Autoclaves

Wet or saturated steam penetrates objects in the autoclave. Condensation creates negative pressure and draws in additional steam. As steam condenses, it transfers heat to the objects in the autoclave and causes cell destruction via coagulation of proteins. Autoclaves can be classified into two different types: gravity displacement and vacuum displacement.

Gravity Displacement

In a gravity displacement autoclave, steam enters the top or side of the chamber and, because steam is lighter than air, it forces the air out of the bottom of the chamber through a drain vent. It is very important that all valves are clear and unobstructed, and the chamber not overfilled for this system to function efficiently. A gravity displacement autoclave can be thought of as a pressure cooker type of system. These often require longer cycle times to achieve sterilization

Vacuum Displacement

 In a vacuum displacement autoclave, a vacuum removes cold air from the chamber prior to the introduction of steam. This allows for nearly instantaneous steam penetration into even porous loads. Vacuum displacement autoclaves typically have two sterilization cycles – 1) fast exhaust or high vacuum cycles which is suitable for sterilization of dry materials, and 2) slow exhaust or pressure pulsing which is suitable for sterilization of liquids such that liquids do not bubble out and evaporate during the cycle.

General Information

• Inspect the autoclave before each use. Do not use equipment that appears damaged, is leaking, or has not completed a prior cycle properly.
• Verify that all materials to be autoclaved are compatible with steam sterilization.
• Do not sterilize clean material together with biohazardous waste in the same load and run.
• Infectious waste for decontamination must be placed in biohazard autoclave bags and be loosely sealed with autoclave tape to allow steam to penetrate.
• Ensure sufficient water in load to allow steam penetration or add 250 mL water to bags containing solids to ensure steam penetration.
• There should only be one bag per pan or enough space between bags to allow adequate steam circulation. Do not overfill containers.
• Document all activities associated with the autoclave on the Autoclave Log Sheet.

Operating Parameters

The parameters for the sterilization cycle will depend upon the amount and type of material. Typically 121 degrees Celsius at 15 pounds-force per square inch (lbf/in² or psi) for a minimum of 30 minutes is recommended. However, the temperature and cycle time can be increased depending on the size and type of load.

Autoclave manufacturers generally provide several pre-set cycle options that vary in the pre-set sterilization temperature, sterilization time and dry time.

Do Not Autoclave

• Polystyrene (PS), polyethylene (PE), and high-density polyethylene (HDPE) plastics
• Materials containing solvents, volatile or corrosive, or flammable chemicals
• Materials contaminated with chemotherapeutic agents or cytotoxic drugs
• Materials containing Bleach
• Anything in a sealed container

Prevent Burns or Spills

To avoid burns and spills, observe the following safety procedures during loading and unloading the autoclave:

• When opening the autoclave, be aware of escaping steam that may cause skin burns
• Check that chamber pressure has returned to zero before opening door
• Stand behind door when opening, keeping face away from escaping steam
• Slowly open autoclave door only a crack to minimize chances of a rush of steam escaping
• If present, keep the autoclave room door closed to prevent steam from escaping into corridor
• Wait five minutes after opening door before removing liquids
• Load the bags into the autoclave and operate the autoclave according to the manufacturer's operating instructions.

Loading the Autoclave

1. Ensure the autoclave is operating properly before commencing
2. Determine the appropriate exposure time, temperature and pressure for the load based on spore testing
3. Loosen screw caps on bottles and tubes of liquids before autoclaving
4. Ensure bags are placed in secondary containers with sufficient space between bags for adequate steam circulation.  
5. Do not allows bags to touch or contact the sides of the autoclave
6. Ensure the autoclave attains the desired temperature (normally 121°C) and pressure (minimum 15 psi) for the desired time (minimum 30 min.)
7. Record daily usage on the Autoclave Log Sheet .
8. Record results of the biological test indicator on the Autoclave Log Sheet monthly. 

Unloading the Autoclave

1. Confirm the chamber pressure gauge reads zero before opening
2. Open the door slightly to allow steam to escape, protecting self from steam exposure
3. Wait 20-30 minutes, more if necessary, for the contents of the autoclave to cool
4. Carefully remove the secondary container with the waste bag to reduce the risk of spillage
5. Allow the autoclaved plastic bag to cool completely before handling
6. Verify that the temperature and duration of the cycle exposure were met
7. Verify that each autoclave tape or strip has changed to black, indicating proper sterilizing conditions were met. Additionally, check the electronic dial to ensure that the recommended temperature is achieved for the proper length of time on each load. If the autoclave tape or strip has not changed color, then the entire load must be re-autoclaved.

Disposal of Autoclaved Biological Waste

Autoclaved bags of biological waste are disposed of in the same manner as infectious waste at UND, through a regulated medical waste vendor. After verification that each autoclave tape or strip changed color by turning black, the operator should unload the bags and place them in a biohazardous waste bin located within the laboratory space.

Do not overfill the waste container. Prepare the autoclavable bag by cinching, twisting, and securing the bag closed when it is no more than three-quarters full.

All individuals who will operate an autoclave on UND property must receive hands-on training by laboratory staff and complete the Office of Safety autoclave safety training before operating any autoclave independently. All training must be documented. Principal investigators are responsible for ensuring that all laboratory personnel under their supervision complete required training before use. Office of Safety will determine training renewal intervals and notify the campus community of any changes to training requirements.

Before Each Run

• Before loading, check the interior chamber drain to be sure it is not clogged.
• Check that door gaskets seal properly.
• Ensure each waste bag or container decontaminated has a heat sensitive indicator, such as autoclave tape, attached to the outside of the bag. 

Weekly

• Flush drain with clean water to assure that drain is not clogged and to remove any impurities in the drain from salt solutions, media, etc.

Monthly 

• Biological testing should be performed monthly, at a minimum. Autoclaves that run more than 10 runs per week (40 loads per month) should perform a biological test weekly. Individual labs sharing an autoclave will monitor the autoclave using a biological indicator containing Geobacillus stearothermophilus once a month, alternating between the participating laboratories. 

Departments must coordinate with the Office of Safety and Facilities Management for autoclave installation, certification, and decommissioning. Autoclave users and laboratory supervisors are responsible for routine maintenance as specified by the manufacturer. Autoclaves are pressure vessels and must be inspected and certified as required by applicable North Dakota state law. Any autoclave malfunction, spill of biohazardous material, or injury involving an autoclave must be reported to the Office of Safety immediately.

The following steps describe the procedure for BT Sure (available from both Fisher Scientific and VWR), steps may vary for other products. Always follow manufacturer's instructions for use.

1. Secure a biological indicator test containing endospores. 
2. Tie a piece of string to the biological indicator vial or secure to an autoclavable tool to facilitate retrieval of the vial after the autoclave cycle. 
3. Bury the vial containing spores within the waste, leaving the string trailing out of the bag opening for retrieval. Use gloves, safety glasses, lab coat, and forceps to minimize exposure during placement and retrieval.
4. Secure the waste bag and start the autoclave cycle.
5. After the cycle and once the bag has cooled, retrieve the vial.
6. Hold the indicator in an upright position using a gloved hand and gently squeeze to break the glass ampoule to activate the media 
7. Incubate at 55‐60° C for up to 48 hours.
8. Read the results of the indicator:
   1. After 24 hours media is yellow = failed test (endospores were not killed; growth leads to fermentation and acid production turning the media yellow).
   2. After 24 hours media is purple = presumptive pass, but continue to incubate until 48 hours.
   3. After 48 hours media is purple = passed test (all endospores were killed).
9. Record date, run parameters, autoclave tested, and test results on the Autoclave Log Sheet .

NOTE: If generating high risk waste that presents a risk for potential exposure, the vial may be run inside a bag of waste that has previously been autoclaved. 

Testing a liquid cycle

Do not submerge the B/T Sure Indicators in liquid during autoclave cycle. Run the indicator in the chamber alongside the load. To run indicators submerged in liquid, use liquid-compatible biological indicators (see Biological Indicators, Incubator and Pouches).

Understand the limits of testing

Testing with autoclave tape or strips does not indicate effective sterilization nor do they indicate how long the temperature was maintained. These temperature indicators are only used to determine if the autoclave reached the approximate desired/operating temperature. Using a recording device or computer to track time and temperature will also not ensure the materials inside the center of the waste bag have been sterilized. Validating performance using a biological indicator is the only way to ensure complete inactivation/sterilization.

Handling an Autoclave that Fails a Biological Indicator

1. Remove the autoclave from service immediately and notify a supervisor and the Office of Safety
2. Confirm the correct biological indicator was used, that it was not expired, and that the results were appropriately interpreted. If an error is noted, repeat the test using appropriate supplies.
3. Verify that the autoclave was used correctly (e.g., correct time and temperature settings). If not, reprocess all inadequately treated materials using appropriate settings.
4. Repeat the biological indicator test in three consecutive cycles as soon as possible. If additional spore tests are positive,  items from the suspect load should be considered non-sterile and be reprocessed. Materials processed since the last acceptable (negative) biological indicator should be recalled/re-sterilized if possible.
5. Check with maintenance to identify any irregularities (electrical, steam supply, etc.). Steam supply should be ≥97% steam and <3% moisture. Report any abnormalities to the person who performs sterilizer maintenance.
6. If steps 1‐5 resolve the problem, and all three repeat biological indicators from three consecutive autoclave cycles are negative, then put the autoclave back into service and document issue on the Autoclave Log Sheet.
7. If one or more biological indicators are positive:

1. 1. Repeat the biological indicator tests using a different manufacturer's indicator.
   2. Discuss the abnormalities with the autoclave manufacturer.
   3. Request an inspection by autoclave maintenance personnel.
   4. Have the steam supply lines inspected.
   5. If there is no resolution, remove the autoclave from service until the manufacturer can confirm it is operating properly. Retest with biological indicators in three consecutive cycles before returning to service,

The individual in charge of the autoclave is responsible for maintaining all records. Users must maintain records of any validation testing they perform on the autoclaves and every autoclave run must be documented in the Autoclave Log Sheet including date, time-in, load type (clean material or waste), quantity treated, printed name and signature of the person responsible for the run, and any relevant information when applicable. Results of biological testing results (growth/no growth), must also be recorded in the Autoclave Log Sheet when appropriate. 

All autoclave records must be maintained for a minimum of three years by the department and made available to the Office of Safety upon request.

The autoclave operator must notify the supervisor and document any incident or problems encountered while operating or monitoring the autoclave.

Supplies such as biohazard autoclavable bags, biohazard sign labels, biological indicators, and autoclave tape or strips must be purchased by each department. Contact the Lab & Research Safety Officer for recommendations.

Spills may occur from a boil-over or breakage of containers. If a spill has occurred, do not operate the autoclave until the spill is cleaned. The operator is responsible for cleaning spills. The spilled material can be contained using materials from the spill kit to absorb or contain the spill.

Wait until the autoclave and materials have cooled to room temperature before starting the cleanup inside the autoclave.

Review the Safety Data Sheet(s) (SDS) of the spilled material(s), if appropriate, to determine which protective equipment, spill cleanup, and disposal protocols are necessary.

If materials have been intermingled, follow the cleanup and disposal protocol for the most hazardous component of the mixture.