Autoclave Guidelines

Rules governing the use and testing of autoclaves are based on Rule 64E-16 of the Florida Administrative Code.


Autoclaves shall be tested before being placed into service and then periodically for effectiveness.


  • 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 other material.)


A commercially available test indicator kit that uses bacterial spores (Geobacillus stearothermophilus) is the approved method of testing autoclave efficiency. 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.

New autoclaves

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.

Autoclaves In Use

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

Storage Information

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.

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?

Confirmation of sterilization:

  • Record the temperature, pressure, and length of time the load is sterilized. Please note that temperature sensitive autoclave tape is not sufficient to indicate that the load reached sterilization conditions because the tape will change color at lower temperatures
  • Save the autoclave print-out (if the autoclave has a working printer)

Autoclave Operating Procedures

A written sterilization procedure shall be in place for each workplace. This shall include the following:


Appropriate parameters for sterilization shall be determined from the testing with spore vials.  The time it takes to sterilize a load will change depending upon the load density and the sterilization cycle one chooses. Tests have been performed which imitate these various situations. Please follow the established guidelines.


Identification of standard treatment containers and proper load placement shall be made.


The autoclave and work areas shall be cleaned after every use and the work area shall be disinfected, as needed.

Autoclave Operation and Safety Training

Autoclave training is given by the EH&S Biological Safety Office upon request. The training is conducted at the autoclave and is geared toward the 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.