Sterilization refers to the killing or elimination of all microorganisms, including highly resistant bacterial spores. Sterilization is an absolute term, i.e. the objects must be sterile which means the absence of all microorganisms in that object. Sterilization can be referred to any process that removes all forms of life, including transmissible agents (such as bacteria, fungi, viruses, spore forms, etc.) present on a surface, in a fluid, drugs or biological culture media. Sterilization can be achieved by applying heat, chemicals, irradiation, high pressure, and filtration or combinations.

There are three methods of sterilization:

1. Physical Sterilization (by heat, filtration or radiation)
2. Chemical Sterilization
3. Physiochemical Sterilization

Methods of sterilization:

1. Physical Sterilization (by heat, filtration or radiation)

A. Heat
The most effective and a rapid method of sterilization and disinfection is Heat. Excessive heat acts by thickening or clotting of cell proteins. Less heat interferes metabolic reactions. The two most common methods for sterilization used in the laboratory are:(a) sterilization by hot air in a hot air oven, (b) sterilization by autoclaving.

Types of Heat:

i. Sterilization by Moist Heat
Moist heat sterilization describes sterilization techniques that utilize saturated steam.Moist heat can denature and coagulate the protein. It can cause breakage of DNA strands and loss of functional integrity of cell membrane. Methods include:
Boiling: Boiling at 100°C is done in a water bath for 30 minutes. By this method Syringes, rubber goods and surgical instruments can be sterilized. The method is effective against all bacteria and some spores.
Steaming: Steam is more effective than dry heat at the same temperature as: (a) Bacteria are more susceptible to steam, (b) Steam has great penetrating power, and (c) Steam has more sterilizing power as more heat is given up during condensation.
Steam Sterilizer works at 100°C under normal atmospheric pressure i.e. without extra pressure. It is suitable for sterilizing the objects which may be damaged at higher than 100°C temperature.

  • Single Exposure for 1/2 hours.
  • Tyndallization
  • Autoclaving:

Moist heat sterilization using autoclave is commonly used for the sterilization of biohazards trash, heat and moisture resistant materials such as aqueous preparation. In this method, sterilization is done by steam under pressure. Steaming at a temperature higher than 100°C is used. When the autoclave is closed, the water starts boiling and the inside pressures increases and now the water boils above 100°C. At 15 lb per sq. inch pressure, 121°C temperatures are obtained which is kept for 15 minutes for proper sterilization and to kill spores.

ii. Sterilization by Dry Heat
Dry heat sterilization is done at 160°C is applied by holding the temperature for one hour, which is required to kill the most resistant spores. The articles remain dry. It is not suitable for clothing which may be spoiled. Mechanisms include (1) Protein denaturation, (2) Oxidative damage, (3)Toxic effect of elevated electrolyte (in absence of water).
Methods include:
Red Heat: Wire loops that are used in microbiology laboratory are sterilized by heating to ‘red’ in bunsen burner or spirit lamp flame. Temperature is above 100°C so it leads to sterilization.
Flaming: The object is passed through flame without allowing it to become red hot, e.g. scalpel. Temperature is not high to cause sterilization.
Sterilization by Hot Air
Hot Air Oven (Sterilizer) It is the most common method used for sterilization. The substances such as Glasswares, swab sticks, all-glass syringes, powder and oily substances are sterilized in a hot air oven. For sterilization, a temperature of 160°C is maintained (holding) for one hour. Spores are killed or removed at this temperature which leads to sterilization.

B. Filtration
Sterilization by filtration is done mainly for thermolabile solutions. These may be sterilized by passage through sterile bacteria-retaining filters, for e.g. membrane filters (cellulose derivatives, etc.), plastic, porous ceramic, or suitable sintered glass filters, or combinations of these. Filters containing Asbestos should not be used. Normally, the membranes of not greater than 0.22 μm having, nominal pore size is used.

Different types of filters
1. Earthenware filters:
2. Asbestos filters:
3. Sintered glass filters:
4. Membrane filters:

C. Radiation:
Two types of radiation, ionizing and non-ionizing.
Non-ionizing rays: Rays having the wavelength longer than the visible light are non-ionizing. A high-pressure mercury vapor lamp is used to generate UV rays. UV rays induce the formation of thymine-thymine dimers and eventually inhibit the replication of DNA. UV radiation induces mutations in cells of bacteria, viruses, yeast, etc. When exposed to the effective UV radiation is inactivated within seconds.
Ionizing rays: ionizing rays are high-energy rays which have good penetrative power. It is termed as “cold sterilization”, as the radiation does not generate heat There are two types of ionizing rays; particulate and electromagnetic rays. Electron beams are particulate in nature while gamma rays are electromagnetic in nature

2. Chemical sterilization:
Sterilization can also be done by the use of chemicals. Although, Heating is the most reliable method to rid objects of all transmissible agents, but it is not always appropriate, because heat-sensitive materials such as biological materials, fiber optics, electronics, and many plastics can be damaged by it. Different types of chemical used are:

Alcohols: Alcohols in combination with distilled water are most effective to facilitate diffusion through the cell membrane. Higher concentration is often used to disinfect wet surfaces
100% alcohol – denature only external membrane proteins.
70% alcohol or diluted Isopropanol – effective against wide range of bacteria
80% ethanol + 5% isopropanol- inactivate lipid-enveloped viruses (such as HIV, hepatitis B, and hepatitis C)

Aldehydes: Aldehydes, such as formaldehyde and glutaraldehyde, have a sporicidal and fungicidal activity.

Oxidizing agents: These chemicals cause oxidation of the cell membrane of microorganisms, which results in loss of structure and leads to cell lysis. Some examples of disinfectants are: Hypochlorites: Sodium hypochlorite, calcium hypochlorite, Electrolyzed water or “Anolyte”, Chloramines, Chlorine dioxide, Hydrogen peroxide

• Phenolics: used in disinfectant, antiseptics, mouthwashes, handwashes, and soaps. E.g. o-Phenylphenol, Chloroxylenol, Hexachlorophene, Thymol etc.

• Quaternary ammonium compounds are used in combination with alcohols to kill encapsulated virus such as norovirus, rotavirus, or poliovirus. Some low-alcohol formulations exhibit high efficacy against bacteria, enveloped viruses, pathogenic fungi, and mycobacteria.

• Silver: Silver has antimicrobial properties but because of its limited shelf-life and instability as disinfectant some chelated forms of silver such as Silver dihydrogen citrate (SDC), are used.

Copper alloy surfaces: Copper alloy surfaces have the ability to destroy a broad range of microorganisms and reduce bacterial contamination to 99.9% within 2 hours

• Thymol-based disinfectant: Thymol is a phenolic chemical obtained from thyme and is considered as an intermediate level of disinfectant.

3. Physiochemical Methods of Sterilization:
A physiochemical method includes both physical and chemical method. Use of steam-formaldehyde is an example of physiochemical method of sterilization.