Many experiments require aseptic operation without any contamination of bacteria. Therefore, it is necessary to sterilise and disinfect the laboratory instruments, culture media and experimental environment before starting the experiments.
Autoclaves are the most common sterilisation equipment in laboratories, with the advantages of being harmless to operators, fast sterilisation and easy to monitor. Autoclave machines are widely used in scientific research, agriculture and medical fields.

Autoclave definition

Autoclaves, also known as steam sterilizers, are a physical method of sterilization. Autoclaving is the process of killing or destroying microorganisms and other life forms present on the interior and surface of an object. It involves sterilizing materials under pressure under moist heat, especially saturated steam. At temperatures above 100°C, pressurized steam sterilization is the most effective, reliable, economical, and least toxic.
Autoclave machines are capable of rapidly heating and penetrating sterilised objects and killing spores quickly. For this reason, they are widely used in medical and laboratory instrument sterilization techniques.

Principle and working atuoclaves

Four main parameters involved in steam sterilization:
Steam, pressure, temperature, time.
Autoclaves work on the principle of hygrothermal sterilization, in which pressurized steam is used to sterilize materials present in the chamber.
Objects to be sterilized are first exposed to saturated steam at a specific pressure and heated to a specified temperature for a particular time.
The pressurized saturated steam sterilizes the object by destroying the microorganisms through irreversible coagulation and denaturing the structural proteins and enzymes in the microorganisms.

Some may think that saturated steam is the same as the water vapor emitted when you boil water at home.

The answer is different. The steam produced by boiling water is also called super-heated steam, and the difference between it and saturated steam is explained next.
Saturated steam is produced when water is heated to its boiling temperature (at a given pressure)
In this state, the liquid and gaseous phases of water co-exist simultaneously.
Saturated steam comes into contact with the object surface, gets condensed on the object surface, and in the process, transfers its stored energy onto the object surface.
Super-heated steam, being dry, cannot condense onto the surface and so cannot transfer its energy to the object, to bring about sterilisation.

Component of steam autoclaves:

Pressure Chamber

The pressure chamber is the main component of the steam steriliser and consists of an inner chamber and a jacket.
The inner chamber is the box in which the material to be sterilized is placed.

Lid/Door

The purpose of the lid is to seal the external atmosphere and create sterile conditions inside the autoclave.

Pressure Gauge

Pressure gauges give a real-time indication of the pressure generated by the autoclave during the sterilization process.
The pressure gauge is essential as it ensures the autoclave’s safety and the working conditions for its operation.

Steam Release or Venting Valves

An outlet valve that completely releases any enclosed air within the sterilization chamber. Removes residual air from the chamber before sterilization so that the air is completely replaced by steam and water vapor. This results in complete and effective steam sterilization.

Safety Valve

Critical in situations where an autoclave cannot operate or the internal pressure increases uncontrollably.
The valve has a thin layer of rubber that ruptures itself to relieve pressure and avoid the risk of explosion.

Steam Generator

An electric steam generator or boiler is located underneath the chamber, and an electric heating system is used to heat water and generate steam in the internal and external chambers.
The water level in the internal chamber is important; the heating system may burn if there is not enough water.
Likewise, if there is too much water, it may interfere with the trays and other components inside the chamber.

Vacuum Generator

In a vacuum autoclave, a separate vacuum generator draws air from the chamber to create a vacuum.

Waste Water Cooler

Many autoclaves have a system that cools the effluent before it enters the drain.
This system prevents any damage to the drain tube due to boiling water being discharged from the autoclave.

Operation of steam autoclaves

If the sterilized items contain reagent bottles with liquid media and screw caps, the caps must be loosened appropriately before sterilization.
If the sterilized items contain containers with liquid media but no cap, the mouth of the bottle needs to be blocked with cotton. And the liquid must not exceed two-thirds of the conical bottle.

Before using the autoclave, check for any items left over from the previous cycle.
An adequate amount of water is then placed in the chamber.
The material to be sterilized is then placed in the chamber.
The lid is then closed, the screws are tightened to ensure an airtight seal and the electric heater is switched on.
The safety valve is adjusted to maintain the required pressure in the chamber.
Once the chamber water boils, the air-water mixture escapes through the discharge pipe, allowing all the air in the chamber to escape. Complete displacement is assured once the water bubbles no longer come out of the pipe.
The drain pipe is then closed, allowing the internal vapor to reach the desired level (15 lbs in most cases).
Once the pressure is reached, a whistle is blown to remove the excess pressure from the chamber.
After the whistle blows, the autoclave runs for some time, usually 15 minutes.
The electric heater is now switched off and the autoclave is allowed to cool until the pressure gauge indicates that the internal pressure has dropped to atmospheric pressure.
Then open the vent tube to allow air to enter the autoclave from the outside.
Finally, open the lid and remove the sterilized material from the chamber.

Commonly adopted sterilization time periods
Gloves : 10-15 mins
Surgical items viz. Forceps, cotton, bandages etc.：20-25mins
Glasswares, autoclavable plastic wares, culture media : 15-20 mins

Autoclave use precautions

  1. The operation and routine maintenance of autoclaves should be the responsibility of well-trained personnel.
  2. Saturated steam should be used and should not contain corrosive inhibitors or other chemicals
  3. All items to be autoclaved should be placed in containers from which air can escape and which have good heat permeability;
  4. In autoclaves without interlocking devices, the main steam valve should be closed, and the door should be opened when the temperature has dropped below 80 °C.
  5. When autoclaving liquids, a slow-discharge setting should be used because the liquid may boil due to overheating when it is removed.
  6. Even if the temperature drops below 80°C, the operator should wear appropriate gloves and a face mask for protection when opening the door.

Conclusion

Autoclaves are essential in medical, laboratory, and industrial environments to ensure sterility and safety. By understanding the principles of autoclaving, its components, and working mechanisms, the user can effectively use its capabilities to achieve optimum sterilization results.
Mastering autoclave operation not only promotes health and safety but also improves the life and performance of instruments and materials used in the autoclaving process.