Autoclave Operation


Regularly inspect your autoclave components for proper operation.  Autoclave door clamps and seals should be inspected for wear and damage.  Remove debris from the autoclave chamber floor drain and rubber door seal.  If a problem is found, promptly notify your area supervisor who will call an authorized service representative. DO NOT OPERATE THE AUTOCLAVE UNTIL IT HAS BEEN REPAIRED PROPERLY.


Only personnel with adequate training on autoclave use should be permitted to operate an autoclave.  Personnel should wear proper personal protective equipment, including heat- and liquid-resistant gloves, a fully-fastened lab coat, eye protection, face protection, and arm protection particularly when unloading the autoclave. Rubberized aprons and full-face shields may also be required.


Each appropriately packaged item in a load must be placed so that steam penetrates into and among all materials to be decontaminated. To be effective, any air should be replaced with steam. Therefore, tightly sealed or stoppered materials may not be effectively decontaminated and may become dangerously pressurized, causing injury when removed. Lids on sealed containers (whether empty or containing liquid) should be loosened to allow passage of steam and to prevent pressure build-up. Any loose items should be packaged or loosely wrapped. Any open containers (where you intend the inside to be decontaminated) should be covered with aluminum foil loosely, so as not to impede the passage of steam into the container. Do not allow any items to touch the walls of the autoclave vessel. If using autoclave tape, place 2-3 inch strips on items to be autoclaved at this point. If using any biological or chemical validation indicators, place in the most appropriate area, as directed by manufacturer’s instructions.


All loose items should be placed into a secondary container and not placed directly on the autoclave racks. Any container used to hold items to be autoclaved should be autoclave-safe. In other words, they should be made of stainless steel or a plastic that will not melt at the high pressure and temperature conditions inside the autoclave. Polypropylene and polycarbonate can resist autoclave temperatures and should be used as secondary containment for smaller items. Polystyrene (PS), polyvinyl chloride (PVC), nylon, acrylic, low-density polyethylene (LDPE), high-density polyethylene (HDPE) labware and polyurethane tubing will generally melt in the autoclave. In particular, pipette tip boxes must be contained in another solid-bottomed secondary containment.  Inspect secondary containers before use; if signs of degradation, discard and use another appropriate secondary container.

Note: use only autoclavable polypropylene bags to package waste to be autoclaved; other bags may melt.


In general, there are 3 commonly-used types of autoclave cycles. Always make sure that you are using the correct cycle, as choosing the wrong cycle could result in ineffective decontamination, liquids boiling over, or glassware breaking. The 3 cycles include:

  • A Fast Exhaust, Gravity, or “Dry” cycle. To be used solely for loads consisting only of dry goods and empty glassware, the pressure vessel is charged at a regular speed to the appropriate pressure and temperature, held there for the set time, and exhausted quickly to room temperature. This cycle should only be used on loads composed solely of dry goods, as a too-rapid exhaust on liquid materials could cause liquids to boil and spill out of their containers. It is recommended that a cup of water be added to a dry load, ensuring that adequate steam is generated in the pressure vessel. In order to avoid the creation of infectious aerosols while adding water, trickle water down the sides of the container instead of pouring water directly onto the material in the container.
  • A pre-vacuum cycle. Generally used for porous materials such as animal bedding, wrapped goods, and caging, the pressure vessel is partially evacuated before steam is introduced, which ensures rapid steam penetration through an often “fluffy” load.
  • A Slow Exhaust, or “Liquid” cycle. Used if there are any liquids in the autoclave load, such as distilled water, LB broth, or culture media. The pressure vessel is charged at a regular speed to the appropriate pressure and temperature, held there for a set time, and exhausted slowly to prevent liquid boil-over.


Decontamination cycle times vary based on type of load, load volume (loosely-packed or tightly-packed), container type (polypropylene, glass, stainless steel), and type of material to be decontaminated.  In general, the larger the load, the longer it will take to decontaminate.

The processing time to decontaminate laboratory and medical waste is at least 60 minutes (unless a shorter interval has proven effective when tested with biological indicators) for materials in metal pans with the lid removed.  Add additional time as necessary if polypropylene containers are used. 

A minimum of at least 90 minutes is recommended for decontaminating waste in low-sided polypropylene containers with bags half-filled and loosely-gathered.  If bags are tightly-closed, a processing time of 120 minutes is recommended.  If your autoclave is equipped to operate at 132oC (270oF), you may be able to reduce processing time if the G. stearothermophilus spores in biological indicators are killed at the shorter cycle time.

EPA Recommended Decontamination Processing (Dwell) Times




60 Minutes


60 Minutes


60 Minutes / Gallon

Animal Bedding

120 Minutes

Animal Carcasses


A drying cycle may be an option, depending on the autoclave model, though this is not actually a decontamination cycle. A vacuum system pulls any steam and condensates out through the steam port for a set time, effectively drying the materials in the chamber. Do not use this cycle with any liquid materials in the autoclave, as this will effectively pull liquid out of the vessels and cause boiling over. 

Routine physical controls such as pressure gauges and thermometers are to be considered secondary methods of ensuring a sterilization cycle. Regular monitoring of temperature and monthly biological monitoring under a standard cycle must be performed and recorded.


A typical cycle will take at least 1 hour to run. At the end of a decontamination cycle, make sure that the pressure in the autoclave chamber is at zero before opening the door.  Stand away from the opening, slowly crack open the autoclave door, and allow the steam to gradually escape from within the autoclave. Opening the autoclave door too quickly may result in glassware breakage and/or steam burns on your skin. The decontaminated materials should be allowed to cool for 10 minutes before they are taken out of the autoclave. Wear appropriate PPE when removing items from the pressure vessel. Move deliberately and slowly to prevent sloshing of liquids.

Things to avoid:

  • Avoid dead air pockets where steam cannot penetrate (eg. closed screw cap tubes) because the temperature within the air pocket is much lower than the saturated steam and therefore sterilization may not be achieved inside the vessel.
  • No sealed liquid containers, radioactive materials, combustible, flammable, volatile chemicals, bleach.