Autoclaving Guidelines

Autoclaving is the most effective and reliable means of sterilizing laboratory materials.  Autoclaving sterilizes material using saturated steam under pressure (“moist heat”).  Due to the use of pressure, steam and high temperatures, there is significant risk for injury, so it's important for individuals to be properly trained on operational procedures.   

Autoclaves may be used to sterilize equipment/products prior to use in an experiment or to render items non-infectious prior to disposal.  The University of Iowa’s Biohazard Waste Guidelines states that cultures, plates, and vials containing pathogenic organisms must be autoclaved prior to disposal.  The reason for autoclaving infectious waste is that it must be handled several times during transport; proper containment and treatment at the source reduces the potential for an accidental exposure. The necessary treatment to achieve sterility will vary in relation to the volume of material treated, its contamination level, moisture content and other factors.

Autoclave compatible material:

Biological cultures and stocks Culture dishes and related devices
Discarded live and attenuated viruses/vaccines Contaminated solid items (pipette tips, gloves, Petri dishes, etc.)
Items for sterilization (glassware, media, equipment, water) Polypropylene (PP) and Polycarbonate (PC) plastics
Borosilicate glass Stainless steel

Autoclave incompatible material:

Materials containing solvents, volatile or corrosive, or flammable chemicals Material contaminated with chemotherapeutic agents, cytotoxic drugs
Radioactive material Material containing bleach (see below)
Carcinogens or Mutagens (ethidium bromide) Phenol and Trizol
Polystyrene (PS), polyethylene (PE), and high density polyethylene (HDPE) plastics Household glassware
  • Neutralize waste containing bleach with equal amounts of 1 percent sodium thiosulfate in water prior to autoclaving.
  • Autoclaving hazardous materials may generate toxic vapors or explosive environments. 

General Guidelines:

  • Every autoclave and sterilizer should be inspected and serviced on a regular basis. This will help ensure the equipment is functioning properly. 
  • Each unit should have a standard operating procedure written in sufficient detail to ensure that operators will use the equipment properly; controls vary between brands, with each having unique loading characteristics, load-sizing requirements, and cycle setting and types.  Principal Investigators and/or lab managers should ensure users are properly trained on the autoclave in use.
  • Units should be tested regularly with a commercial preparation containing Geobacillus stearothermophilus spores (a biological indicator), in particular, any unit in a BSL3 facility. 
  • Tape indicators (autoclave tape) with heat sensitive, chemical indicators should be used in every autoclave load.  Note: the indicators only verify that the autoclave has reached normal operating temperatures; they do not indicate that the contents were heated for the appropriate length of time or at the proper pressure.  Therefore, tape indicators cannot be used to prove organisms are actually killed during an autoclave run.
  • Keep detailed records on biological tests, recording thermometers, and service work performed on the unit. 
  • High density wastes or materials that insulate the agents from heat and steam penetration are not suitable for steam sterilization. Items that are covered with dirt or film require additional retention times. The importance of properly cleaning items to be sterilized cannot be over emphasized. 
  • Place all autoclaved infectious waste into red biohazard bags for disposal.
  • An online training video was developed by Arizona State University that offers safety information, examples of waste to be autoclaved, and procedures for spore testing that may be helpful to any user.  The video is available at: https://www.youtube.com/watch?v=rM_JTgLSKXk&feature=youtu.be

Autoclaving Bags/Container Guidelines

The proper packaging and containment of infectious materials are crucial to achieve effective sterilization.  The most frequent reason for sterilization failure is the lack of contact between the steam and microorganisms.   Dry material should be separated from liquid material to achieve proper sterilization.

Dry material:

  • Ensure only approved autoclave bags are used and are not filled beyond 75% of holding capacity.
  • Most bags that are marketed as autoclavable are not suitable if closed because the steam will not penetrate them.  Steam resistant bags must be left open or have holes punched into the top to allow the steam to penetrate.  Do not transfer open bags to the autoclave. 
  • Never close autoclave bags that have a printed warning stating they are to remain open during sterilization.  If air remains trapped in the bag, the material may not be properly sterilized. 
  • Autoclave bags that allow steam penetration tend to melt or crumble during the sterilization process; autoclavable bags can also leak so they should be placed into a shallow stainless steel pan.  Plastic pans are less effective because they do not transfer heat as fast or efficiently. 

Liquid material:

  • To prevent bottles from shattering during pressurization and to facilitate steam penetration, bottle caps and stoppers must be loosened after placement into the chamber.  If left sealed, they may not be properly sterilized and could burst violently if exposed to extreme heat. 
  • Do not overfill the containers (25-50% of holding capacity) in order to prevent spill and boil over.
  • Bottles/flasks can be placed in an autoclave pan with about 5-10 inches of water for even heating, ensure there are no bubbles under the bottle/flask.
  • Sterilization of bulk liquids requires special care to prevent the containers from exploding.  Do not autoclave bulk liquids without following the manufacturer's written instructions. 
    • Each gallon of infectious liquid must be autoclaved for one hour at 121°C at 15 pounds per square inch.  Closures and lids must be loosened prior to sterilizing. 
    • Bulk solutions must be sterilized separately from all other items in a load dedicated to liquids only.  Solutions are subjected to a cycle designed specifically for liquids. 
    • Sterilized liquids must be allowed to cool before unloading.  Removing hot bottles may cause them to explode.

Appropriate Personal Protective Equipment (PPE)

  • Eye/face protection
  • Gloves (including heat resistant gloves)
  • Laboratory coat

Standard laboratory clothing includes long pants and closed-toed shoes.

Loading the autoclave

  • Add 250ml of water to bags of solid waste in order to create additional steam that drives residual air from the bag. 
  • Transfer infectious waste to the autoclave in a sealed secondary container.  The outer surface should be decontaminated prior to transport.
  • Avoid rough handling of waste containers in order to minimize the formation of infectious aerosols.

Follow the manufacturer’s instructions when loading the chamber.

  • Ensure autoclave is operating properly before commencing cycle and cycle is appropriate for the load.
  • The autoclave bags should be left open during autoclaving to insure steam penetration and sufficient temperatures inside the bag are achieved.
  • Materials should be loosely packed in the chamber for easy steam penetration and air removal.  
  • Ensure the autoclave attains the desired temperature.

Standard autoclave cycles for commonly used material

  • Laundry: 121°C for 30 minutes with 15 minutes pre-vacuum of 27 inches of mercury (in. Hg).
  • Glassware and trash: 121°C for 1 hour with 15 minutes pre-vacuum of 27 in. Hg.
  • Liquids: 121°C for 1 hour for each gallon.

Unloading the autoclave

Follow the manufacturer’s instructions before unloading the chamber.

  • Do not open the autoclave while the chamber is pressurized, released steam can cause severe burns.
  • Verify that the temperature and duration of exposure were met.
  • Wait until the autoclave has cooled prior to opening the door.  Most autoclaves have safety interlocks that prevent the door from opening when the temperature inside is greater than 80°C; however, a puff of steam may be ejected if the autoclave is opened immediately after the cycle.
  • Avoid standing directly in front of the autoclave door when it is opened after a run.
  • Handle waste containers containing liquids with care to avoid being burned by hot liquid splashes or spills.  Liquids should be allowed to cool for 20 minutes before transport to prevent sudden eruption from the containment vessel.

Autoclaved waste can cause odors, the use of autoclave deodorizers may assist if there is a general problem in the area: http://www.argos-tech.com/c-5-p-44-id-5.html or http://scientific-supplies.thomassci.com/scientific-supplies/Autoclave%20Deodorizer