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Sterilization - Accepted Methods (Informational Sheet)

Accepted Sterilization Methods

Informational Sheet: The Office of Animal Resources has provided a set of guidance documents (Policies, Guidelines, and Informational Sheets) for use when planning animal procedures at the University of Iowa. This Informational Sheet provides the current guidance on recommended testing of research biologics for pathogens.


The purpose of this document is to provide guidance on commonly-used methods for sterilization of surgical instruments and other materials for use in IACUC-approved Animal Protocols.

Accepted Methods for Full Sterilization

  • Preferred
    • Autoclave (High pressure/temperature)
  • Other
    • Ethylene oxide gas - for items that cannot withstand high temperature
    • Chemical/Cold Sterilization – for items that cannot be sterilized by other methods


Accepted Method for Re-sterilization Between Animals Using Rodent Aseptic Tip Technique

  • Dry heat (dry bead sterilizer)
    • NOTE: Instruments MUST be fully sterilized by another method between surgical sessions


Autoclave Sterilization

  • Effectiveness of autoclaving must be verified by an integrator strip placed inside the pack
    • Additionally, it is recommended to seal the pack with autoclave tape
  • Autoclave utilizes steam at high heat and pressure which must penetrate the pack to attain sterilization 
    • Materials such as muslin cloth and crepe paper drape material allow the steam to penetrate into the pack
    • Materials such as aluminum foil and wax paper should not be used due to the steam's inability to penetrate them
  • Exposure time in an autoclave will vary based on the type of autoclave
    • For gravity displacement sterilizers, typically need exposure at 121°C (250°F) for at least 30 minutes.
    • For dynamic-air-removal sterilizers, typically need exposure at 132°C (270°F) for at least 4 minutes. [1]
    • Bigger packs require more time to reach appropriate heat and pressure levels
  • Autoclaved pack should be stored in a dry, dust-free, well-ventilated area, preferably in a closed cabinet
    • The length of storage time is indefinite if the pack is stored properly (cabinet, drawer) and does not get wet, torn open or have some other event that compromises its integrity and sterility

Ethylene Oxide Sterilization[2]

  • NOTE: UIHC Central Sterilizing Services no longer performs ethylene oxide sterilization and it is not provided elsewhere on campus[3],[4]
  • Used for heat and/or moisture sensitive items
  • Effectiveness is dependent on gas concentration, temperature, relative humidity and exposure time
  • Items can be sterilized in their final packaging, but effectiveness depends on ability of the gas to freely diffuse through it
  • Requires aeration to desorb ethylene oxide from chamber and items
    • Some materials, such as certain biocompatible polymers and gels, may take weeks to off-gas the chemical completely.
    • Placement of any materials without sufficient time and aeration to desorb may lead to tissue reaction and health concerns
  • Disadvantages
    • Hazard concerns – flammable, explosive, toxic and carcinogenic
    • Length of cycle time
    • Cost


Chemical/Cold Sterilization

  • Examples of common commercial sterilants and their active ingredient(s):
    • NOTE: This information is based on currently available information as of 5/27/20. Refer to the manufacturer’s instructions for how to appropriately prepare and use.
    • CIDEX® Activated Dialdehyde Solution[5]:  Glutaraldehyde
      • Active for up to 14 days once prepared
      • 10 hours of exposure required for sterilization at room temperature
    • Spor-Klenz®:  Hydrogen peroxide and peroxyacetic acid
      • Concentrate Cold Sterilant[6]
        • Dilute at 1% v/v with purified water
        • Use immediately and do not store
        • 11 hours of exposure required for sterilization at room temperature
      • Ready To Use Cold Sterilant[7]
        • May re-use for up to 14 days
        • 5.5 hours of exposure required for sterilization at room temperature
    • Sporicidin® Sterilizing and Disinfecting Solution[8]:  glutaraldehyde, phenol, phenate
      • Active for 14 days once prepared
      • 12 hours of exposure required for sterilization at room temperature
  • Discretion is required in using these agents to assure that they are used with appropriate safety precautions and that the items being sterilized are compatible with the sterilant
  • Factors for effective and proper use of cold sterilization:
    • Chemicals must be classified as “sterilants” 
      • Commonly used disinfectants such as alcohol, iodophors, quaternary ammonium and phenolic compounds are not effective sterilants and are not acceptable for use on items (e.g., catheters, instruments) intended to be used in survival surgical procedures
    • Physical properties of the items being sterilized must be smooth and impervious to moisture
    • All surfaces, both interior and exterior, must be exposed to the sterilant
    • Sterilant solution must be clean and fresh
      • Date of preparation must be labeled on container
    • Chemically sterilized instruments must be thoroughly rinsed both inside and out with sterile saline or sterile water prior to use to avoid tissue damage.
    • Instruments must be handled in an aseptic manner to maintain sterility (e.g. handle with sterile gloves and place on a sterile field)
  • Standard Operating Procedures (SOP) MUST be posted in the laboratory when using chemical sterilization methods and must contain the following information:
    • Agent used (i.e. active ingredient)
    • How sterilant is prepared
    • How long sterilant is active once prepared (expiration time)
    • Exposure time required for sterilization of instruments/supplies
    • How the sterilant is removed prior to use in an aseptic technique
    • Template and an example SOP may be found here: Chemical Sterilization Standard Operating Procedures (SOPs)
  • Consult with an OAR veterinarian if there are any questions relating to the use of chemical/cold sterilants.


Dry Bead Sterilization

  • Used to sterilize the tips of surgical instruments in between multiple surgeries
  • Instruments MUST be fully sterilized by another method between surgical sessions
    • A surgical session includes those surgeries performed on the same day
  • Sterilizer must be activated for a minimum of 20 minutes to reach the appropriate temperature before being used[9]
  • All biological debris (e.g. blood, tissue) must be removed before placing the instruments into the sterilizer
  • Instruments must be inserted into the sterilizer for a minimum of 15 seconds before sterilization is attained[10]
    • Once the instruments are removed from the sterilizer, the tips will be VERY HOT
    • They must be allowed to cool before using to avoid burning the animal
  • Only the tips of the instruments are sterilized and the handles are considered to be contaminated
    • The instruments must be utilized in a fashion that the tips of the instruments remain sterile. See details regarding aseptic tip technique in Rodent Survival Surgery Guidelines


Last Reviewed by the IACUC 6/10/2020