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Sterilization techniques

Chapter 3: Table of contents


Surgical instruments should be free of gross contamination prior to any form of sterilization.  Microorganisms can be concealed by the blood and debris, preventing effective sterilization and contaminating the surgery.  Ultrasonic cleaning is useful for removing blood and debris lodged within the box locks and gripping surfaces of instruments. 

Steam Sterilization: 

Autoclave with pack ready for sterilization (open door)

Steam sterilization is by far the most common method used in veterinary medicine.  Common chemical sterilization techniques include gas sterilization using ethylene oxide or hydrogen peroxide and cold liquid sterilization using aldehydes.  Steam sterilization is extremely popular for disinfecting instruments and other surgical items such as gown packs.  Steam sterilization kills microorganisms by using moisture and extreme heat to cause coagulation of cellular proteins required for survival. Pressure is used to increase the temperature of the steam and allow more efficient sterilization.  Items such as oils and greases cannot be sterilized using steam sterilization.  Three types of steam sterilizers exist including gravity displacement, prevacuum and steam pulsing autoclaves.

Gravity displacement autoclaves are the most common in private practice and operate by entering steam into the top of the autoclave chamber and displacing air towards the bottom for eventual removal. Once all of the air in the chamber has been replaced by steam, a specific temperature is reached in the chamber which is maintained for a period of time to kill the microorganisms.

Prevacuum autoclaves operate by removing all air from the chamber using a vacuum pump and then pumping in steam. This allows for a more rapid sterilization process and also allows for the steam to penetrate instrument packs much faster. As a result, ‘flash’ sterilization can be done to instruments when they are needed quickly (emergency or dropped instrument) – this cycle is done on a non-wrapped instrument and using a fenestrated tray.

Steam pulsing autoclaves operate by pulsing steam into the chamber to a set pressure and removing air. These autoclaves are cheaper but not as fast as prevacuum autoclaves. However, they are faster than gravity displacement autoclaves.

Standard vs. Flash Sterilization:

Single instrument ready for flash sterilization with chemical sterility indicator

In general, all instruments must be thoroughly cleaned of any organic debris before placement in an autoclave. Instruments must be dried, box locks opened and all surfaces exposed for proper sterilization. In the autoclave, containers are placed right side up and instrument packs are loaded vertically and longitudinally with space between them to allow steam to travel all around. The autoclave is set for a temperature of 121°C (250°F). Sterilization time of 13-15 minutes (at 15 psi) is the standard minimum exposure time, an additional 2 to 5 minutes is often added for safety. Typical references will quote a sterilization time of 30 minutes at 121°C for gravity displacement sterilizers.  

Flash sterilization of single instruments can be done at 132°C (270°F) for 3-4 minutes (at 30 psi) in a fenestrated metal tray if a prevacuum autoclave is used.

Cold Sterilization:

Cold sterilization method

Cold sterilization is often used for material and instruments that cannot be steam sterilized, such as instruments with lenses (endoscopes and arthroscopes) and anesthetic equipment.  Gluteraldehyde is a non-corrosive cold sterilant that utilizes alkylation to kill bacteria, viruses and spores.  Gluteraldehyde and other aldehydes (such as formalin) can be irritating to skin and mucous membranes and can cause hypersensitivity reactions.  Instruments soaked in an aldehyde cold sterilant should be rinsed thoroughly with sterile saline prior to use.  

Ethylene Oxide:  

Ethylene Oxide

Ethylene oxide is a gas when at temperatures above 10.9°C.  This sterilization method can kill microorganisms such as all bacteria, spores, fungi and mainly large virus particles. Killing occurs by alkylation; impairing metabolic functions of the organisms.  Effectiveness of sterilization using this compound varies with the gas concentration, temperature, exposure time and humidity of the environment used. Exposure time can vary from as little as 48 minutes upwards to several hours. The gas diffuses through instrument packs and packages killing organisms it contacts.  Before the sterilization is complete, an aeration period must occur to allow the ethylene oxide to diffuse out of the packs. Certain objects such as plastics and rubbers require 168 hours or more before handling and use can occur, whereas stainless steel tools require little time to aerate. Surgical implants can require as much as 14 days aeration before handling and implantation is safe.  Contact with ethylene oxide impregnated objects may cause severe burns to patients and handlers. Ethylene oxide is flammable and explosive in air mixtures and very irritating when inhaled.  Storage of sterilized packs can range from 15 days up to 1 year in duration.  Sterilization using ethylene oxide has been banned in several countries (including Canada) due to release of CFC12 during the sterilization process which damages the ozone layer. Items that have previously undergone radiation sterilization should not be exposed to ethylene oxide sterilization because of the formation of toxic ethylene chlorohydrine. Newer generations of ethylene oxide sterilizers that do not produce CFCs are however permitted in Canada.  Items that have previously undergone radiation sterilization should not be exposed to ethylene oxide sterilization because of the formation of toxic ethylene chlorohydrine.

Plasma Sterilization:  

Hydrogen peroxide gas is used to produce free radicals which kill microorganisms in a closed chamber. A radio frequency is turned on to activate the process and then turned off to revert the chemicals to unharmful oxygen, water and other byproducts.  Sterilization takes approximately 1 hour and no aeration period is necessary after the sterilization cycle is completed.  This method of sterilization is rapidly replacing ethylene oxide sterilization in some countries.  It has the ability to kill a broad spectrum of microorganisms including mycobacteria, resistant bacterial spores, fungi and viruses.  A significant disadvantage is that materials such as linens, gauze sponges, wood, some plastics, and liquids cannot be sterilized using this method.


Manufacturers of prepackaged material for sterile veterinary use often use ionizing radiation.  It is an expensive method of sterilization and typically is reserved to manufacturing facilities and large hospitals.  Commonly used sterile surgical supplies, such as syringes and catheters, are often sterilized by this method.    In veterinary medicine, radiation sterilization is also sometimes used to re-sterilize objects that are meant for single use only and that cannot be easily sterilized using other methods.   Items that have previously undergone radiation sterilization should not be exposed to ethylene oxide sterilization because of the formation of toxic ethylene chlorohydrin.

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