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Chapter: Medical Microbiology: Sterilization and Disinfection

Disinfection: Physical Methods and Chemical Methods

Both live and dead microorganisms can be removed from liquids by positive- or negative-pressure filtration.


Physical Methods


Both live and dead microorganisms can be removed from liquids by positive- or negative-pressure filtration. Membrane filters, usually composed of cellulose esters (eg, cellulose acetate), are available commercially with pore sizes of 0.005 to 1μm. For removal of bacteria, a pore size of 0.2μm is effective because filters act not only mechanically but by electrostatic adsorption of particles to their surface. Filtration is used for disinfection of large volumes of fluid, especially those containing heat-labile components such as serum. For microorganisms larger than the pore size filtration “sterilizes” these liquids. It is not considered effective for removing viruses.


Pasteurization involves exposure of liquids to temperatures in the range 55 to 75°C to re-move all vegetative bacteria of significance in human disease. Spores are unaffected by the pasteurization process. Pasteurization is used commercially to render milk safe and extend its storage quality. With the outbreaks of infection due to contamination with en-terohemorrhagic E. coli , this has been extended (reluctantly) to fruit drinks. To the dismay of some of his compatriots, Pasteur proposed application of the process to winemaking to prevent microbial spoilage and vinegarization. Pasteurization in water at 70°C for 30 minutes has been effective and inexpensive when used to render in-halation therapy equipment free of organisms that may otherwise multiply in mucus and humidifying water.


The use of microwaves in the form of microwave ovens or specially designed units is an-other method for disinfection. These systems are not under pressure, but they but can achieve temperatures near boiling if moisture is present. In some situations, they are being used as a practical alternative to incineration for disinfection of hospital waste. These pro-cedures cannot be considered sterilization only because the most heat-resistant spores may survive the process.

Chemical Methods

Given access and sufficient time, chemical disinfectants cause the death of pathogenic vegetative bacteria. Most of these substances are general protoplasmic poisons and are not currently used in the treatment of infections other than very superficial lesions, having been replaced by antimicrobics . Some disinfectants such as the quater-nary ammonium compounds, alcohol, and the iodophors reduce the superficial flora and can eliminate contaminating pathogenic bacteria from the skin surface. Other agents such as the phenolics are valuable only for treating inanimate surfaces or for rendering conta-minated materials safe. All are bound and inactivated to varying degrees by protein and dirt, and they lose considerable activity when applied to other than clean surfaces. Their activity increases exponentially with increases in temperature, but the relationship be-tween increases in concentration and killing effectiveness is complex and varies for each compound. Optimal in-use concentrations have been established for all available disinfec-tants. The major groups of compounds currently used are briefly discussed next.

     Chemical disinfectants are classified on the basis of their ability to sterilize. High-level disinfectants kill all agents except the most resistant of bacterial spores. Intermediate-level disinfectants kill all agents but not spores. Low-level disinfectants are active against most vegetative bacteria and lipid-enveloped viruses.


The alcohols are protein denaturants that rapidly kill vegetative bacteria when applied as aqueous solutions in the range 70 to 95% alcohol. They are inactive against bacterial spores and many viruses. Solutions of 100% alcohol dehydrate organisms rapidly but fail to kill, because the lethal process requires water molecules. Ethanol (70 – 90%) and iso- propyl alcohol (90 – 95%) are widely used as skin decontaminants before simple invasive procedures such as venipuncture. Their effect is not instantaneous, and the traditional al- cohol wipe, particularly when followed by a vein-probing finger, is more symbolic than effective, because insufficient time is given for significant killing. Isopropyl alcohol has largely replaced ethanol in hospital use because it is somewhat more active and is not subject to diversion to housestaff parties.


Iodine is an effective disinfectant that acts by iodinating or oxidizing essential components of the microbial cell. Its original use was as a tincture of 2% iodine in 50% alcohol, which kills more rapidly and effectively than alcohol alone. This preparation has the disadvantage of sometimes causing hypersensitivity reactions and of staining materials with which it comes into contact. Tincture of iodine has now been largely replaced by preparations in which iodine is combined with carriers (povidone) or nonionic detergents. These agents, termed iodophors, gradually release small amounts of iodine. They cause less skin staining and dehydration than tinctures and are widely used in preparation of skin before surgery. Although iodophors are less allergenic than inorganic iodine preparations, they should not be used on patients with a history of iodine sensitivity.


Chlorine is a highly effective oxidizing agent, which accounts for its lethality to microbes.It exists as hypochlorous acid in aqueous solutions that dissociate to yield free chlorine over a wide pH range, particularly under slightly acidic conditions. In concentrations of less than one part per million, chlorine is lethal within seconds to most vegetative bacteria, and it inactivates most viruses; this efficacy accounts for its use in rendering supplies of drinking water safe and in chlorination of water in swimming pools. Chlorine reacts rapidly with protein and many other organic compounds, and its activity is lost quickly in the presence of organic material. This property, combined with its toxicity, renders it ineffective on body surfaces; however, it is the agent of choice for decontaminating surfaces and glassware that have been contaminated with viruses or spores of pathogenic bacteria. For these purposes it is usually applied as a 5% solution called hypochlorite.


The use of chlorination to disinfect water supplies has proved insufficient in some hospitals because of the relative resistance of Legionella pneumophila to the usual concentrations of chlorine. Some institutions have been forced to augment chlorination with systems that add copper and silver ions to the water.

Hydrogen Peroxide


Hydrogen peroxide is a powerful oxidizing agent that attacks membrane lipids and other cell components. Although it acts rapidly against many bacteria and viruses, it kills bacte- ria that produce catalase and spores less rapidly. Hydrogen peroxide has been useful in disinfecting items such as contact lenses that are not susceptible to its corrosive effect.


Surface-Active Compounds

Surfactants are compounds with hydrophobic and hydrophilic groups that attach to andsolubilize various compounds or alter their properties. Anionic detergents such as soaps are highly effective cleansers but have little direct antibacterial effect, probably because their charge is similar to that of most microorganisms. Cationic detergents, particularly thequaternary ammonium compounds (“quats”) such as benzalkonium chloride, are highly bactericidal in the absence of contaminating organic matter. Their hydrophobic and lipophilic groups react with the lipid of the cell membrane of the bacteria, alter the membrane’s surface properties and its permeability, and lead to loss of essential cell com-ponents and death. These compounds have little toxicity to skin and mucous membranes and, thus, have been used widely for their antibacterial effects in a concentration of 0.1%. They are inactive against spores and most viruses. “Quats” in much higher concentrations than those used in medicine (eg, 5 – 10%) can be used for sanitizing surfaces.

The greatest care is needed in the use of quats because they adsorb to most surfaces with which they come into contact, such as cotton, cork, and even dust. As a result, their concentration may be lowered to a point at which certain bacteria, particularly Pseudomonas aeruginosa, can grow in the quat solutions and then cause serious infec-tions. Many instances have been recorded of severe infections resulting from contamina-tion of ophthalmic preparations or of solutions used for treating skin before transcutaneous procedures. It should also be remembered that cationic detergents are totally neutralized by anionic compounds. Thus, the antibacterial effect of quaternary ammonium compounds is inactivated by soap. Because of these problems, quats have been replaced by other antisep-tics and disinfectants for most purposes.



Phenol, one of the first effective disinfectants, was the primary agent employed by Listerin his antiseptic surgical procedure, which preceded the development of aseptic surgery. It is a potent protein denaturant and bactericidal agent. Substitutions in the ring structure of phenol have substantially improved activity and have provided a range of phenols and cresols that are the most effective environmental decontaminants available for use in hos-pital hygiene. Concern about their release into the environment in hospital waste and sewage has created some pressure to limit their use. This is another of the classic environ-mental dilemmas of our society: a compound that reduces the risk of disease for one group may raise it for another. Phenolics are less “quenched” by protein than are most other disinfectants, have a detergent-like effect on the cell membrane, and are often for-mulated with soaps to increase their cleansing property. They are too toxic to skin and tissues to be used as antiseptics, although brief exposures can be tolerated. They are the active ingredient in many mouthwash and sore throat preparations.

Two diphenyl compounds, hexachlorophene and chlorhexidine, have been extensively used as skin disinfectants. Hexachlorophene is primarily bacteriostatic. Incorporated into a soap, it builds up on the surface of skin epithelial cells over 1 to 2 days of use to pro-duce a steady inhibitory effect on skin flora and Gram-positive contaminants, as long as its use is continued. It was a major factor in controlling outbreaks of severe staphylococ-cal infections in nurseries during the 1950s and 1960s, but cutaneous absorption was found to produce neurotoxic effects in some premature infants. When it was applied in excessive concentrations, similar problems occurred in older children. It is now a pre-scription drug.

Chlorhexidine has replaced hexachlorophene as a routine hand and skin disinfectantand for other topical applications. It has greater bactericidal activity than hexa-chlorophene without its toxicity but shares with hexachlorophene the ability to bind to the skin and produce a persistent antibacterial effect. It acts by altering membrane permeabil-ity of both Gram-positive and -negative bacteria. It is cationic and, thus, its action is neu-tralized by soaps and anionic detergents.

Glutaraldehyde and Formaldehyde

Glutaraldehyde and formaldehyde are alkylating agents highly lethal to essentially all mi-croorganisms. Formaldehyde gas is irritative, allergenic, and unpleasant, properties that limit its use as a solution or gas. Glutaraldehyde is an effective high-level disinfecting agent for apparatus that cannot be heat treated, such as some lensed instruments and equipment for respiratory therapy. Formaldehyde vapor, an effective environmental de-contaminant under conditions of high humidity, is sometimes used to decontaminate labo-ratory rooms that have been accidentally and extensively contaminated with pathogenic bacteria, including those such as the anthrax bacillus that form resistant spores. Such rooms are sealed for processing and thoroughly aired before reoccupancy.

Some risk of infection exists in all health care settings. Hospitalized patients are par-ticularly vulnerable and the hospital environment is complex. The proper matching of the principles and procedures described here to general and specialized situations together with aseptic practices can markedly reduce the risks.

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