Autoclave: Principle, Procedure, Types and Uses

The autoclave is a sealed device (similar to a pressure cooker) that kills microorganisms using saturated steam under pressure. The use of moist heat facilitates the killing of all microorganisms, including heat-resistant endospores which is achieved by heating the materials inside the device at temperatures above the boiling point of water. According to the principle of gas laws, this can be achieved by raising the pressure inside the device.

The boiling point (vapor pressure equals that of the surrounding atmosphere) of water varies depending upon the surrounding environmental pressure. For example, water boils at 100 °C at sea level (higher pressure), but at 93.4 °C at 1,905 metres altitude (lower pressure). So, in an enclosed device, if we raise the pressure, the temperature at which water boils also increases.

The usual procedure is to heat at 1.1 kilograms/square centimeter (kg/cm2) [15 pounds/square inch (lb/in2)] steam pressure, which yields a temperature of 121°C. At 121°C, the time of autoclaving to achieve sterilization is generally considered to be 15-20 min, depending on the volume of the load.  To make sure, sterilization is successful one should ensure:

  1. Air should be evacuated so that the chamber fills with steam.
  2. Articles should be placed in the autoclave so that steam can easily penetrate them.

Note that it is not the pressure of the autoclave that kills the microorganisms but the high temperature that can be achieved when steam is placed under pressure.                         

If bulky objects are being sterilized, heat transfer to the interior will be slow, and the heating time must be sufficiently long so that the object is at 121°C for 15 min. Extended times are also required when large volumes of liquids are being autoclaved because large volumes take longer to reach sterilization temperature.

Components of Autoclave

Autoclave comprises of three parts: a pressure chamber, a lid and an electrical heater.

Gravity Displacement type Autoclave

Pressure chamber consists of –

  • Large cylinder (vertical or horizontal) in which the materials to be sterilized are placed. It is made up of gunmetal or stainless steel and placed in a supporting iron case ay through
  • A steam jacket (water compartment)

The lid is fastened by screw clamps and rendered airtight by an asbestos washer. The lid bears the following-

  • A discharge tap for air and steam discharge
  • A pressure gauge (sets the pressure at a particular level)
  • A safety valve (to remove the excess steam)

An electrical heater is attached to the jacket; that heats the water to produce steam.

Types of Autoclaves

There are different types of autoclaves available.

  1. Gravity displacement type autoclave: It is the most common type used in laboratories and is available in various sizes and dimensions.
    1. Vertical type (small volume capacity)
    1. Horizontal autoclave (large volume capacity)
  2. Positive pressure displacement type autoclave
  3. Negative pressure (vacuum) displacement type.

Procedure

  • Place the material to be sterilized inside the pressure chamber and fill the cylinder with sufficient water
  • Close the lid and put on the electrical heater.
  • Adjust the safety valve to the required pressure.
  • After the water boils, allow the steam and air mixture to escape through the discharge tap till all the air has been displaced
    • This can be tested by passing the steam-air mixture liberated from the discharge tap into a pail of water through a connecting rubber tube.
    • When the air bubbles stop coming in the pail, it indicates that all the air has been displaced by steam.
  • Close the discharge tap. The steam pressure rises inside and when it reaches the desired set level (e.g. 15 pounds (lbs) per square inch in most cases), the safety valve opens and excess steam escapes out.
  • Count the holding period from this point of time, which is about 15 minutes in most cases.
  • After the holding period, stop the electrical heater and allow the autoclave to cool until the pressure gauge indicates that the pressure inside is equal to the atmospheric pressure.
  • Open the discharge tap slowly and allow the air to enter the autoclave.
  • Open the lid of the autoclave and remove the sterilized materials.

Sterilization control

Modern autoclaves have devices to maintain proper pressure and record internal temperature during operation. Regardless of the presence of such a device, autoclave pressure should be checked periodically and maintained.

Several methods are available to ensure that autoclaving achieve sterility. The effectiveness of the sterilization done by autoclave can be monitored by:

  1. Biological indicator: Spores of Geobacillus stearothermophilus (formerly called Bacillus stearothermophilus) are the best indicator because they are resistant to steaming. Their spores are killed in 12 minutes at 121°C. The Centers for Disease Control (CDC) recommends weekly autoclaving of a culture containing heat resistant endospores of Geobacillus stearothermophilus, to check autoclave performance. The spore strip and an ampule of medium enclosed in a soft plastic vial are available commercially. The vial is placed in the center of the material to be sterilized and is autoclaved. Then the inner ampule is broken, releasing the medium, and the whole container is incubated. If no growth appears in the autoclaved culture, sterilization is deemed effective.
  2. Autoclave tapes: Adhesive-backed paper tape with heat-sensitive, chemical indicator marking that change color or display-diagonal stripes, the words “sterile” or “autoclaved” when exposed to effective sterilization temperature (121°C) are used to check the efficacy of autoclaves.
    These tapes are placed inside and near the center of large packages because heat penetration in those areas ensures proper heat penetration (For examples, when a large piece of meat is roasted, the surface can be well done while the center may still remain unheated, and if the center is sufficiently heated then it means the desired temperature is achieved). Autoclave tapes are not fully reliable because they do not indicate how long appropriate conditions were maintained.
  3. Other useful indicators are thermocouple and Browne’s tube. Thermocouple is a temperature measuring device that records the temperature by a potentiometer. Browne’s tube (invented by Albert Browne in 1930) contains a heat-sensitive red dye which turns green after being exposed to certain temperature for a definite period of time. Conversion of dye color gives information about the duration of time and temperature.

Uses of Autoclave

Autoclave is particularly useful for media containing water that cannot be sterilized by dry heat. It is the method of choice for sterilizing the following:

  1. Surgical instruments
  2. Culture media
  3. Autoclavable plastic containers
  4. Plastic tubes and pipette tips
  5. Solutions and water
  6. Biohazardous waste
  7. Glassware (autoclave resistible)

Precautions

Never autoclave any liquid in a sealed container.

The following precautions should be taken while using an autoclave.

  1. Autoclave should not be used for sterilizing waterproof materials, such as oil and grease or dry materials, such as glove powder
  2. Materials are loaded in, such a way that it allows efficient steam penetration (do not overfill the chamber). It is more efficient and safer to run two separate, uncrowded loads than one crowded one.
  3. Wrapping objects in aluminium foil is not recommended because it may interfere with steam penetration. Articles should be wrapped in materials that allow steam penetration.
  4. Materials should not touch the sides or top of the chamber
  5. The clean items and the wastes should be autoclaved separately.
  6. Polyethylene trays should not be used as they may melt and cause damage to the autoclave.

References and further readings

About Nisha Rijal 29 Articles
I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

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