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Tuesday, April 9, 2013

Sterilization - Autoclaving

An autoclave is a pressurized device designed to heat aqueous solutions above their boiling point to achieve sterilization. It was invented by Charles Chamberland in 1879. The term autoclave is also used to describe an industrial machine in which elevated temperature and pressure are used in processing materials.

Introduction to Autoclave:
Under ordinary circumstances (at standard pressure), liquid water cannot be heated above approximately 100 °C (99.99 °C at 101.325 kPa, 99.62 °C at 100 kPa) in an open vessel except for special situations. Further heating results in boiling, but does not raise the temperature of the liquid water. However, when water is heated in a sealed vessel such as an autoclave, it is possible to heat liquid water to a much higher temperature. As the container is heated the pressure rises due to the constant volume of the container (see the ideal gas law). The boiling point of the water is raised because the amount of energy needed to form steam against the higher pressure is increased.

A Lab Scale Autoclave
Lab scale autoclave

An Industrial Scale Autoclave

Uses of Autoclave:Autoclaves are widely used in microbiology, medicine, sterilizing instruments for body piercing, veterinary science, dentistry, podiatry and metallurgy. The large carbon-fiber composite parts for the Boeing 787, such as wing and fuselage parts, are cured in large autoclaves.

Principle of Autoclave
A basic principle of chemistry is that when the pressure of a gas increases, the temperature of the gas increase proportionally. For example, when free flowing steam at a temperature of 100oC is placed under a pressure of 1 atmosphere above sea level pressure – that is, about 15 pounds of pressure per square inch (Psi) --- the temperature rises to 121oC. Increasing the pressure to 20 psi raises the temperature to 126oC. The relationship between temperature and pressure is shown in table 2. In this way steam is a gas, increasing its pressure in a closed system increases its temperature. As the water molecules in steam become more energized, their penetration increases substantially. This principle is used to reduce cooking time in the home pressure cooker and to reduce sterilizing time in the autoclave. It is important to note that the sterilizing agent is the moist heat, not the pressure.


It is essential to remove all the traps from the autoclave otherwise required temperature is not achieved(because air is bad conductor of heat) which leads to non sterility.

• Downward displacement (or gravity type) - As steam enters the chamber, it fills the upper areas as it is less dense than air. This compresses the air to the bottom, forcing it out through a drain.

• Steam pulsing - Some autoclaves remove air by using a series of steam pulses, in which the chamber is alternately pressurised and then depressurised to near atmospheric pressure.

• Vacuum pumps - Some autoclaves use vacuum pumps to suck air or air/steam mixtures from the chamber.

There are 3 sterilization cycles employed in an autoclave to sterilize different loads.

1. Standard cycle {for wrapped containers like glassware, test tubes, utensils}

2. Liquid cycle {for liquid loads like media and buffer in tightly sealed container}

3. HPHV [high pressure high vacuum] cycle {for fabric like lab coats, gloves, flexible tubes}

Limitations and Disadvantages of Autoclave:
Sterilization by autoclave method has certain limitations. For example, some plastic ware melts in the high heat, and sharp instruments often become dull. Moreover, many chemicals breakdown during the sterilization process and oily substances cannot be treated because they do not mix with water. Heat requires extra time to reach the center of solid materials, such as caned meats, because such materials do not develop the efficient heat-distributing convection currents that occur in liquids. Heating large containers also requires extra time. Unlike sterilizing aqueous solutions, sterilizing the surface of a solid requires that steam actually contact it.

Indicator of Sterilization Achievement:
Several commercially available methods can indicate whether sterilization has been achieved by heat treatment. Modern autoclaves have devices to maintain proper pressure and record internal temperature during operations. Regardless of the presence of such a device, the operator should check pressure periodically and maintain the appropriate pressure. Chemical reactions in which an indicator changes color when the proper times and temperatures have been reached. In some designs, the word "sterile" or "autoclaved" appears on wrappings or tapes. These tapes are not fully reliable because they do not indicate how long appropriate conditions were maintained. Tapes or other sterilization indicators should be placed inside and near the center of large packages of determine whether heat penetrated them. In another method, a pellet contained within a glass vial melts. A widely used test consists of preparations of specified species of bacterial endospores such as Bacillus stearothermophilus, impregnated into paper strips. The spore strip and an ampule of medium are enclosed in a soft plastic vial. The vial is placed in the center of the material to be sterilized and is autoclaved. After autoclaving, these can then be aseptically inoculated into culture media. Growth in the culture media indicates survival of the endospores and therefore inadequate processing. Other designs use endospore suspensions that can be released, after heating, into a surrounding culture medium within medium within the same vial.

Important Points to Remember For Autoclaving:
Steam under pressure fails to sterilize when the air is not completely exhausted. This can happen with the premature closing of autoclave's automatic ejector valve. The principles of heat sterilization have a direct bearing on home canning. To sterilize dry glassware, bandages, and the like, care must be taken to ensure that steam contacts all surfaces. For example, aluminum foil is impervious to steam and should not be used to wrap dry materials that are to be sterilized; paper should be used instead. Care should also be taken to avoid trapping air In the bottom of a dry container because trapped air will not be replaced by steam, which is lighter than air. The trapped air is the equivalent of a small hot-air oven, which, as we will see shortly, requires a higher temperature and longer time to sterilize materials. Containers that can trap air should be placed in a tipped position so that the steam will force out the air. Products that do not permit penetration by moisture, such as mineral oil or petroleum jelly, are not sterilized by the same methods that would sterilize aqueous solutions. This precaution is necessary because when an object is exposed to heat, its surface becomes hot much more quickly than its center.