Principle, Procedure, Merits, Demerits and Applications of Physical Methods of Sterilization : Pharmaguideline -->

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Principle, Procedure, Merits, Demerits and Applications of Physical Methods of Sterilization

Moist heat sterilization, Dry heat sterilization, Filtration, Irradiation, Sound (Sonic) waves vibrations, Pressure (pascalization), Sunlight (solar)

Heat Sterilization

  • Heat sterilization, the most effective and widely used method of sterilization, has a bactericidal effect because it destroys enzymes and other essential components of the cell.
  • When a material is fully hydrated, it is more readily sterilized by heat, since it requires less heat energy and a lower temperature, under high humidity conditions where the denaturation and hydrolysis reactions predominate, rather than in the dry state where the oxidation process takes place.
  • Shorter exposure times tend to result in a lower amount of partial thermal degradation, so choosing a temperature range with a higher temperature range will generally minimize the amount of thermal degradation.
  • Thermostable products can be sterilized using this method. While drying (160-180°C) and moist (121-134°C) heating procedures are respectively used for moisture-sensitive and moisture-resistant products, it can be applied to both.

Moist Heat Sterilization

  • One of the most efficient methods of sterilization is moist heat sterilization, in which steam under pressure kills bacteria.
  • In moist heat sterilization, temperatures between 121°C and 134°C are used.
  • When water is under high pressure, its boiling point rises, allowing a higher temperature to be achieved.
  • In addition to rapid heat penetration, high pressure also causes the coagulation of proteins, resulting in irreversible loss of function and activity of microbes due to moisture in the steam.
  • Despite their lower fractional degradation, high temperature-short time cycles also offer the advantage of achieving high levels of sterility assurance, because their inactivation factors are significantly higher.
  • Clinical porous specimens (such as surgical dressings) and bottled fluids are commonly heated for three minutes at 134 degrees and then 15 minutes at 121 degrees.
  • By generating steam under pressure, an autoclave functions as a moist heat sterilizer.
  • In contrast to dry heat sterilization, where bacteria are oxidized, this technique kills microorganisms by coagulating their proteins.
  • As well as ophthalmic preparations, irrigation fluids, and processing contaminated materials, pharmaceuticals and medical industries make use of aqueous injections.
Different temperatures can be used to sterilize with moist heat:

At temperature below 100° C

  • Pasteurization is the standard technique for sterilization at low temperatures.
  • All non-spore-forming microbes in milk are killed using either the holder method or the flash method, where milk is heated to 63°C for 30 minutes.
  • Although pathogenic organisms are killed in pasteurization, not all are eradicated. Pasteurization eliminates disease-causing microbes by reducing their numbers by a logarithmic factor.
  • Almost all mesophilic non-sporing organisms can be killed with a moist heat of 60°C for half an hour, although some require different cycles of temperature and time.
  • In order to keep milk's nutritional value from being destroyed, it is not heated above its boiling point.
  • The milk can also be pasteurized at 60°C for an hour with special water baths, along with other fluids and equipment, such as vaccines of non-sporing bacteria.
  • A water bath is used to sterilize at 56°C for 1 hour serum and body fluids that contain congealable proteins.

At temperature of 100° C

  • The use of hot water at 100°C can provide some sterility, but not complete sterility. Only pathogenic microbes and some spores can be destroyed by hot water.
  • To sterilize these items, they are boiled in distilled water for 30 to 40 minutes.
  • It is recommended that you use distilled water, as hard water might cause calcium salts to form on your instruments.
  • Tyndallization involves sterilizing media with sugar and gelatin at 100°C for 30 minutes for three consecutive days in order to preserve sugar that might decompose at higher temperatures.
  • Temperatures of 100°C can be used for both contaminated dishes, bedding, pipettes, and other objects that require moist heat, in addition to temperature-sensitive objects.

Temperature above 100° C

  • The moist heat method is used to sterilize above 100°C with steam under pressure.
  • When the pressure of the atmosphere is normal (760 mm of Hg), water's boiling point is 100°C. The boiling point increases when the pressure is increased.
  • When water is boiled at 121°C under 15 pounds of pressure or 775 millimeters of mercury, it results in this process.
  • Under pressure, steam penetrates more deeply. Steam gives off latent heat when it is in contact with the surface, killing microbes.
  • As a result of the condensed liquid, microbes are killed moistly.
  • For sterilizing contaminated instruments as well as different culture media, autoclaves are used as they provide complete sterility.

Dry Heat Sterilization

  • Sterilization without moisture is accomplished by applying heat that cannot be contaminated by moisture, which is an effective method for sterilizing moisture-sensitive items.
  • In dry heat sterilization, heat is transferred from the surface of an item to the layers below it by conduction, which is the action of heat absorption by the item. To achieve sterilization, the entire item must reach the appropriate temperature.
  • Heat that is dry and humidity-free destroys microorganisms by causing protein denaturation and by causing protein lysis in many organisms. There is also a generation of free radicals, which can damage cells, sometimes even causing them to burn to ash, as in the case of incineration.
  • Dry heat sterilization can be used on materials that are difficult to sterilize by moist heat sterilization for a multitude of reasons.
  • Humid heat cannot be used to sterilize oil or powder because moisture cannot penetrate deeper parts of these materials, and moisture destroys powders.
  • A similar problem exists with laboratory equipment such as Petri dishes and pipettes when sterilized by moist heat, because of their penetration.
  • It is largely oxidative processes that result in the lethality of dry heat on microorganisms, which are significantly less effective than the hydrolytic damage caused by steam exposure during moist heat sterilization.
  • The temperatures involved in dry heat sterilization range between 160 and 180°C and the exposure times can be up to 2 hours depending upon the temperature used.
  • Hot air ovens and incinerators use this principle to generate very hot, moist-free air.
  • Sterilization of glass bottles with aseptic filling is one of the major industrial applications of dry heat sterilization.
  • Furthermore, by employing this method, it is possible to destroy bacterial endotoxins (which are produced by Gram-negative bacteria and pyrogens, which cause a fever when injected into the body) which are hard to remove by other means.
  • Approximately 250°C is used as a depyrogenating temperature for glass.
Dry heat sterilization can be broken down into different types, as follows:

Red Heat
  • Using a Bunsen flame, instruments are sterilized instantly by rest heat sterilization.
  • Incubation loops, wires, and forceps points are commonly sterilized with dry heat sterilization using this method.
  • Sterilization can be achieved by this process; however, it is limited to substances that can withstand heat until they turn red.
  • Dry sterilization by flame is a type of dry sterilization in which metallic objects are exposed to flames for a period of time, causing the flames to burn microbes and other dust from the instrument.
  • Flaming is the process of dipping instruments in alcohol or spirit and burning them over a gas flame in order to remove their marks.
  • Red hot sterilization is more efficient and ensures sterility, but it isn't as effective as this process.
  • Through incineration, wastes are sterilized, while also being reduced significantly in volume. The process usually occurs when the hospital's or other residue's final disposal is being conducted.
  • Ashes are made from the scraps and then disposed of later.
  • A device called an incinerator is used to conduct this process.
Infrared Radiation
  • Thermal sterilization is accomplished by absorbing infrared radiation (IR) and converting the energy to heat.
  • Tunnels containing IR sources are used to achieve this. A conveyor belt moves the instruments and glassware through a tunnel at a controlled speed, while they are kept in a tray.
  • A temperature of about 180°C will be achieved for about 17 minutes as the instruments are exposed to the radiation.
  • Irradiation can be used to sterilize packaged items such as syringes and catheters in large quantities.
Hot Air Oven
  • A hot air oven can be used to sterilize objects that are not able to be treated with moist heat.
  • Conduction is the process of transferring heat from the outer surface to the inner layer.
  • In order to cook food with hot air, you'll need an insulated chamber containing a fan, thermocouples, thermostat, shelves, and doors with locks.
  • Typically, hot air ovens sterilize materials by heating them to 170oC for 30 minutes, 160oC for 60 minutes, and 150oC for 150 minutes.
  • In addition to sterilizing glassware and Petri plates, these ovens can also be used to sterilize powder samples.


  • When compared with other sterilization techniques, filtration removes instead of destroys microorganisms.
  • Further, it can prevent both viable and nonviable particles from passing through, making it useful for both clarification and sterilization.
  • Filtration is accomplished primarily by sieving, adsorption, and trapping within the matrix of the filter material.
  • A membrane filter is a filter that has tiny pores that allow liquids to pass through while preventing larger particles such as bacteria from passing through. So, with smaller pores, the filter is more likely to block more substances from passing through it.
  • Certain types of filters (membrane filters) can also be employed in sterility testing to trap and concentrate contamination organisms in test solutions.
  • Afterwards, the filters are incubated with a liquid nutrient medium to promote growth and turbidity.
  • In addition to treating injectable and ophthalmic solutions, biological products as well as air and other gases in aseptic areas, sterilizing-grade filters have several other applications.

Filtration Sterilization of Liquids

  • These membrane filters, which come in the form of discs, can either be mounted on syringes and used in-line or they can be used with vacuum filter towers to filter liquids.
  • Filtration under pressure is generally considered the most suitable, because it is possible to fill directly into final containers at high flow rates without experiencing foaming problems, solvent evaporation problems, or air leakage problems.
  • Membrane filters are often combined with fiberglass depth prefilters in order to improve their dirt-handling capacity.

Filtration Sterilization of Gases

  • Normally, the filters used to accomplish this function are made of pleated sheets of glass microfibers separated from one another and supported by corrugated sheets of aluminum or Kraft paper. They are commonly used in air ducts, paneling, or cabinets with laminar air flow.
  • HEPA filters are able to capture up to 99.997% of particles with a diameter of >0.3mm and are thus acting as depth filters.
  • The fact that most bacteria are associated with dust particles means they are better at removing microorganisms.
  • In addition to sterilizing venting or displacement air in tissue culture and microbiology (carbon filters, hydrophobic membrane filters), filters are used to clean the air in mechanical ventilators (glass fiber filters), purify the exhaust air from safety cabinets for use with microbiology (HEPA filters), and clarify and sterilize medical gases (glass wool depth filters, hydrophobic membrane filters).


  • Radiation is a method of sterilizing surfaces and objects by exposing them to various kinds of radiation.
  • The main method of sterilization is electromagnetic radiation.
  • In most cases, these radiations have a major effect on microbial DNA and are primarily ionized and cause free radicals to form (gamma rays and electrons) or excite (UV light) bacterial DNA.

Ultra-violet (non-ionizing) Radiation

  • A wavelength of ultraviolet light consists of rays from 150 to 3900 Å, of which wavelength 2600 is the most bactericidal.
  • The low penetration power of non-ionizing waves causes them to kill microorganisms only on the surface.
  • A wide range of materials absorb these waves, including nucleic acids.
  • Waves cause pyrimidine dimers to form, which result in DNA errors and cause microbes to die by mutation.
  • Since conventional packaging materials are poorly permeable to UV radiation, it is unsuitable for sterilizing pharmaceutical dosage forms.
  • However, it is also used to sterilize air, to surface sterilize aseptic areas, and to treat manufacturing-grade drinking water.

Ionizing Radiation

  • In most cases, ionizing radiation is used for sterilization using gamma rays or x-rays.
  • Radiation from these sources causes the ionization of water as well as various substances.
  • Due to ionization of water, a multitude of metabolites of O2 are formed such as hydroxyl radicals, superoxide ions, and H2O2.
  • Various components of microorganisms are oxidized by these metabolites, causing them to die.
  • The resistance of bacteria to ionizing radiation drops when moisture is presents (a result of the increased production of free radicals) as well as when the temperature is elevated.
  • Radiation sterilization is typically used on dried items such as surgical instruments, sutures, prostheses, unit-dose ointments, plastic syringes, and dry pharmaceuticals.

Sound (Sonic) Waves Vibrations

  • The use of ultrasonic waves for bactericidal purposes utilizes ultrasound (typically between 20 and 40 kHz) to vibrate a fluid.
  • Although ultrasound can be used with just water, using a solvent that is appropriate for the type of dirt and object being cleaned enhances its effectiveness.
  • It has been suggested that airborne sound waves have an antibacterial effect because they may transform acoustic energy into heat.
  • As the nature of the medium through which sound is transmitted greatly affects the absorption of acoustic energy, the impact of airborne sound is different.
  • Through the propagation of sound waves through detergent solutions, alternating tensile and compressive forces are produced that oscillate, leading to the formation of millions of microscopically-sized cavities in the detergent solution.
  • The cavities violently collapse when they reach their maximum size, resulting in the formation of submicroscopic voids that create high-energy hydraulic shock waves.
  • Microorganisms and adhering debris can be physically removed from even the most inaccessible surfaces of a contaminated instrument by shock waves that reach temperatures of 10,000°F and hydrodynamic pressures of 10,000 PSI.
  • Airborne sound waves have antibacterial activity that is influenced by their sound intensity, irradiation period, and distance from the source.
  • Depending on the chemical composition of bacteria spores, sonic waves appear to be effective against them.
  • Lipoidal materials in microbes readily absorb acoustic frequencies, so changes in the lipid level directly affect both how much energy is absorbed by the spore and how effectively those spores are removed.
  • Surgical and dental instruments are routinely cleaned using this method before being sterilized by healthcare facilities.

Pressure (pascalization)

  • A pacalization or high-pressure processing method is used to preserve and sterilize food, in which food is subjected to very high pressure (hundreds of megapascal), resulting in the death of microorganisms and the inactivation of enzymes.
  • In most cases, the shape, color, and nutrients of most foods are not affected by HPP treatments when applied at room temperature.
  • Pressure created by anhydrous pressure is nonthermal, and covalent bonds are unbroken, so flavors are not affected.
  • At a pressure of 400-600 MPa, protein denatures easily, causing cell morphology and ribosomes to be altered.
  • Membrane lipids and proteins undergo changes, as does membrane fluidity, resulting in nucleic acid leakage.
  • Inactivating spores can be achieved through a combination of pacalization and heat, but pacalization alone is not very effective.
  • In acidic foods, such as yogurt and fruit, pacalization is particularly useful because pressure-tolerant spores cannot survive in low pH environments.
  • The treatment can be applied to both liquid and solid products.

Sunlight (Solar Disinfection)

  • With the help of sunlight, solar disinfection can be used to kill microorganisms.
  • Many people use this process for purifying or disinfecting water for drinking.
  • Sunlight's UV-A (wavelength 320–400 nm) energy inactivates pathogenic organisms by generating highly reactive oxygen forms (oxygen free radicals and hydrogen peroxides).
  • In addition to damaging pathogens, these metabolites also interfere with metabolism and destroy bacterial cell structures. Furthermore, all the solar energy (infrared to UV) is present at the surface, heating it up simultaneously.
  • Radiation sterilization uses a similar principle to solar disinfection, but its efficacy is much lower because it requires an extended period of exposure.
  • This process, however, is environmentally friendly and economical.

Advantages and Disadvantages


  • There is a great deal of effectiveness in sterilization
  • Standardized equipment is used to deliver the required heat
  • With various controls such as pressure gauges and temperature meters, the heat delivery system can be monitored effectively.
  • A quality control system has been established.


  • The autoclave cannot sterilize materials like fats, oils, and powders that are impermeable to steam.
  • Materials sensitive to heat cannot be sterilized by heat

Applications of Physical Methods

  • Microbial growth can be controlled with heat, which is a widely used and highly effective method.
  • In laboratories, aseptic techniques commonly use dry-heat sterilization. However, moist-heat sterilization, which penetrates cells better than dry heat, is the more effective method.
  • The process of pasteurization destroys pathogens and reduces the number of microbes causing food spoilage. Milk to be refrigerated is most commonly pasteurized using high-temperature, short-time pasteurization. Milk for long-term storage without refrigeration can be pasteurized using ultra-high temperature pasteurization.
  • Some organisms are killed by freezing, which slows microbial growth. Dry ice and ultra-low temperatures can be used to store and transport laboratory and medical specimens.
  • Microbes in food can be killed by high-pressure processing. Certain infections can also be treated with hyperbaric oxygen therapy.
  • In addition to being used to preserve foods, desiccation can be accelerated by adding salt or sugar, which decreases the amount of water in the food.
  • Sterilizing heat-sensitive and packaged materials with ionizing radiation, including gamma rays, is an effective method. Surfaces cannot be penetrated by nonionizing radiation, like ultraviolet light, but it is useful for sterilizing surfaces.
  • Biological safety cabinets in laboratories and hospital ventilation systems also use HEPA filtration to prevent airborne microbe transmission. Bacteria are commonly removed from heat-sensitive solutions by membrane filtration.
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Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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