Autoclave Use

Autoclaves provide a physical method for disinfection and sterilization. They work with a combination of steam, pressure and time. Autoclaves operate at high temperature and pressure in order to kill microorganisms and spores.
They are used to decontaminate certain biological waste and sterilize media, instruments and lab ware. Regulated medical waste that might contain bacteria, viruses and other biological material are recommended to be inactivated by autoclaving before disposal.


Halina Staniszewska
Sr. Lab Safety Specialist

Autoclave Cycles

  • To be effective, the autoclave must reach and maintain a temperature of 121° C for at least 30 minutes by using saturated steam under at least 15 psi of pressure. Increased cycle time may be necessary depending upon the make-up and volume of the load.
  • The rate of exhaust will depend upon the nature of the load. Dry material can be treated in a fast exhaust cycle, while liquids and biological waste require slow exhaust to prevent boiling over of super-heated liquids.


Recommended for:


Liquids cycle


(Slow Exhaust)

Use with glass containers with vented closures; 2/3 full only

•Liquid media

•Nonflammable liquids

•Aqueous solutions

•Liquid biological waste


Solids or Dry cycle


(Fast Exhaust)

  • Glassware: empty and inverted
  • no tight or impermeable closures
  • Dry hard items, either unwrapped or in porous wrap
  • Metal items with porous parts
  • Other porous materials

Wrapped Goods or Pre vacuum cycle


(Clean: Fast Exhaust

Dirty: Slow Exhaust)

  • Glassware that must be sterilized upright and/or can trap air
  • Wrapped dry items that can trap air
  • Pipette tip boxes
  • Sharps decontamination
  • (in collection containers)
  • Biohazard waste decontamination, in autoclave bags; can be wet or dry


Meagan Fitzpatrick
Biosafety Officer

Halina Staniszewska,
Sr. Lab Safety Specialist

Compatible/Incompatible Materials

AUTOCLAVE-COMPATIBLE MATERIALS                                                 




  • Tissue Culture Flasks
  • Surgical Instruments
  • Glassware
  • Pipette tips
  • Media Solutions
  • Animal food and bedding
  • Waste
  • Polypropylene (Secondary containers)
  • Stainless steel
  • Gloves
  • Acids, bases and organic solvent
  • Chlorides, sulphates
  • Seawater
  • Chlorine, hypochlorite, bleach
  • Non-stainless steel
  • Polystyrene(PS)
  • Polyethylene(PE)
  • Low density (LDPE) and High density polyethylene(HDPE)
  • Polyurethane

Never autoclave:

· Flammable, reactive, corrosive, toxic or radioactive materials

· Household bleach

· Any liquid in a sealed container.

· Any material contained in such a manner that it touches the interior surfaces of the autoclave.

· Paraffin-embedded tissue.



  • Only Pyrex® or Type I borosilicate glass is autoclavable.  When autoclaving liquids in Pyrex® containers, do not fill more than 2/3 full and do not seal the container.


  • Polypropylene is an inexpensive resin that can resist autoclave temperatures. Polypropylene containers are often used as secondary containers to hold materials that are autoclaved.  Polycarbonate can also withstand high temperatures. Polystyrene (PS), polyvinyl chloride (PVC), nylon, acrylic, low-density polyethylene (LDPE), and high-density polyethylene (HDPE) lab ware and polyurethane tubing are not autoclavable under any conditions.


  • Gloves must be placed inside of an autoclavable biohazard bag and exposed to a steam setting; gloves will melt slightly but will not burn when autoclaved in this manner.

Stainless steel

  • Most metals are designed for extreme conditions and are intended to be sterilized. Make sure to remove any plastics, liners and other items that may melt or combust.


  • Paper is combustible and should not be placed directly inside an autoclave. It should be autoclaved in a waste bag on a biobag setting to prevent fire.

Media Solution

  • No liquid should be sealed in a container and autoclaved. Fill 2/3 of the container and loosen caps. They should autoclaved in a steam producing cycle.

Pipette tips

  • Most pipette tips are autoclavable. Some of these tips are plastic, some are high density polyethylene. In general, pipette tips should only enter the autoclave as waste inside of an approved biohazards bag and always sterilized on a steam-producing setting.


Meagan Fitzpatrick
Biosafety Officer

Halina Staniszewska,
Sr. Lab Safety Specialist

Autoclave Procedure

Wear personal protective equipment:

  • Lab coat
  • Eye protection
  • Closed-toe shoes
  • Heat-resistant gloves to remove items, especially hot glassware

Packaging and Loading

  • Only designated individuals should be allowed to set and/or change parameters for the autoclaves.
  • Before using the autoclave, check inside for any items left by the previous user that could pose a hazard.
  • Clean the drain strainer before loading the autoclave.
  • Always place items in a secondary container.
  • Do not overload or package bags too tightly. Leave sufficient room for steam circulation. If necessary, place container on its side to maximize steam penetration and avoid entrapment of air.
  • Use only autoclavable bags to package waste.
  • Do not allow bags to touch the interior walls of the autoclave to avoid melting of plastic.
  • Ensure sufficient liquid is packed with contents of autoclave bags if dry.
  • Place soiled glassware and lab ware in secondary containers and autoclave them in the solids cycle. Do not fill containers more than 2/3 full with liquids. Loosen caps or use vented closures.
  • In case of clean glassware and wrapped instruments, lay them in a secondary container before autoclaving in wrapped goods cycle.
  • For secondary containment, use autoclave trays made out of polypropylene, polycarbonate or stainless steel. The trays should have a solid bottom and sides to contain the contents and catch spills.
  • Choose appropriate cycle for the material.  Incorrect selection of cycle may damage the autoclave, cause liquid to boil over or bottles to break.
  • Start your cycle and fill out the autoclave user log.  A completed cycle usually takes between 1 to 1.5 hours.
  • Check chamber/jacket pressure gauge for minimum pressure of 20 pounds per square inch (psi).
  • Close and lock door.
  • Check temperature for 250⁰F (121⁰C) every load.
  • Do not attempt to open the door while autoclave is operating.


  • Ensure cycle has completed and both temperature and pressure have returned to a safe range.
  • Wear PPE described above, plus an apron and face shield if removing liquids.  Stand back from the door as a precaution and carefully open door no more than 1 inch. This will release residual steam and allow pressure within liquids and containers to normalize.
  • Allow the autoclaved load to stand for 10 minutes in the chamber. This will allow steam to clear and trapped air to escape from hot liquids, reducing risk to operator.
  • Do not agitate containers of super-heated liquids or remove caps before unloading.
  • Place liquids in an area which clearly indicates the items are “hot” until the items cool to room temp.
  • Allow autoclaved materials to cool to room temperature before transporting. Never transport superheated materials.
  • Place cooled autoclaved biohazard bag into regulated medical waste box.  Autoclaved infectious liquids may be disposed of into the sanitary sewer.


Meagan Fitzpatrick
Biosafety Officer

Halina Staniszewska,
Sr. Lab Safety Specialist

Autoclave Validation

Chemical Indicators

Tape Indicators

Tape indicators are adhesive-backed paper tape with heat sensitive, chemical indicator markings.  Tape indicators change color or display diagonal stripes, the words “sterile” or “autoclaved” when exposed to temperatures of 121°C.  Tape indicators are typically placed on the exterior of the waste load.  If the temperature sensitive tape does not indicate that a temperature of at least 121°C was reached during the sterilization process, the load is not considered decontaminated.   If tape indicators fail on two consecutive loads, notify your Department Safety Manager.

Tape indicators are not designed nor intended to prove that organisms have actually been killed. They indicate that a temperature of 121°C has been achieved within the autoclave.  EHS recommends that you DO NOT use autoclave tape as the only indicator of decontamination or sterilization.

Integrated Chemical Indicator Strips

Integrated chemical indicator strips provide a limited validation of temperature and time by displaying a color change after exposure to  normal autoclave operating temperatures of 121ºC for several minutes.  Chemical color change indicators can be placed within the waste load.  If the chemical indicators fail on two consecutive loads, notify your Department Safety Manager.                             

Biological Indicators

Biological indicator vials contain spores from B. stearothermophilus, a microorganism that is inactivated when exposed to 121.1oC saturated steam for a minimum of 20 minutes. Autoclaves used to treat biological waste will be evaluated with a biological indicator by EHS on a quarterly basis.


  • EHS will coordinate biological validation testing with laboratory staff.
  • The indicators will be incubated by EHS for 24 hours at 60°C with a control that has been maintained at room temperature.


  • If the autoclaved indicator exhibits growth, the validation has failed and will be repeated.
  • If the second validation indicator fails, EHS will notify the Department Safety Manager and request service on the autoclave.  Autoclave should not be used until service has been conducted and the validation test passes.
  • Validation tests results are emailed by EHS staff to the appropriate labs and the Department Safety Manager.
  • EHS maintains documentation of all validation tests.
Biological Indicator Test Results


Autoclave Log:

An autoclave log containing the following details should be maintained by lab staff:

  • Date, time, and operator’s name
  • Contact information: Laboratory, room number, phone number
  • Type of material sterilized/cycle
  • Temperature, pressure, and length of time the load is sterilized.

Biological Indicator Test Results

  • EHS maintains all biological indicator test results.


Meagan Fitzpatrick
Biosafety Officer

Halina Staniszewska
Sr. Lab Safety Specialist