The common disinfectants used for disinfection.ppt

The common disinfectants used for disinfection

1. 1
Chemical disinfectants,
antiseptics and preservatives.

2. 2
Disinfectants:
Disinfectantsare chemical agents which are
used to destroy microorganisms on inanimate
(dead) objects.
Disinfectant does not necessarily kill all
microorganisms, but it reduces them to a level
which is acceptable for a define purpose.
The term disinfectant can be used to include
antiseptics, in a wider sense ( but British
Standards Institution consider antiseptic is not a
synonym for disinfectant).
Disinfectants on the other hand are non
selective, irritant, corrosive or toxic to be applied
to the skin or to tissues.

3. 3
Disinfection levels:
1. High Germicidal: (p:286)
Killed all microorganisms unless extreme challenge or resistance exhibited.
M. surviving ( challenge of resistant bacterial spore, prions)
Bactericidal, Sporocidal, Fungicidal & Virucidal
e.g. Ethylene oxide, Formaldehyde, Glutaraldehyde.
2. Intermediate Germicidal:
Killed most vegetative bacteria including M. tuberculosis, most viruses
including hepatitis B virus (HBV), most fungi. M. surviving (bacterial spores,
prions)
Bactericidal, Non-sporocidal, Fungicidal &Virucidal
e.g. Alcohol, Chlorine compounds & Iodine solution.
3. Low Germicidal:
Killed most vegetative bacteria, some viruses, some fungi. M. surviving (M.
tuberculosis, bacterial spore, some viruses, prions)
Bactericidal, Fungicidal & kill lipid viruses only
e.g. Chloroxylenoles, Thiomersal, QACs, Chlorhexidine, Iodophors.

4. 4
Levels of disinfection attainable
Disinfection level
HighIntermediateLow
All
microorganisms
unless extreme
challenge or
resistance
exhibited
Most vegetative
bacteria including
M. tuberculosis
Most viruses including
hepatitis
B virus (HBV)
Most fungi
Most vegetative
bacteria
Some viruses
Some fungi
Microorganisms
killed
Extreme
challenge of
resistant
bacterial spores
Prions
Bacterial spores
Prions
M. Tuberculosis
Bacterial spores
Some viruses and
prions
Microorganisms
surviving
4

5. 5
Somehighleveldisinfectantshavegood
sporicidalactivityandhavebeenascribed
thename‘liquidchemicalsterilant’or
‘chemosterilant’toindicatethattheycan
effectacompletekillofallmicroorganisms,
asinsterilization.
5

6. 6
Disinfectants
6

7. 7
Antiseptics:
Antisepticsare chemical agents which are used
to destroy or inhibition of microorganisms on
living tissues.
Antisepticshave more selectivity in their action
toward the bacteria than the living tissues, at the
recommended concentration ,therefore
antiseptics usually exert a bacteriostaticor low
bactericidal effect at the recommended
concentration.
Antiseptics must not be toxic or irritatingto skin,
and they are often lower concentrations of the
agents used for disinfection.

8. 8
Antiseptics
8

9. 9
Preservatives:
Preservatives:Theseareincludedinpharmaceutical
preparationstopreventmicrobialspoilageoftheproduct
andtominimizetheriskoftheconsumeracquiringan
infectionwhenthepreparationisadministered.
Preservativesmustbeabletolimitproliferationof
microorganismsthatmaybeintroducedunavoidablyinto
non-sterileproducts(oral&topicalmedications)during
theirmanufactureanduse,whileinsterileproducts(eye
dropsandmulti-doseinjections)preservativesshouldkill
anymicrobialcontaminantsintroducedinadvertently
duringuse.

10. 10
Preservative is not toxic, and employed at lower
concentrations, and levels of antimicrobial action lower
order than for disinfectants or antiseptics(European
Pharmacopoeia ).
There are around 250chemicals that have been
identified as active components of microbiocidalproducts
in the European Union.

11. 11
Preservativ
es
11

12. 12
Factors affecting choice of
antimicrobial agent:(P:286)
Properties of the chemical agent.
Microbiological challenge.
Intended application.
Environmental factors.
Toxicity of the agent.

13. 13
Theprocessofkillingorinhibitingthe
growthofmicroorganismsusinganantimicrobial
agentisbasicallythatofachemicalreactionand
therateandextentofthisreactionwillbe
influencedbythefactorsofconcentrationof
chemical,temperature,pHandformulation.
Properties of the chemical agent
13

14. Properties of the chemical agent
Tissue toxicity influences whether a chemical
can be used as an antiseptic or preservative,
and this limits the range of chemicals for these
applications or necessitates the use of lower
concentrations of the chemical.
14

15. 15
Microbiological challenge
•Thetypesofmicroorganismpresentandthe
levelsofmicrobialcontamination(thebioburden)
bothhaveasignificanteffectontheoutcomeof
chemicaltreatment.
•Ifthebioburdenishigh,longexposuretimesor
higherconcentrationsofantimicrobialmaybe
required.
15

16. 16
Microbiological challenge
•Microorganismsvaryintheirsensitivitytothe
actionofchemicalagents.
•Someorganisms,eitherbecauseoftheir
resistancetodisinfectionorbecauseoftheir
significanceincross-infectionornosocomial,
meritattention.
16

17. 17
Microbiological challenge
• ofparticularconcernisthesignificant
increaseinresistancetodisinfectants
resultingfrommicrobialgrowthinbiofilm
formratherthanfreesuspension.
•Microbialbiofilmsformreadilyon
availablesurfaces,posingaserious
problemforHospitalInfectionControl
Committees inadvisingsuitable
disinfectantsforuseinsuchsituations.
17

18. 1818

19. 19
Vegetative bacteria
Atin-useconcentrations,chemicalsusedfor
disinfectionshouldbecapableofkillingmost
vegetativebacteriawithinareasonablecontact
period.Thisincludes‘problem’organismssuchas
listeria,campylobacter,legionella,vancomycin-
resistantenterococci(VRE)andmethicillin-
resistantStaphylococcusaureus(MRSA)..
19

20. 20
Vegetative bacteria
listeria
CHROM agar Listeria
20

21. 21
Vegetative bacteria
campylobacter
21

22. 22
Legionella
Legionellaon BCYE agar plate
22

23. 23
•Antisepticsandpreservativesarealsoexpectedto
haveabroadspectrumofantimicrobialactivitybutat
thein-useconcentrations,afterexertinganinitial
biocidaleffect,theirmainfunctionmaybebiostatic.
•Gramnegativebacilli,whicharethemaincausesof
nosocomialinfections,areoftenmoreresistantthan
Gram-positivespecies.
Vegetative bacteria
23

24. 24
•Pseudomonasaeruginosa,anopportunistpathogen
hasgainedareputationasthemostresistantofthe
Gram-negativeorganisms.However,problemsmainly
arisewhenanumberofadditionalfactorssuchas
heavilysoiledarticlesordilutedordegradedsolutions
areinvolved.
Vegetative bacteria
24

25. 25
Pseudomonas aeruginosa
25

26. 26
Mycobacterium tuberculosis
•M.tuberculosisandothermycobacteriaareresistant
tomanybactericides.
•Resistanceiseither(a)intrinsic,mainlydueto
reducedcellularpermeabilityor(b)acquired,dueto
mutationortheacquisitionofplasmids.
•Tuberculosisremainsanimportantpublichealth
hazard,andindeedtheannualnumberoftuberculosis
casesisrisinginmanycountries.
26

27. 27
Mycobacterium tuberculosis
•Thegreatestriskofacquiringinfectionis
fromtheundiagnosedpatient.
•Equipmentusedforrespiratoryinvestigations
canbecomecontaminatedwithmycobacteriaif
thepatientisacarrierofthisorganism.
•Itisimportanttobeabletodisinfectthe
equipmenttoasafeleveltoprevent
transmissionofinfectiontootherpatients
27

28. 28
Mycobacterium tuberculosis
28

29. 29
Bacterial spores
•Oftheconventionalmicroorganisms,bacterialsporesarethe
mostresistanttochemicaltreatment.
•Themajorityofantimicrobialagentshavenousefulsporicidal
actioninapharmaceuticalcontext.
•However,certainaldehydes,halogensandperoxygen
compoundshaveexcellentactivityundercontrolledconditions
andaresometimesusedasanalternativetophysicalmethodsfor
sterilizationofheat-sensitiveequipment.
•Inthesecircumstances,correctusageoftheagentisof
paramountimportance,assafetymarginsarelowerincomparison
withphysicalmethodsofsterilization
29

30. 30
Bacterial spores
30

31. 31
Fungi
•Thevegetativefungalformisoftenassensitiveasvegetative
bacteriatoantimicrobialagents.
•Fungalspores(conidiaandchlamydospores)maybemore
resistant,butthisresistanceisofmuchlessermagnitudethanfor
bacterialspores.
•Theabilitytorapidlydestroypathogenicfungisuchasthe
importantnosocomialpathogen,Candidaalbicans,filamentous
fungisuchasTrichophytonmentagrophytes,andsporesof
commonspoilagemouldssuchasAspergillusnigerisputto
advantageinmanyapplicationsofuse.
31

32. 32
Fungi
•Manydisinfectantshavegoodactivityagainstthesefungi.In
addition,ethanol(70%)israpidandreliableagainstCandida
species.
32

33. 33
Candida albicans
33

34. 34
Trichophyton mentagrophytes
34

35. 35
Antifungal activity of disinfectants and
antiseptics.
35

36. 36
Viruses
•Susceptibilityofvirusestoantimicrobialagentscandependon
whetherthevirusespossessalipidenvelope.
•Non-lipidvirusesarefrequentlymoreresistanttodisinfectants
anditisalsolikelythatsuchvirusescannotbereadily
categorizedwithrespecttotheirsensitivitiestoantimicrobial
agents.
•Thesevirusesareresponsibleformanynosocomialinfections,
36

37. 37
Viruses
e.g.rotaviruses,picornavirusesandadenovirusesanditmaybe
necessarytoselectanantisepticordisinfectanttosuitspecific
circumstances.
•Certainviruses,suchasEbolaandMarburg,whichcause
haemorrhagicfevers,arehighlyinfectiousandtheirsafe
destructionbydisinfectantsisofparamountimportance.
•HepatitisAisanenterovirusconsideredtobeoneofthemost
resistantvirusestodisinfection.
37

38. 38
Viruses
•Thereismuchconcernforthesafetyofpersonnelhandling
articlescontaminatedwithpathogenicvirusessuchashepatitisB
virus(HBV)andhumanimmunodeficiencyvirus(HIV)which
causesAIDS(acquiredimmunedeficiencysyndrome).
•Disinfectantsmustbeabletotreatrapidlyandreliablyaccidental
spillsofblood,bodyfluidsorsecretionsfromHIV-infected
patients.
•SuchspillsmaycontainlevelsofHIVashighas10
4
infectious
units/ml.Fortunately,HIVisinactivatedbymostchemicalsatin-
useconcentrations.
38

39. 39
Viruses
However,therecommendationistousehighleveldisinfectants
(Table17.2)fordecontaminationofHIV-orHBV-infected
reusablemedicalequipment.
•Forpatient-careareascleaninganddisinfectionwith
intermediateleveldisinfectantsissatisfactory.Floodingwitha
liquidgermicideisonlyrequiredwhenlargespillsofculturedor
concentratedinfectiousagentshavetobedealtwith.
39

40. 40
Protozoa
Acanthamoebaspp.cancauseacanthamoeba
keratitiswithassociatedcornealscarringandlossof
visioninsoftcontactlenswearers.
•Thecystsofthisprotozoanpresentaparticular
probleminrespectoflensdisinfection.Thechlorine-
generatingsystemsinusearegenerallyinadequate.
Althoughpolyhexamethylenebiguanideshows
promiseasanacanthamoebicide,onlyhydrogen
peroxide-baseddisinfectionisconsideredcompletely
reliableandconsistentinproducingan
acanthamoebicidaleffect.
40

41. 41
Acanthamoebas
41

42. 42
Prions
Prionsaregenerallyconsideredtobetheinfectiousagentsmost
resistanttochemicaldisinfectantsandsterilizationprocesses;
strictlyspeaking,however,theyarenotmicroorganismsbecause
theyhavenocellularstructurenordotheycontainnucleicacids.
Prions(smallproteinaceousinfectiousparticles)areaunique
classofinfectiousagentcausingspongiformencephalopathies
suchasbovinespongiformencephalopathy(BSE)incattleand
Creutzfeldt–Jakobdisease(CJD)inhumans.Thereis
considerableconcernaboutthetransmissionoftheseagentsfrom
infectedanimalsorpatients.
42

43. 43
Prions
Riskofinfectivityishighestinbrain,spinalcordandeye
tissues.Therearestillmanyunknownfactorsregarding
destructionofprions.
Andtheyareconsideredresistanttomostdisinfectant
procedures.Forheat-resistantmedicalinstrumentsthat
comeintocontactwithhighinfectivitytissuesorhigh-
riskcontacts,immersioninsodiumhydroxide
(1N)orsodiumhypochlorite(20000ppmavailable
chlorine)for1hourisadvisedinWHOguidelinesand
thismustbefollowedbyfurthertreatmentincluding
autoclaving,cleaningandroutinesterilization.
43

44. 44
Prions
44

45. 45
Intended application
Theintendedapplicationoftheantimicrobial
agent,whetherforpreservation,antisepsisor
disinfection,willinfluenceitsselectionandalso
affectitsperformance.Forexample,inmedicinal
preparationstheingredientsintheformulation
mayantagonizepreservativeactivity.
•Therisktothepatientwilldependonwhether
theantimicrobialisinclosecontactwithabreak
intheskinormucousmembranesoris
introducedintoasterileareaofthebody.
45

46. 46
Intended application
Indisinfectionofinstruments,thechemicalsusedmustnot
adverselyaffecttheinstruments,e.g.causecorrosionof
metals,affectclarityorintegrityoflenses,orchangethe
textureofsyntheticpolymers.Manymaterialssuchasfabrics,
rubberandplasticsarecapableofadsorbingcertain
disinfectants,e.g.quaternaryammoniumcompounds(QACs)
areadsorbedbyfabrics,whilephenolicsareadsorbedby
rubber,theconsequenceofthisbeingareductioninthe
concentrationofactivecompound.Adisinfectantcanonlyexert
itseffectifitisincontactwiththeitembeingtreated.Therefore
accesstoallpartsofaninstrumentorpieceofequipmentis
essential.Forsmallitems,totalimmersioninthedisinfectant
mustalsobeensured
46

47. 47
Environmental factors
Organicmattercanhaveadrasticeffecton
antimicrobialcapacityeitherbyadsorptionor
chemicalinactivation,thusreducingthe
concentrationofactiveagentinsolutionorby
actingasabarriertothepenetrationofthe
disinfectant.Blood,bodyfluids,pus,milk,food
residuesorcolloidalproteins,evenpresentin
smallamounts,allreducetheeffectivenessof
antimicrobialagentstovaryingdegrees,and
someareseriouslyaffected
47

48. 48
Environmental factors
Intheirnormalhabitats,microorganismshaveatendencyto
adheretosurfacesandarethuslessaccessibletothechemical
agent.Someorganismsarespecifictocertainenvironmentsand
theirdestructionwillbeofparamountimportanceintheselectionof
asuitableagent,e.g.Legionellaincoolingtowersandnonpotable
watersupplysystems,Listeriainthedairyandfoodindustryand
HBVinblood-contaminatedarticles.Driedorganicdepositsmay
inhibitpenetrationofthechemicalagent.Wherepossible,objectsto
bedisinfectedshouldbethoroughlycleaned.Thepresenceofions
inwatercanalsoaffectactivityofantimicrobialagents,thuswater
fortestingbiocidalactivitycanbemadeartificially‘hard’by
additionofions.
48

49. 49
Toxicity of the agent
Inchoosinganantimicrobialagentforaparticularapplicationsome
considerationmustbegiventoitstoxicity.Increasingconcernfor
healthandsafetyisreflectedintheControlofSubstances
HazardoustoHealth(COSHH)Regulations(1999)thatspecifythe
precautionsrequiredinhandlingtoxicorpotentiallytoxicagents.In
respectofdisinfectantstheseregulationsaffect,particularly,theuse
ofphenolics,formaldehydeandglutaraldehyde.Toxicvolatile
substances,ingeneral,shouldbekeptincoveredcontainersto
reducethelevelofexposuretoirritantvapourandtheyshouldbe
usedwithanextractorfacility.
49

50. 50
Toxicity of the agent
Limitsgoverningtheexposureofindividualstosuch
substancesarenowlisted,e.g.0.7mg/m3(0.2ppm)
glutaraldehydeforbothshort-andlongtermexposure.
Many disinfectantsincludingthealdehydes,
glutaraldehydelesssothanformaldehyde,mayaffectthe
eyes,skin(causingcontactdermatitis)andinduce
respiratorydistress.Faceprotectionandimpermeable
nitrilerubberglovesshouldbewornwhenusingthese
agents.Table17.4liststhetoxicityofmanyofthe
disinfectantsinuseandotherconcernsoftoxicityare
describedbelowforindividualagents.
50

51. 51
Toxicity of the agent
Wheretheatmosphereofaworkplaceis
likelytobecontaminated,samplingand
analysisoftheatmospheremayneedtobe
carriedoutonaperiodicbasiswitha
frequencydeterminedbyconditions.
51

52. 52
Properties of commonly used disinfectants and
antiseptics
52

53. 53
Properties of commonly used disinfectants and
antiseptics
53

54. 54
Types of compound
Acidsandesters
Antimicrobialactivity,withinapharmaceutical
context,isgenerallyfoundonlyintheorganicacids.These
areweakacidsandwillthereforedissociateincompletely
togivethethreeentitiesHA,H+andA-insolution.Asthe
undissociatedform,HA,istheactiveantimicrobialagent,
theionizationconstant,Ka,isimportantandthepKaofthe
acidmustbeconsidered,especiallyinformulationofthe
agent.
54

55. 55
Types of compound
Benzoicacid
Thisisanorganicacid,C6H5COOH,whichisincluded,aloneorin
combinationwithotherpreservatives,inmanypharmaceuticals.
Althoughthecompoundisoftenusedasthesodiumsalt,the
nonionizedacidistheactivesubstance.Alimitationonitsuseis
imposedbythepHofthefinalproductasthepKaofbenzoicacidis
4.2atwhichpH50%oftheacidisionized.Itisadvisabletolimituseof
theacidtopreservationofpharmaceuticalswithamaximumfinalpHof
5.0andifpossible<4.0.Concentrationsof0.05–0.1%aresuitablefor
oralpreparations.Adisadvantageofthecompoundisthedevelopment
ofresistancebysomeorganisms,insomecasesinvolvingmetabolism
oftheacidresultingincompletelossofactivity.Benzoicacidalsohas
someuseincombinationwithotheragents,salicylicacidforexample,
inthetreatmentofsuperficialfungalinfections.
55

56. 56
Types of compound
Sorbicacid
Thiscompoundisawidelyusedpreservativeasthe
acidoritspotassiumsalt.ThepKais4.8and,aswith
benzoicacid,activitydecreaseswithincreasingpHand
ionization.ItismosteffectiveatpH4orbelow.
Pharmaceuticalproductssuchasgums,mucilagesand
syrupsareusefullypreservedwiththisagent.
56

57. 57
Types of compound
Sulphurdioxide,sulphitesandmetabisulphites
Sulphurdioxidehasextensiveuseasa
preservativeinthefoodandbeverageindustries.
Inapharmaceuticalcontext,sodiumsulphiteand
metabisulphiteorbisulphitehaveadualrole
actingaspreservativesandanti-oxidants.
57

58. 58
Types of compound
Estersofp-hydroxybenzoicacid(parabens)
Aseriesofalkylestersofp-hydroxybenzoicacidwasoriginally
preparedtoovercomethemarkedpHdependenceonactivityof
theacids.Theseparabens,themethyl,ethyl,propylandbutyl
esters,arelessreadilyionized,havingpKavaluesintherange8–
8.5,andexhibitgoodpreservativeactivityevenatpHlevelsof7–
8,althoughoptimumactivityisagaindisplayedinacidicsolutions.
ThisbroaderpHrangeallowsextensiveandsuccessfuluseofthe
parabensaspharmaceuticalpreservatives.Theyareactiveagainst
awiderangeoffungibutarelesssoagainstbacteria,especially
thepseudomonads,whichmayutilizetheparabensasacarbon
source..
58

59. 59
Types of compound
p-Hydroxybenzoates (R is methyl, ethyl,
propyl, butyl, or benzyl).
59

60. 60
Types of compound
Theyarefrequentlyusedaspreservativesofemulsions,
creamsandlotionswheretwophasesexist.
Combinationsofestersaremostsuccessfulforthistype
ofproductinthatthemorewater-solublemethylester
(0.25%)protectstheaqueousphase,whereasthepropyl
orbutylesters(0.02%)giveprotectiontotheoilphase.
Suchcombinationsarealsoconsideredtoextendthe
rangeofactivity.Asinactivationofparabensoccurswith
non-ionicsurfactantsduecareshouldbetakenin
formulationwithbothmaterials.
60

61. 61
the main antimicrobial groups as antiseptics, disinfectants and
preservatives
61

62. 62
the main antimicrobial groups as antiseptics, disinfectants and
preservatives
Also used in combination with other agents, e.g. chlorhexidine, iodine.
†Several forms available having x%chlorhexidine and 10x%cetrimide.
QAC, quaternary ammonium compound. 62

63. 63
Alcohols
Alcoholsusedfordisinfectionandantisepsis
Thealiphaticalcohols,notablyethanolandisopropanol,areusedfor
disinfectionandantisepsis.Theyarebactericidalagainstvegetative
forms,includingMycobacteriumspecies,butarenot
sporicidal.Cidalactivitydropssharplybelow50%concentration.
Alcoholshavepoorpenetrationoforganicmatterandtheiruseis
thereforerestrictedtocleanconditions.Theypossesspropertiessuch
asacleansingactionandvolatility,areabletoachievearapidand
largereductioninskinfloraandhavebeenwidelyusedforskin
preparationbeforeinjectionorothersurgicalprocedures.Theriskof
transmissionofinfectionduetopoorhandhygienehasbeenattributed
tolackofcompliancewithhandwashingprocedures.Analcoholhand-
ruboffersarapideasy-to-usealternativethatismoreacceptableto
personnelandisincreasinglybeingrecommendedforroutineuse.
However,thecontacttimeofanalcohol-soakedswabwiththeskin
priortovenepunctureissobriefthatitisthoughttobeofdoubtful
value.
63

64. 64
Ethanol(CH3CH2OH)iswidelyusedasadisinfectantand
antiseptic.Thepresenceofwaterisessentialforactivity,
hence100%ethanolisineffective.Concentrationsbetween
60%and95%arebactericidalanda70%solutionisusually
employedforthedisinfectionofskin,cleaninstrumentsor
surfaces.Athigherconcentrations,e.g.90%,ethanolisalso
activeagainstmostviruses,includingHIV.Ethanolisalsoa
popularchoiceinpharmaceuticalpreparationsandcosmetic
productsasasolventandpreservative.Isopropylalcohol
(isopropanol,CH3.CHOH.CH3)hasslightlygreater
bactericidalactivitythanethanolbutisalsoabouttwiceas
toxic.Itislessactiveagainstviruses,particularly
nonenvelopedviruses,andshouldbeconsideredalimited-
spectrumvirucide.Usedatconcentrationsof60–70%,itisan
acceptablealternativetoethanolforpreoperativeskin
treatmentandisalsoemployedasapreservativefor
cosmetics.
64

65. 6565

66. 66
Alcohols as preservatives:
The aralkyl alcohols and more highly substituted aliphatic alcohols, are used mostly as
preservatives. These include:
1-Benzyl alcohol.
This has antimicrobial and weak local anaesthetic properties, used as preservative at concentration
of 2% in cosmetics.
2-Chlorbutol(chlorobutanol; trichlorobutanol; trichloro-t-butanol).
Used in concentration 0.5% as preservatives in injections& eye-drops. It is unstable,
decomposition occurring at acid pH during autoclaving, while alkaline solution are unstable at
room temperature.
3-Phenylethanol (phenylethyl alcohol; 2-phenylethanol).
Used in concentration 0.25-0.5% , active against G
-ve
organisms, and employed in conjunction
with another agent.
4-Phenoxyethanol( 2-phenoxyethanol).
Used in concentration 0.1%,active against Ps.aeroginosa, used in combination with
hydroxybenzoates to broaden the spectrum of antimicrobial activity.
5-Bronopol(2-bromo-2-nitropropan-1,3-diol).
Used in concentration 0.01-0.1%, it has broad spectrum. Limitation is, when exposed to light at
alkaline pH, especially if accompanied by an increase in temperature, solution become yellow
or brown. A number of decomposition products including formaldehyde are produced, nitrite
ions may be produced and react with secondary and tertiary amines present forming
nitrosamines( carcinogenic).

67. 67
Aldehydes:
Gluteraldehyde:
It has a broad spectrum activity( high germicidal level), not affected by
organic matter. The gluteraldehyde molecule possesses two
aldehyde groupings which are highly reactive and their presence is
an important component of biocidal activity.
At a pH of 8, biocidal activity is greatest but stability is poor due to
polymerization.
In practice, gluteraldehyde is generally supplied as an acidic 2% or
greater aqueous solution, which stable on prolonged storage, then
activated by addition of suitable alkalizing agent to bring the pH of
the solution to its optimum for activity, these solution have a limited
shelf-life( 2 weeks).
Glutrealdehyde is employed for the cold, liquid chemical sterilization of
medical and surgical materials that cannot be sterilized by other
methods.

68. 68
Aldehydes:
Ortho-phthaldehyde:
OPA is a recent addition to the aldehyde group of
high level disinfectants.
This agent has excellent activity in vitro studies,
showing superior mycobactericidal activity
compared with gluteraldehyde, it requires no
activation, is not a known irritant to the eyes or
nasal passages and has excellent stability over
the pH range 3-9.
It is used for disinfection of endoscopes appears
promising.

69. 69
Formaldehyde:
Formaldehyed used in liquid or gaseous state for
disinfection.
In the vapour phase it has been used for
decontamination of isolators, safety cabinets and rooms;
recent trends have been to combine formaldehyde
vapour with low temperature steam (LTSF) for the
sterilization of heat-sensitive items.
Formaldehyde vapour is highly toxic, carcinogenic if
inhaled, thus its use must be carefully controlled. It is not
very active at 20
0
C and below, and requires a relative
humidity of at least 70%.The agent is not supplied as a
gas but either as a solid polymer, paraformaldehyde, or
a liquid, formalin, which is a 34-38% aqueous solution.
4% formaldehyde, used for disinfecting surfaces.

70. 70
Aldehydes:
Formaldehyde-releasing agents:
Various formaldehyde condensates have been developed to reduce the
irritancy associated with formaldehyde while maintaining activity, and these
are described as formaldehyde-releasing agents or masked-formaldehyde
compounds.
Noxythiolin (N-hydroxy N-methylthiourea):
It has extensive antibacterial and antifungal properties and is used both
topically and in accessible body cavities as an irrigation solution and in the
treatment of peritonitis.
Polynoxylin (poly[ methylenedi( hydroxymethyl) urea]):
It is a similar compound available in gel and lozenge formulations.
Taurolidine ( bis[1,1-dioxoperhydro-1,2,4-thiadiazinyl-4]metane) :
It is a condensate of two molecules of the amino acid taurine and three
molecules of formaldehyde. It is more stable than noxythiolin in solution and
has similar uses.
The activity of taurolidine is greater than that of formaldehyde.

71. 71
Biguanides:
1-Chlorhexidine and alexidine:
Chlorhexidine is a compound related to the biguanide antimalarial
proguanil.
Compounds containing the biguanide structure could be expected to
have good antibacterial effects, thus the major part of the proguanil
structure is found in chlohexidine.
A related compound is the bisbiguanide alexidine, which has use as an
oral antiseptic and anti-plaque agent.
Chlorhexidine base is not readily soluble in water, therefore the freely
soluble salts, acetate, gluconate, and hydrochloride are used in
formulation.
Chlorhexidine exhibits the greatest antibacterial activity at pH 7-8
where it exists exclusively as a dication. The cationic nature of the
compound results in activity being reduced by anionic compounds
including soap due to the formation of insoluble salts.

72. 72
Factors affecting the activity of antimicrobial agents:
1-Temperature:
In general, as the temperature increased in arithmetical
progression, the rate of kill increase geometrically. The effect
of temperature increase on the rate of bactericidal activity at a
fixed concentration and inoculum size is expressed
quantitatively as a temperature coefficient, usually in θ
10
value: which is the change in activity per 10
0
C rise in
temperature, e.g. rising the temperature of phenol from 20
0
C
to 30
0
C increased the killing activity by a factor of 4.
θ
10
value may be calculated by determining the extinction time
at two temperature differing exactly by 10
0
C.
θ
10 =Time require to kill at T
0
Time requiretokillat(T+10)
0
* This value is constant for each compound. Compounds with
high θ
10
are more effective with increases in temperature than
those with low θ
10
.

73. 73
θ
10
Compound
30-50Aliphatic alcohol
3-5Phenols
1.5Formaldehyde

74. 74
2-Concentration:
The rate of kill of bacterial population is directly affects
with concentration or dilution, therefore slightly increase
or decrease in the concentration of certain agents can
increase or decrease their bactericidal effect.
The graph plotting the log
10of a death time(i.e. the time
required to kill a standard inoculums) against the log
10of
the concentration is usually a straight line, the slope of
which is the concentration exponent(η)
η= concentration exponent (dilution coefficient),which is
measure the effect of changes in concentration(or
dilution) on cell death rate
η=
logt2
–
logt1
log c1 –logc2
It is constant for each compound, and the dilution does not
affect the cidal attributes of all disinfectants in a similar
manner. Therefore compounds with low ηare less
affected by dilution, but those with high ηwill be readily
inactivated.

75. 75
Reduce the activity
(the time required)
No. of
dilution
ηCompound
2
1
double the time21Mercuric
chloride
3
1
three times31Formaldehyde
2
6
64 (the time)
3
6
726 (the time)
½
1/3
6Phenol

76. 76
:3-PH
Changes in PH of the medium affect both
bacterial cell and disinfectant activity.
Bacterial growth is optimums at PH 6-8,
any change in this PH will decrease the
bacterial growth as well as change the
physicochemical state of their surface. The
degree of ionization of acidic or basic
disinfectant will obviously depend on the
PH.

77. 77
Evaluation of liquid disinfectants:
1. Suspension Tests:
Essentially are tests of sterility
a. Phenol Coefficient Test:
* Rideal –Walker (RW) test
* Chick –Martin (CM) test
b. Capacity use –dilution test
Kelsey –Sykes (SK) test
•2. Quantitative suspension test

78. 78
The Rideal-Walker Test: 1903
Briefly, dilutions of the disinfectant are
compared with standard dilution of phenol(
from 1 in 95 to 1 in 115) for their lethal activity
against Salmonella typhiNCTC786
To each 5 ml volume of disinfectant or phenol
solution in distilled water held at 17-18
o
C is
added 0.2 ml of 24 hour culture. At intervals
of 2.5,5,7.5 and 10 minutes, subcultures
using a standard loop are made into 5 ml
volume of broth; these are than incubated for
48-72 hours at 37
o
C after which presence or
absence of growth in each broth is recorded.

79. 79
Contact time(min.) of culture and
disinfectant
DilutionDisinfectan
t
107.552.5
___+1 in 250X
__++1 in 300X
_+++1 in 350X
++++1 in 400X
__++1 in 100Phenol

80. 80
The phenol coefficient of the
disinfectant(X) is calculated by dividing the
dilution of X which allows survival of test
organism at 2.5 and 5 but not at 7.5 and 10
minutes by dilution 0f phenol giving the
same response. The Rideal-Walker
phenol coefficient(RW) of disinfectant X is:
RW of X =300= 3
100

81. 81
The Chick-Martin Test:1908
Chick & Martin, realizing that disinfectants usually had to
act in the presence of organic material, suggested the
inclusion of 3% dried human feces in the test. They also
considered the 10 minutes disinfection time allowed in
the Rideal-Walker test was too short and they introduced
a 30 minutes contact time with subcultures in duplicate at
the end of this. A different strain of Salmonella typhifrom
that of (RW) test was used. The human fecal suspension
was later replaced by dead yeast cells and the Chick-
Martin test become the subject of British Standard
Specification BS 808: 1938.

82. 82
Series of dilutions of the unknown disinfectant and of
phenol are made in distilled water in regular diminishing
stages of 10%. To 2.5 ml volume of these held at 20
0
C
are added 2.5 ml of the culture-yeast suspension(2 ml of
a 24 hours Salmonella typhi culture + 48 ml of 5% dry
weight yeast suspension). After a contact time of 30
minutes, a standard loopful of disinfectant-culture-yeast
mixture is transferred in duplicate to 10 ml of broth.
These broth tubes are incubated at 37
0
C for 48 hours,
then the presence or absence of growth is recorded.
The Chick-Martin Coefficient (CM) is calculated by
dividing the mean of the highest concentration of phenol
permitting growth in both subcultures and the lowest
concentration showing absence of growth in both
subcultures by the corresponding mean concentration of
the unknown disinfectant.

83. 83
Subculture
tubeDisinfectant
X(%)
Subculture
tubePhenol(
%)
21 21
__1.00__2.00
__0.90__1.80
++0.81++1. 62
++0.73++1.46

84. 84
Mean of phenol concentration is:
½ ( 1.80+ 1.62)=1.71
Mean of disinfectant X concentration is:
½ (0.90+ 0.81)=0.85
Chick-Martin Coefficient of X
=1.71
=2.0
0.85
•If growth occurs in one, but not in the other, of
the a pair of subculture tubes the concentration
value corresponding to that pair is used.
Chick-Martin Coefficient of X
=1.62
=2.0
0.81

85. 85
Subculture tube
DisinfectantX(%)
Subculture tube
Phenol(%)
2121
__1.00__2.oo
__0.90__1.80
_+0.81_+1.62
++o.73++1.46

86. 86
Capacity use-dilution tests:
There are four test organisms,
Pseudomonas aeroginosa, Proteus vulgaris, E.coli and
Staphylococcus aureus; the bacteria are suspended in
standard hard water for the test under clean conditions,
and in a yeast suspension for the test under dirty
conditions, the disinfectant is diluted in hard water. To 3
ml of each disinfectant dilution add 1 ml of the bacterial
suspension prepared in broth, yeast, or serum as
required, and shake gently. After 8 minutes, remove the
sample of the mixture with a dropper, pipette and transfer
one drop to each of five tubes of the liquid recovery
medium.

87. 87
Alternatively, five drops may be placed separately on a
nutrient agar plate. Two minutes later, i.e. 10 min. after
the first inoculation, re-inoculate the disinfectant mixture
with a further 1 ml of bacterial suspension, and 8 min.
later subculture as before. A further 2 min. later ,i.e. 20
min. after the first inoculation, repeat the process again.
The initial test is carried out at 20 to 22
o
C, and all
subcultures are incubated at about 32
o
C for 48 hours.
Record the number of tubes showing growth in the liquid
medium or the number of colonies growing on the
surface plate cultures.
A capacity test (the Kelsey-Sykes): use-dilution 1.o%

88. 88
Number of subculture broth ( out of
five) showing growth after
Disinfectant
Concentration
(%)
28 min.18 min.8 min.
3002.0
5201.0
5410.5
555o.25

89. 89
Quantitative suspension tests:
The number of survivors expression as the
percentage remaining viable at the end of a giving
time may be determined by viable counts & this
parameter is often used in assessing bactericidal.
After exposure of bacterial cells to the disinfectant,
surviving organisms can be counted by two
techniques , either by direct culture or by membrane
filtration.
The basic principle of the quantitative suspension
tests using direct culture is as follows: after contact
with the disinfectant, a sample of the reaction
mixture is inoculated on a solid nutrient medium;
after incubation the number of survivors is counted
and compared with initial inoculums size.

90. 90
The pour-plate technique as well as
surface plates may also be used for
subculturing.
The decimal reduction rate, or Microbial
Effect(ME) can be calculated using the
formula:
ME= log NC-log ND
( NC: CFU in control, ND: CFU after
exposure to disinfectant).
•ME= ( 4+ 2.04) –(1+ 1.94)
= 3.10 ( after 5 min.)

91. 91
No. of CFUDilution of
subculture
in
disinfectant
in control
_tntc _tntc10
0
1.9488_tntc10
-1
0.786_tntc10
-2
_o_tntc10
-3
_02.0411010
-4

92. 92
Methods which measure only growth inhibition(
bacteriostasis) are:
1. Serial dilution:
Graded doses of the test substance are incorporated
into broth and the tubes inoculated with the test
organisms and incubated. The point at which no growth
occurs is taken as the bacteriostatic concentration(
Minimum Inhibitory Concentration, MIC). It is essential
when performing these tests to determine the size of
the inoculum at the position of the endpoint varies
considerably with inoculum size, which should always
be defined in any description of results.

93. 93
The test is carried out in practice by mixing the
appropriate volume of the solution under test with double-
strength broth and making it up to volume with water as
illustrated in table below:
Tube contents for determining the MIC of phenol
Final volume
555555Double-strength broth
5432100.5% phenol solution
012345Sterile distilled water
0.250.20.150.10.050Final con. phenol
(%w/v)

94. 94
2. Ditch-plate technique:
The test solution is placed in a ditch cut in nutrient
agar contained in a petridish, or it may be mixed with
a little agar before pouring into the ditch. The test
organisms( as many as six may be tested) are
streaked up to the ditch. The plate is then incubated.
•3. Cup-plate technique:
•The solution is placed in contact with agar, which is
already inoculated with the test organism and after
incubation, zones of inhibition observed. A method
used widely in antibiotic assays. The solution may be
placed in a small amount in a well cut from agar with
a sterile cork-borer.

95. 95
4. Disc tests:
These are modification of the earlier cup or ditch-plate procedures
where filter-paper discs impregnated with the antimicrobial agents.
For disc tests, standard suspensions are prepared and inoculated
onto the surface of appropriate agar plates. Commercially available
filter-paper discs containing known concentrations of antimicrobial
agent.
5. E-tests:
•The most presently accepted method of determining bacterial MICs,
however, is the E(Epsilometer)-test. Basically this is performed a
similar manner to the disc test except that nylon strips that have a
linear gradient of antimicrobial lyophilized on one side and the on the
other side of the nylon strip are a series of lines and figures denoted
MIC values.

96. 96
Cup-plate Disc-plate

97. 97
E-Test:

98. 98
6. Solid dilution method:
In this method the dilutions of the substance under
test are made in agar instead of broth. The agar
containing the substance under test is subsequently
poured onto a petridish. It has the advantage that for
any one concentration of the test substance, several
organisms, may be tested.
7. Gradient-plate technique:
•In this technique the concentration of a drug in an
agar plate may be varied infinitely between zero and
a given maximum. To perform the test, nutrient agar
is molted, the solution under test added, and the
mixture poured into a sterile petridish and allowed to
set in the form of a wedge(A). A second amount of
agar is then poured onto the wedge and allowed to
set with the petridish flat on the bench.

99. 99
8. NCCLS:
Regularly updated guidelines have been provided by the
National Committee for Clinical Laboratory Standards
(NCCLS) and are widely used in many countries.