CHAPTERCHAPTER – – IVIV

DISINFECTIONDISINFECTION

CONTENT OF THE CHAPTERCONTENT OF THE CHAPTER

GENERALGENERAL IntroductionIntroduction TerminologiesTerminologies ClassificationClassification Properties of disinfectantProperties of disinfectant Factors Affecting disinfectant actionFactors Affecting disinfectant action

EVALUATIONEVALUATION Bacteriostatic, Bacteriocidal evaluation Bacteriostatic, Bacteriocidal evaluation Phenol Coefficient testPhenol Coefficient test

DIS - INFECTIONDIS - INFECTION

Destruction of microorganisms, but not usually Destruction of microorganisms, but not usually spores: not necessarily killing all spores: not necessarily killing all microorganisms but reducing them to a level of microorganisms but reducing them to a level of not harmful to human health.not harmful to human health.

Commercially for the treatment of inanimate objects Commercially for the treatment of inanimate objects and materilas.and materilas.

Commercially Available Disinfectants Commercially Available Disinfectants And Their ApplicationsAnd Their Applications

s.nos.no EnzymeEnzyme Application(s)Application(s)

0101 Dettol Dettol Anti-infectiveAnti-infective

0202 LysolLysol Floor CleaningFloor Cleaning

0303 PhenolPhenol Gargles, mouth washesGargles, mouth washes

0404 ChlorxylenolChlorxylenol Active against Active against staptococcistaptococci

0505 ChlorhexidineChlorhexidine Skin disinfectantSkin disinfectant

TERMINOLOGIES TERMINOLOGIES

BactericideBactericide Killing bacteriaKilling bacteria

BacteriostatBacteriostat Preventing the growth of bacteriaPreventing the growth of bacteria

FungicideFungicide Killing fungi incl. SporesKilling fungi incl. Spores

FingistatFingistat Prevents fungal proliferation Prevents fungal proliferation

Germicide Germicide Kills all microorganismsKills all microorganisms

VirucideVirucide Kills virusKills virus

SporicideSporicide Kills sporesKills spores

AlgicideAlgicide Kills algaeKills algae

AntibioticAntibiotic Produced by and act against microbesProduced by and act against microbes

Antisepsis Antisepsis Destruction of m/o but not bac.spores on Destruction of m/o but not bac.spores on living tissueliving tissue

Classification of DisinfectantsClassification of Disinfectants

PhenolPhenol Phenol, xylenol, chlorphenols, Phenol, xylenol, chlorphenols, chlorxylenols,,etc….chlorxylenols,,etc….

AlcoholsAlcohols Etanol, isopropanol,Iso propyl alcohol, Etanol, isopropanol,Iso propyl alcohol, amyl alcohol, etc….amyl alcohol, etc….

AldehydesAldehydes Formaldehyde, glutaraldehyde, Formaldehyde, glutaraldehyde,

DyesDyes Acridine, triphenylmethane, acriflavine, Acridine, triphenylmethane, acriflavine, proflavine, crystal violet, etcproflavine, crystal violet, etc

Surface Surface active agentsactive agents

Cetremide, sodium stearate, sorbiton, Cetremide, sodium stearate, sorbiton, tween 80, etc…tween 80, etc…

Groups of DI Examples

HalogensHalogens Chlorine, bromine, iodine, etc…Chlorine, bromine, iodine, etc…

MetalsMetals Copper, aluminium, silver, mercuryCopper, aluminium, silver, mercury

Guanidines & Guanidines & amidinesamidines

Chlorhexidine, dibromopropamidine Chlorhexidine, dibromopropamidine isothionate , etc…isothionate , etc…

Furan Furan derivativesderivatives

Nitrofurazone,furan, etc…Nitrofurazone,furan, etc…

Quinolines and Quinolines and isoquinolinesisoquinolines

Hydroxyquinoline, dequalinium Hydroxyquinoline, dequalinium chloride, etc….chloride, etc….

Groups of DI Examples

Mechanism of action of disinfectants

PhenolPhenol Low conc.- Disrupt cytoplasmic membrane. – Low conc.- Disrupt cytoplasmic membrane. – leakage of cellular constituentsleakage of cellular constituents

High conc. - Act as protoplasmic poisonHigh conc. - Act as protoplasmic poison

AlcoholsAlcohols Denature the protein – 60-90% best active.Denature the protein – 60-90% best active.

AldehydesAldehydes Combine with amino group of proteinCombine with amino group of protein

DyesDyes Inactivate bacteria by reacting with acidic Inactivate bacteria by reacting with acidic group of cell. Inhibit glutamine synthesis. group of cell. Inhibit glutamine synthesis.

Inhibit cell wall systhesisInhibit cell wall systhesis..

Surface Surface active active agentsagents

+vely charged Quart.amm.comp strongly +vely charged Quart.amm.comp strongly absorbed on the –velty charged bacterial absorbed on the –velty charged bacterial

surface. – damage cell membrane – leakage…surface. – damage cell membrane – leakage…

DISINFECTANT MECHANISM OF ACTION

HalogensHalogens Halogenate or oxidize the vital cell wall Halogenate or oxidize the vital cell wall components.components.

MetalsMetals Hg, Ag, Cu – protein precipitants. Toxic Hg, Ag, Cu – protein precipitants. Toxic part react with enzyme and affects the part react with enzyme and affects the

protein structureprotein structure

Guanidines & Guanidines & amidinesamidines

Disrupt cell wall followed by Disrupt cell wall followed by destruction.destruction.

Furan Furan derivativesderivatives

Inhibits H2 and electron transport chain Inhibits H2 and electron transport chain (uncertain).(uncertain).

Quinolines Quinolines and and

isoquinolinesisoquinolines

Form a complex with iron , copper to Form a complex with iron , copper to form toxic complex.form toxic complex.

DISINFECTANT MECHANISM OF ACTION

Properties of DisinfectantsProperties of Disinfectants

PhenolPhenol Produced From Coaltar Distillaries.Activity Produced From Coaltar Distillaries.Activity Reduced By Dilution. More active in acidicpH. Reduced By Dilution. More active in acidicpH.

Organic matter – dimnishes its actionOrganic matter – dimnishes its action

AlcoholsAlcohols Most active against vegetative bacteria. Most active against vegetative bacteria. Methanol vapour is fungicidalMethanol vapour is fungicidal

AldehydesAldehydes Gas or solution – sporicidal. Virus infectivity Gas or solution – sporicidal. Virus infectivity can be reduced in vaccine preparationcan be reduced in vaccine preparation

DyesDyes Inhibit Staphylococci, Streptococci. Crystal Inhibit Staphylococci, Streptococci. Crystal violet destroy only staphylo not strepto – violet destroy only staphylo not strepto –

selective mediumselective medium

Surface Surface active active agentsagents

Soaps – anitbacterial, cetrimide- bactrecidal.Soaps – anitbacterial, cetrimide- bactrecidal.

DISINFECTANT PROPERTIES OF DISINFECTANT

HalogensHalogens Treatment of water supplies, swimming Treatment of water supplies, swimming pool, deodorant in sewage. pool, deodorant in sewage.

MetalsMetals Slow in action. Corrosive in tissue and Slow in action. Corrosive in tissue and metals. Acetates and nitrates of Hg more metals. Acetates and nitrates of Hg more

effective antibactericidal, antifungaleffective antibactericidal, antifungal

Guanidines & Guanidines & amidinesamidines

Wide rage of G+ve, G-ve. Skin disinfectant Wide rage of G+ve, G-ve. Skin disinfectant – against pseudomonas, proteus– against pseudomonas, proteus

Furan Furan derivativesderivatives

Antibactericidal against most vegetative Antibactericidal against most vegetative bacteria. – included-ointment preparationbacteria. – included-ointment preparation

Quinolines Quinolines and and

isoquinolinesisoquinolines

Bactericidal, fungicidal . Lozenges, mouth Bactericidal, fungicidal . Lozenges, mouth washes, throat infections.washes, throat infections.

DISINFECTANT PROPERTIES OF DISINFECTANT

Factors Affecting Disinfectant ActionFactors Affecting Disinfectant Action

Time of contactTime of contact

Conc. of disinfectantConc. of disinfectant

TemperatureTemperature

Type of m/o. present, its number & conditionType of m/o. present, its number & condition

Hydrogen ion conc. Hydrogen ion conc.

Surface tensionSurface tension

The formulation if the disinfectantThe formulation if the disinfectant

The chemical structure of the disinfectantThe chemical structure of the disinfectant

The nature of surface to be disinfected.The nature of surface to be disinfected.

Potentiation, Synergism and Antagonism of disinfectantPotentiation, Synergism and Antagonism of disinfectant

1.Time of contact 1.Time of contact

Viable count Vs Time – Viable count Vs Time – Death curveDeath curve

TIME

LO

G N

O. O

F S

UR

VIV

AL

S

AB

C

a

b

c

DEATH CURVEDEATH CURVE

A- Curve often obtained with high conc. A- Curve often obtained with high conc. B- Disinfection Process Obeying First B- Disinfection Process Obeying First

Order KineticsOrder Kinetics C- Sigmoid curve C- Sigmoid curve

a- slow initial kill of more sensitive org.a- slow initial kill of more sensitive org. b- faster “near linear” average resistant org.b- faster “near linear” average resistant org. c- slower death rate of more resistant org.c- slower death rate of more resistant org.

Rate k = 1/ t log N / nRate k = 1/ t log N / n

ConcentrationConcentration2. Concentration 2. Concentration

TIME - MINUTES

LO

G N

UM

. OF

SU

RV

IVA

LS

A = 0.66% w/v

B = 0.55% w/v

C = 0.50% w/v

D = 0.44% w/v

0.001

0.1

1.0

10

0100

A B C D

Concentration Concentration

61

1

1

A

B

Log C

Log t

Comparison of increased and decreased Comparison of increased and decreased concentration on killing rates of Phenol and concentration on killing rates of Phenol and

FormaldehydeFormaldehyde

Phenol : n = 6Phenol : n = 6

0.7% kills in 100 mins.0.7% kills in 100 mins.

Doubling :1.4% kills in Doubling :1.4% kills in

100/2100/2nn 100/2100/26 = 6 = 1.6 min1.6 min

Halving : 0.35% kills Halving : 0.35% kills

in 100X2n in 100X2n 100X26 = 6400mins100X26 = 6400mins

Formaldehyde : n = 1Formaldehyde : n = 1

0.7% kills in 100 mins0.7% kills in 100 mins

Doubling: 1.4% kills in Doubling: 1.4% kills in

100/2100/2nn

100/2100/211 = 50mins = 50mins

Halving : 0.35% kills in Halving : 0.35% kills in

100 X 2100 X 2nn

100X2100X211 = 200 mins = 200 mins

Temperature Temperature

Increase in the temp, increases the velosity, increases the Increase in the temp, increases the velosity, increases the

bactericidal action bactericidal action ӨӨ(T2-T1)(T2-T1) = K = K22/K/K11

K1, = reaction velocity at temp T1, K1, = reaction velocity at temp T1,

K2 = reaction velocity at temp T2K2 = reaction velocity at temp T2

Reaction velocity K = 1 / extinction timeReaction velocity K = 1 / extinction time

(Extinction time = time at which no living cells can be detected from the sample taken)(Extinction time = time at which no living cells can be detected from the sample taken)

temperature

Extinction time

0.5

1.0

1.5

2.0

10 20 30

2.5

Type Of Organism Present, Its Number, Cndn,Type Of Organism Present, Its Number, Cndn,

Aldehyde + Halogen + Aldehyde + Halogen + ββ--Propiolactone - ANTIVIRALPropiolactone - ANTIVIRAL

Presence Of Organic Matter, Any In activatorsPresence Of Organic Matter, Any In activators

Extracellular protein – reduction in bactericidal actionExtracellular protein – reduction in bactericidal action

Oils & Fats – Decreases Phenol activityOils & Fats – Decreases Phenol activity

Hydrogene ion ConcentrationHydrogene ion Concentration

Affects the rate of growth of microbesAffects the rate of growth of microbes

Degree of ionization of disinfectant & potencyDegree of ionization of disinfectant & potency

Adsorption of disinfectant at cell surfaceAdsorption of disinfectant at cell surface

Phenol, Benzoine, Salycilates are active in acidic pHPhenol, Benzoine, Salycilates are active in acidic pH

Acridine - active in ionized fromAcridine - active in ionized from

Surface tensionSurface tension

The formulation of disinfectantThe formulation of disinfectant

Increase in conc. of soap will increase the extinction time.Increase in conc. of soap will increase the extinction time.

Alcoholic formulation increases the activityAlcoholic formulation increases the activity

The chemical structure of disinfectantThe chemical structure of disinfectant

Alkyl chain up to 6 increases the activityAlkyl chain up to 6 increases the activity

Alkyl chain more than 6 decreases the activityAlkyl chain more than 6 decreases the activity

Straight alkyl chain give greater activity than branchedStraight alkyl chain give greater activity than branched

Halogenation increases the activityHalogenation increases the activity

Nitration increases the activityNitration increases the activity

The nature of surface to be disinfectedThe nature of surface to be disinfected

Uneven, porous, cracked surface resist chemical disinfectantUneven, porous, cracked surface resist chemical disinfectant

Plain, clean impenetrable surface increases the activityPlain, clean impenetrable surface increases the activity

Potentiation, Synergism and AntagonismPotentiation, Synergism and Antagonism

EVALUATION OF DISINFECTANTEVALUATION OF DISINFECTANT

Evaluation of DisinfectantEvaluation of Disinfectant

Evaluation of Bacteriostatic disinfectantEvaluation of Bacteriostatic disinfectant Serial dilution in liquid mediaSerial dilution in liquid media

Serial dilution in solid mediaSerial dilution in solid media

Fish Spine bead methodFish Spine bead method

Cylinder plate methodCylinder plate method

Filter paper disc methodFilter paper disc method

Cup and Plate methodCup and Plate method

Gradient Plate techniqueGradient Plate technique

Ditch Plate methodDitch Plate method

Serial dilution in liquid mediaSerial dilution in liquid media

Media + 0.2ml of Culture + Test DI DilutionMedia + 0.2ml of Culture + Test DI Dilution

controlcontrol I st DilI st Dil 2nd Dil2nd Dil 3rd Dil3rd Dil 4th Dil4th Dil 5th Dil5th Dil

Serial dilution in solid mediaSerial dilution in solid media

Solid media + culture + Test DISolid media + culture + Test DI

controlcontrol Ist dilutionIst dilution 22ndnd dilution dilution

33rdrd dilution dilution 44thth dilution dilution 55thth dilution dilution

I st DilI st Dil IInd DilIInd Dil

5th Dil5th Dil 4th Dil4th Dil 3rd Dil3rd Dil

controlcontrol

Fish spine bead, Cylinder plate, Cup Fish spine bead, Cylinder plate, Cup and plate, Filter paper disc methodand plate, Filter paper disc method

Fish Spine Fish Spine BeadBead

Cup Shape Cup Shape

Beaker Beaker containing Test containing Test DisinfectantDisinfectant

Beaker Beaker containing Test containing Test DisinfectantDisinfectant

Paper discPaper disc

Zone of InhibitionZone of Inhibition

Assessment of Assessment of Bacteriostatic activityBacteriostatic activity

Growth of Growth of organism on organism on Agar Media Agar Media

Zone of Zone of Inhibition Inhibition

Dilution of Test Dilution of Test disinfectant disinfectant

0.50.5

1.01.0

1.51.5

2.02.0 2.52.5

controlcontrol

Evaluation of DisinfectantEvaluation of Disinfectant

Evaluation of Bactericidal disinfectantEvaluation of Bactericidal disinfectant Phenol Coefficient testPhenol Coefficient test

End point techniqueEnd point technique

Extinction time methodExtinction time method

Rideal –Walker Coefficient methodRideal –Walker Coefficient method

FDAFDA

Chick Martin testChick Martin test

AOACAOAC

Crown Agent techniqueCrown Agent technique

Schematic Diagram of Phenol Schematic Diagram of Phenol Coefficient testCoefficient test

00

0.50.5

1.01.0

1.51.5

2.02.0

S.Typhi S.Typhi

24 hrs24 hrs

cultureculture

ControlControl

Phenol Phenol

1 in 1051 in 105

1 in 10001 in 1000

1 in 11001 in 1100

1 in 12001 in 1200

1 in 13001 in 1300

Reaction mixtureReaction mixture

Broth tubesBroth tubes

Incubate 2-3 days at 37Incubate 2-3 days at 3700CC

Temp 17.5 Temp 17.5 2.52.5 5.05.0 7.57.5 1010

R.W.COEFFICIENT R.W.COEFFICIENT

There are two types of extinction time There are two types of extinction time

methodmethod

In which the extinction time is fixed and the conc. In which the extinction time is fixed and the conc.

Of disinfectant needed to kill in the specified time Of disinfectant needed to kill in the specified time

is estimated.is estimated.

In which the conc. of bactericide is fixed and the In which the conc. of bactericide is fixed and the

extinction time is estimated.extinction time is estimated.

R.W.COEFFICIENTR.W.COEFFICIENT Standard: Rideal Walker BrothStandard: Rideal Walker Broth Organism: Organism: Salmonella TyphiSalmonella Typhi Dilution of Phenol 1 in 105Dilution of Phenol 1 in 105 Dilution of test DI = 1in 1000, 1 in 1100, 1 in 1200, 1 in Dilution of test DI = 1in 1000, 1 in 1100, 1 in 1200, 1 in

1300.1300. Test DI + 0.2 ml of Test DI + 0.2 ml of S.Typhi S.Typhi Reaction mixture should be at 17.5Reaction mixture should be at 17.500C temp.C temp. Subculture of each reaction mixture are taken and Subculture of each reaction mixture are taken and

transformed to broth after 2.5, 5, 7.5 and 10 mins.transformed to broth after 2.5, 5, 7.5 and 10 mins. Broth tubes are incubated at 37Broth tubes are incubated at 3700C, for 48 to 72 hrs, C, for 48 to 72 hrs,

examined.examined. Presence or absence of growth was notedPresence or absence of growth was noted

Observed results in table Observed results in table Minutes culture exposed to Minutes culture exposed to

disinfectantdisinfectant

DilutionDilution 2.52.5 5.05.0 7.57.5 1010

Test Test disinfecdisinfectanttant

1 in 10001 in 1000

1 in 11001 in 1100

1 in 12001 in 1200

1 in 13001 in 1300

++

++

++

++

--

++

++

++

--

--

++

++

--

--

--

++

PhenolPhenol 1 in 1051 in 105 ++ ----++

+ = Growth - = No growth+ = Growth - = No growth

R.W. CoefficientR.W. Coefficient

R.W. Coefficient = R.W. Coefficient =

Dilution of test disinfectant killing in 7.5 Dilution of test disinfectant killing in 7.5 mins, but not in 5 mins. / Dilution of Phenol mins, but not in 5 mins. / Dilution of Phenol killing in 7.5 mins, but not in 5 mins.killing in 7.5 mins, but not in 5 mins.

1100/105 = 10.47.1100/105 = 10.47.

Essential difference between phenol Essential difference between phenol coefficient testscoefficient tests

Medium pHMedium pH

Medium Medium volumevolume

Rxn Mix Rxn Mix VolumeVolume

DiluentDiluent

Reaction Reaction TempTemp

OrganismOrganism

Sampling Sampling Time Time (Mins)(Mins)

CalculationCalculation

RWCRWC FDA`FDA` CMCCMC AOACAOAC Crown AgentsCrown Agents

7.3-7.57.3-7.5

5.0ml5.0ml

5.0ml5.0ml

WaterWater

17.517.5

S.TyphiS.Typhi

2.5,5.0,7.5,102.5,5.0,7.5,10

Dil of test Dil of test killing in 7.5 killing in 7.5 but not in 5 but not in 5 min / Same for min / Same for phenolphenol

6.86.8

10.0ml10.0ml

10.0ml10.0ml

WaterWater

2020

S.TyphiS.Typhi

5, 10, 155, 10, 15

Dil of test Dil of test killing in 10 killing in 10 but not in 5 but not in 5 min / same min / same for phenolfor phenol

7.3-7.57.3-7.5

10.0ml10.0ml

5.0ml5.0ml

Yeast Suspn.Yeast Suspn.

3030

S.TyphiS.Typhi

3030

Mean of high Mean of high phenol conc. phenol conc. Inhibit and Inhibit and low permit low permit growth / growth / same for same for phenolphenol

6.86.8

10.0ml10.0ml

5.0ml5.0ml

WaterWater

37 or 2037 or 20

S.Typhi, StphS.Typhi, Stph

5, 10, 155, 10, 15

Greatest Dil Greatest Dil of test killing of test killing in 10min / in 10min / Same for Same for phenolphenol

7.3-7.57.3-7.5

5.0ml5.0ml

5.0ml5.0ml

Sea waterSea water

19.019.0

S.TyphiS.Typhi

1010

Greatest Dil of Greatest Dil of test killing in test killing in 10min / same 10min / same for phenolfor phenol

Bentley's text book of Bentley's text book of PharmaceuticsPharmaceutics

REFERENCEREFERENCE

Kindly refer the text book referred by The TN Dr. MGR Medical UniversityKindly refer the text book referred by The TN Dr. MGR Medical University

I ACKNOWLEDGE MY MISTAKES. SORRY