Sterilization Sterilization is a term referring to any process that eliminates or kills all forms of microbial life, including transmissible

agents, such as fungi, bacteria, viruses, and spore forms that present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media. Sterilization can be achieved by applying the proper combinations of heat, chemicals, irradiation, high pressure, and filtration.

Method of sterilization : Autoclave

What is Autoclave ?

Autoclave is most commonly- used device in sterilization process. Autoclaves are widely used in microbiology, medicine, tattooing, body piercing, veterinary science, mycology, dentistry, chiropody and prosthetics fabrication. They vary in size and function depending on the media to be sterilized. In its most basic form, the autoclave is a pressure cooker. Water is heated in a pressurized environment to create steam. Using pressure makes it possible to heat to higher temperatures with less energy. Autoclaves are usually made of steel and have various configurations for removing air prior to pressurization.

Working principle of Autoclave A basic principle of chemistry is that when the pressure of a gas increases, the temperature of the gas increase proportionally. An autoclave is a large pressure cooker, it operates by using steam under pressure as the sterilizing agent. High pressures enable steam to reach high temperatures, thus increasing its heat content and killing power. Most of the heating power of steam comes from its latent heat of vaporization. This is the amount of heat required to convert boiling water to steam. This amount of heat is large compared to that required to make water hot. For example, it takes 80 calories to make 1 liter of water boil, but 540 calories to convert that boiling water to steam. Therefore, steam at 100 C has almost seven times more heat than boiling water. Steam is able to penetrate objects with cooler temperatures because once the steam contacts a cooler surface, it immediately condenses to water, producing a decrease in steam volume. This creates negative pressure at the point of condensation and draws more steam to the area. Condensations continue so as long as the temperature of the condensing surface is less than that of steam; once temperatures equilibrate, a saturated steam environment is formed. As the water molecules in steam become more energized, their penetration increases substantially. It is important to note that the sterilizing agent is the moist heat, not the pressure. Moist heat is thought to kill microorganisms by causing coagulation of essential proteins. Another way to explain this is that when heat is used as a sterilizing agent, the vibratory motion of every molecule of a microorganism is increased to levels that induce the cleavage of intramolecular hydrogen bonds between proteins. Death is therefore caused by an accumulation of irreversible damage to all metabolic functions of the organism.

Types of Autoclave Generally, autoclaves are divided into their types based on method of achieving their air removal. It is very important to ensure that all of the trapped air is removed from the autoclave before activation, as hot air is a very poor medium for achieving sterility. i. Downward Displacement

The downward displacement autoclave is also referred to as a gravity displacement unit. This is because of the method of air removal in the sterilization chamber. A heating element is submerged in a pool of water, which, when heated, becomes steam. As steam is lighter than air, it forces the air in the sterilization chamber downward and out through a drain hole. Once the temperature in the sterilization chamber is sufficient, the drain hole is closed automatically and the sterilization process begins. ii. Positive Pressure Displacement

A positive pressure displacement autoclave is an improvement upon the design of a downward displacement unit. Steam is created in a second, separate chamber and held until the proper amount to displace all of the air in the sterilization chamber is accumulated. The steam is then released into the sterilization chamber in a pressurized blast, forcing the air out through the drain hole and starting the sterilization process. This has the effect of a more accurate displacement of air than a downward displacement unit can achieve. iii. Negative Pressure Displacement

A negative pressure displacement autoclave is one of the most accurate types of unit available. Once the sterilization chamber door is closed, a vacuum pump removes the air. Steam is created in a second, separate chamber. Once the air has been completely removed from the sterilization chamber, the steam is then released into the sterilization chamber in a pressurized blast much like that of a positive pressure displacement unit. The negative pressure displacement unit is able to achieve a high "Sterility Assurance Level" (SAL), but the system can be quite large and costly.

iv.

Triple Vacuum Autoclave

A triple vacuum autoclave is set up in a similar fashion to a negative pressure displacement unit in that there is a vacuum pump to remove air from the sterilization chamber and steam is created in a second, separate chamber or unit. The process begins by the vacuum removing the air, then a pulse of steam. This is repeated three times, hence the name "triple vacuum" autoclave. This type of autoclave is suitable for all types of instruments and is very versatile.

Preparation of items for autoclaving In preparing items for autoclaving, containers should be unsealed and articles should be wrapped in materials that allow steam penetration. Large packages of dressings and large flasks of media require extra time for heat to penetrate them. Likewise, packing many articles close together in an autoclave lengthens the processing time to as much as 60 minutes to ensure sterility. It is more efficient and safer to run two separate, uncrowded loads than one crowded one. Wrapping objects in aluminum foil is not recommended because it may interfere with steam penetration.

Operating instructions 1) 2) 3) 4) 5) Ensure that there is sufficient water inside the chamber. Load the autoclave and fasten the lid keeping the discharge tap open. Adjust the safety valve to the required temperature and turn the heat on. Allow the mixture of air and steam to pass out freely till all air has been discharged. Close the air discharge tap and let the steam pressure rise within the chamber till it attains a temperature of 121C (1.5 kg/cm2). 6) 7) 8) Hold on the pressure for 15 minutes. Turn off the heat and let the autoclave cool. Slowly open the air and steam discharge taps after the pressure gauge has reached zero. 9) Allow the material to cool before these are handled (usually agar bottles take hours before these become safe to handle).

Safety/Physical Concerns When loading the autoclave with contaminated items, it is prudent to wear a labcoat and gloves as you would exposed with any potential exposure of biohazards. When the autoclave cycle is finished, keep exposed skin away from the door as it is opened. Remove items from the autoclave wearing a labcoat and using something to protect hands from heat (special gloves or hot pads.) Steam under pressure is inherently dangerous. Major maintenance of the autoclave should only be performed by trained service technicians. The autoclave should be situated in an area where traffic is kept to a minimum. Only health department staff, never clients or children, should have access to the area where the autoclave is running. The autoclave should only be operated by trained personnel, following all manufacturers instructions. A strict schedule of maintenance should be followed, again as recommended by the instrument manufacturer. The area where the autoclave is situated should be properly vented, as the release of water vapor after the sterilizing cycle results in increasing humidity.

Routine cleaning and maintanence The following are suggested actions for autoclaves in general. For exact cleansing and maintenance, refer to the instructions provided by the manufacturer in the autoclave manual.

i.

Each day of use, check the filter or plug screen on the water flow that keeps foreign matter from entering the autoclave. The danger here is that the chamber discharge system (venting and water drainage) will become clogged.

ii. iii.

Check the water reservoir level prior to each run. Check the door gasket every three months for steam leaks while the autoclave is fully pressurized.

iv.

Clean the interior and exterior of the autoclave thoroughly every three months, using only cleaning materials approved by the manufacturer.

v.

Some users prefer to enclose particularly messy items in autoclave bags. Be sure to use a high-quality bag and follow the instructions for sealing it. There are reports of poor quality bags bursting during the autoclave cycle, creating an even bigger mess.

vi.

Check the following every three months for any visible sign of wear: a. door hinges and general door structure, b. exterior structure, c. chamber drain (should completely drain within 3 minutes), d. slow exhaust (should completely vent between 12 and 20 minutes), e. fast exhaust (three minutes maximum), f. timer.

References :

i. ii. iii. iv. v.

Technical Bulletin. North Carolina State Lab of Public Health. Laboratory Improvement http://en.wikipedia.org/wiki/Autoclave http://www.ehow.com/about_5199725_autoclave-machine_.htm http://en.wikipedia.org/wiki/Sterilization_(microbiology) http://www.searo.who.int/en/Section10/Section17/Section53/Section482_1780.htm