Sterilization is defined as a process by which an article, surface, or medium is freed of all living microorganisms.

Any material that has been subjected to this process is said to be sterile.

Methods of sterilization can be broadly classified as:

  1. Physical methods of sterilization.
  2. Chemical methods of sterilization.

Physical methods of sterilization include the following:

  1. Sunlight
  2. Heat
  3. Filtration
  4. Radiation
  5. Sound (sonic) waves


  • Direct sunlight is a natural method of sterilization of water in tanks, rivers, and lakes.
  • Direct sunlight has an active germicidal effect due to its content of ultraviolet and heat rays.
  • Bacteria present in natural water sources are rapidly destroyed by exposure to sunlight.


  • Heat is the most dependable method of sterilization.
  • Two types of physical heat are used in sterilization.
    • Sterilization by moist heat
    • Sterilization by dry heat

Sterilization by moist heat

  • Moist heat occurs in the form of hot water, boiling water, or steam (vaporized water).
  • The temperature of moist heat usually ranges from 60 to 135°C.
  • Moist heat kills microorganisms by denaturation and coagulation of proteins.

Sterilization by moist heat can be classified as follows:

  1. Sterilization at a temperature more than 100°C
  2. Sterilization at a temperature of 100°C
  3. Sterilization at a temperature less than 100°C
  4. Intermittent sterilization

Sterilization at a temperature more than 100°C:

Pasteurization is an example of sterilisation at a temperature more than 100°C.


  • Pasteurization is a technique in which heat is applied to liquids to kill potential agents of infection and spoilage, while at the same time retaining the liquid’s flavor and food value.
  • This technique is named after Louis Pasteur who devised this method.
  • This method is extensively used for sterilization of milk.
  • Pasteurization inactivates most viruses and destroys the vegetative stages of 97–99% of bacteria and fungi.
  • Newer techniques have now been used to produce sterile milk that has a storage life of 3 months.
  • In this method, milk is processed with ultrahigh temperature (UHT) of 134°C for 1–2 seconds.

Sterilization at a temperature of 100°C:

Sterilsation at a temperature of 100ºC includes (a) boiling and (b) steam sterilizer at 100°C.


  • Simple boiling of water for 10–30 minutes kills most of the vegetative forms of bacteria but not bacterial spores.
  • Exposing materials to boiling water for 30 minutes kills most nonspore-forming pathogens including resistant species, such as the bacillus and staphylococci.
  • This method cannot be used for sterilization but only for disinfection.
  • This method is not recommended for sterilizing instruments used for surgical procedure.
  • The greatest disadvantage of this method is that the items sterilized by boiling can be easily recontaminated when removed from water after boiling.

Steam sterilizer at 100°C:

  • Koch’s or Arnold’s steam sterilizer is used for heat-labile substances that tend to degrade at higher temperatures and pressure, such as during the process of autoclaving.
  • These substances are exposed to steam at atmospheric pressure for 90 minutes during which most vegetative forms of the bacteria except for the thermophiles are killed by the moist heat.

Sterilization at a temperature less than 100°C:

  • This method is known as sterilization by steam under pressure.
  • The principle of sterilization by steam under pressure.
  • Such pressure temperature combinations can be achieved only with a special device that can subject pure steam to pressures greater than 1 atmosphere.
  • Health and commercial industries use an autoclave for this purpose and a comparable home appliance is the pressure cooker.


  • It is a cylindrical metal chamber with an airtight door at one end and racks to hold materials.
  • Heating is usually carried out by electricity.
  • Steam circulates within the jacket and is supplied under pressure to the inner chamber where materials are loaded for sterilization.
  • The water in the autoclave boils when its vapor pressure equals that of surrounding atmosphere.
  • The increase of pressure inside the closed vessel, the temperature at which the water boils inside the autoclave also increases.
  • Sterilization is achieved when the steam condenses against the objects in the chamber and gradually raises their temperature.
  • The condensed water facilitates moist conditions that ensures killing of microbes.
  • The most efficient pressure temperature combination for achieving sterilization by autoclave is 15 psi, which yields 121°C.

Intermittent sterilization:

  • Certain heat-labile substances (e.g., serum, sugar, egg, etc.)
  • That cannot withstand the high temperature of the autoclave can be sterilized by a process of intermittent sterilization, known as tyndallization.
  • Tyndallization is carried out over a period of 3 days and requires a chamber to hold the materials and a reservoir for
    boiling water.
  • Items to be sterilized are kept in the chamber and are exposed to free-fl owing steam at 100°C for 20 minutes, for each of the three consecutive days.
  • On the first day, the temperature is adequate to kill all the vegetative forms of the bacteria, yeasts, and molds but not sufficient to kill spores.
  • The surviving spores are allowed to germinate to vegetative forms on the second day and are killed on re-exposure to steam.
  • The third day re-ensures killing of all the spores by their germination to vegetative forms.
  • Intermittent sterilization is used most often to sterilize heat-sensitive culture media, such as those containing sera
    (e.g., Loeffl er’s serum slope), egg, or carbohydrates (e.g., serum sugars) and some canned foods.

Sterilization by dry heat:

  • Sterilization by dry heat makes use of air with a low moisture content that has been heated by a flame or electric heating coil.
  • The temperature of dry heat ranges from 160°C to several thousand degrees Celsius.
  • The dry heat kills microorganisms by protein denaturation, oxidative damage, and the toxic effect of increased level of electrolytes.
  • The temperature and time employed in dry heat vary according to the particular method, but in general they are greater than with moist heat.
  • Sterilization by dry heat includes sterilization by (a) flaming, (b) incineration , and (c) hot air oven


  • Sterilization of inoculating loop or wire, the tip of forceps, searing spatulas, etc., is carried out by holding them in the flame of the Bunsen burner till they become red hot.
  • Glass slides, scalpels, and mouths of culture tubes are sterilized by passing them through the Bunsen flame without allowing them to become red hot.


  • Incineration is an excellent method for safely destroying infective materials by burning them to ashes.

It has many uses:

  • Incinerators are used to carry out this process and are regularly employed in hospitals and research labs to destroy hospital and laboratory wastes.
  • The method is used for complete destruction and disposal of infectious material, such as syringes, needles, culture material, dressings, bandages, bedding, animal carcasses, and pathology samples.
  • This method is fast and effective for most hospital wastes, but not for metals and heat-resistant glass materials.

Hot-air oven:

  • The hot-air oven provides another means of dry heat sterilization and is the most widely used method.
  • The hot-air oven is electrically heated and is fitted with a fan to ensure adequate and even distribution of hot air in the chamber.
  • It is also fitted with a thermostat that ensures circulation of hot air of desired temperature in the chamber.
  • Circulated air transfers its heat to the materials inside the chamber.
  • While sterilizing by hot-air oven, it should be ensured that the oven is not overloaded.
  • The materials should be dry and arranged in a manner which allows free circulation of air inside the chamber.
  • Sterilization by hot-air oven requires exposure to 160–180°C for 2 hours and 30 minutes, which ensures thorough heating of the objects and destruction of spores.


  • Filtration is an excellent way to reduce the microbial population in solutions of heat-labile material by use of a variety of filters.
  • Filters are used to sterilize these heat-labile solutions.
  • Filters simply remove contaminating microorganisms from solutions rather than directly destroying them.
  • The filters are of two types: (a) depth filters and (b) membrane filters.

Depth filters:

  • Depth filters consist of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter.
  • The solution containing microorganisms is sucked in through this layer under vacuum and microbial cells are removed by physical screening or entrapment and also by adsorption to the surface of the filter material.

Depth filters are of the following types:

  1. Candle filters
  2. Asbestos filters
  3. Sintered glass filters

Membrane filters:

  • Membrane filters are made up of (a) cellulose acetate, (b) cellulose nitrate, (c) polycarbonate, (d) polyvinylidene fluoride, or (e) other synthetic materials.
  • These filters are now widely used and have replaced depth filters.
  • These filters are circular porous membranes and are usually 0.1 mm thick.
  • A wide variety of pore sizes (0.015–12 m) are available, membranes with pores about 0.2 m are used, because the pore size is smaller than the size of bacteria.
  • These filters are used to remove most vegetative cells, but not viruses.


The ionizing and nonionizing radiations are the two types of radiation used for sterilization.

Ionizing radiations:

  • Ionizing radiation is an excellent sterilizing agent with very high penetrating power.
  • These radiations penetrate deep into objects and destroy bacterial endospores and vegetative cells, both prokaryotic and eukaryotic.
  • These are, however, not that effective against viruses.
  • Ionizing radiations include (a) X-rays, (b) gamma rays, and (c) cosmic rays.
  • Gamma radiation from a cobalt-60 source is used for sterilization of antibiotics, hormones, sutures, catheters, animal feeds, metal foils, and plastic disposables, such as syringes.
  • This has also been used to sterilize and “pasteurize” meat and other food items.

Nonionizing radiations:

  • Nonionizing radiations include infrared and ultraviolet radiations.
  • Infrared radiations are used for rapid and mass sterilization of disposable syringes and catheters.
  • Ultraviolet (UV) radiation with wavelength of 240–280 nm is quite lethal and has a marked bactericidal activity.
  • It acts by denaturation of bacterial protein and also interferes with replication of bacterial DNA.
  • UV radiation is used primarily for disinfection of closed areas in microbiology laboratory, inoculation hoods, laminar flow, and operating theaters.
  • It kills most vegetative bacteria but not spores, which are highly resistant to these radiations.
  • UV radiations on exposure tend to burn the skin and cause damage to the eyes, UV lamps should be switched off while people are working in such areas.

Sound (sonic) waves:

  • High-frequency sound (sonic) waves beyond the sensitivity of the human ear are known to disrupt cells.
  • Sonication transmits vibrations through a water-filled chamber (sonicator) to induce pressure changes and create intense points of turbulence that can stress and burst cells.
  • Sonication also forcefully dislodges foreign matter from objects.
  • Heat generated by the sonic waves (up to 80°C) also appears to contribute to the antimicrobial action.
  • Gram-negative rods are most sensitive to ultrasonic vibrations, while Gram-positive cocci, fungal spores, and bacterial spores are resistant to them.
  • Ultrasonic devices are used in dental and some medical offices to clear debris and saliva from instruments before sterilization and to clean dental restorations.

Chemical Methods of Sterilization:

Several chemical agents are used as antiseptics as well as disinfectants. All these chemical agents (e.g., alcohols, aldehydes, etc.)


  • Alcohols are among the most widely used disinfectants and antiseptics.
  • They are bactericidal and fungicidal but not sporicidal.
  • They have no action against spores and viruses.
  • Ethyl alcohol and isopropyl alcohol are the two most popular alcohol germicides.
  • They are effective at a concentration of 60–70% in water.
  • They act by denaturing bacterial proteins and possibly by dissolving membrane lipids.
  • They are used as skin antiseptics. Isopropyl alcohol is used for disinfection of clinical thermometers.
  • A 10–15 minute soaking is sufficient to disinfect thermometers.
  • Methyl alcohol is effective against fungal spores.


  • Formaldehyde and glutaraldehyde are the two most commonly used aldehydes that are used as disinfectants.
  • They are highly reactive molecules that combine with nucleic and alkylating molecules.
  • They are sporicidal and can also be used as chemical sterilants.


  • Formaldehyde is usually dissolved in water or alcohol before use.
  • In aqueous solution, it is bactericidal, sporicidal, and also effective against viruses.
  • Formalin solution is 40% aldehyde in aqueous solution.

It is used to:

  • Preserve fresh tissue specimens
  • Destroy anthrax spores in hair and wool
  • Prepare toxoids from toxins
  • Sterilize bacterial vaccines
  • Kill bacterial cultures and suspensions



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