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Policy#: 529-260

Title: ANIMAL USE GUIDANCE: Aseptic Rodent Surgery

Effective: 5/17/2021



The guidelines for rodent surgery are based on the Guide for the Care and Use of Laboratory Animals. Survival surgery on rodents should be performed using aseptic procedures, including sterile instruments, sterile gloves, masks and aseptic techniques. A dedicated space is required for rodent surgery.

Related Policies and Guidance

Policy 529-323: Policy on Animal Surgery


  1. All animals should receive a pre-anesthetic assessment. The assessment should include a thorough visual assessment for any signs of illness or abnormal behavior. An accurate weight should be obtained for appropriate drug dosing.
  2. Aseptic surgery should be conducted in an uncluttered area dedicated to the procedure while it is being performed. The area should be disinfected before use. (Table 1)
  3. Withholding food prior to surgery is not necessary in rodents unless the protocol or the surgical procedure requires it. Water should not be withheld prior to anesthesia.
  4. Surgeons should wear a clean lab coat, head covers, gloves, and face masks.
  5. The animal should be anesthetized using an appropriate inhalant or injectable anesthetic. The use of a local anesthetic such as bupivacaine at incision sites should be considered. Administration of analgesics prior to the beginning of surgery (preemptive analgesia) can enhance their effectiveness. Contact the Campus Veterinarian for more information on the use of various anesthetics and analgesics.
  6. Ophthalmic ointment should be applied to the eyes to protect the corneas as soon as the animal is anesthetized.
  7. The area of the incision must be prepared aseptically. The animal's fur must be removed from the area where the incision will be made leaving a clear border of at least 1 cm around the incision site. The clipping should be done in an area separate from where the surgery is to be conducted. Fine bladed clippers, a razor blade or plucking may be used. Loose fur should be removed with a vacuum or damp towel.
  8. The skin around the incision area must be prepared with an appropriate skin disinfectant (Table 2) using a gauze pad or cotton-tip applicator. Work from the center to the periphery of the site. The standard procedure is to alternate between surgical soap (e.g., betadine) and alcohol scrubs three times. Alcohol, by itself, is not an adequate skin disinfectant. Care should be taken not to wet the animal excessively as this may contribute to increased heat loss (hypothermia).
  9. Procedures must be in place to maintain the animal's body temperature with supplemental heat. This can be supplied with a warm water circulating heating pad. Electric heating pads and heat lamps are not recommended because of their potential to cause burns.
  10. Surgeons must wash their hands before aseptically donning sterile surgical gloves.
  11. When feasible, the surgical site should be draped aseptically with sterile material prior to making an incision to create a sterile surgical field. Sterile gauze sponges or plastic drapes (Glad Press-N-Seal, a food grade quality product that has specifically been tested for this application) with an opening can be used as a drape over the incision site.


  1.  The animal must be maintained in a surgical plane of anesthesia throughout the surgery.
  2. Surgery must begin with sterile instruments (Table 3) which are then handled aseptically.
  3. Instruments and gloves may be used for a series of similar surgeries provided they are kept clean and disinfected between animals.
    • If a “tips-only” technique is utilized (only the tips of instruments have touched the sterile surgical field or the animal’s tissues), then remove any blood any debris from instruments and disinfect them by dipping them in a hot glass bead sterilizer for approximately 30 seconds. Let them cool completely before touching tissues (sitting at room temperature for several minutes or dipping in sterile saline). Alternatively, instruments can be soaked in 70% isopropyl alcohol between surgeries for two minutes. A new set of sterile instruments should be used after 5 animals.
    • Gloves can be decontaminated by soaking them in 70% isopropyl alcohol for a minimum of 30 seconds and used on up to five animals.
  4. Anesthetized animals must be observed continuously throughout the duration of the anesthetic procedure until they have fully recovered. Anesthetic monitoring includes responsiveness to painful stimuli, character of respiration, and skin or mucous membrane color as seen by observing the ears, tail, and oral mucosa or foot pads. Pedal withdrawal reflex (toe or footpad-pinch on the hind foot) is recommended for assuring adequate depth of anesthesia prior to first incision and at least every 15 minutes during the procedure. Depending on the surgical procedure, other monitoring may be indicated such as heart rate, blood pressure, body temperature, and tissue oxygenation.
  5. Tissues exposed for long periods of time should be kept moist with warm saline or lactated Ringer’s solution. If the surgery is prolonged or the animal has lost more than a small amount of blood, these same fluids should be administered subcutaneously or intraperitoneally to prevent dehydration and volume depletion.
  6. The surgical incision should be closed using appropriate technique and sterile absorbable or non-absorbable material as appropriate. (Table 4)


  1. After the surgery is completed, move the animal to a warm, dry area and monitor it during recovery. Supplemental heat, using a heating pad or warm water blankets to maintain body heat, should be provided until the animal has recovered from anesthesia Heat lamps or electric blankets are discouraged as they may become too hot. Space should be available so that an awake animal can escape the heat if it becomes too hot. It is generally acceptable to place a heating pad under ½ to 1/3 of the cage during recovery.
  2. During recovery rodents should be placed either in a clean empty cage or on a paper towel placed on top of the bedding substrate to help protect against a suffocation hazard. All animals should be continuously observed while recovering from anesthesia until they are both sternal and ambulatory. Only at this time should the animal be returned to its routine housing.
  3. Provide analgesics as appropriate and approved in the AUP.
  4. Dehydration should be mediated by providing warmed isotonic fluids (0.9% Sodium Chloride or Lactated Ringer’s Solution) SC or IP (0.5-2mL for mice; 3-10mL in rats) following procedures lasting longer than 15 to 20 minutes.
  5. Sutures, staples, or wound clips must be removed 7-14 days following surgery. Absorbable sutured do not need to be removed. If animals will be euthanized within 14 days following surgery, removal of sutures prior to euthanasia is not necessary.”
  6. Observe the animal daily for at least the first 5 days. Maintain a detailed surgical record in lab notebook. Annotate cage card with procedure and date. Record anesthetic and analgesic administration and animal's condition on surgical cage card.

Table 1. Hard Surface Disinfectants





70% Ethyl alcohol, 85% isopropyl alcohol

Contact time required is 15 minutes. Contaminated surfaces take longer to disinfect.

Quaternary Ammonium

Sani Cloth®, Roccal®, Quatricide®, Tec-Surf II®

Compounds may support growth of gram-negative bacteria.


Sodium hypochlorite
(Clorox® 10% solution)
Chlorine dioxide
(Clidox®, MB-10®)

Corrosive. Solutions need to be made up fresh daily to maintain activity.


(Cetylcide®, Cide Wipes®)

Rapidly disinfects surfaces.


Lysol®, TBQ®

Less affected by organic material than other disinfectants.


Nolvasan® , Hibiclens®

Rapidly bactericidal and persistent. Effective against many viruses. Presence of blood does not interfere with activity.

Hydrogen peroxide Peracetic acid

Spor Klenz, Virkon S®


Contact time 10 minutes.

*For all the agents, organic matter must be removed prior to disinfection (presence of organic matter reduces/inactivates activity).

Table 2. Skin Disinfectants





Betadine®, Prepodyne®, Wescodyne®

Reduced activity in presence of organic matter. Wide range of microbicidal action. Works best in pH 6-7.


Nolvasan®, Hibiclens®

Presence of blood does not interfere with activity. Rapidly bactericidal and persistent. Effective against many viruses. Excellent for use on skin.



Table 3. Sterilization Procedures for Surgical Instruments & Equipment




Steam sterilization


Effectiveness dependent upon temperature, pressure and time.

Dry heat

Hot bead sterilizer

Dry chamber

Only tips of instruments are sterilized with hot beads. Only for re-sterilization between animals when conducting surgery on multiple animals. Instruments must be cooled before contacting tissue.

Gas sterilization

Ethylene oxide

Gas is irritating to tissue and all materials require safe airing time. Significant occupational safety hazard.

Appropriate sterilization indicators must be used to ensure sterility.

Cold chemical sterilants*

Chlorine dioxide (Clidox®, Alcide®)

Sodium hypochlorite (Clorox® 10% solution)

Glutaraldehyde (Cidex®, Cetylcide®, Metricide®)

Hydrogen peroxide/Acetic acid products
(Actril®, Spor-Klenz®)

To ensure adequate sterilization, these products must be used according to the manufacturer's recommendations for sterilization. This may require several hours of contact time.

Subsequent instrument removal and handling must be done using aseptic techniques in a sterile field or the items may be recontaminated.

Most cold sterilants are corrosive so will limit lifespan of instruments and are not compatible with all materials.

Instruments must be clean and free of organic material prior to sterilization.

Instruments must be rinsed with sterile saline or sterile water to remove the chemical sterilant before use.

Chemical expiration dates must be followed.

* Only products classified as sterilants can be used for sterilizing instruments and implants for surgery. Common disinfectants (alcohol, chlorhexidine, iodine, phenols) are not sterilants.


Table 4. Recommended Suture and Wound Closure






Polyglactin 910 (VicrylR), Polydiaxanone (PDSR), Polypropylene (ProleneR)

3-0, 4-0, 5-0, 6-0

Taper point (round)

Subcutaneous tissues

Polyglactin 910 (VicrylR), Polydiaxanone (PDSR),

5-0, 6-0


Reverse cutting


Polyglactin 910 (VicrylR), Polydiaxanone (PDSR), Nylon (EthilonR)

3-0, 4-0, 5-0, 6-0


Reverse cutting


Wound clips (Autoclips)

7 mm or 9 mm



Cyanoacrylate*** (VetbondR,NexabandR, Tissue MendR)

Surgical glue. For non-tension bearing wounds.


* Use the smallest gauge suture material that will perform adequately.

**Silk is not recommended for skin closure as it causes inflammatory tissue reaction and is associated with a higher incidence of wound infection.

*** Skin glue is generally used in addition to skin sutures or incisions less than 1 cm in length. Many rodents will rip the glue out making it difficult to close the incision. Thus, the use of surgical glue only is generally not recommended for a surgical wound.



·         Anesthesia and Analgesia in Laboratory Animals, Second Edition. R.E. Fish, et al Eds. Academic Press, 2008. 

·         CDC Guideline for Disinfection and Sterilization in Healthcare Facilities

·         Emmer, KM, Celeste, NA, Bidot, WA, Perret-Gentil, MI, Malbrue, RA. Evaluation of the sterility of Press’n Seal cling film for use in rodent surgery. J Am Assoc Lab Ani Sci 58:235-239. 2019.

·         Experimental and Surgical Technique in the Rat, Second Edition. H.B. Waynforth, P.A. Flecknell. Academic Press, 1992. 

·         Guide for the Care and Use of Laboratory Animals, National Research Council, National Academy Press, 2011. 

·         Hoogstraten-Miller, SL and Brown, PA. Techniques in aseptic rodent surgery. Current protocols in immunology chapter 1, Unit-1.1.14, 2008.

·         Keen, JN, Austin, M., Huang, L-S, Messing, S. and Wyatt, JD. Efficacy of soaking in 70% isopropyl aocohol on aerobic bacterial decontamination of surgical instruments and gloves for serial mouse laparotomies. J Am Assoc Lab Ani Sci 49:832-837, 2010.

·         Laboratory Animal Anesthesia, Fourth Edition. P.A. Flecknell. Academic Press, 2016. 



Updated: 12/9/04, 4/6/09, 8/13/12, 5/17/21