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.

Before using any autoclave for the first time, read and thoroughly understand the owner’s manual. The different makes and models of autoclaves ensure that each will have unique characteristics with respect to operation and maintenance. If you cannot locate a manual, contact the manufacturer and have a copy sent to you.

Modes of Operation

The autoclave creates different patterns of high heat, vacuum and pressure to sterilize its load. The type of materials you sterilize will determine the type of sterilization “runs” you use. The general types of “runs” are “Liquids” for any type of water based solutions, “dry goods with vacuum” and dry goods without vacuum”. Autoclaves often have an additional “drying cycle” in which hot air is blown through the chamber to dry items before they are removed. These controls settings may be different for different brands of autoclave, so always refer to the instruction manual for guidance on loading, load sizes, and cycle types.

The “liquids” run is longer than the other two modes, but it operates at lower temperatures so as to minimize the evaporation of the liquids being sterilized.. Make sure seals on containers are loose so vapor expansion during the heating process will not explode the container. Never autoclave any flammable or volatile liquids!

The “dry goods with vacuum” run moves steam and heat into the deepest parts of large bags or bundles of material and produces the best conditions for killing persistent organisms. During this type of run, the chamber alternates between cycles of vacuum and high pressure. Then the chamber is pressurized with steam for a long period, followed by a short vacuum cycle. It is important that steam and pressure be able to reach the entire load, so carefully loosen autoclave bag closures once they are in the autoclave.

The “dry goods without vacuum” run simply pressurizes the chamber with steam for the duration of the cycle, and then returns to normal. This process is used primarily for items that have been cleaned, but need to be sterilized. Materials should be packed so that the heat and pressure can readily reach the whole load.

Routine Maintenance

It is a good practice to use a Biological Indicator (e.g., Amsco’s Proof System, BBK’s Kilit), monthly to confirm that the autoclave is working properly. If either the dye or biological indicator fails, the autoclave must be examined to identify and correct the problem and also re-autoclave the load to ensure sterility. Labs will be notified anytime an autoclave fails to pass tests with Biological Indicators.

The best way to ensure your autoclave is working properly is to have regular maintenance performed. The regularity of this maintenance is usually determined by the age of the unit, the type of water supply (filtered water, unfiltered/unsoftened water or steam-fed) and frequency of use. Autoclaves that are water fed systems with hard water sources require more frequent maintenance and inspection to ensure that the water softeners, or water filtration systems are tended to appropriately so as to ensure the minimum of buildup on the units heating elements. Excessive build-up on the heating elements force the excessive use of energy, created additional expense due to frequent failure of heating elements, and may result in un-sterilized loads. In addition, users should perform the daily and weekly maintenance procedures described in the owner’s manual. Also make sure that any drain strainers are clean before each run.

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, or toxic 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.

Autoclave Compatible and Incompatible Material

The following are examples of compatible and incompatible materials. This is not an exhaustive list.

Important Notice: Autoclaving hazardous materials may generate toxic vapors or explosive environments.

Acceptable Materials

Obviously, the extreme conditions created by the use of high heat and pressure inside of the autoclave will require careful consideration of the type of material that goes into the autoclave. Information below should be considered as general information on this subject. For additional information with respect to suitability for autoclaving, consult with your laboratory supervisor, Principal Investigator, or refer to manufacturer’s technical services department.

Glass: In general, the only glass you should place inside the autoclave is Pyrex or Pyrex type material. Most vendor guides will indicate if a particular brand of glassware may be autoclaved. However, even Pyrex type glassware will explode if it contains a sufficient volume of liquid and the container is securely closed. Never autoclave liquids in a closed container.

Polypropylene: Polypropylene is an inexpensive resin, which can withstand autoclaving temperatures. Polypropylene containers are available in a variety of shapes and forms including bags, pans and trays. The bags must be open to allow steam to penetrate. It is advisable to pour a small amount of water into the bag prior to autoclaving to facilitate heat transfer to items being sterilized.

Latex or Vinyl: These items will generally melt inside of an autoclave. Combustion is possible. However as waste items, placed inside of an autoclavable biohazards bag and with a steam setting, these items will melt slightly, but will not combust resulting in fire or smoke. Since biohazard bags inside of an autoclave often contain latex or vinyl safety gloves as well as paper and plastic culture plates, some melting should be observed with respect to these items. As paper is also frequently present always use a “wet” (steam producing) setting for autoclaving all bags of waste. Failure to do so has resulted in fires!

Paper: Paper should not be placed inside of an autoclave except as a waste item inside of a red or clear biohazardous autoclave waste bag. Never sterilize waste containing paper on a “dry” setting. Always use “wet” (steam producing) settings to prevent a fire.

Plastic pipette tips and culture plates: Some of these tips are plastic, some are high density polyethylene. The degree to which melting will occur will depend upon the setting of the autoclave, density of the material, and the type of plastic. 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.

Metals: Most metals used in laboratories are designed for extreme conditions and are intended to be sterilized. With few exceptions metal objects may be autoclaved. Even small cages used in housing animals may be autoclaved, just be sure to remove any plastics, liners or other items that may melt or combust.

Plant Material: Plant material, especially transgenic plant material must be inside of a red or clear biohazard bag, with a cup (or more) of water added and with the bag unsealed.

Unacceptable Materials

It should go without saying, but every year campuses have fires or situations of excessive smoke production in an autoclave because someone forgot to be mindful of what was being placed inside the autoclave. There are a few items that under no circumstance should EVER be placed inside of an autoclave.

These items include:

Radioactive material or any material that may be contaminated by radiation.
Combustible, flammable or volatile liquids.
Any liquid in a sealed container.
Any material contained in such a manner that it touches the interior surfaces of the autoclave.

Causes of Autoclave Failure

Autoclaves are valuable tools that benefit a wide range of industries. In healthcare settings, autoclaves protect patients by eliminating pathogens from tools and equipment. In the laboratory, autoclaves provide scientists with assurance that they have eliminated biological factors that may interfere with their results. In manufacturing facilities, autoclaves are used in a variety of innovative ways to create new products and materials such as aerated concrete.

When autoclaves don’t work properly, however, they’re not benefiting anybody. Here are some of the most common reasons that steam autoclave sterilization cycles fail:

Choosing the Wrong Cycle for Autoclave Contents

Nobody wants to hear that they’re the problem, but human error is at the root of many cases of sterilization failure.

While a steam autoclave can process a variety of different objects, not all materials can be sterilized in the same manner. For example, if you are sterilizing wrapped medical tools using your autoclave’s gravity cycle, sterilizing steam may not be reaching every part of your instruments.

If you experience a sterilization failure, begin by looking back at where you started and consider if you chose the correct cycle for the autoclave’s contents.

Inappropriate Packing or Loading

While some objects, such as large glassware, may go into the autoclave completely uncovered, many smaller objects must be packed or wrapped prior to sterilization. Wrapping ensures that sterilizing steam reaches all materials in an even manner.

When a packet of materials is not wrapped correctly, though, it may interfere with your autoclave’s ability to sterilize. This is another example of how human error may be interfering with your sterilization cycle.

Additionally, when loading the autoclave chamber, technicians should place materials based on the equipment manufacturer’s guidance. If your autoclave is not loaded the way the manufacturer intended, it may not work properly.

Proper wrapping and loading protocols should be part of training for medical and dental assistants, sterilization engineers, laboratory technicians or any other professionals who may be responsible for preparing materials to be placed into an autoclave.

Poor Steam Quality

If you’re seeing wet packets and materials after a sterilization cycle, your autoclave may be suffering from poor steam quality.

Low steam quality can be created by a variety of factors. If the “weight” (water content) of the steam is outside of desired parameters, it may interfere with the autoclave’s ability to function as designed. Superheated steam – steam heated above the point at which all liquid has vaporized – is also considered low quality for sterilization.

To understand your steam production, it’s important to have a clear picture of both temperature and pressure within the autoclave chamber. Measurement devices, such as data loggers, are useful in creating these profiles.

Vacuum Failure

If you’re sterilizing small, porous or irregularly-shaped materials, you need to be using a vacuum sterilization cycle. In a vacuum cycle, all air is forcibly removed from the autoclave during preconditioning. This allows sterilizing steam to reach every nook and cranny of your contents.

If your autoclave is not able to create adequate vacuum conditions, your sterilization cycle will fail. The most straightforward way to learn if you have a vacuum failure is to use a Bowie-Dick test. The Bowie-Dick test is a specialized test sheet that uses steam and air barriers to validate that your autoclave is producing the proper vacuum environment for steam production and sterilization.

Inadequate Temperatures

Steam sterilization simply doesn’t work without high temperatures. The specific temperature at which you will be sterilizing will depend on a variety of factors such as the materials you’re sterilizing, the nature of your work and good manufacturing or laboratory practices.

Temperatures within the autoclave can be double-checked or validated using a temperature data logger or similar device. If your autoclave isn’t reaching 100 °C, it can’t produce steam at all! If it’s not reaching 121 °C, it isn’t reaching the threshold required for sterilization.

Create a study of autoclave temperatures throughout your sterilization cycle to determine if you’re reaching adequate conditions for sterilization.

Whether it’s human error or equipment malfunction, sterilization failures can cost businesses and laboratories valuable time and money. To keep this from happening to you, make sure you have an autoclave validation plan in place and train any autoclave users in proper preparation and use of the device.