3 Sterilization.ppt

Microbiology practice lecture

1. Practical Bacteriology -General Bacteriology -2009 -3
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Lesson 3
Sterilization and
Disinfection
Definitions
Heat
Filtration
Radiation
Chemical agents
Sterilization methods

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1-Sterilization
Isaprocessinwhichalllivingvegetativeandspore
formingmicroorganismsaredestroyed.
2-Disinfection
Isaprocessinwhichvegetative,nonsporing
microorganismsaredestroyed.
Definitions

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A-Physical methods
B-Chemical agents
Two main types of sterilization are used.
Sterilization methods

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1.Phenol and phenolic
2.Alcohols
3.Halogens
4.Heavy metals
5.Gaseous agents
6.Soap and detergents
A-Physical methods
1.Heat
2.Filtration
3.Radiation
B-Chemical agents

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1-Heat
Click over the section
Heat
A
Dry heat
B
Moist heat
Flaming
Short time
flaming
Hot
Air oven
Pasteurization Tyndalization
Steam
sterilization
Boiling

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Themosteffectivemethodfordestroyingmicroorganisms,itsoftwotypes,
dryandmoistheat.
A-Dryheat
1-Flaming:
•Thismethodiscommonlyusedinmicrobiologylabs.
•UsedforsmallmetalorglassobjectsInoculatingloops,
needles,forcepsandscissors,butnotforlargeobjects.
•Inoculatingloopsandneedlesshouldbeheateduntil
theyarered-hot.Beforetheyareintroducedinto
cultures,theymustbeallowedtocooldownsufficiently
topreventkillingorganismsthataretobetransferred.
1-Heat

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•Usedforflamingtesttubeopenings,
flasksandpipettesinordertoprevent
contamination.
•Thismethodusedforsterilizingdry
glassware,forceps,scalpsand
scissors.
•Notsuitableforheatsensitivematerials
likemanyplasticandrubberitems
•Thestandardsettingforahotairoven
isatleast2hoursat160°C.
2-Short time flaming:
3-Hot air oven:

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Hot air oven

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B-Moist heat:
Classified according to the temperature of water to:
1-Pasteurization
•Theprocesswasnamedafteritscreator,Frenchchemistand
microbiologistLouisPasteur.
•Itisaprocesswhichslowsmicrobialgrowthinfood.
•Pasteurizationusuallydestroyallthenon-sporeformerpathogens.

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Therearetwomainlymethodsforpasteurization:
•Holdingmethod(batchpasteurization):Involvesheatinglarge
batchesofmilktoalowertemperature,typically63-66ºCfor30
minutesfollowedbyquickcoolingtoabout4ºC.
•Flashmethod:Involvesheatingto72ºCfor20seconds.

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•Itisusefulforreducingviablelevelsifno
bettermethodisavailable.
•Boilinginwaterat100ºCfor15minuteswillkillmostvegetative
bacteriaandinactivateviruses,butboilingisineffectiveagainst
manybacterialandfungalspores.
2-Boiling
•Boilingisasimpleprocess,Itisanoptionavailabletomost
people,requiringonlywater,enoughheat,andacontainer.

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•Theprocessinvolvesboilingforaperiod(typically20minutes)at
atmosphericpressure,cooling,incubatingforadayThisprocess
repeatedtwotimes.(totally3times),finallyboilingagain.
•NamedafterJohnTyndall,itisalengthyprocessdesignedtoreducethe
levelofactivityofsporulatingbacteriathatareleftbyasimpleboiling
watermethod.
3-Tyndallization

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•Thethreeincubationperiodsaretoallowheat-resistantsporessurviving
thepreviousboilingperiodtogerminatetoformtheheat-sensitive
vegetative(growing)stage,whichcanbekilledbythenextboilingstep.
•Thisiseffectivebecausemanysporesarestimulatedtogrowbytheheat
shock.
•Theprocedureonlyworksformediathatcansupportbacterialgrowth.

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Awidely-usedmethodforheatsterilization.Itiscarriedoutwith
anautoclave,adevice,somewhatlikeapressurecooker.
Theprinciple:
Steamunderpressure.
Pressure,15lbs/in
2
(1atmosphere).
Temperature,121ºC.
Time,15-20min.
Thismethodusedforsterilizationofculturemedia,clothesand
surgicalmaterials.
4-Steam sterilization

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Bench top autoclave

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Top loading autoclave

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1.Temperature:Consideredasthemainfactorthataffectstheprocess
ofautoclaving.
2.Steam:Usedforcoagulationofbacterialprotoplasm(proteinsand
enzymes)thiscouldbeoccursat121ºC.
3.Pressure:Ithasnoeffectonautoclaving,butitisnecessaryforrising
temperatureover100ºC.
4.Time:Mainly15-20minutes,additionalsterilizingtimeisusually
requiredforliquidsandinstrumentspackedinlayers.
5.Trappedair:Mustbeexhaustedtotheoutsideasitaffectsthe
sterilizationquality.
6.Materialnature:Packedmaterialsneedmoretime,makesuretoleave
adequatedistancesbetweenLoadedmaterials,don’tsecurethebottles,
leavethemloosetopreventexplosion.
Factors affect sterilization by autoclave

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•Clearliquidsthatwouldbedamagedbyheat,
irradiationorchemicalsterilizationcanbesterilized
bymechanicalfiltration.
•Itdependsonseparationbyspecialfilters,Afilter
withporesize0.2µmwilleffectivelyremove
bacteria.Ifvirusesmustalsoberemoved,amuch
smallerporesizearound20nmisneeded.
•Thismethodiscommonlyusedforsensitive
pharmaceuticalsandproteinsolutionsthatcanbe
affectedbyheatliketoxins,serum,sugarsolutions
andantibiotics.
2-Filtration

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Typesoffilterused:
1.Membranefilters:
•Itismostcommonusedfiltersin
microbiologylab.
•Membranefiltersremovemicroorganisms
byscreeningthemout.
•Itismadeofcelluloseacetate,cellulose
nitrate,chemicallyinertandautoclavable.
•Widevaryofporesizesareavailable,the
mostusedoneis0.22µmwithaspecial
holder.

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2.Seitzfilters:Madeofasbestospadlayers.
3.Sinteredglassfilters:Madeofglasslikeplateswithporesthat
materialscanpassthrough.
4.Chamberlandfilters:Madeofkaolin.

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Two main types of sterilization by radiation.
3-Radiation
Ultraviolet Visible Infrared
4
nm
1000
nm
0
100
%
Killed
200
nm
400
nm
600
nm
800
nm265
nm
1-UltravioletlightorU.V.light(240–280nm)
•Ultravioletlightirradiationisusefulonlyforsterilizationofsurfaces
(benches,biologicalsafetycabinetsBSC).
•Itdoesn’tpenetrateglass,dirtfilms,waterandothersubstances
effectively(ineffectiveinshadedareas,includingareasunderdirt).
•Itburnstheskinanddamageseyes.
•MakesuretheUVlampsareoffwhentheareasareinuse.

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2-Ionizingradiation
•IthasaveryshortwavelengthlikeXraysandGammarays.
•TheyinducedamageinDNAbyvariousmechanismsincludingthe
productionoffreeradicals(H
2O
2,HO
2andHO).
•Itisanexcellentsterilizingagentandpenetratesdeepintoobjects.
•Gammaradiationisusedinthecoldsterilizationofantibiotics,
hormones,plasticdisposablesuppliessuchassyringesthatcouldbe
damagedbyheat.

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•Manydifferentchemicalsareavailableforuseas
disinfectantsorantiseptics,andeachhasitsown
advantagesanddisadvantages.
•Oftencalled“coldsterilization".
•Ideallythedisinfectantmustbeeffectiveagainstawide
varietyofinfectiousagentsathighdilutionsandinthe
presenceoforganicmatter.
•Theeffectofchemicalagentsiseitherbactericidalthat
cankillallmicroorganismsorbacteriostaticthatjust
inhibitthegrowthofbacteria.
B-Chemical agents

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1-Phenolandphenolics
•Thepurephenolarenotusednowduetoitsirritation
effectandbadodor.
•Phenolderivativeslikecresols,Lysoland
hexachlorophenearemostpopularantiseptics.
•Phenolicsactbydenaturatingproteinsanddisturbing
cellmembranes.
•Effectiveinthepresenceoforganicmaterial.
•Remainactiveonsurfaceslongafterapplication.

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•Alcoholsareamongthemostwidelyuseddisinfectant
andantiseptics.
•Theyarebactericidalandfungicidalbutnotsporicidal;
somelipid-containingvirusesarealsodestroyed.
•Ethanolandisopropanolalcoholareusedinabout
70-80%concentration.
•Theyactbydenaturatingproteinsanddissolving
membranelipids.
2-Alcohols

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•They are important antimicrobial agents like chlorineand iodine.
•Act by oxidation of cellular materials.
•Chlorine ( sodium hypochlorite) is the usual disinfectant for diary and
food industries, municipal water supplies and swimming pool.
•Iodine can be used as a dye in concentration of 2%.
•More recently iodine has been complexed with an organic carrier to form
iodophorwhich is water soluble, nonstaining and used in hospitals and
laboratories for disinfecting.
3-Halogens

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4-Heavy metals
•Mostofheavymineraldisinfectantscontainsmercuryandsilver,
includingorganicandinorganiccompounds.
•Commercialcompound(mercurochrome)usedasantisepsis.

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5-Gaseousagents
•Theformaldehydeandethyleneoxideareconsideredthemost
commongasesusedinsterilization.
•Theformaldehyde(formalinsolutions40%)isusedfordisinfectionof
halls,heatsensitivematerialssuchasplastictools.Itisalsousedin
vaccineindustries.
•Ethyleneoxidepenetrateswell,movingthroughpaper,cloth,andsome
plasticfilmsandishighlyeffective.

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6-Soapanddetergents
Itreducesthesurfacetensionand
characterizedbymoisturewithhighlywater
solubility.

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