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4.14 Autoclave Use

4.14.1 Hazards

Potential safety risks for autoclave users include:

  • Burns from touching hot autoclave chamber surfaces
  • Steam burns from residual steam when door is opened at end of cycle
  • Hot fluid scalds from boiling or overboiling liquids/spillage
  • Hand and arm injuries related to door operation
  • Explosion of the pressurized chamber could injure or kill persons nearby
  • Inhalation or other exposure to fumes/vapors from chemicals that should not have been autoclaved
  • Exposure hazards from handling biohazardous materials before autoclaving, or after autoclaving if material was unsuccessfully decontaminated


4.14.2 Training Requirement

Successful completion of autoclave training (EHS online module Safe Autoclave Use and hands-on training in your facility) is required prior to operating an autoclave at Virginia Tech due to the serious hazards associated with use of this equipment.


4.14.3 Overview of Autoclave Operation

Autoclaves are essential for bioresearch, providing a reliable means for sterilizing equipment and supplies, and for decontaminating biohazardous waste. Autoclaves are designed to accomplish this by applying steam to items in a pressurized chamber; steam penetration and actual surface contact with the steam is required for sterilization/decontamination of materials to take place.

Examples of lab materials that can be autoclaved: metal items, glass items, heat-resistant plastic items (e.g., Nalgene, polypropylene, polycarbonate), pipette tips, aqueous solutions, water, animal food and bedding, soil, biohazardous waste.

Biohazardous waste types to be decontaminated by autoclaving:

  • Solid waste in autoclave bags – gloves, paper towels, empty tubes, agar media in Petri dishes, other solid lab debris contaminated with biohazards.
  • Liquid biological waste: exhausted culture media, supernatants, etc.
  • Sharps waste in rigid containers: glass slides, needles, syringes, pipettes, tips, blades, etc.

Examples of items that must not be autoclaved: chemical solvents, corrosives, flammable liquids, other chemicals or chemical containers, kit chemical bottles, antibiotic bottles, vials or tubes with chemical residue, radioactive material, any sealed container.

Longer sterilization/decontamination times are needed as load sizes/densities/volumes increase.

The Solid/Gravity cycle supplies steam to the chamber with no mechanical vacuum assistance, i.e., by gravity. When process time is complete, steam is quickly exhausted from the chamber. Items such as upright containers that trap air within them cannot be fully sterilized/ decontaminated using this cycle because the trapped air prohibits full steam penetration and surface exposure.

  • Use the Solid/Gravity cycle for: Pyrex/borosilicate glassware (empty, inverted, no closures), dry hard items (unwrapped or in porous wrap), metal items with porous parts, other porous materials.
  • Use the Solid/Gravity cycle for decontamination of solid biological waste if you have no Pre-vacuum cycle available on your autoclave; compensate for poor steam penetration in the waste by lengthening the process time as needed.
  • Do not use this cycle for liquids or media that require a slow exhaust.

The Liquid cycle also supplies steam to the chamber with no mechanical vacuum assistance. When process time is complete, steam is slowly exhausted from the chamber to prevent boil-over of liquids. NOTE: Because this slow exhaust phase takes extra time to complete, plastic materials that will withstand fast-exhaust cycles can melt using the Liquid cycle due to prolonged exposure to heat.

  • Use the Liquid cycle for: Pyrex/borosilicate glass containers up to 2/3 full of liquid (liquid media, aqueous solutions, liquid biowaste)
  • Do not use this cycle for items that could melt during slow exhaust.

The Pre-vacuum cycle mechanically removes air from the chamber, then supplies steam to the chamber in a series of pulses which allows full steam penetration of dense materials. Thus it is the preferred cycle for decontaminating solid biological waste. When process time is complete, steam is quickly exhausted from the chamber.

  • If available on your autoclave, use the Pre-vacuum cycle for: decontamination of solid biowaste and sharps biowaste; sterilization of glassware that must be processed in an upright position, and other dry items that can trap air (e.g., pipette tip boxes)
  • Make sure that bags containing agar plates have only a one-inch opening so that the bag can properly contain melted agar during processing.
  • Do not use this cycle for liquids, media, lighter weight plastic containers, dry items that will collapse in a vacuum.


4.14.4 Things to Check Before You Autoclave a Load

  • Wear required PPE to protect yourself from burns through direct contact with autoclave surfaces when loading items, and contact with hot items, splashes, etc. when removing loads from autoclave: buttoned lab coat, closed-toe shoes, heat-resistant gloves, safety glasses/ goggles.
  • Use heat-resistant packaging material and containers.
  • Label items to be autoclaved so you can identify them in case they are removed after cycle completion and placed elsewhere.
  • Make sure the autoclave drain is clear of debris.
  • Check to see if the autoclave is functioning properly (no ABORT message, etc.).
  • Include a verification device with every waste load (see Section 4.13).
  • Adjust closures on bags of solid waste so they have at least a one-inch opening.
  • Check to see that closures on liquid-containing vessels are loosened.
  •  Arrange items in the load so they are spaced evenly, allowing room between items (no stacking/crowding).
  •  Make sure containers of liquids are no more than 2/3 full.
  • Load items so that they do not touch sides or top of autoclave chamber.
  • Properly place bags of waste and liquid items in heat-resistant pans or trays, e.g., bags do not overflow the pans; vessels of liquid are in a pan deep enough to contain potential boil-over.
  • Do not over-pack load items into the autoclave chamber.
  • Make sure that items for sterilization are being processed in separate loads from waste loads for decontamination.


4.14.5 Response to Malfunctions

If you see an error message, evidence of an aborted cycle or other operational problems, always record your observations on the autoclave user log, and promptly report them to the person responsible for the autoclave.

If you discover that the autoclave is dysfunctional, and/or if you think it is unsafe to use, immediately post an “Out of Service” sign on the unit and report it to the person responsible for the autoclave. Never attempt repairs on a malfunctioning autoclave.

If you feel an emergency situation is developing or has developed, immediately remove yourself and others from the area and report the situation to the appropriate authorities/emergency response providers.


4.14.6 Standard Pre-Run Procedure:

  1. Sign in on autoclave use log.
  2. For a waste load, put on disposable gloves and place a verification device into proper position within the waste, (or in an empty, like-sized container if running a liquid waste load). Only one bag/ container per load needs a device placed in it. After placing the device, adjust bag openings to approximately one inch to allow steam penetration.
  3. Wearing appropriate PPE for burn protection, place load into chamber.
  4. Close chamber door and ensure that it closes completely.
  5. Select and start cycle.
  6. Ensure door has sealed and cycle is successfully underway before leaving the autoclave facility.
  7. Set a personal timer to remind you when your cycle will end.


4.14.7 Standard Post-Run Procedure:

  1. Ensure that cycle is completely finished and chamber pressure is zero before proceeding.
  2. Wearing PPE, open autoclave door and stand aside to avoid contact with any escaping steam.
  3. Allow load to cool somewhat with door open; let liquids cool in autoclave for at least 15 minutes.
  4. Wearing PPE, carefully remove load from chamber. If liquids begin to boil over, stop and allow more cooling time before removal.
  5. Close autoclave chamber door when finished.
  6. If running a waste load, check the verification result. Results of autoclave verification must be documented in a BI log (BI results), or on the Autoclave Use log sheet (CI results).