Autoclave Definition, Working Principle, Components, Operating procedure.

What is autoclave? An autoclave is a large vessel containing steam under high pressure. It mainly used to sterilize different materials in...

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This article writter by MN Editors on November 29, 2020

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What is autoclave?

An autoclave is a large vessel containing steam under high pressure. It mainly used to sterilize different materials in the laboratory. Autoclave heating up the material at a specific temperature for a specific period of time. It is also termed as steam sterilizers or steam autoclave.

Hence autoclave is based on the moist heat sterilization method, it considered the most reliable and effective method of sterilization among all physical methods.

Autoclave definition medical

Autoclave is a laboratory instrument to help in the physical method of sterilization by killing harmful bacteria, viruses, fungi, and spores. These are mainly used in industrial and scientific processes to perform sterilization of different materials.

  • In 1879, Charles Chamberland first invented the autoclave. For 150 years the methods and principles of steam sterilizer or autoclave have still remained unchanged.
  • This method of sterilization is nontoxic, inexpensive 826, rapidly microbicidal, sporicidal, and rapidly heats and penetrates fabrics.
  • Autoclave uses saturated steam under pressure to kill microbes.
  • Heat-resistant endospores can be killed by heating them at temperatures above the boiling point of water. These can be accomplished by raising the pressure inside the device.
  • It handles saturated vapor under pressure of almost 15 pounds per square inch to produce a chamber temperature of at least 250°F (121°C) for a designated time—usually 30–60 minutes.

Autoclave Temperature

Differen tempareture rang are used for steam sterilization such as 250° F (121° C), 270°F (132°C) or 275°F (135° C). But, 121°C (250°F) and 132°C (270°F) are the two most common steam-sterilizing temperatures uses to kill microorganisms.

Autoclave Working Principle

YouTube video

Video demonstrating how autoclaves work

An autoclave works on a basic principle. The material is directly exposed to steam at the required temperature and pressure for a specific time. The boiling point of water will increase with the rising pressure as a result high temperatures will be generated for sterilization.

High-pressure helps in the rapid penetration of heat within the material and the pressure help in the coagulation of proteins.

Within the autoclave, the water boils at 121°C at the pressure of 15 psi or 775 mm of Hg. When the microbes come in contact with the steam it kills them by giving off latent heat. The specified temperature ensures the microbicidal activity.

Autoclave principle
Autoclave principle | Image Source:

Mode of Action

Moist heat destroys cells and viruses by degrading nucleic acids and denaturing enzymes and other essential proteins. It also disrupts cell membranes. Exposure to boiling water for 10 minutes is sufficient to destroy vegetative cells and eukaryotic spores. 

To destroy bacterial endospores, moist heat sterilization must be carried out at temperatures above 100°C, and this requires the use of saturated steam under pressure.

Components of Autoclave 

A simple autoclave contain these following components such as;

1. Pressure Chamber

  • A pressure chamber has a capacity of 100 L to 3000 L.
  • It contains an inner chamber and an outer jacket. The material which is needed to sterilize is put inside the inner chamber.
  • The inner chamber consists of stainless steel or gunmetal whereas the outer chamber is consists of an iron case.
  • In healthcare laboratories, autoclaves are covered with an outer jacket. This jacket contains steam which helps to reduce the time taken to reach the sterilization temperature.

2. Lid/ Door

The lid is fastened by screw clamps and rendered airtight by an asbestos washer.  It contains different components such as;

  • Pressure releasing unit/ Whistle: Unlike a pressure cooker an autoclave contains a Pressure releasing unit or a Whistle. It maintain the pressure within the autoclave by releasing a certain amount of vapor.
  • Safety valve: It plays an important role in the autoclave. When the pressure of the autoclave increases uncontrollably the Safety valve bursts itself to release the pressure and to avoid the danger of explosion. It is made up of a thin layer of rubber.
  • Pressure gauge: It is an essential component for the safety of the autoclave. During sterilization, it indicates the pressure created by the autoclave.

3. Steam generator/ Electrical heater

  • The main function of a Steam generator or Electrical heater is to produce steam from water.
  • It is located underneath the chamber.
  • It is important to keep the Electrical heater under the water. The coil of the Electrical heater will burn If the water level within the inner chamber is not sufficient.

4. Vacuum generator (if applicable)

  • Some autoclaves contain a vacuum generator. It generates a vacuum within the autoclave by pulling out the air from the inside of the chamber.
  • By creating a vacuum it prevents the growth of different microorganisms.

5. Wastewater cooler

  • Some autoclaves contain a Wastewater cooler, which cools the effluent before it enters the draining pipes.
  • It protects the drainage pipe from being damaged which may be caused by the boiling water.
autoclave diagram
autoclave diagram | Image Source:

Approximate Conditions for Autoclave sterilization

OrganismVegetative CellsSpores
Yeasts5 minutes at 50-60 degree centigrade.5 minutes at 70-80 degree centigrade.
Molds30 minutes at 62 degrees centigrade.30 minutes at 80 degrees centigrade.
Bacteria10 minutes at 60-70 degrees centigrade.2 to over 800 minutes at 100 degrees centigrade. 0.5 – 12 minutes at 121 degrees centigrade.
Viruses30 minutes at 60 degrees centigrade.

Autoclave Pressure and Temperature Chart

Steam autoclave 121 C (250 F)15psi15min
unwrapped items 132 C (270 F)30psi3min
lightly wrapped items 132 C (270 F)30psi8min
heavily wrapped items 132 C (270 F)30psi10min
Dry heat wrapped 170 C (340 F)
160 C (340 F)
150 C (300F)
140 C (285F)
121 C (250F)
60 min
Dry heat (rapid flow) unwrapped items190 C (375F)6min
Dry heat (rapid flow) packaged items190 C (375 F)12min
Ethylene oxide Ambient8-10 hours
Chemical vapor 132 C (270 F)20-40 psi20min
As published by Jada, (Journal of American Dental Association)

Autoclave Machine

Behind a successful steam sterilization, three important factors are responsible such as time, temperature, and steam quality. To fulfill these requirements there are 3 steps to the autoclave process such as:

  1. Conditioning Phase 
  • In the first phase of the sterilization cycle, the air must be removed from the autoclave by using the vacuum system. Otherwise, it will inhibit the sterilization.
  • The air can be removed from the autoclave by a series of steam flushes and pressure pulses.
  1. Exposure Phase
  • After that the lid is closed as a result the steam continuously increases with pressure and temperature. From here the exposure phase begins.
  • The item which is needed to be sterilized is held within the autoclave for a fixed amount of time.
  1. Exhaust Phase
  • At the final stage of sterilization, the lid is opened and releases the steam, depressurizing the vessel and allowing the items in the load to dry.

Operating Procedures of Autoclave

YouTube video
  • Before run a autoclave check if an item is left inside the autoclave from the previous batch.
  • Then check the water level in the autoclave. If the coil is visible then add sufficient water to it and make the coil invisible.
  • Now place the material within the autoclave which is needed to sterilize.
  • Then close the lid and make it airtight by tightening the screws. After that switched on the autoclave.
  • To maintain the required pressure adjust the safety valves.
  • Then close the drainage pipe and allow the steam to reach the desired levels (15 lbs).
  • After reaching the desired level of pressure the whistle will blow to remove excess pressure from the autoclave chamber.
  • After the whistle holds the autoclave for 15 minutes, this is known as the holding period.
  • Switch off the autoclave and allow it to cool down until the pressure gauge shows that the inside pressure of the autoclave has lowered down to that of the atmospheric pressure.
  • Open the discharge pipe to allow the outside air to enter into the autoclave.
  • Then take out the sterilized material from the autoclave by opening the lid.

Classification of Autoclave

There are four types of autoclave such as;

  1. Positive pressure displacement type (B-type): This type of autoclave can generate steam within seconds. They contain a separate steam generator, where steam is generated and then pass it into the autoclave.
  2. Negative pressure displacement type (S-type): This type of autoclave carries both the steam generator and a vacuum generator. The steam generator generates steam and then passes it into the autoclave. Vacuum generates pull out the air from the autoclave to create vacuum. This is the most expensive autoclave among all.
  3. Gravity displacement type autoclave: Gravity displacement type autoclave mainly used in laboratories. The steam generated inside the autoclave by a heating unit. It is cheaper than other types.
  4. Pressure cooker type/ Laboratory bench autoclaves (N-type): It contains an air and steam discharge tap, pressure gauge, and safety valve. This autoclave also contains an electric immersion heater at the bottom. The modern form of the autoclave contains a secure metal lid that can be fastened and sealed with a rubber gasket.
Types of autoclave
Types of autoclave

Application of Autoclave

  • In laboratories it is used to sterilize glasswares, instruments, and media.
  • In medical laboratories autoclave is used for the sterilization of medical equipment, glassware, surgical equipment, and medical wastes.
  • Autoclave also used to decontaminate medical wastes which contains bacteria, viruses, and other biological materials.


During operating an autoclave you should follow these following rules of precautions;

  1. Oil or powders like water proof or water-resistant substances can be used in autoclaves for sterilization.
  2. Don’t overload the autoclave, make sure there is sufficient space inside the autoclave.
  3. The materials needed to sterilize should always be placed in a secondary container.
  4. Avoid autoclave of a combustible substance such as paper. Palace them in a waste bag on a bio bag setting to prevent fire and then autoclave them.
  5. Avoid autoclave of flammable, reactive, corrosive, toxic or radioactive materials, household bleach, or paraffin-embedded tissue.
  6. Avoid the use of Plastic or polyethylene trays or containers they might melt and damage the autoclave.
  7. The water level within the containers should be 2/3rd of the total volume. It will prevent the spilling of the liquid.
  8. Never open the lid when the autoclave is working.
  9. Autoclave the waste materials and clean items separately.
  10. Use autoclavable bags to pack the wastes and items.

Sterilization control

Modern autoclaves come with equipment to keep the pressure at a constant level and to record internal temperature throughout the operation. No matter the presence of such a device the pressure of the autoclave should be monitored frequently and kept at a constant level.

There are a variety of methods available to make sure that autoclaves ensures the goal of sterility. The efficiency of sterilization process that is performed by the autoclave can be monitored through:

1. Biological indicator

Spores of Geobacillus Stearothermophilus (formerly called Bacillus Stearothermophilus) are the most reliable indicator because they resist steaming. The spores die within 12 minutes when temperatures reach 121 degrees Celsius. It is recommended that the Centers for Disease Control (CDC) suggests weekly autoclaving of an organism that has heat-resistant endospores from Geobacillus Stearothermophilus to test the effectiveness of autoclaves. The spore strip as well as the ampule of media contained inside a soft plastic vial are sold on the market. The vial is put in the middle of the material that is to be sterilized before being autoclaved. Then , the ampule inside is broken and the medium is released, and the entire container is then incubated. If there is no growth within the autoclaved cult, sterilization is considered efficient.

2. Autoclave tapes

Adhesive-backed tape that is chemical indicator markings which change the color of display-diagonal stripes or colors, with the wording “sterile” as well as “autoclaved” in the event of exposure to temperatures that are effective for sterilization (121degC) can be utilized to determine the effectiveness of autoclaves.

The tapes are placed in the middle of large packaging since heat absorption in these areas is guaranteed. (For instances, when the meat of a huge size is roasted, the outside is likely to be done, but the middle may remain unheated. And if the center is sufficiently heated , then it indicates that the desired temperature is reached). Autoclave tapes may not be 100% reliable as they don’t provide a time frame for how long the conditions were in place.

3. Other useful indicators are thermocouple and Browne’s tube.

Thermocouple is a temperature-sensing device that measures temperature using the use of a potentiometer. The Browne’s Tube (invented in 1930 by Albert Browne in 1930) is made up of a red heat-sensitive dye that changes color after being exposed to a specific temperature for a specified period of time. The conversion of color information gives details about the time period and temperature.

Stages of Autoclave Sterilization

The sterilization process involves various phases, which are which are listed below.

  1. Purge Phase: During the purge period, steam dispenses air from an autoclave’s chamber. Temperature and pressure start to rise.
  2. Sterilization Phase: The exhaust is kept closed to allow the pressure and temperature to quickly increase to the desired levels. In this stage, that the autoclave’s extreme conditions eliminate spores, bacteria, and other pathogens.
  3. Exhaust Phase: The pressure is removed from the chamber but temperatures remain high. Technicians must be cautious when taking hot liquids out of the autoclave.

Autoclave Limitations

Autoclaves are extremely versatile instruments, certain materials and substances aren’t compatible with autoclaves , and could be hazardous if they are included in the load and then placed into a sterilization process. For instance, certain chemicals like bases and acids shouldn’t be autoclaved or reconstituted from polyurethane, polyethylene or polystyrene. It is essential that autoclave technicians fully comprehend the principles of sterilization in autoclaves so that they can be able to avoid any safety risks during the use.

FAQ on Autoclave

1. What are autoclave bags made of?

Autoclave bags made of two-millimeter-thick Polypropylene (PP).

2. Why are autoclave indicators used?

Autoclave indicators are used to make sure that articles have been sterilized. Autoclave tape, sensitivity marks on bags or wraps, and indicator capsules.

3. What are autoclave bags used for?

Autoclave bags are used in high heat sterilization applications in order to prevent low temp plastics inside the bag from sticking to the walls of the sterilizer

4. Can autoclave kill endospores?

Yes, By increasing the pressure, the autoclave reaches a boiling point of 100°C or higher (121°C) and kills endospores.

5. Can autoclave kill prions?

Yes, by exposing them to effective sterilisation temperatures for around 14 minutes longer than the standard 134°C cycle.

6. How autoclave kill microorganisms?

Autoclaves kill microorganisms by degrading nucleic acids and denaturing enzymes and other essential proteins.

Further Reading


  • (ANSI) American National Standards Institute Inc./(AAMI) Association for the Advancement of Medical Instrumentation
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Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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