18 - Disinfection

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DisinfectionS. SpecterD. JeffriesVirucidal agents may inactivate viruses eitherbecause of their physical or chemical proper-t ies. These agents general ly are effect ive onviruses outside of their host cel ls. The abil i tyof these agents to inactivate virus is veryclosely associated wi th the condi t ions underwhich they are used. Depending on the agent ,this may include temperature, pH, moisturecontent of the sample, concentrat ion andcomposi t ion of the vi rus, concentrat ion of theagent, and t ime of exposure. Thus, viruseskept at room temperature wi l l become inact i -vated more rapidly than those held at 4~Likewise some vi ruses maintained at neutralpH may be inactivated less rapidly than ateither lower or higher pH (Lancz 1976), othervi ruses su ch as enteroviruses m ay be stable athighly acidic pH. So me vi ruses are also kno wnto lose infectivi ty as the sample in which theyare col lected dries (Parker et al 1944). Thus,vi rus maintained in mucus containing secre-t ions on an environmental surface is l ikely toretain infect ivi ty longer than the same amountof virus in sal iva, since the latter wil l dry morequickly (Dunham 1977).

An important factor in the ef fect iveness ofdisinfection is the virus t i ter present at theoutset of addi t ion of the disinfectant . Theloss of infectivi ty over t ime is such that i f asample that is treated with a virucidal agentis held for 15 min i t may lose 50% of i ts infec-t ivi ty, after 30 min 90% is lost, and by 60 minthere is a 99% loss. I f a sample contains 1000infectious part icles per mil l i l i ter at outset, after15 min 0.1 ml wil l conta in app roxim ately 5infectious units, where as a fter 60 min 0.1 mlVirology Methods ManualISBN 0-12-465330-8

has a 10% chance of containing infect iousmaterial. By contrast, i f the original concentra-tion of virus is 106 particles per mill i l iter, theneven a 99% inactivation after 1 h may leavesuff icient infectious material to cause infec-t ion; i .e. 1000 infectious units wil l be presentin 0.1 ml.

The abil i ty of various agents to inactivateviruses is also dependent on the composi t ionof the virus. Ma ny viruses co ntain a l ipid envel-ope that is sensit ive to l ipid solvents such asether , chloroform or detergents, whereasnaked (non-enveloped) viruses are resistantto such agents (Dunham 1977). Final ly, thenature of the disinfect ion agents can deter-mine the extent of inactivation. This wil ldepend upon whether the agent is physicalor chemical and if i t is organic or inorganic. I tshould be noted that other organic material insolut ions containing vi ruses may inhibi t thevirucidal ef fects of many disinfectants. Wied-brauk and Johnston (1993) have del ineatedwhich compounds are par t icular ly useful forvi ruses that are commonly encountered bothin the laboratory and on environmental sur-faces. Inact ivat ion of commonly used disinfec-tants is shown in Table 18.1.

The methods for test ing vi rucidal agents arewell del ineated (Koski and Chen 1977).

Inorganic chemicalagentsThe most commonly used inorganic disinfec-tants are halophi l ic compounds, includingCopyright 9 1996 Academic Press LtdAll rights of reproduction in any form reserved


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V i ro l o g y m e t h o d s m a n u a lTable 18.1 . Inactivation of com monly used disinfectants

Protein Hard Water DetergentsHypoch lorites ++++ + CPhenolics + + CAlcohols* + + --Formaldehyde + + -Glutaraldehyde + + -

. . . . . . , . . , . . m . . .* Alcohols are extremely poor in penetrating proteinaceous material and are, therefore, ofvery limited use as disinfectants.C, Cationic detergentschlorine and bromine (Brown et a11963; Clarkeand Kabler 1964; Lund 1964). The most readilyavailable antiviral for use is common house-hold bleach (sodium hypochlori te), whichusually can be used as a 10% solut ion (thesolut ion must contain 10,000 ppm avai lablechlorine), to inactivate many enveloped andnaked viruses. The bleach is capable ofdestroying nucleic acids, making i t a univer-sal ly effect ive disinfectant for viruses. Thus, i tis the recommended disinfectant for rout ineuse in the cl inical laboratory, the physician'sof f ice, and any set t ing where concerns aboutviral contamination exist. I ts abi l i ty to inacti-vate nucleic acids makes household bleachan ideal agent for deconta minat ion of a labora-tory whe re polyme rase chain react ions are per-formed, and carry over of nucleic acids is aconcern (Prince and Andrews 1992). Chlorineand bromine are useful for the inactivation ofvi ruses in swimm ing pools when used at about0.5 parts per mil lion. Iodine also can be use d asa disinfectant of contaminated water suppl ies(Dunham and MacNeal 1944; Kabler 1962).

Ozone is considered to be safer and moreeffective than chlorine for the disinfection ofwater, but is not practical for widespread usedue to cost (Sl i ter 1974). Similar ly, hydrogenperoxide can be used to inactivate viruses.However, the presence of other organic mate-r ials in f luids wil l compete with the viruses andblock the ef fects of the H202. Acid solut ions,such as 1 N HCI, and alkaline co mpo und s,such as 2% NaOH are also useful disinfec-tants for inactivation of viruses.

Metals and metal salts also have antiviralpropert ies. Copper salts, mercury, potassium

permanganate and si lver ni t rate have beenused as virucidal agents (Dunham 1977).

Organic chemical agentsThe l ist of organic compounds wi th vi rucidalactivi ty is quite long, and includes quaternaryammonium compounds, a lcohols, a ldehydes,ether, glycerol, beta-propiolactone, ethyleneoxide, digest ive enzymes and a preparat ionknown as l iquor ant isept icus.

Benzalkonium chloride (Zephiran) has beendemonstrated to be useful for the disinfect ionof skin. I t is the preparation of choice forcleansing animal bites, due to i ts abi l i ty toinactivate rabiesvirus when used at a 1:1000di lut ion. By contrast this compound is nothighly effect ive against naked viruses. O-phe-nylphenol is l ikewise act ive aga inst envelopedbut not naked viruses (Klein and Deforest1963). Diethyl ether is also an effective agentfor inactivating l ipid containing viruses, show-ing vi rucidal act iv i ty at concentrat ions of 3-10% for these viruses. However, ether is notef fect ive against pol iovi rus (a non-envelopedvirus), even at 95% concentrat ion.

Seventy percent ethanol is an ef fect ive dis-infectant for naked and e nveloped vi ruses, butcan be decreased in act ivi ty by substancesthat precipi tate alcohol . This can be over-come by the addi t ion of 0.005 N NaOH to thealcohol solut ion. Whi le lower conce ntrat ions ofethanol may be effectively virucidal, the higherconcentrat ion is more ef fect ive, wi th mostvi rus destroyed wi thin a few minutes by 70%.Methanol also may be used to inact ivate vi rus,

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Organic disinfection agentsbut is considerably less effective than ethanol(Cox et al 1947). However, alcohols penetrateorganic matter rather poorly and are not effec-t ive for disinfection of such materials. Further-more, the inactivation of HIV and other virusesby alcohols is slow and the alcohol is l ikely toevaporate, i f used for surface decontamina-t ion, before the virus is inactivated (Hansonet al 1989). Liquor antisepticus is a compounddescribed in the National Formulary that canbe used as a mouthwash (National Formulary1965). Its principal ingredient is ethyl alcohol(28.5% by volume).Form aldeh yde is an excellent virucidal agent,and can be use d to inactivate viruses as a 0.3%solut ion (Dunham 1977; Wiedbrauk and John-ston 1993). Similar ly gluteraldehyde is veryuseful for cold steri l izat ion and a 2% solut ionwil l inactivate most virus in 10 min when buf-fered wi th sodium bicarbonate at pH 7.5-8.5.Beta-propiolactone is an effective virucidalagent, even when virus is in plasma, that haslow toxicity in mammalian systems. I t has beenused in the preparation of vaccines, such asthe Merieux rabies vaccine.

Physical agentsElevated temperature has a dist inct antiviraleffect, increasing the rate of virus inactiva-t ion. The inactivation rate due to high tempera -ture varies for dif ferent viruses and can bechanged greatly by the pH of a solut ion. Thepresence of a protein stabi l izer such as serumdecreases the abil i ty of heat to inactivate virusin a suspension. Most viruses lose some infec-t ivi ty when heated at 56~ for 30 min. Allv i ruses are destroyed by appropr iate autoclav-ing, 121~ und er 15 psi press ure for 15 min.Ultraviolet radiat ion is also a highly effect ivevirucidal physical agent, as long as it is ofsuitable wavelength and intensity. Wave-lengths of a pproximately 250 nm have beendemonstrated to be effective against inf lu-enza virus (Dunham 1977). The effectivenessis signif icantly diminished when the virus sus-pension is moved away f rom the source ofradiation. In addition, ultraviolet rays aremore effective through air than water, andpart iculate matter (dust part icles or salt crys-tals) can further decrease effectiveness.

Ionizing radiat ion, including X-rays, gammarays, high energy electrons, deuterons andalpha part icles, is capable of inactivatingviruses. This is effected by the damaging ofviral nucleic acids. However, radical scaven-gers are able to inhibit the effects of ionizingradiation. Viruses have different levels of resis-tance to ultraviolet and gamma radiat ion,depending on the target size of the nucleicacid. Many of the doses necessary for inacti-vation of mammalian viruses are l isted byMcCrea (1960). Ionizing radiat ion has beenused for many years for steri l izat ion of dispo-sable medical suppl ies, and has become morepopular of late for more common consumableitems including food.Fi l ters may also be used for the steri l izat ionof l iquids or air. While mo st c om mo n f i lters thatare used to remove bacteria from solut ions donot remove viruses, small pore f i l ters can beused to hold back viruses. This practice ismost commonly used for f i l ter ing air in bio-logical safety cabinets or 'clean rooms' usinghepa-filters. The use of such filters is generallyreserved for areas where t issue culture is per-formed, to provide steri le air, or in Biosafetylevel 3 and 4 rooms, to remove the r isk ofcontaminat ion of the environment when patho-genic viruses are studied.SummaryThere are a wide variety of methods that canbe used for steri l izat ion, disinfection and anti-sepsis. When possible, the recommendedmethod for steri l izat ion is steam heating usingan autoclave. An alternative effective methodof l imit ing contamination is to use disposablesterile items (needles, syringes, pipettes,tubes, f lasks, etc.) that come in a sealed ster-i le container. Such items, i f not autoclavable,can be steri l ized using y-irradiat ion or ethyleneoxide gas after packaging, to achieve steri l iza-t ion. The recommended disinfectants fordecontaminat ion of sur faces or solut ions con-taining viruses are bleach or gluteraldehyde.Instruments that are reused should becleaned with detergents that wil l removeorganic matter, pr ior to disinfection with thesesubstances, as such material promotes survi-val of viruses.

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ReferencesBrown JR, M cLean DM, Nixon MC (1963) Can J Pub

Hlth 54: 267-270.Clarke NA, Kabler PW (1954) Am J Hyg 59: 119 -127.Cox HR, van der Scheer J, Aiston S, B ohnel E (1947)J Immunol 56: 149-166.Dunham WB (1977)In: Disinfection, Sterilization andPreservation, 2nd Edn, Bloc k SS (Ed.) Lea andFebiger, Philadelphia, pp. 426-441Dunham W B, M acNeal WJ (194 4) J Immunol 49:123-128.Hanson PJV, Gor D, Jeffries DJ, Collins JV (1989)Brit Med J 298: 862-864.Kabler PW (1962) Ann Rev Microbiol 16: 127-140.Klein, M D eforest A (1963) Soap Che m Spe c 39: 70-72.

Koski TA, Chen JHS (1 977) In: Disinfection, Steril-ization and Preservation 2nd Ed n, Block SS (Ed.)Lea and Febiger, Philadelphia, pp. 116-134.Lancz GJ (1976) Virology 75: 488 -491.Lund E (1964) Am J Hyg 80: 1 -10.McCrea JF (1960) Ann NY Acad Sci 83: 692-705.National Formulary, 12th Ed. (NF XlI) (1965 ) Am er-ican Pharmaceutical Assoc, Washington, pp. 36-37.Parker ER, Dunham WB, MacN eal WJ (19 44) J LabClin Med 29: 37-42.Prince AM, A ndrus L (1992) BioTechniques 12: 358-360.Sliter JT (1974) J Water P ollut Control Fed 46: 4-6.

Wiedbrauk DL, Johnston SLG (19 93) Manual ofClinical Virology, Raven Press, Ne w York, 273 pp.

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18 - Disinfection

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