Guidelines for Aseptic Survival Surgery on Rodents

Background:
The United States Public Health Service Guide for the Care and Use of Laboratory Animals states that "Aseptic technique includes preparation of the patient, such as hair removal and disinfection of the operation site: preparation of the surgeon, such as the provision of decontaminated surgical attire, surgical scrub, and sterile surgical gloves; sterilization of instruments, supplies, and implanted material and the use of operative techniques to reduce the likelihood of infection". While it is thought by some that rodents are somehow uniquely resistant to infection, there is no body of scientific evidence to substantiate this perception. In fact, rodents are often used in surgical infection research.

Surgical Facilities:
Surgical procedures can be done in a dedicated surgical facility or in a laboratory. If done in a laboratory, the surgery must be conducted on a clean, uncluttered lab bench or table surface in a low traffic area. The surface should be wiped with a disinfectant before and after use and covered with a clean drape.

Animal Preparations:

  • After anesthesia induction, the hair should be removed from the site where the incision will be made.
  • After the hair has been removed, the site should be thoroughly scrubbed with a detergent solution to remove superficial flora, soil, or debris which interfere with antisepsis. There are several antiseptic agents to choose from each with its own indications and contraindications:
    • Iodine in Alcohol or Iodophors: A good choice for a surgical preparation with a broad spectrum of activity, including Mycobacterium. Action is rapid and persistent if not removed
    • Tinctures of Chlorohexidine: The 4% aqueous solution effectively cleans the skin with a rapid onset of activity primarily due to the alcoholic component of the tincture. It has the added benefits of a prolonged activity that alcohol alone does not provide. Chlorhexidine has a broad spectrum of activity with minimal inactivation.
    • Alcohol: 70% ethyl or isopropyl alcohol are less effective than other agents, but probably still effective under most circumstances for rodents. One should be cautioned that evaporation of alcohol decreases the patients' body temperature and may induce hypothermia.
  • When detergents are used, they must be rinsed from the skin with sterile water or sterile saline prior to surgery. Alcohol may be used, but note the potential for decreased body temperature.
  • Application of antiseptic solution to kill or inhibit more adherent, deep bacterial residents may be indicated. Povidone iodine or iodophor solutions may be sprayed or daubed onto the surgical site after the initial scrub and rinse.
  • Ideally, where major survival surgery is done the surgical site should be covered with a sterile drape. This should be done once the surgeon is gloved.

Instrument Preparation:
All instruments must be cleaned and sterilized prior to use. First, clean instruments of any debris by hand washing or by mechanical washer/sterilizer. The method of choice may be determined by the procedure or the delicacy of the surgical instruments or the devices being used.

Smooth, Hard Metal Instruments and Polyethlene Tubing and Catheters:

  • Heat Sterilization:
    • Steam Autoclave: The instruments should be placed in a specially designed pack or wrapped in sterile in drapes or cloths. This should be secured with a thermosensitive tape. Use of such tape provides some indication that the autoclave procedure was effective. Instruments should be autoclaved for 4 minutes at 132oC (270oF) and dried for 3 minutes in a vacuum autoclave. Different times are required for gravity autoclaves. Once autoclaved, packs or wrapped instruments should be stored in closed cabinets. Double wrapped packs can be considered sterile for six weeks. Storage on open shelves reduces this number to three weeks. Single wrapped packs maintain sterility for 3 weeks. Note that steam is unsuitable for plastic with a low melting point, powders or anhydrous solutions.
    • Flash Steam: Used to sterilize articles intended to be used immediately. The temperature must reach 132oC for three to five minutes.
    • Sterile Bead Sterilizer: These are handy accessories that will sterilize a metal instrument in 10 seconds. However, clean instruments must be used and the beads should be cleaned or replaced monthly. Instruments should also be appropriate size for the unit. This type of sterilization is ideal for multiple cage surgeries. NOTE: Most sterile bead sterilizers take thirty minutes to heat up.
  • Cold (Chemical) Sterilization/Disinfectants:
    • Effective and proper use of chemical sterilization is dependent on many factors, including the use of chemicals classified as sterilants (not disinfectants), physical properties of the item(s) being sterilized (i.e., smooth, impervious to moisture, clean) and assurance of proper exposure. Chemical sterilants have finite shelf lives and must be used, depending on the agent, within one to four weeks. Furthermore, the solutions must be protected from contamination. Effective cold sterilization requires thorough cleaning of instruments prior to processing because blood and organic debris may inactivate chemical germicides and/or shield microorganisms from the sterilization process. Clean Tupperware type containers with secure lids or stainless steel instrument trays and lids are recommended for cold sterilization procedures and instrument storage. Sterile water or saline should be used to rinse the instruments, implants and tubing (inside and outside) prior to use to avoid tissue damage to the animals. The following are acceptable chemical sterilants/disinfectants for rodents:
      • AlcideR Active ingredient: Chorine dioxide 1.37%. Exposure time must exceed 6 hours. Shelf life is 14 days.
      • Ethyl Alcohol: 70 90%; requires 16 hour contact time; may rust or corrode instruments; alcohol is not fungicidal, virucidal or sporicidal. Of controversial value.
      • Cetylcide G: Active ingredient: 3.2% denatured glutaraldehyde. Exposure time of 20 minutes will kill all but bacterial spores. Full sterilization requires 10 hours. Shelf life is 28 days.
      • CidexR : Active ingredient: 2% glutaraldehyde. Exposure time must exceed 10 hours for sterilization. Shelf life is 14 or 28 days depending on the product.
      • EndosporeR : Active ingredient: stabilized hydrogen peroxide 6%. Not acceptable for metallic items.
      • SporicidinR : Active ingredient (activator + buffer): phenol 7.05%, glutaraldehyde 2%, sodium phenate 1.2%. Exposure time must exceed 6.75 hours for sterilization. Shelf life is 28 days.
      • Ethylene Oxide Gas: This is only to be used with instruments that will be damaged by heat or steam sterilization. This process is toxic, expensive and is regulated by federal law. Fine gauge catheters may be sterilized with ethylene oxide gas on the cool cycle.

Rubber Tubing:
The following should be considered:

  • Heat sterilization
  • Ethylene oxide gas
  • 6% hydrogen peroxide solution

Surgeon Preparation:
The surgeon should wash his/her hands with an antimicrobial detergent, for example, chlorohexidine or iodophor and rinse with water. Then, place on sterile surgical latex gloves. If working alone, the surgeon should have the animal anesthetized and positioned prior to gloving. If the instruments are in a sterile pack, that first layer of the double wrapped instrument pack should be opened before gloving.

The surgeon should also wear a face mask to prevent contamination of the surgical field. The wearing of gowns and surgical bonnets is optional, but recommended.

Multiple Surgeries:
The first animal in the group of animal surgeries should be done using the above aseptic protocol. This includes using sterile instruments and good surgeon and animal preparations. After the first animal surgery, clean the instruments of gross debris and use suitable disinfectant and rinses or a hot bead sterilizer. Continue to the next surgery. The IACUC recommends a new set of autoclaved instruments after every 5 animals.


Approved by the Institutional Animal Care and Use Committee on May 6, 2003