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