Autoclave Use and Waste Validation

What Can Be Autoclaved: Lab Materials and Biowaste

• Examples of lab materials:
  • Metal items;
  • Borosilicate (Pyrex) glass items;
  • Heat-resistant plastic items (Nalgene, polypropylene, polycarbonate);
  • Pipette tips;
  • Aqueous solutions;
  • Water;
  • Animal food and bedding; and/or
  • Soil.
• Biohazardous waste: 
  • Solid waste in autoclave bags: gloves, paper towels, empty tubes, agar media in Petri dishes, other solid lab debris contaminated with biohazards.
  • Water-based biological waste: exhausted culture media, supernatants, etc. containing no chemicals and no heat-stable antibiotics.
  • Sharps waste in rigid containers: glass slides, needles, syringes, pipettes, tips, blades, etc.
  • NOTE: Ensure that biowaste is autoclaved in a secondary container such as an autoclave safe pan.

Items that Must NOT Be Autoclaved

• Chemical solvents;
• Corrosives;
• Flammable liquids;
• Other chemicals or chemical containers;
• Kit chemical bottles;
• Antibiotic bottles;
• Vials or tubes with chemical residue;
• Radioactive material;
• Any sealed container; and/or
• Any material that cannot withstand 121˚C for ≥18 minutes and/or 15-18 PSI, such as polyethylene plastics (PE, recycle #1), or high-density polyethylene plastics (HDPE, recycle #2).

Autoclave Cycles

• Solid/gravity:
  • Supplies steam to the chamber with no mechanical vacuum assistance, i.e., by gravity. When process time is complete, steam is quickly exhausted from the chamber. 
  • Items such as upright containers that trap air within them may not be fully sterilized/ decontaminated using this cycle because the trapped air can prohibit full steam penetration and surface exposure.
  • Use the solid/gravity cycle for: 
    • Pyrex/borosilicate glassware (empty, inverted, no closures);
    • Dry hard items (unwrapped or in porous wrap); and/or
    • Metal items with porous parts, other porous materials.
  • Use the solid/gravity cycle for decontamination of solid biological waste if you have no Pre-vacuum cycle available on your autoclave; compensate for poor steam penetration in the waste by lengthening the processing time as needed.
  • Do not use this cycle for liquids or media that require a slow exhaust.
• Liquid:
  • Supplies steam to the chamber with no mechanical vacuum assistance. When process time is complete, steam is slowly exhausted from the chamber to prevent boil-over of liquids.
  • NOTE: Because this slow exhaust phase takes extra time to complete, plastic materials that will withstand fast-exhaust cycles can melt using the Liquid cycle due to prolonged exposure to heat.
  • Use the liquid cycle for: 
    • Pyrex/borosilicate glass containers up to 2/3 full of liquid (liquid media, aqueous solutions, liquid biowaste).
    • Do not use this cycle for items that could melt during slow exhaust.
• Pre-vacuum (available on newer autoclaves):
  • Mechanically removes air from the chamber by pulling a vacuum in a series of pulses, then supplies steam to the chamber. The vacuum allows full steam penetration of dense materials. When process time is complete, steam is quickly exhausted from the chamber.
  • Is the preferred cycle for decontaminating solid biological waste.
  • If available on your autoclave, use the Pre-vacuum cycle for: 
    • Decontamination of solid biowaste and sharps biowaste;
    • Sterilization of glassware that must be processed in an upright position; and/or
    • Sterilization of dry items that can trap air (e.g., pipette tip boxes).
  • Make sure that bags containing agar plates have only a one-inch opening so that the bag can properly contain melted agar during processing.
  • Do not use this cycle for liquids, media, lighter-weight plastic containers, dry items that will collapse in a vacuum.

Hazards for Autoclave Users

• Burns: 
  • From contact with hot autoclave chamber surfaces;
  • From residual steam when the door is opened at end of the cycle; and/or
  • Hot fluid scalds from boiling or overboiling liquids/spillage.
• Hand and arm injuries related to door operation.
• Injury or death from the explosion of the pressurized chamber.
• Inhalation or other exposure to fumes/vapors from chemicals that should not have been autoclaved.
• Exposure to biohazardous materials before autoclaving, or after autoclaving if the material was unsuccessfully decontaminated.

Training Requirements for Autoclave Users

• Successful completion of the EHS online module Safe Autoclave Use.
• Hands-on training given by an experienced individual in your facility is also required prior to operating an autoclave due to the serious hazards associated with use of this equipment, and because operating procedures differ from building to building, depending on the specific autoclave provided.
• Hands-on training must cover the topics provided on the EHS Autoclave Training Record.
• It is highly recommended that departments which provide autoclaves for research should designate trainers from their pool of people who are experienced in autoclave use, and can knowledgeably provide the hands-on training required for new personnel.

Safe Work Practices for Autoclave Users

• Wear required personal protective equipment: Buttoned lab coat, closed-toe shoes, heat-resistant gloves (make sure they are dry; if wet, they transfer heat), safety glasses.
• Use only autoclavable packaging material and containers.
• Make sure the autoclave drain is clear of debris before each use.
• Before operating, check to see if the autoclave is functioning properly (no ABORT message, etc.).
• Include a verification device such as a chemical Integrator (CI) with every waste load.
• Adjust closures on bags of solid waste so they have at least a one-inch opening.
• Check to see that closures on liquid-containing vessels are loosened.
• Arrange items in the load so they are spaced evenly, allowing room between items (no stacking/ crowding/ overpacking).
• Make sure containers of liquids are no more than 2/3 full.
• Load items so that they do not touch each other, or sides and top of the autoclave chamber.
• Properly place bags of waste and liquid items in autoclavable pans or trays: bags must not overflow the pans; vessels of liquid are in a pan deep enough to contain potential boil-over.
• Process items for sterilization in separate loads from waste for decontamination.

Standard Pre-Run Procedure

1. Sign in on autoclave use log and check that drain is clear of debris.
2. For a waste load, put on disposable gloves and place a verification device into proper position within the waste, (or in an empty, like-sized container if running a liquid waste load). Only one bag/ container per load needs a device placed in it. After placing the device, adjust bag openings to approximately one inch to allow steam penetration.
3. Wearing appropriate PPE for burn protection, place load into chamber.
4. Close chamber door and ensure that it closes completely.
5. Select and start cycle.
6. Ensure door has sealed and cycle is successfully underway before leaving the autoclave facility.
7. Set a personal timer to remind you when your cycle will end.

Standard Post-Run Procedure

1. Ensure that cycle is completely finished and chamber pressure is zero before proceeding.
2. Wearing PPE, open autoclave door and stand aside to avoid contact with any escaping steam.
3. Allow load to cool somewhat with door open; let liquids cool in autoclave for at least 15 minutes.
4. Wearing PPE, carefully remove load from chamber. If liquids begin to boil over, stop and allow more cooling time before removal.
5. Close autoclave chamber door when finished.
6. If running a waste load, check the verification result. Results of autoclave verification must be documented; record CI results on autoclave user log, and BI results in BI log.

Malfunctions

• If you see an error message, evidence of an aborted cycle or other operational problems, always record your observations on the autoclave user log, and promptly report them to the person responsible for the autoclave.
• If you discover that the autoclave is dysfunctional, and/or if you think it is unsafe to use, immediately post an 'Out of Service' sign on the unit and report it to the person responsible for the autoclave. Never attempt repairs on a malfunctioning autoclave.
• If you feel an emergency situation is developing or has developed with an autoclave, immediately remove yourself and others from the area. Secure the area, and post a sign to warn others to stay away. Immediately report the situation to the appropriate authorities/emergency response providers.

Certification as a Pressurized Vessel

• Autoclaves at Virginia Tech which achieve >175 PSI or have a chamber volume of 60 gallons/8 cubic feet, which includes most floor models and built-in units, are inspected as pressurized vessels every two years by Virginia state inspectors through the Office of Risk Management. Smaller tabletop models that do not meet these criteria are not subject to this inspection.
• Current inspection certificates are posted on/at the equipment.
• University departments are responsible for arranging for this certification. 
• EHS checks autoclave certification when conducting general building inspections, and will notify building managers and/or department heads if autoclave certifications have expired, or are not present.

Verifying Autoclave Performance

• Autoclaves used for decontamination of biohazardous waste must be regularly tested to verify performance in reaching proper decontamination conditions for hazardous biological waste. This is necessary to ensure that infectious agents in waste are inactivated prior to leaving the university for final disposal.
• NOTE: Performance verification is highly recommended for autoclaves used to sterilize media, reusable glassware, medical devices, etc. This is considered a best practice and improves the quality and reliability of research data and laboratory operations.
• Verification testing methods:
  • So-called "autoclave tape" reacts to exposure to heat with a light-to-dark color reaction. It can serve as an easy visual means of identifying items that have been through an autoclave cycle from those that have not. However, this tape it is NOT a definitive indicator of sterilization/ decontamination conditions having been met when an item is autoclaved, and must not be used as a verification testing method.
  • Approved testing devices include Biological Indicators (BIs), and Chemical Integrators (CIs).
  • BIs require incubation to determine results; CIs provide immediate results after a cycle is run.
  • CIs are not as accurate as BIs; however, they are reliable. They are also less expensive.
  • BIs must be used when testing autoclaves:
    • For routine performance verification.
    • Following autoclave installation and repair.
    • When new autoclave cycles are added, or when cycle parameters are changed.
    • When a new load configuration or packaging material is introduced.
  • An autoclave verification program must include both testing methods:
    • Verify autoclave performance monthly with BIs and record results in BI Log.
    • Verify decontamination of each waste load with CIs and record results on Autoclave Use Log.
  • Location of the test BI or CI in a waste load is important. Placement of the test device on the outside of an autoclave bag of solid biological waste will not yield the information needed about conditions on the inside of the bag. Instead, the test device must be placed within the bag, preferably in the center of the waste where steam will have the greatest penetration challenge, but not so deeply buried that it cannot be safely and successfully retrieved after decontamination in the autoclave.
  • Depending on the size of your autoclave chamber, a 'load' of waste in need of decontamination may consist of multiple items (e.g., several bags of waste, or a bag and several sharps containers, etc.). You only need to use one test device per load, not in every bag or container in the load.
  • When decontaminating liquid biological waste:
    • Use BIs that are designed for use with liquid wastes.
    • Place BI ampule directly in liquid for the autoclave run, then retrieve the ampule after the liquid has cooled.
    • DO NOT place CIs directly in liquid; they are not designed for direct use in liquids. Place CI in an empty vessel similar or identical in size to the vessel(s) containing the liquid waste (e.g., bottle, flask, etc.) and cap the vessel with the same type of closure as used on the waste container(s). Then place the test vessel in the autoclave with the waste vessel locating it in load position that will pose the greatest challenge to the CI.
• BI and CI Verification Testing Procedures

Biological Indicators (BI):

• Label each BI with the date, autoclave identification, and the cycle type you are testing.
  1. Place the BI inside a bag of solid biological waste. Alternatively, the BI can be placed within a bag of mock waste which approximates the density of actual waste; the mock waste can be run as a test load on its own, or along with other bags of actual waste (depending on the size of the autoclave). Retrieval of BIs from mock waste is often easier, and less messy.
  2. Label one extra BI as a positive control for the lot number being used. This BI will not be autoclaved; it will be activated and incubated to confirm that the bacterial spores of the test organism will germinate under favorable conditions.
  3. Run the autoclave cycle.
  4. Fill out the monthly autoclave verification log.
  5. Upon cycle completion and cooling of the load, retrieve the BI.
  6. Wearing appropriate PPE, follow product instructions to release the culture media in all test vials to put it into contact with the bacterial spores.
  7. Make sure the vial incubator is turned on and set at the recommended temperature. Place vials in the incubator.
  8. Observe and record any color changes at 24 and 48 hours. The positive control should grow (and vial liquid should change color); test vials that have been exposed to successful decontamination conditions in the autoclave should not grow (and vial liquid will not change color).

Chemical Integrators (CI):

• Make sure your CIs have been stored in a manner that protects them from exposure to light, which can affect their performance.
  1. Check the expiration date on a CI before use.
  2. Wearing appropriate PPE, place a CI within a bag of waste, then adjust the closure of the bag to approximately a one-inch opening. If autoclaving liquid waste, place CI in a like-sized container.
  3. Run the load on an appropriate cycle for the waste type.
  4. When the cycle is complete and load has been removed and cooled, retrieve the CI, interpret the reaction according to information supplied with the CI product.
  5. Record results on Autoclave Use log.

BI or CI failure to verify:

• When BIs or CIs indicate decontamination conditions were not met, an investigation of autoclave performance must take place; the machine must be taken out of service until its status can be determined, and malfunction corrected.
• Waste that has been autoclaved with a verification device that failed to validate kill conditions must not be discarded, but re-autoclaved in a different autoclave, or held until the cause of the problem is identified and it can be determined that the waste was in fact sufficiently decontaminated.
• Possible causes of BI or CI failure to verify (other than autoclave malfunction):
  • Test devices "Use By" dates have expired.
  • The wrong kind of test device was used (e.g., a test device designed to test dry heat or gas sterilization equipment); the correct test device will specify that it is meant to be used for steam autoclaves.
  • CIs which have been overexposed to light, completely soaked with water, or crimped/bent during use in an autoclave run can result in their performance being compromised.
  • Steam did not fully penetrate the load because 1) the bag was packed too densely with material; 2) the bag was not opened at least one inch before the decontamination cycle was run; 3) the autoclave chamber itself was packed too densely which prohibited steam penetration.