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Sterilization Techniques - Different Methods of Sterilization


Sterilization is a process by which all viable forms of micro-organisms are removed or destroyed.
Sterilization (or sterilization), referring to any process that eliminates (removes) or kills (deactivates) all forms of life and other biological agents including transmissible agents (such as fungi, bacteria, viruses, spore forms, unicellular eukaryotic organisms such as Plasmodium, etc.) present in a specified region, such as a surface, a volume of fluid, medication, or in a compound such as biological culture media.


For sterilization of product/material, various methods of sterilization are used. These can be classified as under:

A. Physical Methods

a) Dry heat sterilization
b) moist heat sterilization
c) sterilization by radiation

B. Chemical Methods

a)  Gaseous sterilization
b) Sterilization with disinfectants

C. Mechanical Methods

a) sterilization by filtration.


A. Physical Methods

a) Dry heat sterilization

Sterilization by dry heat is usually carried out in an apparatus known as ‘Hot Air Oven’. It is a metallic chamber made up of steel or aluminum separated from the outer case by a thick layer of glass ber insulation. The door is double-walled and the inner side has asbestos gasket which makes it airtight and prevents heat loss. Heat is transferred from source to arrival in hot air oven by conduction, convection, and radiation.
A thermometer is fitted in front of the oven to note down the temperature during sterilization. Hot air oven should satisfy the following requirement:
1. Every item inside the oven must receive correct exposure of heat.
2. The temperature of sterilization must be attained quickly and be maintained with little variation.

At the top of the oven, there is a ventilator and on the bottom, there is a chamber in which heating elements are fitted.
The method is used for the sterilization of oil, petroleum jellies, talcum powder, glass apparatus. In this method the destruction micro-organisms by dry heat. The micro-organisms are more resistant to dry heat as compared to moist heat.


1. During sterilization, the material to be sterilized is placed in the oven properly and the door is closed. Extra time is always allowed to penetrate heat into the material
2. The temperature is adjusted to 150°C and the ventilator is allowed to remain open until the temperature of the oven reaches 115°C. It helps in the removal of moisture from the container/material to be sterilized
3. When it reaches 115°C, the ventilator is closed and the oven temperature is allowed to rise up to 150°C. The hear is transferred from the source mainly by radiation and convection. After one hour, the oven is switched o and allowed to cool to about 60°C.
4. During dry heat sterilization, all the living micro-organisms and their spores get destroyed due to oxidation if proteins present in the living cells. Although heating at 250°C can destroy all types of micro-organisms and their spores, but this can spoil the product also. Hence dry heat sterilization at a temperature of 150°C- 160°C gives the most satisfactory results.


1. For sterilization of injectable, needles, and syringes.
2. Glass apparatus such as a flask, pipettes, bottles, beakers, and test tubes are sterilized by this method.
3. Generally metallic instruments like scalpels, scissors, knives, spatula, blades.
4. Drugs that are stable at 150°C and thermostable.
5. The equipment used for aseptic processing such as mortars, pestles, tiles, etc.
6. This method is of particular value in case of oily injection, ointment bases, powders, pharmaceutical lubricants, etc.


1. This method cannot be used for thermolabile medicament,  rubbers, and plastics.
2. It is unsuitable for surgical dressing because it destroys the natural moisture of fibers and makes them brittle and discolored.


1. Overloading should be avoided.
2. There should be sufficient space between the article to provide a uniform distribution of heat.
3. To prevent breakage of glass apparatus it should be kept in mind to cool down the oven temperature.
4. The opening of glass items should be plugged with nonabsorbable cotton wool and wrapped further in a paper.

b) Moist heat sterilization

Moist heat in the form of saturated steam under pressure in which micro-organisms are destroyed by Moist heat due to the coagulation or denaturation of proteins in the living cell of micro-organisms.
Steam under pressure provides more heat than boiling water. The higher the steam pressure, the higher will be the temperature. Steam under means enough to destroy micro-organisms.
Autoclave consists of aluminum or stainless steel of about 15liters capacity. Pressure within the autoclave is medicated by a pressure gauge. The penetration power of steam is more as compared to dry heat.
The thermal capacity of steam is much greater than the thermal capacity of dry heat.

About apparatus

An autoclave is a strong cylindrical chamber made up of aluminum alloy/stainless steel. On its lid, there are various controls, like a steam vent, pressure gauze, safety value, and sometimes a thermometer. The inner side of the lid has a rubber gasket which makes it airtight. To keep its cover in a position, it is provided with wing nuts and bolts inside the chamber. There is a perforated metallic basket or wire container in which the material to be sterilized is packed inside, and a gasket in which electrically heated elements are fitted.


1) The perforated metallic chamber is removed out and water is put to a level so that it does not touch the bottom of the perforated chamber. The material to be sterilized is loosely packed so as to leave space for expansion and prevent breakage and then it is placed into the chamber.
2) Caps of bottled fluid should be screwed down tightly so that there is no danger of explosion as the internal pressure is approximately balanced by the steam pressure outside.
3) The lid is put in position with the help of wings nuts and bolts. The vent is opened. The autoclave is switched on and the water is allowed to boil. The steam is allowed to pass freely from the vent for 5 minutes.
4) When the whole of the air is removed, the steam vent is closed and pressure is allowed to rise up to 10pounds per square inch. B.P. allows exposure of the whole of contents to 115°C to 116°C for 30 min. Then the autoclave is switched o and is allowed to cool until the pressure falls to zero. Now the vent can be opened otherwise if the internal pressure is high, it may lead to vigorous boiling and bursting of sealed bottles and ampoules.
5) The sterilized material should be carefully collected by opening the lid.
Moist heat sterilization on a small scale can be done by using pressure cookers.


This method cannot be used for thermolabile substances.


1) This method is used for sterilization of glass apparatus and container (115°C for 30).
2) plastic screw caps, rubber liners, closures, rubber gloves must be autoclaved.
3) it is a suitable method of sterilization for injectable solutions (except oily injection) and suspensions.
4) The surgical dressing can be autoclaved for sterilization.

c) Sterilization by radiation

This is another physical method of sterilization. Radiation can be classified as:
1. Electromagnetic waves
a. Infrared radiation
b. X-ray
c. Ultraviolet light
d. Gamma rays
2. Minute particles
a. Alpha particles
b. Beta particles

UV radiation

It posses the greatest activity in the destroying of micro-organisms. It is commonly employed in the reduction of air born contamination.
The most common source of UV radiation is UV lamps. UV light is absorbed by the nucleic acids of the cell and where it does the greatest damage.


1. Uv light is helpful to maintain an aseptic condition.
2. UV lamps are mounted over the doors of rooms, above the heads of personnel, on the walls, ceiling to prevent cross-infection in hospitals.

Gamma rays

This is produced from the radioactive isotope of cobalt and cesium. The time and dose of sterilization depend upon density and quantity of material. Gamma rays have great penetration power. It is mainly used for sterilization of rubber catheters, needles, adhesive dressing, plastic lm, aluminum caps, plastic syringes, blades, etc.
Although radiation sterilization appears an attractive method, but it may lead to many undesirable effects decomposition, alterations in color, texture, potency, and solubility.


1. As the sterilization time is very small hence it is a continuous process.
2. This method is reliable for bacterial and viral vaccines.
3. A rise in temperature is negligible.
4. Dry, Moist, and frozen materials can be sterilized.


1. The investment cost for the plant is very high.
2. To prevent deleterious effects and hazards, elaborate and expensive precautions are taken.
3. The process once started can’t be controlled until the whole of the radioactive isotope is used.

B. Chemical Methods

a) Gaseous sterilization

Gaseous sterilization may be defined as the destruction of all living micro-organisms with a chemical in the Gaseous or vapor state.
The method of Gaseous sterilization is important particularly when solid material may not be sterilized by either dry or Moist heat because of the material or the product. May be damaged or destroyed. All these gases are toxic to human beings above a certain concentration.
Commonly used Gaseous sterilization agent are discussed below:–
1) Ethylene oxide:- Ethylene oxide is a reaction, flammable, and colorless gas. The pure form of this gas is highly explosive therefore the gas is diluted with carbon dioxide or freon to make it non- flammable. This gas is highly diffusible in nature and can penetrate areas that are not accessible to liquids or steam.
2) Ozone:- It is a pleasant-smelling but irritant and toxic gas. It is highly reactive with organic substances like aldehyde, fats, amino acids, proteins, and enzymes.
Ozone is used mainly for the disinfection of water, and the preservation of foods.

b) Sterilization by disinfectants

In an emergency, all surgical instruments are sterilized using disinfectants. The instruments to be used are dipped in a disinfectant and are washed with apyrogenic water for use, e.g., phenol, cresol with soap, Chlorine, alcohol, formaldehyde, dyes and mercury compounds, etc..

C. Mechanical Methods

a) Sterilization by filtration

Sterilization by filtration is another old method used for injectable preparations. By this method, all living and dead bacteria get removed when the solution is filtered through bacteria proof filtration media. Various filter media are used for bacteria proof filtration which includes
a. Sintered ceramics
b. Fibrous pad
c. Sintered glass
d. Microporous plastic.
The filters function by pores which are formed by the fusion of the porcelain, sintered glass, metal, cellulosic, or plastic polymer matrix. The filtration involves the bacteria to be entrapped in the pores and get removed from the solution. As the pores are of very small size, hence requires a lot of time. Vacuum or pressure or both are used to increase the rate of filtration.


1. These filters are useful for thermolabile medicaments.
2. All living and dead bacterias are removed.


As the process is not reliable, hence the sterility test is necessary. It can’t be used for suspensions and oily preparations.

Also Read:
Parenteral Dosage Forms - Sterile Pharmaceutical Dosage Forms