Sterilization Unit 2.pdf their physical chemical and mechanical process inshort

Sterilization Unit 2.pdf their physical chemical and mechanical process inshort

1. Sterilization
Presented by Ass. Prof. Miss Priya S. Talekar
Samarth College of Pharmacy, D. Raja

2. Physical
Method
01
Evaluation of
efficiency of
Sterilization
method
04
Gaseous
Method
02
Equipments used
in Large scale
Sterilization
05
Chemical
Method
03
Sterility
indicator
06

3. Introduction
•Sterilizationisaprocessbywhichanarticle,surfaceormediumis
freedofallmicroorganismseitherinthevegetativeorsporestate.
•Sterility:Theabsenceofviablemicroorganismiscalled‘sterility’
andpreparationfreefromviablemicroorganismsarecalled‘sterile’

4. Objective
•Topreventthecontaminationofunwantedmicrobesinpure
cultures,othermicrobiologicalresearchstudies,antibiotic,
enzyme,vitaminfermentationandindustrialprocessaswell
asinasepticareaswhichareusedforpreparationofsterile
dosageformsandsterilitytesting
•Topreventioncontaminationinsterileproductswhichare
responsibleforcausingdiseases
•Topreventdecompositionandspoilageoffoodproducts

5. Methods of Sterilization
Physical
Method
Dry heat sterilisation,
Moist heat sterilisation,
Radiation sterilisation,
Chemical
method
Mechanical
Method
FiltrationSterilisation by heating with
bactericide,
Gaseous Sterilisation

6. PHYSICAL METHODS
•Allmicroorganismsincludingbacterialsporescanbedestroyedbyheat
•Dryheatingat100ºCkillsallvegetativebacteriainonehourbutitdoes
notkillspores
•AccordingtoPharmacopoeia,sterilisationbydryheatingiseffectedby
heatingattemperatureof160ºCfortwohours
•Methods:Flaming,RedHeat,Incineration,Hotairoven
•Itisusedforsterilisationofpharmaceuticalproducts&Equipments,double
walledchambermadeofsteelfilledwithInsulationmaterialtoavoidheat
loss
HOT AIR OVEN
DRY HEAT STERILISATION

7. Principle
•Chambermadeofaluminiumorstainlesssteel,Withashelves
madeofaluminium,thermostat,fans,temperaturesensor.
•Whenelectricityispassedthroughheatingcoil,electricalenergy
isconvertedtoheatenergy.
•Hotaircirculatesinthechamberduetoairconvection.
•Theovenuseconductiontosterilizeitemsbyheatingtheoutside
surfacesoftheitems,whichthenabsorbstheheatandmovesit
towardsthecentreoftheitem.
•Thetemperatureiscontrolledbythermostat

8. PRECAUTIONS
•Glassapparatusmustbewrappedwithcleanclothorpaperand
containermustbepluggedwithcotton
•Thesubstancewhicharetobesterilisedshouldnotbeplacedat
thefloorasitreceivesdirectheat
APPLICATIONS
•Itismainlyusedforsterilisationofglasswares
•Usedforsterilisepowder&granulessuchastalc,zincoxide,
starchetc.
•Dryingofglasswares
•Todrygels

9. ADVANTAGES
•Usedforsubstancewhichgetspoiledduringmoistheat
sterilisatione.g.Oilymaterialandpowders
•Themethodissuitableforglasssyringes
DISADVANTAGES
•Thismethodisnotsuitableforsurgicaldressing
•Thismethodisnotsuitableformostofthemedicaments,
rubberandplasticgoods
•SlowspeedthenAutoclave

10. •Moistheatsterilisationismoreeffectivethandryheatmethod
•Bczsteamhasmorepenetrationpowerandthermalcapacity
thandryheat
•Themethodisveryusefulforkillingofbacterialspores
MOIST HEAT STERILISATION

11. Autoclave
•ItisinventedbytheCharlesChamberlandin1879.
PressurizedDevicewhichlookslikeCookerandsterilize
thermostablematerial
DEFINITION
•Theautoclaveisaequipmentusedtoremove
microorganisms(Virus,Bacteria,fungusetc.)andspores
usinghighpressureandhightemperature
•Autoclaveisapressurizeddevicedesignedtoheataqueous
solutionsabovetheirboilingpointatnormalatmospheric
pressuretoachievesterilization

12. Principal
•Boilingpointofwaterisdirectlyproportionaltothepressure
whenthevolumeisconstant
•PressuredirectlyproportionaltoTemperature
•Whenpressureisincreasedinaclosedvesselthe
temperatureincreases
•Thispressureandtemperatureiskeptconstantfor15-20
minutesduringautoclaving
•Itissufficienttokillallthevegetativeformsandsporesofthe
organism

13. Mechanism
•Bycoagulatinganddenaturingenzymesandstructural
proteinSterilizationtakeplace
•Resistantsporesgenerallyrequires121°Cfor15-30minutes
•Usedforthesterilizationofculturemedia,andallother
materialsthroughwhichsteamcanpenetrate
•Moistheatismoreeffectivethandryheat
•Lowertemperaturesinagiventimeatashorterduration

14. Diagram

15. WORKING
•Theautoclaveisswitchedontoheatthewater
•Theventisopenedandsafetyvalveissetattherequired
pressure
•Whensteamstartscomingoutfromtheventanditcontinues
for5minutes,itisthenclosed
•Thesteampressurestartsraisinganditcomestodesired
pressurei.e.10Psiwithcorrespondingtemperature116ºC
•Afterstatedperiod,switchofftheautoclave
•Allowittocoolbeforeopening
•Whensteaminsidetheautoclaveisremoved,thelidisopened
andthesterilisedmaterialistakenout

16. Pressure correspondence to temperature

17. Advantages
•Autoclavingdestroysmicroorganismsmoreefficientlythan
dryheat
•Fasterthendryheating
•Easytouse
•Itisusedforsterilisationofalargenumberofofficial
injections
Disadvantages
•Unsuitableforsterilisationofpowdersandoilsieanhydrous
materials
•Cannotbeusedforsterilisationofplasticswhichgetspoiled
at115-116ºcfor30minorabove100oc

18. Application
•Itisusedforsterilizationofsurgicaldressing,surgical
instruments,thermostableliquids,culturemedia,ampules...
•Thecontainerandclosurecanbesterilizedbyautoclave
•Itisusedforthesterilizationofmajorityofofficialinjection
•Itisusedforsterilizationarticlesmadeofrubberplasticglass

19. OTHER METHODS OF STERILISATION
BY MOIST HEAT
1.Tyndallisationmethod
•Thisisfractionalsterilisationmethod.
•Solutiontobesterilisedispacked&sealedinitsfinalcontaineran
heatedat80ºcforonehouroneachofthreesuccessivedays
•Thefirstheatingdestroysthevegetativecellsbutnotthebacterial
spores
•Thesebacterialsporesgerminateintothevegetativeformsinthe
intervalbetweenfirstandsecondheatingandarekilledinthe
secondheating
•Thethirdheatingprovidesasafeguardagainstanysporeswhich
maynotgerminateuntilthesecondinterval

20. 2.PASTEURISATION
•Itispartialsterilisationmethodwhichisusedtomakemilksafeand
alsotoimproveitskeepingproperties
•Theprocesskillsonly97to99%microorganismbutitdoesnotkill
bacterialspores
3.STERILISATIONOFVACCINES
•Sterilisationiscarriedoutatatemperaturebetween55ºCto60ºCfor
onehour
•Thestrictasepticprecautionsareobservedtoexcludeanypossibility
ofcontamination

21. RADIATION STERILISATION
•Killingorremovalofmicroorganismswiththehelpofradiation
powerlikegamma,Xrays,UVrays..whichisactas
antimicrobialagentdependsonwavelength
•Onthebasisoftheirwavelengthandpenetrationpowerthey
areclassifiedin2type
1.Ionizationradiation:Thisisverylethalradiationwhichhave
higherenergyandhighpenetrationpower,causesmutationof
DNAie.Gamma,cathoderays,Xrays..
•Ionizingradiationcanbeaserioushealthhazard,causingburns,
radiationsickness,cancer,andgeneticdamage

22. •Highenergyradiationwhichcausesionizationofvarious
substancesalongwithwater
•Theionizationresultsintheformationofalargenumberof
toxicO2metaboliteslikehydroxylradical,superoxideion,and
H2O2throughionizationofwater
•Radiationsterilizationisgenerallyexposedtoitemsinthe
driedstatewhichincludesurgicalinstruments,sutures,unit-
doseointments,plasticsyringes,anddrypharmaceutical
products
•Solardisinfection(sun):Principletheefficacyofsolar
disinfectionissignificantlylowasitrequiresalongperiodof
exposure,thisprocessiseconomical&environment-friendly
option

23. 2.Nonionizationradiation:Thisradiationwhichhavelow
energyandlowpenetrationpowerie.UVrays,IR,microwave...
Whenradiationpassesthroughthemicroorganismstheyabsorb
bynucleicacidofcellandcausedamageordeathdueto
abnormalityinnucleotideswhichinterferethereplicationand
mutationofDNA
APPLICATIONS
•Itisusedforsterilisationofairtopreventcrossinfectionin
hospitalsoperatingrooms
•Usedforsurfacesterilisationandmaintenanceofasepticarea
inthepharmaceuticalindustry
•Itisusedforsterilisationbeforepackingorpackagedfood

24. Ultraviolet radiation
•Includeslightraysfrom150-3900Å,ofwhich2600Åhasthe
highestbactericidaleffect
•Non-ionizingwaveshaveaverylittlepenetrationpower,so
microorganismsonlyonthesurfacearekilled.
•Thesewavesareabsorbedbymanymaterialslikenucleicacids,
CauseserrorinDNAreplicationandcausethedeathof
microbesbymutation
•ApplicationtoSterilizationofair&forthesurfacesterilizationof
asepticworkareas,andthetreatmentofmanufacturing-grade
water

26. ADVANTAGE
•Bestforheatsensitiveproducts
•Highpenetrationpower
•Effectivenessresult
•Cleanprocess
DISADVANTAGES
•Itislethalforworker’seyes&skinshouldbeprotectedfrom
directcontactwithultravioletradiation
•Requirequalifiedperson
•Noneconomical

27. CHEMICAL METHODS
1.ChemicalSterilizationistheprocessofremovalof
microorganismsbytheuseofchemicalbactericidalagents
2.Evenifphysicalmethodsofsterilizationaremoreappropriatefor
effectivesterilization,itisnotalwaysappropriatetouseforheat-
sensitivematerialslikeplasticsandbiologicalspecimens
3.Thechemicalmethodofsterilizationcanbecategorizedas
•liquidsterilization
•gaseoussterilization

29. GASEOUS STERILISATION
•Sterilisationisdonewiththehelpofchemicalingaseousform
iscalledgaseousSterilization
•Gaseoussterilizationisamoreeffectivetechniqueasgasescan
passthroughatinyorificeandprovidemoreeffectiveresults

30. Ethylene Oxide
•Itiscolourlessgasatroomtemperatureandcanbeliquefiedeasily
&actasbactericidalagent
•MOA:Asaalkylatingagentsreactwithamino,hydroxyl,carboxyl,
nitrogrpspresentinproteinsandDNAofmicroorganisms,the
DNAreplicationinhibited
•widerangeofcompatibilitytousealmost70%ofallsterilizations
andaround50%fordisposablemedicaldevicesevenwhen
repeatedlyappliedalsooftenreplacesothersterilization
techniqueslikeheat...
•diffusesintomanypackagingmaterialslikerubber,plastics,fabric,
andpaper
•Ethyleneoxidekillsallmicroorganisms,suchasbacteria(including
spores),viruses,andfungi

31. Formaldehyde
•Thegasisobtainedbyheatingformalin(37%w/v)toa
temperatureof70-80°C.Itisabroad-spectrumbactericidal
agent
•MOA:Thegaspenetratedthroughcellanddestroythecell
proteinsandbyinterferingwithnitrogenbasepresentonDNA
•Application:tosterilizationofreusablesurgicalinstruments,
specificmedical,diagnosticandelectricalequipment...
•Formaldehydeworksontheprincipleofmodificationofprotein
andnucleicacid.
•Asaresultofthelowpenetratingpower,itsuseisoftenlimited
topaperandcottonfabrics

32. Nitrogen dioxide
•Rapidandeffectivesterilantthatcanbeusedfortheremovalof
commonbacteria,fungi,andevenspores.
•NO2hasalowboilingpoint(20°C)whichallowsahighvapor
pressureatstdtemperature
•MOA:Thebiocidalactionofthisgasinvolvesthedegradationof
DNAbythenitrationofphosphatebackbone,whichresultsin
lethaleffects
•Advantage-lowlevelofgasusedandthehighvapourpressure

33. Ozone
•Highlyreactiveindustrialgasthatiscommonlyusedtosterilize
airandwaterandasadisinfectantforsurfaces
•Thehighreactivityofozoneallowstheremovalofwasteozone
byconvertingtheozoneintooxygenbypassingitthrougha
simplecatalyst.
•However,becauseozoneisanunstableandreactivegas,ithas
tobeproducedon-site,whichlimitstheuseofozonein
differentsettings
•Itisalsoveryhazardousandthusonlybeusedata
concentrationof5ppm,whichis160timeslessthanthatof
ethyleneoxide

34. Liquid Sterilisation
•Liquidsterilizationistheprocessofsterilizationwhich
involvesthesubmergingofequipmentintheliquidsterilant
tokillallviablemicroorganismsandtheirspores
•Althoughliquidsterilizationisnotaseffectiveasgaseous
sterilization,itisappropriateinconditionswherealowlevel
ofcontaminationispresent

35. Hydrogen peroxide
•Liquidchemicalsterilizingagentwhichisastrongoxidant
andcandestroyawiderangeofmicroorganisms
•Ithasashortsterilizationcycletimeas28minutes
•However,ithasdrawbackslikelowmaterialcompatibility,
lowercapacityofpenetration,andassociatedhealthrisks
•Vaporizedhydrogenperoxide(VHP)isusedtosterilize
largelyenclosedandsealedareas,suchasentireroomsand
aircraftinteriors

36. Glutaraldehyde
•Itisanacceptedliquidsterilizingagentwhichrequires
comparativelylongimmersiontime
•Fortheremovalofallspores,itrequiresaslongas22hoursof
immersiontime
•Thepenetrationpowerisslowthususeinlimitedtocertain
surfaceswithlesscontamination.
•Itisanoxidizingagentandactasoxidizingorganiccompounds
whichresultsinthemodificationofproteinsinmicrobeswhich
mightultimatelyleadtodeath&toreachsterilization
submersionfor20-24hoursisrequired.
Hypochlorite

37. APPLICATION
•Usedtosterileoperationtheatre,asepticareas
•Usefultoheataswellasmoistsensitiveproducts
•Usetopreventfood,books,Cloths,Heavyequipment’s
•Highpenetrationaswellaseffectiveprocess
ADVANTAGE
•Ethyleneoxidehasagoodpenetrationpower
•Usetosterilemoistsensitivesubstanceandequipmentbcz
onlyalowhumidityisrequired
DISADVANTAGE
•Carcinogenicagentgases
•Toxictoeye,Nasalirritation

38. MECHANICAL METHODS
Thesolutioncanbesterilisedbyfiltrationthroughbacteriaproof
filters.Filtrationmethoddoesn’tdestroybutremovesthe
microorganism
Thesefiltersretainthebacteriaandthesterilefiltrateiscollectedin
sterilisedreceive.Thewholeapparatusmustbesterile
TYPESOFBACTERIAPROOFFILTER
1.Ceramicfilters/filtercandles:
•ThesearemadeofporcelainWhenvacuumisapplied,pressure
insidethecandleisdecreased
•Duetothedifferenceinpressurebetweenoutside&insideofthe
candle,thesolutionmovesintocandle

40. 2.SEITZFILTER
•Theasbestossheetismadeupofasbestosfibres(magnesium
trisilicate)with0.01to5micronsporesize
3.SINTEREDGLASSFILTERS
•Thesearemadefromborosilicateglass
4.HEPAFILTER
•Usetosterileairorgasesinindustries

42. MEMBRANE FILTER
•Thesearemadeofcellulose
acetateorcellulosenitrate.These
arefixedinmetallicholdersalso
calledmilliporeFilter
APPLICATIONS
•Methodisusefulforsterilisationof
parenteralsolutionswhich
contains heat sensitive
medicaments or biological
productsi.e.Insulin,bloodserum...

43. ADVANTAGES
•SuitableforsterilisationofSolutionformulationlikeeyedrops,
injections,antibioticsolutionsetc
•Bothclarificationandsterilisationaredonesidebyside
DISADVANTAGES
•Themethodisn’treliableoneandsterilitytestrequired
•Thesuspensionandoilypreparationscannotbesterilisedby
thismethod
•Theyarechancesofabsorptionofmedicamentfromsolutionby
thefilter
•AseptictechniqueisnecessaryandHighlytrainedstaffis
required

44. Evaluation of efficiency of the
Sterilization method
•Toensuretheabilityofsterilizationprocesstokillthebacteria
•TotakeHighdegreeofqualityassurance
•Thisprovesthatanyprocedures,equipment,materialactivityor
systemleadstotheexpectedresultandproducequalityproduct
•DifferentQAtechniquesareusedforevaluationofsterilization
methods
STERILITYCRITERIA
•Bioburden/MicrobialLimitTestingisgivesthenumberortype
ofbacterialiving(Viable)onasurfacethathasn’tbeensterilized,
whichisperformedonp’ceuticals&medicaldevicesforquality
controlpurpose

45. •Productsuseinpharmaceuticalfieldrequiredtocontrol
microbiallevelsduringprocessingandhandling
•Themaximumpermittedconcentrationofcontaminantsmay
bespecifiedbyvariouspharmacopoeiaorthelevels
establishedbythemanufacturerduringproduced
development
•ThebioburdenquantificationisexpressedinColony
FormingUnitandresultexpressasCFU/mlforliquidsand
CFU/gmforsolids
•Performedbymembranefiltermethod,platecountmethod

46. D-ValueOrDecimalReductionTime
•Timeinminutesatanydefined
temperaturetodestroy90%viable
microorganismsiscalledD-value
•DValueistimerequiredtoreduce
themicrobialpopulationbyone
decimalor10%oforiginal
population
•Dvalue=t/log(No-Nt)
•Noisinitialno.Ofmicrobes
•Ntisafterexposuretime

47. Z-ValueOrThermalReductionTime
•Zisthenumberofdegreeoftemperaturechangerequiredto
produce10foldchangeinD-value
•Zvaluemaybedefineasthetemperaturerequiredforonelog
reductioninDvalue
•Zvalue=T2-T1/(logD1-logD2)
•WhereT1isinitialtemperature
•T2isfinaltemperature
•Disdecimalreductiontime

48. F-Value
•Usedinthefoodindustry,alsocalled“UnitofLethality”
•Definedastheequivalentinminutesof121˚Cheatconsider
withrespecttoitscapacitytodestroysporesorvegetativecell
oforganisms
•Itisatimeinminutesrequiredtokillaknownpopulationof
microorganismsinagivenconditionsalsousedtocalculatethe
no.ofbacteria'sremainaftertheprocess
•F=D(logNo–logN)
•Where,
Disdecimalvalueat121
o
C
Noisinitialpopulationnumber
Nisfinalpopulationnumber

49. Q
10ValueorTemperaturecoefficient
•Itistheincreaseinthereactionorkillingratebroughtabout
byariseintemperatureof10degreeCelsius
•Q
10=K
T+10/K
T
•Kisconstant
InactivationFactor
•Inactivationfactorisadegreetowhichtheviablepopulation
oforganismisreducedbythetreatmentappliedandis
obtainedbydividingtheinitialviablecountbythefinal
viablecount
•IF=10
t/D
•Where,tisholdingtime&Dvalue

50. STERILITYINDICATORS
•Changingappearancesincolourorpattern
•Indicatorsareusedinclinicalandresearchenvironmentswhere
contaminationeliminationiscrucial
•Types-PhysicalIndicators,Chemical&BiologicalIndicator
PHYSICALINDICATOR
1.MoistheatIndicator:AMasterProcessRecord(MPR)isprepared
aspartofthevalidationprocedureforaparticularautoclaveThe
MPRshouldbecheckedatannualintervalsandwhenever
significantchangesoccurintheBPR(BatchProductionRecords)

51. 1.Dryheat:Atemperaturerecordchartismadeofeach
sterilizationcycleandiscomparedagainstamastertemperature
record
2.RadioSterilization:Aplasticdosimetergraduallydarkensin
proportiontotheradiationabsorbsgivesanaccuratemeasureof
theradiation
3.Gaseousmethods:Temperaturesaremonitoredandrecordedfor
eachsterilizationcyclebytemperatureprobes,androutineleak
testsareperformedtoensuregas-tightseals
4.Filtration:Bubblepointpressuretestisatechniqueemployedfor
determiningtheporesizeoffilters

52. CHEMICALINDICATORS
•Theyareappliedeithertooutsideorplacedinsidetheinstrument
unite.g.packs,peelpouches,containers,etc…
•Theydon’tprovethatsterilizationhasbeenachieved,buttheycan
provideanearlyindicationofaproblem
1.Browne’stubes:Mostcommonlyusedchemicalindicator,small
sealedcolouredtubeshavingareactionmixture.Exposeto
temperatureresultinginthechangeofcolour(Redtogreen)
2.WittnessTubes:Consistofsinglecrystallinesubstancesofknown
meltingpointcontainedinglasstubeslikeSulphur(115°C),
Succinicanhydride(120°C),Benzoicacid(121°C)

53. 3.HeatSensitiveTape
•Autoclavetapeworksbychangingcolorafterexposureto
temperaturescommonlyusedinsterilizationprocesses,typically
121°Cinasteamautoclave(usuallybeigetoblack)uponheating
BIOLOGICALINDICATORS
•Autoclavingat121°C-Bacillusstearothermophilus,B.Coagulans
&Clostridiumsporogenes
•Dryheatat160°C–B.subtilisvar.niger
•EthyleneOxide-Bacillussubtilisvar.niger
•Ionizingradiations-Bacilluspumilus
•Membranefilter(0.22micrometer)-Pseudomonasdiminuta