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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.

Preferred Methods


  • High pressure/temperature (autoclave)
  • Ethylene oxide gas for items that cannot withstand high temperature
  • Dry heat (dry bead sterilizer) – used for aseptic tip technique only
    • NOTE: Instruments must be fully sterilized by another method between surgical sessions




  • 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 
    • Muslin and paper (crepe) 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 these materials
  • Exposure time in an autoclave is normally 13 minutes at 121°C (250°F)
    • 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 some other event that compromises sterility


Dry Bead Sterilization


  • Used to sterilize the tips of surgical instruments in between multiple surgeries
  • Sterilizer must be activated for a minimum of 20 minutes to reach the appropriate temperature before being used
  • 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
    • 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 [LINK: IACUC Guidelines: Survival Surgery]


Chemical/Cold Sterilization


  • Common commercial sterilants and their active ingredient(s):

    • Cidex:  glutaraldehyde
      • Active for 15 days once prepared
      • 10 hours of exposure required for sterilization
    • Spor-Klenz:  Hydrogen peroxide
      • Use immediately, do not store
      • 11 hours of exposure required for sterilization
    • Sporicidin:  glutaraldehyde (1.12%), phenol (1.92%), sodium phenate (0.01%)
      • Active for 28 days once prepared
      • 6.75 hours of exposure required for sterilization
  • 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 long agent 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
    • SOPs for Cidex, Spor-Klenz, and Sporicidin may be found here: Chemical Sterilization Standard Operating Procedures (SOPs)


Last Reviewed by the IACUC 06/14/2017