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Introduction

The autoclave that uses saturated steam under pressure is one of the most dependable methods available in the laboratory for the inactivation of all forms of microbial life. To ensure safety and quality control, all biohazardous materials and items contaminated with potentially infectious agents should be decontaminated before use or disposal. Such items include, but are not limited to: culture media, surgical instruments, laboratory equipment, glassware, and biomedical waste including sharps. Steam sterilization is not recommended for anhydrous substances, flammable materials, electrical equipment, radioactive material, or any item that may be damaged in the autoclaving process.

How the Steam Sterilizer Works

The most common steam sterilizer found in laboratories is the gravity displacement type. Saturated steam enters the top of the chamber by a steam pressure control valve. As the steam enters, it pushes the air out through a trap in the drain line. Once all the air is evacuated, the trap closes. Steam continues to fill the autoclave chamber until a preset temperature and pressure is reached. Common autoclave operating conditions are 121 oC and 15 pounds per square inch gauge pressure (psig).

The sterilization procedure consists of three phases. These are the autoclave heat-up time, the contact time, and the cool-down time. Once the temperature has come to equilibrium, a minimum of twenty minutes contact time for all surfaces that require sterilization is necessary to insure complete biological inactivation. Usually the heat-up time section of the sterilization procedure is the time given for the autoclave chamber to heat up to the prescribed temperature; therefore, the run time used must be long enough for the entire package to equilibrate at 121 oC and still give the load a twenty minute contact time. Approximate total run times for solid materials are given in Table 1. Liquids will take even longer to sterilize. A good approximation for liquid run times is 1.5 – 2 times the time necessary to sterilize the given weight if it was a solid material. Remember, these data are for single items. For large loads, a rule of thumb is that if a space greater than six inches is between each item, the run time can be set for the weight of the heaviest item; however, if the items are less than six inches apart, they are considered to be one. An example would be if a researcher is autoclaving two bags of biomedical waste: one is six pounds, and one is eleven pounds. If the autoclave is large enough that the bags can be placed seven inches apart, the run time can be set for sixty minutes. However if the bags are less than six inches apart, there will not be enough room for adequate steam mixing, and the load must be considered to be the total of both bags- seventeen pounds. From Table 1, it can be seen that a run time of ninety minutes would be required for this load.

General Safety Procedures

All potentially infectious materials must be autoclaved before being washed, stored, or disposed as biomedical waste. Personnel who operate the autoclave must be trained to understand proper packaging, loading, labeling, as well as operation and emergency procedures. This training should also be documented by the principal investigator in charge of the laboratory. Autoclaves generate high heat and pressure; therefore, all users must understand and respect the associated risks. Always manipulate hot items with a thick glove designed for this purpose. At the completion of the cycle, allow the autoclave unit to cool down before opening. Then stand back and crack the door slowly so as to allow the excess steam to escape. Some older autoclaves have little or no heat shielding.

Warning signs alerting users to these hot surfaces should be placed next to the autoclave to remind personnel of this hazard. Do not stack or store combustible materials such as cardboard or plastic containers, or flammable liquids next to the autoclave.

Most autoclaves have a safety interlocking system which prevents the instrument from working if the door is not properly closed; however, same older units may not have such a built in safety mechanism. Should the autoclave that services your laboratory not have an interlocking system, special precautions must be observed to insure that the door is properly sealed before the process begins. If steam is leaking around the door during the sterilization process, the door has not been sealed properly. In this event shut down the system as safely as possible. Let the unit cool, and reset the door. Give special attention to making sure that the door is sealed tight, and restart the run. If this problem persists, the unit needs to be serviced by a qualified technician.

Biomedical waste must be labeled as such using the universal biohazard symbol, and should if possible be sterilized by the end of each work day. Never leave unsterilized material inside the autoclave, or sitting in the autoclave room overnight. Never autoclave materials that contain toxic agents, volatile chemicals, or radionuclides. The Environmental Health and Safety Department must be contacted should one require the sterilization of these types of materials.

Packaging and Loading Considerations

All biomedical waste that is to be autoclaved must be first placed in an approved and labeled autoclave bag. Sharps such as needles and scalpels must be placed in an approved, labeled, and ridged sharps container before sterilizing. Do not place sharp pipettes or broken glass in bags. These waste items should go into boxes or pails to be autoclaved, or be sterilized using a liquid sterilizing agent such as 10% sodium hypochlorite. Biomedical waste that is to be sterilized in an autoclave must have an indicator that demonstrates that the waste has been autoclaved before it can be disposed. Many autoclave bags come with an indicator that produces the word “AUTOCLAVED” when the sterilization cycle is completed. Such bags are available at no cost to the investigator through EH&S. Boxes and pails will have to have an indicator applied to them before they are autoclaved and disposed in the waste dumpster. Potentially infectious waste must be labeled “BIOHAZARD”, and have the biohazard label present on the container. When sterilizing non-hazardous materials such as research animal necessities, media, and laboratory equipment that will be used in a sterile environment, plain, unmarked containers will be adequate.

Approximately 50 ml – 100 ml of water should be placed in the autoclave bag prior to the start to facilitate steam production in the bag. If this water is naturally occurring in the load, additional water will not need to be added. Autoclave bags should be wrapped to prevent spillage should the bag tip over; however, if the tape is wrapped too tight, steam pressure can build up inside the bag causing it to rupture. Do not double bag waste. Autoclave bags are designed to be permeable to steam, but a double bag will retard the flow of the steam through the bag. Do not overfill bags or the autoclave unit, as this may interfere with the sterilization process due to poor steam circulation. It is best not to place bags directly into the autoclave. They should be placed on stainless steel or polypropylene trays. Stainless steel trays conduct heat much more efficiently than polypropylene, and therefore will enhance the sterilization process.

Do not fill the container more than 75 percent capacity when autoclaving liquids. This will ensure that the fluid has room for expansion when heated. Since sterilizing liquids using steam can be problematic, it is best to sterilize liquid waste using a chemical agent if possible. Should the investigator elect to use steam to sterilize the liquid, the container housing the liquid must be capable of withstanding the autoclave conditions. Borosilicate glass such as Kimax or Pyrex, or polypropylene containers can be used. Be sure to loosen caps on the vessels to allow for pressure build up during the process. Always place loose glassware and liquid containers inside a secondary container during the autoclaving process. When liquids are being removed from the autoclave, wear thick heat resistant gloves, splash goggles, and a rubber apron. The autoclave exhaust cycle must be set very slow for liquid loads. This allows time for the liquid to cool below 100 °C so the liquid does not boil as pressure is reduced. Upon the completion of the run, open the door slowly and stand back until the steam has cleared.

Autoclave Operation Parameters

The parameters for the sterilization cycle will depend upon the amount and type of material. The times given in Table 1 are good guidelines to follow. The cycle time should be developed for a worst case scenario. Denser loads will take longer than less dense loads of the same weight. The exact operating procedure for each autoclave model and various loads will differ; therefore, the responsible user should write a complete Standard Operating Procedure (SOP) for the steam sterilization operation. This SOP should include the sterilization procedure for each type of load autoclaved.

It is prudent practice to document each autoclave load in an autoclave log. This log should include the type of load (media, waste, animal necessities, etc), amount, run program, date, time, and operator’s name. Keep charts or printout strips in the logbook as documentation of the autoclave operation.

Each autoclave unit should have preventative maintenance, including autoclave spore testing procedures, and be placed on a preventative maintenance schedule with a qualified repair technician. It should be inspected annually. The service technician will insure that the unit operates safety, and properly. Any problems should be reported directly to the maintenance contractor so that the situation can be remedied as soon as possible. Responsible operator preventative maintenance includes: removal and cleaning of the drain strainer, if applicable, once per week, and monthly visually inspecting the autoclave gaskets, doors, shelves, and walls for residue buildup or wear. Any problems or deterioration should be remediated as soon as possible after they are observed.

A false sense of security can prevail unless a quality control monitoring program is implemented in every laboratory using an autoclave. A visible indicator must be used in every biomedical waste load that is sterilized. It is prudent practice to include an indicator, such as autoclave tape, on all autoclave runs. In addition, Washoe County mandates that all autoclaves are challenged with a biological indicator, and documented, monthly. The documented test results must be kept for at least one year. Biological indicators use heat resistant Bacillus stearothermophilus spores as a control to test for an effective kill during the autoclave process. Spore vials are placed in a challenging location in a medical waste bag during a run. A negative growth demonstrates that the autoclave is working properly. If the tester obtains a positive growth, try the run again with a fresh vial. Consistent positive growth indicates a problem that must be repaired before the autoclave can continue in service.

Conclusions

Though the autoclave is an effective instrument for steam sterilization, it must be maintained properly, and all users must be trained in proper usage and awareness of hazards while using the autoclave. Should one have any questions about sterilization, they may be communicated to Chet Carpenter at chestonc@unr.edu or (775) 784-4342.