Autoclave Sterilization: Principle, Procedure, Types, Uses

The autoclave is a sealed device (similar to a pressure cooker) that kills microorganisms using saturated steam under pressure.

Principle

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. To ensure effective steam penetration, articles should be properly positioned inside the autoclave before sterilization.

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 (Parts) of Autoclave

Gravity Displacement type Autoclave
  1. Lid/Door: It is at the top of the large-scale autoclave but in the case of a horizontal autoclave, it may be in the front part. The lid should be sealed tightly to prevent contamination and proper sterilization. The top is sealed tightly with the help of an airtight screw.
  2. Pressure Chamber: It is a vessel whose outer part is made of stainless steel, and the outer coat covers the inner part. The autoclavable materials are placed inside the inner part of the vessel, and the lid is closed tightly.
  3. Power Switch: It is present at the side of the autoclave and controls the electricity supplied to the auoclave.
  4. Control Panel: It controls the pressure and temperature inside the vessel and is present beside the main switch.
  5. Water Level Indicator: It helps indicate the water level of the autoclave. The correct level of water is essential. 
  6. Pressure gauge: It indicates the pressure inside the chamber and is on top of the lid. 
  7. Whistle: It is only present in some types of autoclave, like pressure cooker type, and is on the top of the lid. It helps release the pressure of the chamber before opening the lid after sterilizing the materials.  
  8. Safety valve: This type of valve helps to avoid an accident when the pressure inside the vessel is exceptionally high. It is also present in the lid of the autoclave.
  9. Electrical heater: It is the heating element attached to the jacket; that heats the water to produce steam.
  10. Water releasing valve: It helps remove water for replacing and cleaning the water inside the autoclave chamber.
  11. Thermometer: It is at the top of the lid and displays the temperature inside the chamber.  
  12. Stand: The part present helps the autoclave stand upright and forms the base of the autoclave. 

Extra accessories

  1. Autoclave bag: It is a bag where waste materials are placed for sterilization inside the autoclave chamber.  
  2. Autoclave baskets: Cans or baskets help to safely transfer sterilized material to and from the autoclave. It is available in various sizes; some can have lids or holes.  
  3. Sterilization box: These are stainless steel boxes with lids and venting holes that can be used while sterilizing inside the autoclave.

Types of Autoclaves

There are different types of autoclaves available.

Based on size

  1. Large-scale autoclave: It is more significant in size. Some may have double chambers and can have 500 liters to more than 1500 liters chamber. The capacity of the autoclave depends on the manufacturers. Companies like Systec provide large autoclaves ranging from 510 liters to 1580 liters. It is suitable for hospitals and clinical and research laboratories.  
  2. Small-scale autoclave: It is smaller in size. It has chambers that can simultaneously fit 20-300 liters of autoclavable materials. But the size range varies based on the companies. It is suitable for university and college laboratories. 

Types of Autoclave on the basis of Working Principle

  1. Gravity displacement autoclave: The hot steam enters the chamber and forces all the air through a vent. It is unsuitable for autoclave bags because it creates air pockets. It is generally of two types; horizontal and vertical autoclave.
    • Horizontal autoclave: The door/lid of this type of autoclave open outwards towards the handler. It is usually available in large sizes.
    • Vertical autoclave: The autoclavable material is loaded from the top side of the autoclave. It is usually available in small sizes.
  2. Positive pressure displacement autoclave: Here, the steam is generated in a separate steam generator unit, and then the moisture is transferred into the autoclave. It is faster because it takes only a few seconds to generate steam.
  3. Negative pressure (vacuum) displacement autoclave: In this type of autoclave, a vacuum generator creates a vacuum that removes air inside the chamber before beginning the sterilization cycle. This type of autoclave has both a steam and vacuum generator inside it. 

Procedure

Using autoclave to sterilize materials

  1. Place the material to be sterilized inside the pressure chamber and fill the cylinder with sufficient water

  2. Close the lid and put on the electrical heater.

  3. Adjust the safety valve to the required pressure.

  4. 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.

  5. 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.

  6. Count the holding period from this point of time, which is about 15 minutes in most cases.

  7. 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.

  8. Open the discharge tap slowly and allow the air to enter the autoclave.

  9. 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 achieves sterility. The effectiveness of the sterilization done by autoclave can be monitored by:

Biological indicator

Spores of Geobacillus stearothermophilus (formerly called Bacillus stearothermophilus) are the best indicator because they are resistant to steam. 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.

Autoclave tapes

Adhesive-backed paper tape with heat-sensitive chemical indicator marking that changes 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 example, 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.

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 that turns green after being exposed to a 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 aluminum 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

Nisha Rijal

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.

2 thoughts on “Autoclave Sterilization: Principle, Procedure, Types, Uses

  1. Greeting,

    I want autocleave wast lab contain a plates, tubes, ect…
    Can I put these materials in container then autocleave. so I can be sure the old media will not melt outside.
    I wonder if this technique affect the autocleave.

    Zainab Almahdi

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