personnel are in a hurry to remove flasks or bottles from the autoclave, the superheated liquids may boil out of their containers or explode.

Never autoclave the following:

  • Sharps: It is not necessary to autoclave discarded sharps (used/unused needles and syringes, contaminated broken glass, microscope slides and coverslips, Pasteur pipettes, scalpel or razor blades) prior to disposal in a sharps disposal container. For a pickup of full sharps containers, fill out the online form.
  • Hazardous chemicals (including items contaminated hazardous chemicals). Do not autoclave flammable, reactive, corrosive, ortoxic chemicals (e.g., alcohols, chloroform, acetic acid, formalin, or fixed tissues). Lab coats that have been contaminated with chemicals should not be autoclaved but cleaned by an approved laundry service or disposed of as chemical waste. To schedule a chemical waste pick-up, fill out the online Chemical Waste Pickup form.
  • Dried bleach and bleach-associated materials, or nitrocellulose; both compounds pose a fire or explosion risk. 
  • Radioactive materials: Contact the DRS Radiation Safety Program for information on proper disposal of radioactive materials. To schedule a pickup, fill out a Radioactive Waste Pick-up Request online.
  • Pathological waste: Includes animal carcasses, tissues, and organs and human tissues and organs. University policy requires that certain types of pathological waste be disposed of by incineration. Refer to the online information on how to dispose of pathological waste for incineration or e-mail the Division of Research Safety.
  • Low Molecular Weight (LMW) biotoxins and prions: Some biohazards will not be inactivated by autoclaving, as the material is extremely stable. Contact the Division of Research Safety via e-mail if you are disposing of these types of materials.

Preparing Materials

To ensure adequate steam penetration, pack solid materials loosely; do not intentionally compact waste or overfill biohazardous waste bags. Bags/containers should be placed in a large, leak-proof, non-glass, shallow pan to contain spills. Stainless steel pans or plastics that can be autoclaved repeatedly at high temperatures (e.g., polypropylene, polypropylene copolymer or fluoropolymers) are recommended. Before processing, open the bags/containers so that the steam can penetrate and effectively raise the temperature for adequate sterilization. A small amount of water may be added to ensure heat transfer inside the bag/container. If the bag is closed during autoclaving, the temperature of the contents may not be raised sufficiently for decontamination. If processing more than one tray, make sure that there is ample room between the trays so as to not impede steam circulation. 

Place containers of liquid (e.g., bottles, beakers, flasks) topped with a cotton plug or steam-penetrable bung in a large, leak-proof, shallow pan. Inspect the glass to make sure there are no cracks. Do not fill containers to the top, and leave plenty of head room. Bottles with narrow necks may boil over if filled too full. Avoid the use of bottles if possible, but if it is necessary, make sure that the screw-cap is nearly unscrewed to allow for pressure changes or it may explode. Water should be added to the pan to help prevent heat shock to the containers.

Basic Operating Instructions

The following are basic instructions for autoclave use but do not replace the manufacturer's operating instructions and hands-on training. Before using any autoclave for the first time, read and thoroughly understand the owner's manual because many makes and models have unique characteristics.

  1. Place the items to be autoclaved in the chamber.
  2. Check the drain screen to make sure that it is not plugged or obstructed.
  3. For bench top units that do not have inline steam, check and fill the reservoir with deionized water to the fill line (see manufacturer's instructions).
  4. Close and seal the autoclave door.
  5. On the autoclave keypad, or dials, select:
    a. The type of load: gravity or liquid.
    b. The sterilization time: The sterilization time will vary according to the contents and how the load is packaged and should be measured after the temperature of the materials reaches 121°C and 15 pounds per square inch (PSI). It can be variable with a minimum of 12-15 minutes. Several trays with large bags or containers loaded in the autoclave will require a longer time to reach 121°C and should be set accordingly.
    c. The sterilization temperature. Unless specifically instructed, the chamber temperature is set to 121°C (250°F).
    d. A dry cycle if desired.
    e. Or select a preprogrammed cycle, i.e,. the “waste” cycle if your autoclave has this option. Preprogrammed cycles are either entered at the factory or by the autoclave responsible party. 
  6. Run the autoclave cycle. Fill out the autoclave log if this is required.
  7. At the completion of the cycle, don appropriate PPE before opening the door. Wear safety glasses, a lab coat with long sleeves, closed-toed shoes, and heat-resistant gloves.
  8. Open the door slowly and only slightly and allow steam to escape.
  9. Allow items to cool in the autoclave for at least 10 minutes before fully opening the door.
  10. Check the autoclave tape for a color change, and the print-out from the recorder to see if the time and temperature were attained. If not, the load should be re-autoclaved in another autoclave.
  11. Any bag displaying the biohazard symbol must be over bagged with an opaque trash bag and sealed prior to disposal in the regular waste stream. Bags with the biohazard symbol, regardless of use, must not be placed in the regular waste stream without being over bagged.

Guide to Temperatures and Times

Downloadable version of the Autoclave Safety Poster  (8.5X11)

Downloadable version of the Autoclave Safety Poster  (11X17)

    Items

    Biological Waste (Gravity Cycle)

    Liquids(Liquid Cycle)

    Dry Items (Gravity Cycle)

    Glassware (Gravity Cycle)

    Preparation

    Open the bag >2", Place in tray,
    Place indicator if needed

    Loosen caps or use a vented closure,
     Fill containers no more than 75% capacity

    Fabrics Wrap; Instruments: Clean, dry, lay in pan

    Dirty: Place in middle of the pan;
     Clean: wash, rinse, wrap

    Placement in Autoclave

    In the center

    Upright in pan

    Fabrics: Separated, on edge;
    Instruments: Flat

    Dirty: In detergent and pan;
    Clean: On side or inverted

    Temperature

    121oC

    121oC

    121oC

    121oC

    Treatment Time in Minutes

    60-120 min. depending on load size and packing density

    22 min. for volumes <100mL;
     40 min. for volumes >100mL

    30-60 min.

    30-60 min.

    Exhaust Cycle

    Slow exhaust

    Slow exhaust

    Fast exhaust and dry

    Dirty: Slow exhaust; Clean: Fast/dry

    Notes:

    Avoid puncturing bags. Overbag and dispose of properly.

    Hot bottles may explode. Let cool before moving.

    Check reference for proper packaging methods

    Glassware with cracks or deep scratches may crack

      Last Update: 5/22/2017