Autoclave Operation and Safety

Training

If an autoclave is not operated correctly, your research or clinical materials could be compromised, the unit can become damaged and taken out of service, or worse, you could be badly injured. Autoclave training is given by the EH&S Biological Safety Office upon request. The training is conducted at the autoclave and is geared for research staff. The proper use and maintenance of autoclaves as well as safety training is covered. Please contact the Biosafety Office at 392-1591 to schedule a training session.

Standard Procedures

  • Collect biohazardous waste in autoclave bags. Close the bags and place them in a sturdy, leak-proof tray or container for transport to the autoclave. The tray or container is used as secondary containment; do not overload these. Use a wheeled cart to transport bags/trays to and from autoclave. ALWAYS bring autoclave gloves with you to the unit.
  • Autoclave bags or must be closed, yet vented, to allow proper sterilization. Loosen the closure on the bags as you load the autoclave; sealed bags will not allow penetration of steam and may explode during exhaust step.
  • The inner contents of tightly sealed or covered containers will not be adequately sterilized. Sealed containers may also explode in the autoclave or when you attempt to remove them.
  • Do not place items directly on racks. Use autoclave trays for all sterilization of biohazardous waste. Trays must be designated for use at 250ºF.
  • Autoclave trays, Sharps boxes & biohazardous waste bags must be labeled with PI name and laboratory room number.
  • Do not leave biohazardous waste unattended. If the sterilizer is in use, please return the biohazardous waste to your laboratory.
  • DO NOT change autoclave temperature to >250ºF.
  • DO NOT turn off the autoclave at the end of your cycle.
  • DO NOT adjust water/steam settings or valves.
  • DO NOT autoclave liquids containing bleach, formalin or glutaraldehyde.
  • Do NOT autoclave flammable or volatile liquids.
  • Do NOT autoclave radioactive materials.
  • Do NOT autoclave items containing corrosives (acids, bases, phenol).
  • Warning! Burn Hazard – clogged lines, equipment malfunction or a failure in the steam supply may cause the autoclave chamber to fill with scalding water. If water leaks from front of the autoclave, DO NOT OPEN the chamber door. Failure to heed this warning may result in second- or third-degree burns from scalding water!
  • IMPROPER USE OF THE AUTOCLAVE IS DANGEROUS TO YOU & CAN RESULT IN EXPENSIVE REPAIRS.
  • Training is essential to use the autoclave and minimize the risk of injury. Training is given by EH&S Biological Safety Office at the autoclave. Please contact the Biosafety Office at 392-1591 to schedule a training session.
  • EMERGENCIES: During work hours, call EH&S at 353-392-1591 & speak with Idania Alvarez regarding any mechanical problems/failures. EH&S will notify the approved vendor for service. Please contact Physical Plant after hours or weekends.

Specific Procedures

The Biosafety Office has detailed instruction sheets for UF’s more heavily used autoclaves. Click on the applicable link below. The instruction sheets are NOT  SUBSTITUTES for PROPER TRAINING. Schedule a training session by contacting our office.

Recommended Sterilization Times [Drying Time not Included]

121ºC / 250ºF & 15PSI

BIO-WASTE IN AUTOCLAVE BAGS, VENTED

  • MULTIPLE BAGS ― 100 MINUTES OR LONGER
  • SINGLE BAG ― 90 MINUTES
  • PARTIAL BAG ― 60 MINUTES

DRY GOODS

  • GLASSWARE, EMPTY, INVERTED ― 15 MINUTES
  • INSTRUMENTS, WRAPPED ― 30 MINUTES
  • UTENSILS, WRAPPED ― 30 MINUTES

LIQUIDS (BOTTLES WITH VENTED CAPS, ½ FULL

  • 75mL ― 25 MINUTES
  • 250mL ― 30 MINUTES
  • 500mL ― 40 MINUTES
  • 1000mL ― 45 MINUTES
  • 1500mL ― 50 MINUTES
  • 2000mL ― 55 MINUTES

Please note that Liquid cycles will take ~30 minutes longer to run than the set sterilization time due to slow charge and exhaust.

Record Keeping

The following records regarding autoclave use must be kept:

  1. Maintenance records
  2. Autoclave use log – must be available near the autoclave
    Each load of material inactivated shall be logged as follows.
    - Date, time, and operator’s name
    - Contact information: Lab, room number, phone number
    - Is this biohazardous material?

Testing

Autoclaves shall be tested before being placed into service and then periodically to ensure they are functioning as required:

Every 40 hours of use (Required for autoclaves that are used to inactivate human or non-human primate blood, tissues, clinical samples, or human pathogens.)

Every 6 months (Required for autoclaves that are used to inactivate or sterilize other material.)

A commercially available test indicator kit that uses bacterial spores (Geobacillus stearothermophilus) is the approved method of testing autoclave efficacy. Most spore vial test kits require 560 to 600 C incubation of the autoclaved test vial along with a non-autoclaved control vial. Incubation causes surviving spores to grow, indicated by a color change.

Please read the product information sheet for appropriate storage information, but, in general, spore vials should not be frozen. Each batch of vials has an expiration date. Vials should not be used after their expiration date.

Before placing an autoclave into service, a test load approximating the weight and density of the type of waste generated shall be autoclaved with test spore vials. The spore vials should be placed at the bottom, top, front, rear, and center of the autoclave chamber. This can be achieved by either:

  • placing vials at those positions within one large test load, OR
  • making several smaller test packs with 1 vial at the center of each and placing the packs at those locations within the chamber.

The appropriate parameters for sterilization including temperature, pressure, and treatment time shall be determined in this way.

For periodic testing, place a spore vial in the very center of a test load prior to autoclaving.