PRIOR TO USE:

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.

PROPER PERSONAL PROTECTIVE EQUIPMENT AND TRAINING:

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.

LOADING THE AUTOCLAVE:

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.

SECONDARY CONTAINMENT AND PLASTICS:

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.

CHOOSING A CYCLE:

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.

DWELL TIMES:

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 132^oC (270^oF), 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

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.

END OF CYCLE AND REMOVING ITEMS:

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.

Chemical and biological indicators are used to validate whether an autoclave is working properly. Chemical indicators are quick to read, but generally, only demonstrate that the autoclave has reached a certain minimum temperature.

They do not give an indication of how long that high temperature was held or whether the procedure was sufficient to provide adequate sterilization. Biological indicators, in contrast, take more time to read but measure the growth or inhibition of bacterial spores generally known to have high resistance to steam and vaporized hydrogen peroxide.

Note that autoclave tape only indicates that a certain temperature threshold has been met and therefore is not sufficient as the only indicator of sterilization. 

Biological Indicators

Biological indicator systems are designed to validate that an autoclave run was successful at killing microorganisms. This is achieved by demonstrating actual effectiveness on bacterial spores, which are generally regarded as difficult to kill. Specifically, Geobacillus stearothermophilus spores are highly resistant to steam and vaporized hydrogen peroxide and are therefore most commonly used in biological indicator tests. G. stearothermophilus spores are inactivated when exposed to temperatures of 121.1^oC for at least 20 minutes and therefore can be used as an indicator both of temperature reached as well as the length of time the minimum temperature was held. 

Typical biological indicator systems consist of spore strips that must be plated on growth media for incubation or small glass ampules of growth medium with spores and indicator dye that act as self-contained biological indicators.  The indicator should be placed in the most challenging location to sterilize, which is commonly on the bottom shelf near the drain. After the autoclave load has been run, the indicator is incubated at the manufacturer-specified temperature for 2-3 days and evaluated for spore growth. A control vial, which was not run in the load, should be incubated at the same time and compared against the test vial. If spores survived and proliferated in the test vial, rendering the contents turbid and cloudy, the validation has failed as bacterial spores were still viable. If no spores survived, the test vial will remain clear and validation was successful. If indicator dyes are included in the growth media, there may be a color change in the media with relation to spore growth, due to a change in pH. The control tube, which was not autoclaved, should be turbid, as spores would be expected to grow. If there is no growth in the control tube, the test is invalid and must be repeated. If validation has failed, notify your supervisor or floor administrator, as the autoclave may require servicing and/or settings may need to be changed.

Examples of biological indicators include the Duo-Spore Biological Indicator (VWR Catalog # 15425-032), Sterikon-Bioindicator EM (VWR ), and the Mesa Labs Prospore Self-Contained Biological Indicator (Fisher Scientific Catalog #12-001-1). Whichever product you purchase, follow manufacturers’ instructions for use. 

Chemical Indicators

Chemical indicators for steam autoclaves irreversibly change color after being exposed to normal autoclave operating temperatures of 121^oC (250^oF) at 15 psi. Note that there is no time factor that causes this color change. Chemical indicators can give you a quick visual reference for heat penetration inside the load.  They are frequently sold as paper or card strips that are positioned near the center of each load, and toward the bottom front of the autoclave. Some indicators may be multi-parameter, and change color with temperature and ethylene oxide, while other indicators may be manufactured for other temperature and time ranges (eg. 275^oC, 3min) than standard. Ensure that you are using the appropriate indicator for your sterilization technique.

Chemical indicators alone are not designed nor intended to prove that organisms were actually killed during a decontamination cycle.

Chemical indicators are manufactured by many companies and come in a wide variety of sizes, shapes, and colors.  Some examples include Verify Steam indicator strips and 3M Comply Steam Chemical Indicator strips.

Chemical Tape Indicators

Chemical Tape Indicators commonly appear on adhesive-backed paper tape or on sterilization pouches, where the areas darken upon reaching a specified temperature to form dark diagonal lines and may display words, such as “sterile” or “autoclaved”. These markings only appear when the tape has been exposed for a few minutes to normal autoclave decontamination temperatures.  Tape indicators should be used on all material decontaminated by autoclaving to show that the material has been processed.  A three to four inch strip of autoclave tape placed on the outside of the autoclave pan, bag, or individual container is sufficient. If the temperature sensitive tape does not indicate that a temperature of at least 250° F (121° C) was reached during the sterilization process, the biomedical waste is not considered decontaminated.

A durable notebook should be used as a permanent record of autoclave use.  The autoclave logbook should be located in an easily accessed location near the autoclave.  The autoclave logbook should have at least the following information entered:

The main section of the autoclave record should have a log including:

Autoclave User, Date Used, Materials Decontaminated, Process Type, Run Duration (Cycle Time), Chemical/Biological Indicator Used, Chemical/Biological Indicator Results, Envelope for "Wheel Graphs" or "Data Strips".

Materials that are to be decontaminated should be carried to the autoclave in closed, leak-proof containers.  Containers used to hold material while being autoclaved are described below. 

SECONDARY CONTAINERS:

Plastic Containers

Polypropylene is a durable inexpensive plastic resin which is commonly used to contain material during autoclaving.  Polypropylene plastic pans with 6-12 inch sides are favored over polyethylene and polystyrene because polypropylene can withstand autoclaving without melting.

Note: When using polypropylene containers, add extra processing time to the autoclave decontamination cycle because polypropylene does not conduct heat well.

Stainless Steel Containers

Stainless steel containers are durable and come in a variety of sizes and shapes.  Stainless steel is a good conductor of heat.  Where waste containment is mandatory, stainless steel containers may be the container of choice because it is durable.  Restaurant supply companies are a good source for these pans.

PRIMARY CONTAINERS:

Plastic Autoclave Bags

Polypropylene bags are used to contain materials during decontamination cycles within autoclaves.  Also known as “biohazard bags”, “autoclavable bags”, or [VWR bags, bio check] come in a wide variety of sizes, shapes, and colors. 

Autoclave bags are usually placed in polypropylene or stainless steel pans during decontamination cycles to catch liquids that may drain out of the bag.

Autoclave bags must be left open or loosely closed during decontamination procedures to allow steam to penetrate into the bag.

Some waste material has an extremely noxious odor, i.e., anaerobic bacteria, feces or decaying organic materials.  When decontaminating these materials, it may become necessary to add an odor control additive to the load.  A scoop-full of unused animal bedding (cedar shavings) works quite well.  These additives may produce chemical exposure symptoms for some people; if this is an issue, an alternative means of odor control should be used.

It is imperative that supervisors provide training to qualify their staff for the use of autoclaves to decontaminate materials.  Qualified autoclave users should understand the time, temperature, and pressure relationships required for proper material decontamination.  Additional training on handling materials to be decontaminated should also be provided.  Supervisors should maintain a permanent record of the training provided to their staff.

Additional training support on the effective use of autoclaves is available from the EHS office.  For assistance, call the Biosafety Office at (410) 706-7055.