第3章-消毒灭菌与病原微生物实验室生物安全课件

第3章消毒与灭菌与

病原微生物实验室生物安全§1常用术语§2.消毒灭菌的方法§3.消毒灭菌的运用（自学）§4.影响消毒灭菌效果的因素

（自学）§5.病原微生物实验室生物安全复习题基本概念2.消毒(disinfection)1.灭菌(sterilization)3.防腐(antisepsis)5.无菌(asepsis)和无菌操作4.清洁(cleaning)SterilizationSterilizationisaprocessusedtoachievesterility,anabsolutetermmeaningtheabsenceofallviablemicro-organisms,includingviruses,bacteriaandtheirspores,andfungiandtheirspores.UsesofsterilizationSterilizationisrequiredforinstrumentsandmaterialsusedinproceduresthatinvolvedpenetrationintonormallysterilepartsofthebody,e.g.insurgicaloperations,intravenousinfusions,hypodermicinjectionsanddiagnosticaspirations.Itisalsorequiredformedia,reagentsandequipmentusedinlaboratorypractice.UseofdisinfectionDisinfectionisappliedincircumstancesinwhichsterilityisunnecessaryorsterilizingproceduresareimpracticable.AntisepsisAntisepsisisusedtodescribedisinfectionappliedtolivingtissuesuchasawound.Somewouldrestrictthetermantiseptictopreparationsappliedtoopenwoundsorabradedtissueandpreferthetermskindisinfectionfortheremovaloforganismsfromthehandsandintactskinsurfaces.CleaningCleaningisasoil-removingprocesswhichremovesmanymicro-organisms.Theyhavewideapplicationinthehospitalenvironmentandasthenecessaryprerequisitetosterilizationanddisinfection.Thedetergentsusedincleaninghavesomedisinfectionproperties,andthedilutioneffectofthoroughrinsingfurtherreducesthemicrobialload.Removalofdebrisdriedoninstrumentsorotheritemsthataredifficulttocleanmaybeenhancedbytheuseofultrasonicbaths.无菌操作：

防止微生物进入人体或其它物品的操作方法。消毒灭菌的方法物理消毒灭菌法化学消毒灭菌法（自学）消毒灭菌方法的选择考虑：待处理物品的性质、可能污染的微生物、接触者被感染的风险热力消毒灭菌法辐射杀菌法滤过除菌法干燥与低温抑菌法首选SterilizationanddisinfectionbyheatTheonlymethodofsterilizationthatisbothreliableandwidelyapplicableisbyheatingundercarefullycontrolledconditionsattemperaturesabove100℃toensurethatbacterialsporesarekilled.Heat:

Moistheat→usingsteamorwater.Itkillsmicro-organismsbycoagulatinganddenaturingtheirenzymesandstructuralproteins,aprocessinwhichwaterparticipates.

Dryheat→usingflameorhotair.Itkillsmicro-organismsbycausingadestructiveoxidationofessentialcellconstituents.Temperatureandtimeareinverselyrelated.MinimumrecommendedholdtimesforheatsterilizationProcessTemperature(℃)Holdtime(min)Dryheat1601201706018030Moistheat12115126101343Thosedonotincludetimeforheatinguporcoolingtheloadforsafehandling.Thusthetotalcycletimeforprocessingasterilizedarticlewillbemuchlonger.Saturatedsteamismoreefficientthandryheat

Thegreaterlethalactionofmoistheat.Steamisquickerinheatingupthearticletobesterilized.Whendrysaturatedsteammeetsacoolersurfaceitcondensesintoasmallvolumeofwaterandliberatestheheatofvaporization.

e.g.6litersofsteam(134℃)→10mlofwater→2162Jofheatenergy;6litersofair(134℃)→100Jofheatenergy;

（一）干热灭菌法

1.焚烧：废弃物、尸体

2.烧灼：接种环、试管口

3.干烤：160～170℃，2h.

油、粉末、炭钢手术器械、实验用玻璃器皿

4.红外线：医疗器械pathologicalwastematerialswastesharpneedlesInoculatingwires,loops,andpointsofforceps（二）湿热灭菌法1.巴氏消毒法(pasteurization)61.1～62.8℃，30min或71.7℃,15-30s主要用于牛乳和酒类消毒

2.煮沸法:

煮沸100℃，5-10min,杀灭细菌繁殖体，细菌芽胞经1-2h被杀死。常用于食具、刀剪等。如加入2%碳酸氢钠……3.流动蒸汽消毒法：一个大气压下100℃的水蒸汽消毒。细菌繁殖体15-30min.可被杀死，但芽胞不一定被全部杀灭4.间歇蒸汽消毒法(fractionalsterilization)：利用反复多次的流动蒸汽间歇加热消毒，100℃，15-30min.37℃过夜后,再蒸,如此反复3次.

如：血清培养基、含糖培养基血清凝固器5.高压蒸汽灭菌法：用于所有耐高温、耐湿物品103.4kPa(1.05kg/cm2)

121.3℃，15-20min.Atsea-level,boilingwateratatmosphericpressure(1bar)willproducesteamat98-100℃,2.4bar→125℃,3.0bar→134℃.1.紫外线（ultraviolet,UV）

波长240-300nm的紫外线具有杀菌作用以265-266nm最强。其主要作用于DNA，使一条DNA链上相邻的两个胸腺嘧啶共价结合形成二聚体，导致细菌变异和死亡。

Theshortestultravioletraysthatreachtheearth’ssurfaceinquantityhaveawavelengthofabout290nm.Moreeffectiveradiationof240-280nmisproducedbymercurylamps(ultravioletlamp).穿透力弱，故仅用于空气（如手术室、传染病房、无菌室）和不耐热物品表面消毒。对芽胞无作用；可逆性。DonotlookdirectlyintotheUVlightduringoperationofthedevice.ExposuretoUVlight,evenforabriefperiod,candamageyoureyes.Expositionoftheskinwithultravioletradiation(UV)isthemaincauseofskincancerdevelopment.Theconsistentlyincreasingincidencesofskintumorsisassociatedwithrecreationalsunexposure.Epidemiologicaldataindicatethatexcessiveorcumulativesunlightexpositiontakeplaceyearsanddecadesbeforethetumoroccurs.2.电离辐射IonizingirradiationUses:Itisusedtosterilizelargeamountsofprepackedsingle-useitemssuchasplasticsyringesandlatexcathetersandsurgicalgloves.薄膜滤菌器滤过除菌：用于分离细菌生长过程产生的毒素或其它可溶性产物、制备不耐高温的抗生素溶液、放射性药物、血清等。滤膜孔径≤0.45μm.滤过除菌不能除去大多数病毒和支原体

生物洁净技术高效分子空气过滤器（HEPAfilters）生物洁净室冷冻真空干燥法：是目前保存菌种的最好方法。在低温下快速将含菌样冻结，并减压抽真空，使水升华将样品脱水干燥，形成完全干燥的固体菌块。并在真空条件下立即融封，造成无氧真空环境，最后置于低温下，使微生物处于休眠状态，而得以长期保藏。

病原微生物的分类病原微生物实验室的分级高致病性病原微生物BSLBiosafetyLevel1(BSL-1)Laboratory

BiosafetyLevel2(BSL-2)Laboratory

SafetysuitsinthebiosafetylevelIIIfacilityBiosafetylevelIIIgloveboxEbolainfectionsperformedinabiosafetycabinetunderbiosafetylevel4conditionsTakingoutarackfromtheliquidnitrogentank,whichcontainsthevirusesforworkinbiosafetyle