沉降菌的测定Determinationofsettlingmicrobe课件

沉降菌的测定

Determinationofsettlingmicrobe重庆医药高等专科学校

ChongqingMedicalandPharmaceutialCollege学习目标

Learningobjective知识目标

KnowledgeObjectives能力目标

Abilityobjective1、掌握洁净室（区）相关基本概念；悬浮粒子、浮游菌、沉降菌的测定方法。

1.Masterthebasicconceptsrelatedtothecleanroom(area);thedeterminationofairborneparticles,airbornemicrobe,settlingmicrobe.2、熟悉空气洁净度标准。

2.Familiarwithaircleanlinessstandards.3、了解悬浮粒子、浮游菌、沉降菌的测定仪器。

3.Understandtheinstrumentsforthedeterminationofairborneparticles,airbornemicrobeandsettlingmicrobe.能正确、熟练、规范实施悬浮粒子、沉降菌和浮游菌的测定。

Beabletoperformthedeterminationofairborneparticles,settlingmicrobe,andairbornemicrobecorrectly,proficiently,andinastandardizedmanner.素质目标

Qualityobjective具备无菌观念，培养良好的工作习惯。

Possessanasepticmindsetanddevelopgoodworkhabits.重点难点

Keypointanddifficulty重点悬浮粒子、浮游菌、沉降菌的测定方法。Keypoint

Testmethodsforairborneparticles,airbornemicrobe,andsettlingmicrobe.难点洁净室悬浮粒子的测定和浓度计算。Difficulty

Determinationofairborneparticlesinacleanroomandcalculationofconcentration.二、沉降菌的测定

II.Determinationofsettlingmicrobe（一）测试依据

(I)Testbasis《中华人民共和国国家标准医药工业洁净室（区）沉降菌测试方法》（GB/T16294－2010）

Testmethodforsettlingmicrobeincleanroom(area)ofthepharmaceuticalindustry（GB/T16294－2010）（二）测试方法

(II)Testmethods采用沉降法，即通过自然沉降原理收集在空气中的生物粒子于培养基平皿，经若干时间，在适宜的条件下让其繁殖到可见的菌落进行计数，以平板培养皿中的菌落数来判定洁净环境内的活微生物数，并以此来评定洁净室（区）的洁净度。Adoptthesedimentationmethod,thatis,collectthebiologicalparticlesintheairthroughtheprincipleofnaturalsedimentationtomediumplatepetridish,afteracertainperiodoftime,letitpropagatetothevisiblecoloniesforcountinginsuitableconditions,anddeterminethenumberoflivemicroorganismsinthecleanenvironmentbythecolonyformingunitsintheplatepetridish,anduseittoassessthecleanlinessofthecleanroom(zone).二、沉降菌的测定

II.Determinationofsettlingmicrobe（三）测试仪器

(III)Testinstruments01培养皿：90mm×15mm。

Petridish:90mmx15mm.02培养基：大豆酪蛋白琼脂培养基（TSA）或沙氏培养基（SDA）。

Medium:tryptosesoyaagar(TSA)orsabourauddextroseagar(SDA).恒温培养箱。

Constanttemperatureincubator.03高压蒸汽灭菌器。

Autoclavesterilizer二、沉降菌的测定

II.Determinationofsettlingmicrobe（四）测试规则

(IV)Testrules二、沉降菌的测定

II.Determinationofsettlingmicrobe（1）测试条件：温度、湿度应与生产及工艺要求相适应，如无特殊要求，温度控制在18～26℃，相对湿度控制在45％～65%之间为宜。(1)Testconditions:Temperatureandhumidityshouldbecompatiblewiththeproductionandprocessrequirements.Iftherearenospecialrequirements,itisappropriatetocontrolthetemperatureat18to26℃andtherelativehumiditybetween45%and65%.（2）测试状态：静态和动态均可。(2)Teststatus:At-restandoperationalareallallowed.二、沉降菌的测定

II.Determinationofsettlingmicrobe（四）测试规则

(IV)Testrules（3）测试时间

(3)Testtime:净化系统、功能完备，生产设备、原料和人员均未入场Purificationsystem,functionalityiscomplete,productionequipment,rawmaterialsandpersonnelarenotinthefield.生产条件准备就绪，人员未入场（生产前）Productionconditionsareready,personnelarenotinthefield(beforeproduction).正常的生产状态（生产中）Normalproductionstatus(duringproduction)空态As-bulit静态aAt-resta动态Operational静态bAt-restb生产结束后，人员撤离现场（生产后）Afterproduction,personnelevacuatethesite(afterproduction).测试时间Testtime:单向流Unidirectionalairflow净化Purification10min非单向流Non-unidirectionalairflow净化Purification30min净化Purification10min净化Purification30min记录生产开始时间及测试时间Thetimeoftheproductstartingandthetesttimeshallberecorded.净化Purification10min净化Purification20min（五）测试步骤

(V)Teststeps二、沉降菌的测定

II.Determinationofsettlingmicrobe01培养基严格消毒。

Themediumisstrictlysterilized.02采样：将已制备好的培养皿按采样点布置图逐个放置，然后从里到外逐个打开培养皿盖，使培养基表面暴露在空气中。静态测试时，培养皿暴露时间为30分钟以上；动态测试时，培养皿暴露时间为不大于4小时。Sampling:Thepreparedpetridishesareplacedonebyoneaccordingtothelayoutofthesamplingsite,andthenthelidsofthepetridishesareopenedonebyonefromtheinsidetotheoutsidesothatthesurfaceofthemediumisexposedtotheair.Forat-resttesting,petridishesareexposedfor30minutesormore;Foroperationaltesting,petridishesareexposedfornomorethan4hours.二、沉降菌的测定

II.Determinationofsettlingmicrobe（五）测试步骤

(V)Teststeps①最少采样点数目可参考悬浮粒子测试方法。

Theminimumnumberofsamplingpointscanrefertotheairborneparticletestmethod.②采样点的位置可参考悬浮粒子测试方法。

Thelocationofthesamplingpointscanrefertotheairborneparticletestmethod.③最少培养皿数（下表）。

MinimumnumberofPetridishes(tablebelow).④采样次数：每个采样点一般采样一次。

Samplingtimes:eachsamplingpointisnormallysampledonce.最少培养皿数

MinimumnumberofPetridishes洁净度级别

Cleanlinessclass所需90mm培养皿数（以沉降0.5小时计）

Numberof90mmPetridishesrequired(basedonsedimentationfor0.5hours)A14B2C2D2二、沉降菌的测定

II.Determinationofsettlingmicrobe（五）测试步骤

(V)Teststeps采样点的布置

Layoutofsamplingpoints二、沉降菌的测定

II.Determinationofsettlingmicrobe（五）测试步骤

(V)Teststeps采样

Sampling二、沉降菌的测定

II.Determinationofsettlingmicrobe（五）测试步骤

(V)Teststeps03培养Culture全部采样结束后，将培养皿倒置于恒温培养箱中培养。采用TSA配制的培养皿经采样后，在30～35℃培养箱中培养，时间不少于2天；采用SDA配制的培养基经采样后，在20～25℃培养箱中培养，时间不少于5天。每批培养基应有对照试验，检验培养基本身是否污染。可每批选定3只培养皿作对照培养。

Afterallsamplingwascompleted,thepetridishesareinvertedandincubatedinaconstanttemperatureincubator.Aftersampling,thepetridishpreparedwithTSAisculturedinanincubatorat30-35℃fornotlessthan2days;Aftersampling,themediumpreparedwithSDAisculturedinanincubatorat20~25℃fornotlessthan5days.Eachbatchofmediumshouldhaveacontroltesttocheckforcontaminationofthemediumitself.Threepetridishesperbatchmaybeselectedforcontrolculture.二、沉降菌的测定

II.Determinationofsettlingmicrobe（五）测试步骤

(V)Teststeps04菌落计数Colonycount用肉眼对培养皿上所有的菌落直接计数、标记或在菌落计数器上点计，然后用5～10倍放大镜检查，有否遗漏。若平板上有2个或2个以上的菌落重叠，可分辨时仍以2个或2个以上菌落计数。

Allcoloniesonthepetridisharecounteddirectlywiththenakedeye,labeled,orcountedonacolonycounter,andthenexaminedwitha5-to10-powermagnifyingglasstoseeifanythinghasbeenmissed.If2ormorecoloniesoverlapontheplate,theyarestillcountedas2ormorecolonieswhendistinguishable.二、沉降菌的测定

II.Determinationofsettlingmicrobe（五）测试步骤

(V)Teststeps05结果计算Calculationofresults平均菌落数按下列公式计算。

Theaveragecolonyformingunitsiscalculatedbythefollowingformula.为平均菌落数；

istheaveragecolonyformingunits;m1为1号培养皿菌落数；m2为2号培养皿菌落数；mn为n号培养皿菌落数；n为培养皿总数。

m1isthecolonyformingunitsinPetridish1;m2isthecolonyformingunitsinPetridish2;mnisthecolonyformingunitsinPetridishn;nisthetotalnumberofpetridishes.二、沉降菌的测定

II.Determinationofsettlingmicrobe（五）测试步骤

(V)Teststeps06结果评定Resultsevaluation用平均菌落数判断洁净室（区）空气中的微生物。每个测试点的平均菌落数必须低于所选定的评定标准。在静态测试时，若某测点的沉降菌平均菌落数超过评定标准，则应重新采样两次，两次测试结果均合格才能判为符合。

Determinethemicroorganismsintheairofacleanroom(zone)byusingtheaveragecolonyformingunits.Theaveragecolonyformingunitsforeachtestpointmustbebelowtheselectedevaluationstandard.Inthestatictest,iftheaveragecolonyformingunitsofsettlingmicrobeatatestingpointexceedstheevaluationstandard,thenitshouldbere-sampledtwice,andtheresultsofthetwotestsshallbequalifiedinordertobejudgedascompliant.洁净度级别

Cleanlinessclass浮游菌

AirbornemicrobeCFU/m3沉降菌90mm

Settlingmicrobe90mmCFU/4h表面微生物

Surfacemicroorganisms接触55mm

Contact55mmCFU/碟

CFU/dish5指手套

5-fingerglovesCFU/手套

CFU/glovesA级

LevelA1111B级

LevelB10555（六）注意事项

(VI)Notes二、沉降菌的测定

II.Determinationofsettlingmicrobe测试用具要做灭菌处理。

Sterilizetestinstruments.防止人为对样本的污染。

Preventhumancontaminationofsamples.计数时不要漏计培养皿边缘生长的菌落，并注意细菌菌落与培养基沉淀物的区别，必要时用显微镜鉴别。Whencounting,donotmissthecoloniesgrowingattheedgeofthepetridishandnotethedifferencebetweenthebacterialcoloniesandthemediumprecipitate,usingthemicroscopetoidentifythemifnecessary.采样。

Sampling.对照试验。

controlledtrial.二、沉降菌的测定

II.Determinationofsettlingmicrobe（七）日常监测

(VII)Routinemonitoring（八）沉降菌测试报告

(VIII)Settlingmicrobetestreport测试单位

Testunit环境温度（