CLINICAL MICROBIOLOGY REVIEWS,
`0893-8512/99/$04.00⫹0
`Copyright © 1999, American Society for Microbiology. All Rights Reserved.
`
`Jan. 1999, p. 147–179
`
`Vol. 12, No. 1
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`Antiseptics and Disinfectants: Activity, Action, and Resistance
`GERALD MCDONNELL1* AND A. DENVER RUSSELL2
`STERIS Corporation, St. Louis Operations, St. Louis, Missouri 63166,1 and Welsh School
`of Pharmacy, Cardiff University, Cardiff CF1 3XF, United Kingdom2
`
`INTRODUCTION .......................................................................................................................................................148
`DEFINITIONS ............................................................................................................................................................148
`MECHANISMS OF ACTION ...................................................................................................................................148
`Introduction.............................................................................................................................................................148
`General Methodology .............................................................................................................................................148
`Alcohols ....................................................................................................................................................................151
`Aldehydes .................................................................................................................................................................151
`Glutaraldehyde ....................................................................................................................................................151
`Formaldehyde ......................................................................................................................................................153
`Formaldehyde-releasing agents.........................................................................................................................153
`o-Phthalaldehyde.................................................................................................................................................153
`Anilides.....................................................................................................................................................................153
`Biguanides................................................................................................................................................................153
`Chlorhexidine ......................................................................................................................................................153
`Alexidine...............................................................................................................................................................154
`Polymeric biguanides..........................................................................................................................................154
`Diamidines ...............................................................................................................................................................155
`Halogen-Releasing Agents .....................................................................................................................................155
`Chlorine-releasing agents ..................................................................................................................................155
`Iodine and iodophors .........................................................................................................................................155
`Silver Compounds...................................................................................................................................................155
`Silver nitrate........................................................................................................................................................156
`Silver sulfadiazine...............................................................................................................................................156
`Peroxygens ...............................................................................................................................................................156
`Hydrogen peroxide..............................................................................................................................................156
`Peracetic acid ......................................................................................................................................................156
`Phenols .....................................................................................................................................................................156
`Bis-Phenols ..............................................................................................................................................................157
`Triclosan ..............................................................................................................................................................157
`Hexachlorophene.................................................................................................................................................157
`Halophenols .............................................................................................................................................................157
`Quaternary Ammonium Compounds ...................................................................................................................157
`Vapor-Phase Sterilants ..........................................................................................................................................158
`MECHANISMS OF RESISTANCE..........................................................................................................................158
`Introduction.............................................................................................................................................................158
`Bacterial Resistance to Antiseptics and Disinfectants ......................................................................................158
`Intrinsic Bacterial Resistance Mechanisms........................................................................................................158
`Intrinsic resistance of bacterial spores............................................................................................................159
`Intrinsic resistance of mycobacteria ................................................................................................................160
`Intrinsic resistance of other gram-positive bacteria......................................................................................161
`Intrinsic resistance of gram-negative bacteria ...............................................................................................161
`Physiological (phenotypic) adaption as an intrinsic mechanism.................................................................162
`Acquired Bacterial Resistance Mechanisms .......................................................................................................164
`Plasmids and bacterial resistance to antiseptics and disinfectants ............................................................164
`(i) Plasmid-mediated antiseptic and disinfectant resistance in gram-negative bacteria......................164
`(ii) Plasmid-mediated antiseptic and disinfectant resistance in staphylococci .....................................165
`(iii) Plasmid-mediated antiseptic and disinfectant resistance in other gram-positive bacteria..........166
`Mutational resistance to antiseptics and disinfectants.................................................................................166
`Mechanisms of Fungal Resistance to Antiseptics and Disinfectants ..............................................................167
`Mechanisms of Viral Resistance to Antiseptics and Disinfectants .................................................................168
`Mechanisms of Protozoal Resistance to Antiseptics and Disinfectants..........................................................169
`Mechanisms of Prion Resistance to Disinfectants.............................................................................................169
`
`* Corresponding author. Present address: STERIS Corporation,
`5960 Heisley Rd., Mentor, OH 44060. Phone: (440) 354-2600. Fax:
`(440) 354-7038. E-mail: gerry_mcdonnell@steris.com.
`
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`MCDONNELL AND RUSSELL
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`CONCLUSIONS .........................................................................................................................................................169
`REFERENCES ............................................................................................................................................................170
`
`INTRODUCTION
`
`MECHANISMS OF ACTION
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`Introduction
`Considerable progress has been made in understanding the
`mechanisms of the antibacterial action of antiseptics and dis-
`infectants (215, 428, 437). By contrast, studies on their modes
`of action against fungi (426, 436), viruses (298, 307), and pro-
`tozoa (163) have been rather sparse. Furthermore, little is
`known about the means whereby these agents inactivate prions
`(503).
`Whatever the type of microbial cell (or entity), it is probable
`that there is a common sequence of events. This can be envis-
`aged as interaction of the antiseptic or disinfectant with the cell
`surface followed by penetration into the cell and action at the
`target site(s). The nature and composition of the surface vary
`from one cell type (or entity) to another but can also alter as
`a result of changes in the environment (57, 59). Interaction at
`the cell surface can produce a significant effect on viability (e.g.
`with glutaraldehyde) (374, 421), but most antimicrobial agents
`appear to be active intracellularly (428, 451). The outermost
`layers of microbial cells can thus have a significant effect on
`their susceptibility (or insusceptibility) to antiseptics and dis-
`infectants; it is disappointing how little is known about the
`passage of these antimicrobial agents into different types of
`microorganisms. Potentiation of activity of most biocides may
`be achieved by the use of various additives, as shown in later
`parts of this review.
`In this section, the mechanisms of antimicrobial action of a
`range of chemical agents that are used as antiseptics or disin-
`fectants or both are discussed. Different types of microorgan-
`isms are considered, and similarities or differences in the na-
`ture of the effect are emphasized. The mechanisms of action
`are summarized in Table 2.
`
`General Methodology
`
`A battery of techniques are available for studying the mech-
`anisms of action of antiseptics and disinfectants on microor-
`ganisms, especially bacteria (448). These include examination
`of uptake (215, 428, 459), lysis and leakage of intracellular
`constituents (122), perturbation of cell homeostasis (266,
`445), effects on model membranes (170), inhibition of en-
`zymes, electron transport, and oxidative phosphorylation (162,
`272), interaction with macromolecules (448, 523), effects on
`macromolecular biosynthetic processes (133), and microscopic
`examination of biocide-exposed cells (35). Additional and use-
`ful information can be obtained by calculating concentration
`exponents (n values [219, 489]) and relating these to mem-
`brane activity (219). Many of these procedures are valuable for
`detecting and evaluating antiseptics or disinfectants used in
`combination (146, 147, 202, 210).
`Similar techniques have been used to study the activity of
`antiseptics and disinfectants against fungi, in particular yeasts.
`Additionally, studies on cell wall porosity (117–119) may pro-
`vide useful information about intracellular entry of disinfec-
`tants and antiseptics (204–208).
`Mechanisms of antiprotozoal action have not been widely
`investigated. One reason for this is the difficulty in cultur-
`ing some protozoa (e.g., Cryptosporidium) under laboratory
`conditions. However, the different life stages (trophozoites
`and cysts) do provide a fascinating example of the problem
`
`Antiseptics and disinfectants are used extensively in hospi-
`tals and other health care settings for a variety of topical and
`hard-surface applications. In particular, they are an essential
`part of infection control practices and aid in the prevention of
`nosocomial infections (277, 454). Mounting concerns over the
`potential for microbial contamination and infection risks in the
`food and general consumer markets have also led to increased
`use of antiseptics and disinfectants by the general public. A
`wide variety of active chemical agents (or “biocides”) are
`found in these products, many of which have been used for
`hundreds of years for antisepsis, disinfection, and preservation
`(39). Despite this, less is known about the mode of action of
`these active agents than about antibiotics. In general, biocides
`have a broader spectrum of activity than antibiotics, and, while
`antibiotics tend to have specific intracellular targets, biocides
`may have multiple targets. The widespread use of antiseptic
`and disinfectant products has prompted some speculation on
`the development of microbial resistance, in particular cross-
`resistance to antibiotics. This review considers what is known
`about the mode of action of, and mechanisms of microbial
`resistance to, antiseptics and disinfectants and attempts, wher-
`ever possible, to relate current knowledge to the clinical envi-
`ronment.
`A summary of the various types of biocides used in antisep-
`tics and disinfectants, their chemical structures, and their clin-
`ical uses is shown in Table 1. It is important to note that many
`of these biocides may be used singly or in combination in a
`variety of products which vary considerably in activity against
`microorganisms. Antimicrobial activity can be influenced by
`many factors such as formulation effects, presence of an or-
`ganic load, synergy, temperature, dilution, and test method.
`These issues are beyond the scope of this review and are
`discussed elsewhere (123, 425, 444, 446, 451).
`
`DEFINITIONS
`
`“Biocide” is a general term describing a chemical agent,
`usually broad spectrum, that inactivates microorganisms. Be-
`cause biocides range in antimicrobial activity, other terms may
`be more specific, including “-static,” referring to agents which
`inhibit growth (e.g., bacteriostatic, fungistatic, and sporistatic)
`and “-cidal,” referring to agents which kill the target organism
`(e.g., sporicidal, virucidal, and bactericidal). For the purpose of
`this review, antibiotics are defined as naturally occurring or
`synthetic organic substances which inhibit or destroy selective
`bacteria or other microorganisms, generally at low concentra-
`tions; antiseptics are biocides or products that destroy or in-
`hibit the growth of microorganisms in or on living tissue (e.g.
`health care personnel handwashes and surgical scrubs); and
`disinfectants are similar but generally are products or biocides
`that are used on inanimate objects or surfaces. Disinfectants
`can be sporostatic but are not necessarily sporicidal.
`Sterilization refers to a physical or chemical process that
`completely destroys or removes all microbial life, including
`spores. Preservation is the prevention of multiplication of mi-
`croorganisms in formulated products, including pharmaceuti-
`cals and foods. A number of biocides are also used for cleaning
`purposes; cleaning in these cases refers to the physical removal
`of foreign material from a surface (40).
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`TABLE 1. Chemical structures and uses of biocides in antiseptics and disinfectants
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`of how changes in cytology and physiology can modify re-
`sponses to antiseptics and disinfectants. Khunkitti et al. (251–
`255) have explored this aspect by using indices of viability,
`leakage, uptake, and electron microscopy as experimental tools.
`
`Some of these procedures can also be modified for study-
`ing effects on viruses and phages (e.g., uptake to whole cells
`and viral or phage components, effects on nucleic acids and
`proteins, and electron microscopy) (401). Viral targets are
`
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`TABLE 1—Continued
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`predominantly the viral envelope (if present), derived from
`the host cell cytoplasmic or nuclear membrane; the capsid,
`which is responsible for the shape of virus particles and for
`the protection of viral nucleic acid; and the viral genome.
`Release of an intact viral nucleic acid into the environment
`
`following capsid destruction is of potential concern since
`some nucleic acids are infective when liberated from the cap-
`sid (317), an aspect that must be considered in viral disin-
`fection. Important considerations in viral inactivation are
`dealt with by Klein and Deforest (259) and Prince et al.
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`TABLE 1—Continued
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`(384), while an earlier paper by Grossgebauer is highly rec-
`ommended (189).
`
`Alcohols
`
`Although several alcohols have been shown to be effective
`antimicrobials, ethyl alcohol (ethanol, alcohol), isopropyl alco-
`hol (isopropanol, propan-2-ol) and n-propanol (in particular in
`Europe) are the most widely used (337). Alcohols exhibit rapid
`broad-spectrum antimicrobial activity against vegetative bacte-
`ria (including mycobacteria), viruses, and fungi but are not
`sporicidal. They are, however, known to inhibit sporulation
`and spore germination (545), but this effect is reversible (513).
`Because of the lack of sporicidal activity, alcohols are not
`recommended for sterilization but are widely used for both
`hard-surface disinfection and skin antisepsis. Lower concen-
`trations may also be used as preservatives and to potentiate the
`activity of other biocides. Many alcohol products include low
`levels of other biocides (in particular chlorhexidine), which
`remain on the skin following evaporation of the alcohol, or
`excipients (including emollients), which decrease the evapora-
`tion time of the alcohol and can significantly increase product
`efficacy (68). In general, isopropyl alcohol is considered slightly
`
`more efficacious against bacteria (95) and ethyl alcohol is more
`potent against viruses (259); however, this is dependent on the
`concentrations of both the active agent and the test microor-
`ganism. For example, isopropyl alcohol has greater lipophilic
`properties than ethyl alcohol and is less active against hydro-
`philic viruses (e.g., poliovirus) (259). Generally, the antimicro-
`bial activity of alcohols is significantly lower at concentrations
`below 50% and is optimal in the 60 to 90% range.
`Little is known about the specific mode of action of alcohols,
`but based on the increased efficacy in the presence of water, it
`is generally believed that they cause membrane damage and
`rapid denaturation of proteins, with subsequent interference
`with metabolism and cell lysis (278, 337). This is supported by
`specific reports of denaturation of Escherichia coli dehydroge-
`nases (499) and an increased lag phase in Enterobacter aero-
`genes, speculated to be due to inhibition of metabolism re-
`quired for rapid cell division (101).
`
`Aldehydes
`
`Glutaraldehyde. Glutaraldehyde is an important dialdehyde
`that has found usage as a disinfectant and sterilant, in partic-
`ular for low-temperature disinfection and sterilization of en-
`doscopes and surgical equipment and as a fixative in electron
`
`TABLE 2. Summary of mechanisms of antibacterial action of antiseptics and disinfectants
`
`Target
`
`Antiseptic or disinfectant
`
`Mechanism of action
`
`Cell envelope (cell wall, outer membrane)
`
`Glutaraldehyde
`EDTA, other permeabilizers
`
`Cross-linking of proteins
`Gram-negative bacteria: removal of Mg2⫹, release of some LPS
`
`Cytoplasmic (inner) membrane
`
`Cross-linking of macromolecules
`
`DNA intercalation
`
`QACs
`Chlorhexidine
`
`Diamines
`PHMB, alexidine
`Phenols
`
`Formaldehyde
`Glutaraldehyde
`
`Acridines
`
`Generalized membrane damage involving phospholipid bilayers
`Low concentrations affect membrane integrity, high concentrations
`cause congealing of cytoplasm
`Induction of leakage of amino acids
`Phase separation and domain formation of membrane lipids
`Leakage; some cause uncoupling
`
`Cross-linking of proteins, RNA, and DNA
`Cross-linking of proteins in cell envelope and elsewhere in the cell
`
`Intercalation of an acridine molecule between two layers of base
`pairs in DNA
`
`Interaction with thiol groups
`
`Silver compounds
`
`Membrane-bound enzymes (interaction with thiol groups)
`
`Effects on DNA
`
`Oxidizing agents
`
`Halogens
`Hydrogen peroxide, silver ions
`
`Inhibition of DNA synthesis
`DNA strand breakage
`
`Halogens
`Peroxygens
`
`Oxidation of thiol groups to disulfides, sulfoxides, or disulfoxides
`Hydrogen peroxide: activity due to from formation of free hydroxy
`radicals (䡠OH), which oxidize thiol groups in enzymes and pro-
`teins; PAA: disruption of thiol groups in proteins and enzymes
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`TABLE 3. Mechanism of antimicrobial action of glutaraldehyde
`Target
`microorganism
`
`Glutaraldehyde action
`
`Bacterial spores ..........Low concentrations inhibit germination; high con-
`centrations are sporicidal, probably as a conse-
`quence of strong interaction with outer cell layers
`Mycobacteria...............Action unknown, but probably involves mycobacte-
`rial cell wall
`
`Other nonsporulat-
`ing bacteria..............Strong association with outer layers of gram-positive
`and gram-negative bacteria; cross-linking of
`amino groups in protein; inhibition of transport
`processes into cell
`Fungi............................Fungal cell wall appears to be a primary target site,
`with postulated interaction with chitin
`Viruses.........................Actual mechanisms unknown, but involve protein-
`DNA cross-links and capsid changes
`Protozoa ......................Mechanism of action not known
`
`icroscopy. Glutaraldehyde has a broad spectrum of activity
`against bacteria and their spores, fungi, and viruses, and a
`considerable amount of information is now available about the
`ways whereby these different organisms are inactivated (Tables
`2 and 3). Earlier reviews of its mechanisms of action have been
`published (179, 182, 374, 482).
`The first reports in 1964 and 1965 (182) demonstrated that
`glutaraldehyde possessed high antimicrobial activity. Subse-
`quently, research was undertaken to evaluate the nature of its
`bactericidal (339–344, 450) and sporicidal (180, 181, 507, 508)
`action. These bactericidal studies demonstrated (374) a strong
`binding of glutaraldehyde to outer layers of organisms such as
`E. coli and Staphylococcus aureus (179, 212, 339–341, 343, 344),
`inhibition of transport in gram-negative bacteria (179), inhibi-
`tion of dehydrogenase activity (343, 344) and of periplasmic
`enzymes (179), prevention of lysostaphin-induced lysis in S. au-
`reus (453) and of sodium lauryl sulfate-induced lysis in E. coli
`(340, 344), inhibition of spheroplast and protoplast lysis in
`hypotonic media (340, 344), and inhibition of RNA, DNA, and
`protein synthesis (320). Strong interaction of glutaraldehyde
`with lysine and other amino acids has been demonstrated (450).
`Clearly, the mechanism of action of glutaraldehyde involves
`a strong association with the outer layers of bacterial cells,
`specifically with unprotonated amines on the cell surface, pos-
`sibly representing the reactive sites (65). Such an effect could
`explain its inhibitory action on transport and on enzyme sys-
`tems, where access of substrate to enzyme is prohibited. Partial
`or entire removal of the cell wall in hypertonic medium, lead-
`ing to the production of spheroplasts or protoplasts and the
`subsequent prevention of lysis by glutaraldehyde when these
`forms are diluted in a hypotonic environment, suggests an ad-
`ditional effect on the inner membrane, a finding substantiated
`by the fact that the dialdehyde prevents the selective release of
`some membrane-bound enzymes of Micrococcus lysodeikticus
`(138). Glutaraldehyde is more active at alkaline than at acidic
`pHs. As the external pH is altered from acidic to alkaline,
`more reactive sites will be formed at the cell surface, leading to
`a more rapid bactericidal effect. The cross-links thus obtained
`mean that the cell is then unable to undertake most, if not all,
`of its essential functions. Glutaraldehyde is also mycobacteri-
`cidal. Unfortunately, no critical studies have as yet been un-
`dertaken to evaluate the nature of this action (419).
`The bacterial spore presents several sites at which interac-
`tion with glutaraldehyde is possible, although interaction with
`a particular site does not necessarily mean that this is associ-
`ated with spore inactivation. E. coli, S. aureus, and vegetative
`cells of Bacillus subtilis bind more glutaraldehyde than do rest-
`
`ing spores of B. subtilis (377, 378); uptake of glutaraldehyde is
`greater during germination and outgrowth than with mature
`spores but still lower than with vegetative cells. Low concen-
`trations of the dialdehyde (0.1%) inhibit germination, whereas
`much higher concentrations (2%) are sporicidal. The alde-
`hyde, at both acidic and alkaline pHs, interacts strongly with
`the outer spore layers (508, 509); this interaction reduces the
`release of dipicolinic acid (DPA) from heated spores and the
`lysis induced by mercaptoethanol (or thioglycolate)-peroxide
`combinations. Low concentrations of both acidic and alkaline
`glutaraldehyde increase the surface hydrophobicity of spores,
`again indicating an effect at the outermost regions of the cell.
`It has been observed by various authors (182, 374, 376, 380)
`that the greater sporicidal activity of glutaraldehyde at alkaline
`pH is not reflected by differences in uptake; however, uptake
`per se reflects binding and not necessarily penetration into the
`spore. It is conceivable that acidic glutaraldehyde interacts
`with and remains at the cell surface whereas alkaline glutaral-
`dehyde penetrates more deeply into the spore. This contention
`is at odds with the hypothesis of Bruch (65), who envisaged the
`acidic form penetrating the coat and reacting with the cortex
`while the alkaline form attacked the coat, thereby destroying
`the ability of the spore to function solely as a result of this
`surface phenomenon. There is, as yet, no evidence to support
`this theory. Novel glutaraldehyde formulations based on acidic
`rather than alkaline glutaraldehyde, which benefit from the
`greater inherent stability of the aldehyde at lower pH, have
`been produced. The improved sporicidal activity claimed for
`these products may be obtained by agents that potentiate the
`activity of the dialdehyde (414, 421).
`During sporulation, the cell eventually becomes less suscep-
`tible to glutaraldehyde (see “Intrinsic resistance of bacterial
`spores”). By contrast, germinating and outgrowing cells reac-
`quire sensitivity. Germination may be defined as an irreversible
`process in which there is a change of an activated spore from
`a dormant to a metabolically active state within a short period.
`Glutaraldehyde exerts an early effect on the germination pro-
`cess. L-Alanine is considered to act by binding to a specific
`receptor on the spore coat, and once spores are triggered to
`germinate, they are committed irreversibly to losing their dor-
`mant properties (491). Glutaraldehyde at high concentrations
`inhibits the uptake of L-[14C]alanine by B. subtilis spores, albeit
`by an unknown mechanism (379, 414). Glutaraldehyde-treated
`spores retain their refractivity, having the same appearance
`under the phase-contrast microscope as normal, untreated
`spores even when the spores are subsequently incubated in
`germination medium. Glutaraldehyde is normally used as a 2%
`solution to achieve a sporicidal effect (16, 316); low concen-
`trations (⬍0.1%) prevent phase darkening of spores and also
`prevent the decrease in optical density associated with a late
`event in germination. By contrast, higher concentrations (0.1
`to 1%) significantly reduce the uptake of L-alanine, possibly as
`a result of a sealing effect of the aldehyde on the cell surface.
`Mechanisms involved in the revival of glutaraldehyde-treated
`spores are discussed below (see “Intrinsic resistance of bacte-
`rial spores”).
`There are no recent studies of the mechanisms of fungicidal
`action of glutaraldehyde. Earlier work had suggested that the
`fungal cell wall was a major target site (179, 182, 352), espe-
`cially the major wall component, chitin, which is analogous to
`the peptidoglycan found in bacterial cell walls.
`Glutaraldehyde is a potent virucidal agent (143, 260). It
`reduces the activity of hepatitis B surface antigen (HBsAg) and
`especially hepatitis B core antigen ([HBcAg] in hepatitis B
`virus [HBV]) (3) and interacts with lysine residues on the
`surface of hepatitis A virus (HAV) (362). Low concentrations
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`(⬍0.1%) of alkaline glutaraldehyde are effective against puri-
`fied poliovirus, whereas poliovirus RNA is highly resistant to
`aldehyde concentrations up to 1% at pH 7.2 and is only slowly
`inactivated at pH 8.3 (21). In other words, the complete po-
`liovirus particle is much more sensitive than poliovirus RNA.
`In light of this, it has been inferred that glutaraldehyde-in-
`duced loss of infectivity is associated with capsid changes (21).
`Glutaraldehyde at the low concentrations of 0.05 and 0.005%
`interacts with the capsid proteins of poliovirus and echovirus,
`respectively; the differences in sensitivity probably reflect ma-
`jor structural variations in the two viruses (75).
`Bacteriophages were recently studied to obtain information
`about mechanisms of virucidal action (298–304, 306, 307). Many
`glutaraldehyde-treated P. aeruginosa F116 phage particles had
`empty heads, implying that the phage genome had been eject-
`ed. The aldehyde was possibly bound to F116 double-stranded
`DNA but without affecting the molecule; glutaraldehyde also
`interacted with phage F116 proteins, which were postulated to
`be involved in the ejection of the nucleic acid. Concentrations
`of glutaraldehyde greater than 0.1 to 0.25% significantly af-
`fected the transduction of this phage; the transduction process
`was more sensitive to the aldehyde than was the phage itself.
`Glutaraldehyde and other aldehydes were tested for their
`ability to form protein-DNA cross-links in simian virus 40
`(SV40); aldehydes (i.e., glyoxal, furfural, prionaldehyde, acet-
`aldehyde, and benzylaldehyde) without detectable cross-link-
`ing ability had no effect on SV40 DNA synthesis, whereas
`acrolein, glutaraldehyde, and formaldehyde, which formed
`such cross-links (144, 271, 297), inhibited DNA synthesis (369).
`Formaldehyde. Formaldehyde (methanal, CH2O) is a mono-
`aldehyde that exists as a freely water-soluble gas. Formalde-
`hyde solution (formalin) is an aqueous solution containing ca.
`34 to 38% (wt/wt) CH2O with methanol to delay polymeriza-
`tion. Its clinical use is generally as a disinfectant and sterilant
`in liquid or in combination with low-temperature steam. Form-
`aldehyde is bactericidal, sporicidal, and virucidal, but it works
`more slowly than glutaraldehyde (374, 482).
`Formaldehyde is an extremely reactive chemical (374, 442)
`that interacts with protein (156, 157), DNA (155), and RNA
`(155) in vitro. It has long been considered to be sporicidal by
`virtue of its ability to penetrate into the interior of bacterial
`spores (500). The interaction with protein results from a com-
`bination with the primary amide as well as with the amino
`groups, although phenol groups bind little formaldehyde (155).
`It has been proposed that formaldehyde acts as a mut