Sterilization and Disinfection


November 13, 2021


Microorganisms are found everywhere. Many of them spoil food and cause diseases. So, it is necessary to kill them or inhibit their growth to minimize their destructive effects. Disinfection and sterilization are needed in healthcare and hospitals, during surgical procedures, in microbiology, food industry, and public health services such as providing safe drinking water, disposal of sewage.

Frequently Used Terms

Sterilization

Sterilization is a process which destroy all types of living microbes from an article, surface or a medium including their spores. It is carried out in healthcare facility mostly with a physical agent. Some chemicals called sterilants because of their ability to destroy spores.

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Disinfection

Disinfection is a process which destroy or inhibit the growth of microorganisms on inanimate objects. It do not eliminate spores. It is carried out by physical or chemical means. Chemicals can be toxic to humans when used in higher concentration so normally done on nonliving objects.

Antiseptic

Sepsis is defined as growth of microorganisms in  the body. Asepsis is any practice that prevents the entry of infectious agent into a sterile tissues. Antiseptics are chemical agents that destroy or inhibit the growth of microorganisms in or on living tissue. (e.g. skin or mucous membranes) wounds, and surgical incisions.

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Sanitization

Sanitization is any cleaning technique that mechanically removes microorganisms. Objects washed with soaps and detergent may not be completely free of microbes but they are considered safe for normal use. Sometimes degerming procedure is necessary like scrubbing hands with chemicals before surgery, using alcohol wipes or by using germicidal soaps.

Bactericidal or Bacteriostatic Agents

Bactericidal agents kills bacteria while bacteriostatic agents prevent multiplication of bacteria without killing them.

Methods of sterilization and Disinfection are broadly classified as

  • Physical methods
  • Chemical methods
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Physical Methods of Sterilization

  • Sunlight
  • Drying
  • Heating
  • Filteration
  • Radiation
  • Ultrasonic waves

Sunlight and Drying

Direct sunlight is a natural method of sterilization of water in tanks, rivers and lakes. Sunlight possesses appreciable bacteriocidal activity. It kill germs due to its ultraviolet and heat rays. Drying in air kills many bacteria, as they need moisture to grow. Bacterial spores are not affected by drying.

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Heating

Heat is the most dependable method of sterilization. Heat can be moist or dry.

Sterilization by Moist Heat

Moist heat occurs in form of hot water, boiling water or steam. Temperature usually ranges 60-135°C in practice.

  • Steriliazation at a temperature < 100 °C
  • Steriliazation at a temperature 100° C
  • Steriliazation at a temperature >100°C
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Sterilization at a temperature below 100°C is also called pasteurization. This technique is used for fresh beverages such as milk and juices. It kills 97-99 % of germs while preserving the flavor and food value. It does not kill spores or thermoduric species ( mostly non-lactobacillus, micrococci, and yeast). In fact, there are still 20,000 microbes per ml which is why they get spoiled after 2-3 days. Newer techniques have been used to produce sterile milk that has a storage life of up to 3 months. In this method, milk is processed with an ultrahigh temperature (UHT) of 134°C for 1-2 seconds. In this way, more resistant pathogens such as coxiella and tubercle bacilli get killed.

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Boiling or by steaming for 10-30 minutes kills most microbes  including resistant species such as tubercle bacillus and staphylococcus but it does not kill the spores. Steam sterilizers is used for substances that tend to degrade by higher temperatures and pressure. These substances are exposed to steam at atmospheric pressure for 90 minutes. Most bacteria are killed except the thermophilic bacteria.

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Sterilization at a temperature above 100°C is done by increasing pressure. In healthcare facilities, it is carried out in an autoclave. Surgical instruments, heat-resistant plastics, and glassware are sterilized in an autoclave. In an autoclave, materials are exposed to 121°C temperature for 15-20 minutes at 15 lb pressure per square inch (psi). The autoclave is used for sterilizing culture media, instruments, syringes, gowns, and dressings, etc. 

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Intermittent sterilization or Tyndallization is used for certain heat-labile substances which can not withstand the high temperature of an autoclave. Items to be sterilized are kept in a chamber and are exposed to free-flowing steam at 100°C for 20 minutes, for 3 consecutive days. Spores are allowed to germinate and killed by re-exposure to steam.

Sterilization by Dry Heat

Temperature of dry heat ranges from 160 °C to several thousand°C. Dry heat is not as widely used as moist heat, but it has several important sterilization applications.

Sterilization by dry heat include:

  • Red heat
  • Flaming
  • Incineration
  • Hot air oven
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Red heat is used to sterilize metallic objects by holding them in flame till they get red hot or an article is passed over flame without allowing it to become red hot such as glass slides.

Incineration is an excellent method for rapidly destroying contaminated materials. The drawback of incinerators are air pollution. Toxic fumes are harmful for environment and unhealthy. Hot air ovens are used to sterilize glasswares, dressings and metal instruments. Items are sterilize at 160 -180°C for 2 hour and 30 minutes. It is most widely used method in laboratories and clinics.


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Filteration is used in sterilizing heat labile solutions such as antibiotic solutions, sera, blood products, and purification of water etc. Filteration remove microorganisms without killing them. There are two types of filters.

  • Depth filters
  • Membrane filters

Depth Filters

Depth Filters consist of fibrous or granular material that have been bonded into a thick layer filled with twisting channels of smaller diameters. There are three types of depth Filters such as candle filters, Asbestos filters, Sintered glass filters.

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Membrane Filters

They are made of cellulose esters. They are widely used and have replaced depth filters. A wide variety of pore sizes from 0.015,0.12 and 0.22 micrometer are available. They can remove most of the microorganisms but not viruses. Membrane Filters have many uses in addition to sterilizing pharmaceutical preparations it is also used to isolate viruses or toxins from bacteria.

Air can also be sterilized by filteration. Two common examples are surgical masks and cotton plugs on culture vessels to prevent entry of most microbes.

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Radiation

X-rays and other ionizing radiation are used for the sterilization of disposable materials like sutures, disposable syringes, adhesive dressings. Ionizing radiation is an excellent sterilizing agent with very high penetrating power. These are however not effective against viruses. Ionizing radiation includes X-rays, gamma rays, cosmic rays. This method is often referred to as cold sterilization. Gamma radiation from cobalt-60 source also has Ben used for sterilization of meat and food items. Both the FDA and WHO have approved food irritation and declared it safe.

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Non-ionizing radiation includes infrared and ultraviolet radiation. Spores are not killed by these radiations. Ultraviolet radiation with a wavelength of 240-280 nm has marked bactericidal activity. It acts by denaturing bacterial proteins and also interfering with bacterial DNA. Air sanitization with ultraviolet lamps in hospitals, veterinary clinics, and laboratories reduces airborne microbes. Commonly used UV lamps are of low-pressure mercury vapor type.

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Ultrasonic and Sonic Vibrations

They are bactericidal causing mechanical agitation and rupture of microbes. Gram-negative bacteria are most sensitive to ultrasonic vibration, while gram-positive cocci, fungal and bacterial spores are resistant to them. The heat generated by sonic vibration (up to 80 °C) also contributes to microbial action.

Chemical Methods of Sterilization

The chemical substances kill bacteria by different methods. They coagulate (heavy metals) or oxidize (halogens) bacterial protoplasm. Some disrupt the cell membrane or binds with enzymes and coenzyme systems thus causing interference with metabolism. Disinfecting chemicals in common use are:

  • Phenolic compounds
  • Halogens
  • Aldehydes
  • Alcohols
  • Oxidizing agents
  • Acid and alkalis
  • Dyes
  • Heavy metals
  • Surface active agents
  • Aerosol and gaseous disinfectants
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Phenolic Compounds

Phenolic compounds are the most widely used antiseptic and disinfectant worldwide in laboratories and hospitals. They act by denaturing proteins and disrupting cell membranes. They remain active on surfaces long after application. Different phenolic compounds are phenol, cresol, Thymol and halogenated diphenyl compounds such as chloroxylenol (Dettol), Chlorocresol.

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Halogens

They are among the best bactericidal. Halogens kill fungi and viruses but not spores. Chlorine and iodine are only two routinely used because fluorine and bromine are dangerous to handle. Chlorine and its compounds have been used for disinfection and antisepsis for approximately 200 years. The major forms used are liquid and gaseous chlorine and hypochlorite. In solution, these compounds combine with water and release hypochlorous acid (HOCL), which oxidizes the sulfhydryl group on Amino acid cysteine and interferes with disulfide bridges on numerous enzymes. Gaseous and liquid chlorine are used almost exclusively for large-scale disinfection of drinking water, sewage, and wastewater from agriculture and industries. Chlorination at a concentration of 0.6-1.0 part of chlorine per million parts of water makes water portable and safe to use.

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Hypochlorites such as sodium hypochlorite (Dakin ‘s solution), Calcium hypochlorite (Eusol), Chlorinated Lime are most extensively used of all chlorine compounds. They are used for

  • Sanitization and disinfection of food equipment in dairies and restaurants.
  • Treatment of swimming pools, spas, drinking water and even fresh food.
  • Treatment of wounds
  • Disinfection of equipments bedding and instruments.

Common household bleach is a weak solution of 5% of sodium hypochlorite that is used as an all-around disinfectant and stain remover. It is frequently used as an alternative to chlorine in treating water supplies.

Iodine and its compounds: kills all types of microorganisms if optimum concentration and exposure time is used. The two primary iodine preparations are free iodine in solution and iodophors.

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Iodine is used chiefly for the skin. Aqueous iodine solution (Lugol’s solution) with 2% free iodine and 2.4 % sodium iodide is used as an antiseptic before surgery and also occasionally as a treatment for burnt and infected skin. A stronger iodine solution with double concentration is used primarily as a disinfectant for plastic items, cutting blades, etc. Iodine tincture is a weak iodine solution of iodine and sodium iodide in 70 % alcohol that can be used as a skin antiseptic. Because iodine can be extremely irritating to skin and toxic when absorbed so 5-7% strong solutions are no longer considered safe for antisepsis. Iodine tablets are available for disinfecting water during emergencies or for destroying pathogens in impure water supplies.

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Iodophors are complexes of iodine and a neutral polymer, such as polyvinyl alcohol. It allows the slow release of free iodine. These compounds largely replaced free iodine solution because they cause less staining and are less irritating. Betadine, povidone, and Isodine are the common iodophors compounds that contain 2-10 % of available iodine. They are used as antiseptic before surgery and for hand scrubbing. They are also used to treat burns and disinfect equipment.

Alcohols

Alcohols are among the most widely used disinfectant. They are bactericidal and fungicidal but have no action against spores and viruses. Ethyl alcohol and isopropyl alcohol are the two most popular germicides. They are effective at a concentration of 60-70 % in water. Methyl alcohol is effective against bacterial spores. They act by denaturing bacterial proteins and dissolving membrane lipids.

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Aldehydes

Formaldehyde and glutaraldehyde are the two most commonly used aldehyde that is used as disinfectants. They are active against spores also so can be used as a chemical sterilant. Formaldehyde is usually dissolved in water or alcohol before use. In aqueous 5-10% solution it is bactericidal, sporicidal, and also active against viruses. Formalin solution is 40 % aldehyde in an aqueous solution. It is used to preserve fresh tissue specimens, prepare toxoids, sterilize bacterial vaccines, kill bacterial culture and suspension, and for fumigation.

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For fumigation of 100 cubic feet of air space, 50 ml of 40 % formalin is used. This gives 2 mg gas/ L of air. Formalin with sufficient water is heated in the room with its windows and doors closed and sealed. The room is exposed to fumes for 4 hours. Diluted formalin can be sprayed for disinfecting spaces.

Glutaraldehyde : A 2%  buffered solution is an effective disinfectant. Used to disinfect hospital and laboratory equipment. Usually it disinfects in 10 minutes but may need 12 hours to kill all spores.

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Aerosol and Gaseous Disinfectants

Various gaseous agents are used for sterilization of large volumes of heat-sensitive disposable items and also instruments, Plastic and rubber items, heart and lung machines, etc. Ethylene Oxide, formaldehyde gas, and Beta-Propiolactone (BPL) are frequently used, gaseous agents. The formaldehyde gas is used for fumigation of operation theater, wards, sick rooms, and laboratories. Beta-propiolactone is a powerful disinfectant. It is a condensation product of ketone and formaldehyde. It is more efficient than formaldehyde for fumigation purposes. It destroys microorganisms more readily than Ethylene oxide but does not penetrate materials well and maybe carcinogenic so not used as extensively as Ethylene Oxide. Ethylene Oxide can kill all kinds of microbes and spores. Other sterilant gases are Chlorine Dioxide, Paracetic Acids, Glutaraldehyde and Hydrogen Peroxide vapors, etc.

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Surface Active Agents

Surface active agents such as soaps and detergents are good cleaning agents. Detergents acts by disrupting the cell membrane or it may denature proteins and enzymes. Surfactants used as disinfectant are such as benzalkonium chloride solution, Sodium Lauryl Sulphate, Cetrimide etc.

Oxidizing Agents

Halogens, Hydrogen peroxide, Zinc Peroxide, Potassium Permanganate, Potassium Chlorate, and Sodium Perborate are oxidizing agents. They are good disinfectants and antiseptics but are less effective in presence of organic matter. Hydrogen Peroxide 3% solutions are used for cleaning wounds and for mouth wash and gargling. Potassium Permanganate is bactericidal and it is active against viruses too.

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Dyes

The dyes cause staining and have a narrow spectrum so they have limited applications. They are more active against gram-positive than gram-negative bacteria. They act by interfering with the synthesis of nucleic acids and proteins in bacterial cells. Dyes used as antiseptics are, for example, Acridine Dyes, Aniline Dyes.

The yellow Acridine Dyes include Acriflavine, Eufavine, Proflavine, and Aminacrine are used for antisepsis and wound treatment in medical and veterinary clinics. Acriflavine is bacteriostatic for staphylococcus in 1: 3000,000 concentration. Aniline dyes (such as gentian violet, crystal violet, and malachite green) are also more active against gram-positive bacteria. They are also active against fungi.

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Acid and Alkalis

Strong acid and alkalis are germicidal in nature. They are rarely used as a disinfectant. Organic acids are widely used in food preservation because they prevent spores germination and prevent the growth of bacteria and fungi and they are safe to eat. For example, vinegar is used in pickles. Propionic Acid is commonly added to bread and cakes to prevent fungal growth. Benzoic Acid and Sorbic Acid are used in beverages, syrups, and margarine to prevent yeast. Other acids used are Mandelic Acid or Salicylic Acid.

Heavy Metals

Soluble salt of mercury, silver, copper, arsenic, zinc, and other metals have antibacterial activity. They combine with proteins and inactivate them. They may also precipitate cell proteins. Silver compounds are widely used as antiseptics. Silver sulfadiazine is used for burns. Silver nitrate is used as a prophylactic agent in ophthalmia neonatorum in newborn infants. Copper sulfate is an effective algicide in lakes and swimming pools. Mercuric Chloride is used as a disinfectant. These compounds are however are increasingly replaced by less toxic and more effective germicidal.

Newer techniques of disinfection and sterilization are use of Surfacine, super oxidant water, Endoclens and plasma sterilizers. They are safer to environment, non toxic and have prolong action.

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