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Chapter: Microbiology and Immunology: Sterilizationand Disinfection

Types of Disinfectants

Disinfectants include the following: (a) phenolic compounds, (b) halogens, (c) alcohols, (d) aldehydes, (e) gases, (f) surface active agents, (g) oxidizing agents, (h) dyes, (i) heavy metals, and (j) acids and alkalis.

Types of Disinfectants

Disinfectants include the following: (a) phenolic compounds, (b) halogens, (c) alcohols, (d) aldehydes, (e) gases, (f) surface active agents, (g) oxidizing agents, (h) dyes, (i) heavy metals, and (j) acids and alkalis.

Phenolic compounds

In 1867, Joseph Lister employed phenolic compounds to reduce the risk of infection during operations. Phenolic compounds are the most widely used antiseptics and disinfectants in laboratories and hospitals worldwide. They are bactericidal or bacteriostatic and some are fungicidal also. They act by denaturing proteins and disrupting cell membranes. They are effective in the pres-ence of organic material and remain active on surfaces long after application. Different phenolic compounds are as follows:

     Phenol: It is effective against vegetative forms of bacteria,Mycobacterium tuberculosis, and certain fungi. It is an excellentdisinfectant for feces, blood, pus, sputum, etc. It has a low degree of activity as compared to other derivatives. It is not suitable for application to skin or mucous membrane.

     Cresol: Cresols are more germicidal and less poisonousthan phenol but corrosive to living tissues. They are used for cleaning floors (1% solution), for disinfection of surgical instruments, and for disinfection of contaminated objects. Lysol is a solution of cresols in soap.

     Halogenated diphenyl compounds: These compoundsinclude hexachlorophene and chlorhexidine. They are highly effective against both Gram-positive and Gram-negative bacteria. They are used as skin antiseptics and for the clean-ing of wound surfaces.

Hexachlorophene has been one of the most popular antiseptics because once applied it persists on the skin and reduces growth of skin bacteria for longer periods. However, it can cause brain damage and is now used in hospital nurseries only after a staphylococcal outbreak.


Halogens are fluorine, bromine, chlorine, and iodine—a group of nonmetallic elements that commonly occur in minerals, sea water, and salts. Although they can occur either in the ionic (halide) or nonionic state, most halogens exert their antimicro-bial activity primarily in their nonionic state, but not in the halide state (e.g., chloride, iodide).

     These agents are highly effective disinfectants and anti-septics, because they are microbicidal and not just microbi-static. They are also sporicidal with longer exposure. For these reasons, halogens are the active ingredients in nearly one-third of all antimicrobial chemicals currently marketed. Chlorine and iodine are the only two routinely used halogens because fluorine and bromine are dangerous to handle.

Chlorine and its compounds: Chlorine has been used for dis-infection and antisepsis for approximately 200 years. The major forms used in microbial control are (a) liquid and gaseous chlo-rine and (b) hypochlorites. In solution, these compounds com-bine with water and release hypochlorous acid (HOCl), which oxidizes the sulfhydryl (S–H) group on the amino acid cysteine and interferes with disulfide (S–S) bridges on numerous enzymes. The resulting denaturation of the enzymes is permanent.

      Gaseous and liquid chlorine are used almost exclusively for large-scale disinfection of drinking water, sewage, and wastewa-ter from sources, such as agriculture and industry. Chlorine kills not only bacterial cells and endospores but also fungi and viruses. Treatment of water with chlorine destroys many patho-genic vegetative microorganisms without unduly affecting its taste. Chlorination at a concentration of 0.6–1.0 part of chlorine per million parts of water makes water potable and safe to use.

Common household bleach is a weak solution (5%) of sodium hypochlorite that is used as an all-around disinfectant, deodor-izer, and stain remover. It is frequently used as an alternative to pure chlorine in treating water supplies. However, the major limitations of chlorine compounds are that they are:

a.     Ineffective if used at an alkaline pH,

b.     Less effective in the presence of excess organic matter, and

c.      Relatively unstable, especially if exposed to light.

Iodine and its compounds: Iodine is a pungent black chemicalthat forms brown-colored solutions when dissolved in water or alcohol. Iodine rapidly penetrates the cells of microorganisms, where it apparently disturbs a variety of metabolic functions. It acts by interfering with the hydrogen and disulfide bonds of proteins (similar to chlorine). It kills all types of microorgan-isms if optimum concentrations and exposure times are used. Iodine activity, unlike chlorine, is not as adversely affected by organic matter and pH. The two primary iodine preparations are free iodine in solution and iodophors.

      Free iodine in solution:Aqueous iodine contains 2% freeiodine in solution and 2.4% sodium iodide. It is used as a topicalantiseptic before surgery and also occasionally as a treatment for burnt and infected skin. A stronger iodine solution (5% iodine and 10% potassium iodide) is used primarily as a disinfectant for plas-tic items, rubber instruments, cutting blades, and thermometers.

     Iodine tincture is a 2% solution of iodine and sodium iodide in 70% alcohol that can be used in skin antisepsis. Because iodine can be extremely irritating to the skin and toxic when absorbed, strong aqueous solutions and tinctures (5–7%) are no longer considered safe for routine antisepsis.

Iodine tablets are available for disinfecting water during emer-gencies or for destroying pathogens in impure water supplies.

Iodophors:Iodophors are complexes of iodine and a neutralpolymer, such as polyvinyl alcohol. This formulation permits the slow release of free iodine and increases its degree of pen-etration. These compounds have largely replaced free iodine solutions in medical antisepsis because they are less prone to staining or irritating tissues.

·        Betadine, povidone, and isodine are the common iodophor compounds that contain 2–10% of available iodine. They are used to prepare skin and mucous membranes for surgery and in surgical hand scrubs.

·        They are also used to treat burns and to disinfect equipments.

·        A recent study has shown that betadine solution is an effective means of preventing eye infections in newborn infants, and it may replace antibiotics and silver nitrate as the method of choice.


Alcohols are among the most widely used disinfectants and antiseptics. They are bactericidal and fungicidal but not spori-cidal. 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 antisep-tics. Isopropyl alcohol is used for disinfection of clinical ther-mometers. 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: Formaldehyde is usually dissolved in water oralcohol before use. In aqueous solution, it is bactericidal, spo-ricidal, 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, and

§  Kill bacterial cultures and suspensions.

Glutaraldehyde: A 2% buffered solution of glutaraldehyde isan effective disinfectant. It is less irritating than formaldehyde and is used to disinfect hospital and laboratory equipments. Glutaraldehyde usually disinfects objects within time frame of 10 minutes but may require as long as 12 hours to destroy all spores. Glutaraldehyde is especially effective against tubercle bacilli, fungi, and viruses. It can be used for cleaning cysto-scopes and bronchoscopes, corrugated rubber anesthetic tubes and face masks, plastic endotracheal tubes, metal instruments, and polythene tubing.


Various gaseous agents are used for sterilization of large volume of heat-sensitive disposable items and also instruments. Ethylene oxide, formaldehyde gas, and betapropiolactone are frequently used gaseous agents.

Ethylene oxide: Ethylene oxide is a colorless liquid used forgaseous sterilization. It is active against all kinds of bacteria, spores, and viruses. It kills all types of microorganisms by inhibiting proteins and nucleic acids. It is both microbicidal and sporicidal. It is a highly effective sterilizing agent because it rapidly penetrates packing materials, including plastic wraps. It is used to sterilize disposable plastic Petri dishes, sutures, syringes, heart-lung machine, respirators, and dental equip-ments. Ethylene oxide is highly inflammable and carcinogenic. Extensive aeration of the sterilized materials is necessary to remove residual ethylene oxide gas, which is toxic.

Formaldehyde gas: The formaldehyde gas is used for(a) the fumigation of operation theaters, wards, sick rooms, and laboratories; and (b) the sterilization of instruments and heat-sensitive catheters, clothing and bedding, furniture, books, etc. The formaldehyde gas is produced by adding 150 gm of potassium permanganate to 280 mL formalin in 1000 cu ft of room volume. The room to be sterilized is completely closed and sealed at least for 48 hours after fumigation with formalin gas. Sterilization is achieved by condensation of gas on exposed surface. The gas is toxic when inhaled and is irri-tant to eye, hence its effect should be nullified by exposure to ammonia. It is highly inflammable and carcinogenic.

Beta-propiolactone: Beta-propiolactone (BPL) is a condensa-tion product of ketone and formaldehyde. It is active against all microorganisms and viruses. It is more efficient than formaldehyde for fumigation purpose. In the liquid form, it has been used to sterilize vaccines and sera. BPL destroys microorganisms more readily than ethylene oxide but does not penetrate materials well and may be carcinogenic. For these reasons, BPL has not been used as extensively as ethylene oxide. Recently, vapor-phase hydrogen peroxide has been used to decontaminate biological wastes.

Surface active agents

Surface active agents, such as detergents are the substances that alter energy relationship at interfaces producing a reduction in surface tension. Detergents are organic mol-ecules that serve as wetting agents and emulsifiers because they have both polar hydrophilic and nonpolar hydrophobicends. Due to their amphipathic nature, detergents solubilize and are very effective cleansing agents. They are different from soaps, which are derived from fats. Surface active agents are of four types:

          Cationic surface active agents: The cationic detergentsare effective disinfectants. Cationic detergents like benzal-konium chloride and cetylpyridinium chloride kill most bacteria but not M. tuberculosis, endospores, or viruses. They do have the advantages of being stable and nontoxic, but they are inactivated by hard water and soap. These are often used as skin antiseptics and also as disinfectants for disinfection of food utensils and small instruments. Qua-ternary ammonium compounds, such as cetrimide are the most popular cationic detergents. They act by disrupting microbial membranes and possibly by denaturing proteins.

        Anionic surface active agents: These include soaps preparedeither from saturated or unsaturated fatty acids, which act better at acidic pH. The soaps prepared from saturated fatty acids are more effective against Gram-negative organisms, whereas those prepared from unsaturated fatty acids are more active against Gram-positive bacilli and Neisseria.

    Nonionic surface active agents: These are nontoxic andsome of them may even promote the growth of bacteria.

   Amphoteric or ampholytic compounds: These are activeagainst a wide range of Gram-positive and Gram-negative bacteria and also against a few viruses. These are known as “Tego” compounds.

Oxidizing agents

This group includes halogens, hydrogen peroxide, potassium permanganate, and sodium perborate. They are good disinfec-tants and antiseptics but are less effective in the presence of organic matter. Hydrogen peroxide, used as 3% solution, is a weak disinfectant. It is useful for cleaning of the wounds and for mouth wash or gargle. Potassium permanganate is bacteri-cidal in nature and active against viruses also.


The dyes that have been used extensively as skin and wound antiseptics include (a) acridine dyes and (b) aniline dyes. The acridine dyes include acriflavine, euflavine, proflavine, andaminacrine. They show more activity against Gram-positive bac-teria than against Gram-negative organisms. They act by interfer-ing with the synthesis of nucleic acids and proteins in bacterial cells. The yellow acridine dyes, acriflavine and proflavine, are sometimes used for antisepsis and wound treatment in medi-cal and veterinary clinics. Aniline dyes (such as gentian violent, crystal violet, and malachite green) are also more active against Gram-positive bacteria than against Gram-negative organisms. They are also effective against various fungi, hence are incorpo-rated into solutions and ointments to treat fungal skin infec-tions, such as ringworm.

          The dyes, nevertheless, have limited applications because they stain and have a narrow spectrum of antimicrobial activ-ity. They also have no activity against tubercle bacilli. Their actions are also inhibited by the presence of organic matter.

Heavy metals

Soluble salts of mercury, silver, copper, arsenic, and other heavy metals have antibacterial activity, both bactericidal and bacte-riostatic. They combine with proteins, often with their sulfhy-dryl groups 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 ophthalmianeonatorum in newborn infants. Copper sulfate is an effective algicide in lakes and swimming pools. Mercuric chloride is used as disinfectant. These compounds, however, are increasingly replaced by other less toxic and more effective germicides.

Acids and alkalis

Acids (such as sulfuric acid, nitric acid, hydrochloric acid, and benzoic acid) and alkalis (like potassium and sodium hydroxide and ammonium hydroxide) are germi-cidal in nature. They kill microorganisms by hydrolysis and altering the pH of the medium. They are rarely used as disinfectants.

          Organic acids are widely used in food preservation because they prevent spore germination and bacterial and fungal growth, and because they are generally regarded as safe to eat. Acetic acid, in the form of vinegar, is a pickling agent that inhibits bacterial growth. Propionic acid is com-monly added into breads and cakes to retard molds; lactic acid is added to sauerkraut and olives to prevent growth of anaerobic bacteria, especially the clostridia; and benzoic and sorbic acids are added to beverages, syrups, and margarine to inhibit yeasts.

          Activities of commonly used disinfectants against various microorganisms are summarized in Table 3-5.

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