沉降菌的測定

Determinationofsettlingmicrobe重慶醫(yī)藥高等專科學校

ChongqingMedicalandPharmaceutialCollege學習目標

Learningobjective知識目標

KnowledgeObjectives能力目標

Abilityobjective1、掌握潔凈室（區(qū)）相關(guān)基本概念；懸浮粒子、浮游菌、沉降菌的測定方法。

1.Masterthebasicconceptsrelatedtothecleanroom(area);thedeterminationofairborneparticles,airbornemicrobe,settlingmicrobe.2、熟悉空氣潔凈度標準。

2.Familiarwithaircleanlinessstandards.3、了解懸浮粒子、浮游菌、沉降菌的測定儀器。

3.Understandtheinstrumentsforthedeterminationofairborneparticles,airbornemicrobeandsettlingmicrobe.能正確、熟練、規(guī)范實施懸浮粒子、沉降菌和浮游菌的測定。

Beabletoperformthedeterminationofairborneparticles,settlingmicrobe,andairbornemicrobecorrectly,proficiently,andinastandardizedmanner.素質(zhì)目標

Qualityobjective具備無菌觀念，培養(yǎng)良好的工作習慣。

Possessanasepticmindsetanddevelopgoodworkhabits.重點難點

Keypointanddifficulty重點懸浮粒子、浮游菌、沉降菌的測定方法。Keypoint

Testmethodsforairborneparticles,airbornemicrobe,andsettlingmicrobe.難點潔凈室懸浮粒子的測定和濃度計算。Difficulty

Determinationofairborneparticlesinacleanroomandcalculationofconcentration.二、沉降菌的測定

II.Determinationofsettlingmicrobe（一）測試依據(jù)

(I)Testbasis《中華人民共和國國家標準醫(yī)藥工業(yè)潔凈室（區(qū)）沉降菌測試方法》（GB/T16294－2010）

Testmethodforsettlingmicrobeincleanroom(area)ofthepharmaceuticalindustry（GB/T16294－2010）（二）測試方法

(II)Testmethods采用沉降法，即通過自然沉降原理收集在空氣中的生物粒子于培養(yǎng)基平皿，經(jīng)若干時間，在適宜的條件下讓其繁殖到可見的菌落進行計數(shù)，以平板培養(yǎng)皿中的菌落數(shù)來判定潔凈環(huán)境內(nèi)的活微生物數(shù)，并以此來評定潔凈室（區(qū)）的潔凈度。Adoptthesedimentationmethod,thatis,collectthebiologicalparticlesintheairthroughtheprincipleofnaturalsedimentationtomediumplatepetridish,afteracertainperiodoftime,letitpropagatetothevisiblecoloniesforcountinginsuitableconditions,anddeterminethenumberoflivemicroorganismsinthecleanenvironmentbythecolonyformingunitsintheplatepetridish,anduseittoassessthecleanlinessofthecleanroom(zone).二、沉降菌的測定

II.Determinationofsettlingmicrobe（三）測試儀器

(III)Testinstruments01培養(yǎng)皿：90mm×15mm。

Petridish:90mmx15mm.02培養(yǎng)基：大豆酪蛋白瓊脂培養(yǎng)基（TSA）或沙氏培養(yǎng)基（SDA）。

Medium:tryptosesoyaagar(TSA)orsabourauddextroseagar(SDA).恒溫培養(yǎng)箱。

Constanttemperatureincubator.03高壓蒸汽滅菌器。

Autoclavesterilizer二、沉降菌的測定

II.Determinationofsettlingmicrobe（四）測試規(guī)則

(IV)Testrules二、沉降菌的測定

II.Determinationofsettlingmicrobe（1）測試條件：溫度、濕度應與生產(chǎn)及工藝要求相適應，如無特殊要求，溫度控制在18～26℃，相對濕度控制在45％～65%之間為宜。(1)Testconditions:Temperatureandhumidityshouldbecompatiblewiththeproductionandprocessrequirements.Iftherearenospecialrequirements,itisappropriatetocontrolthetemperatureat18to26℃andtherelativehumiditybetween45%and65%.（2）測試狀態(tài)：靜態(tài)和動態(tài)均可。(2)Teststatus:At-restandoperationalareallallowed.二、沉降菌的測定

II.Determinationofsettlingmicrobe（四）測試規(guī)則

(IV)Testrules（3）測試時間

(3)Testtime:凈化系統(tǒng)、功能完備，生產(chǎn)設備、原料和人員均未入場Purificationsystem,functionalityiscomplete,productionequipment,rawmaterialsandpersonnelarenotinthefield.生產(chǎn)條件準備就緒，人員未入場（生產(chǎn)前）Productionconditionsareready,personnelarenotinthefield(beforeproduction).正常的生產(chǎn)狀態(tài)（生產(chǎn)中）Normalproductionstatus(duringproduction)空態(tài)As-bulit靜態(tài)aAt-resta動態(tài)Operational靜態(tài)bAt-restb生產(chǎn)結(jié)束后，人員撤離現(xiàn)場（生產(chǎn)后）Afterproduction,personnelevacuatethesite(afterproduction).測試時間Testtime:單向流Unidirectionalairflow凈化Purification10min非單向流Non-unidirectionalairflow凈化Purification30min凈化Purification10min凈化Purification30min記錄生產(chǎn)開始時間及測試時間Thetimeoftheproductstartingandthetesttimeshallberecorded.凈化Purification10min凈化Purification20min（五）測試步驟

(V)Teststeps二、沉降菌的測定

II.Determinationofsettlingmicrobe01培養(yǎng)基嚴格消毒。

Themediumisstrictlysterilized.02采樣：將已制備好的培養(yǎng)皿按采樣點布置圖逐個放置，然后從里到外逐個打開培養(yǎng)皿蓋，使培養(yǎng)基表面暴露在空氣中。靜態(tài)測試時，培養(yǎng)皿暴露時間為30分鐘以上；動態(tài)測試時，培養(yǎng)皿暴露時間為不大于4小時。Sampling:Thepreparedpetridishesareplacedonebyoneaccordingtothelayoutofthesamplingsite,andthenthelidsofthepetridishesareopenedonebyonefromtheinsidetotheoutsidesothatthesurfaceofthemediumisexposedtotheair.Forat-resttesting,petridishesareexposedfor30minutesormore;Foroperationaltesting,petridishesareexposedfornomorethan4hours.二、沉降菌的測定

II.Determinationofsettlingmicrobe（五）測試步驟

(V)Teststeps①最少采樣點數(shù)目可參考懸浮粒子測試方法。

Theminimumnumberofsamplingpointscanrefertotheairborneparticletestmethod.②采樣點的位置可參考懸浮粒子測試方法。

Thelocationofthesamplingpointscanrefertotheairborneparticletestmethod.③最少培養(yǎng)皿數(shù)（下表）。

MinimumnumberofPetridishes(tablebelow).④采樣次數(shù)：每個采樣點一般采樣一次。

Samplingtimes:eachsamplingpointisnormallysampledonce.最少培養(yǎng)皿數(shù)

MinimumnumberofPetridishes潔凈度級別

Cleanlinessclass所需90mm培養(yǎng)皿數(shù)（以沉降0.5小時計）

Numberof90mmPetridishesrequired(basedonsedimentationfor0.5hours)A14B2C2D2二、沉降菌的測定

II.Determinationofsettlingmicrobe（五）測試步驟

(V)Teststeps采樣點的布置

Layoutofsamplingpoints二、沉降菌的測定

II.Determinationofsettlingmicrobe（五）測試步驟

(V)Teststeps采樣

Sampling二、沉降菌的測定

II.Determinationofsettlingmicrobe（五）測試步驟

(V)Teststeps03培養(yǎng)Culture全部采樣結(jié)束后，將培養(yǎng)皿倒置于恒溫培養(yǎng)箱中培養(yǎng)。采用TSA配制的培養(yǎng)皿經(jīng)采樣后，在30～35℃培養(yǎng)箱中培養(yǎng)，時間不少于2天；采用SDA配制的培養(yǎng)基經(jīng)采樣后，在20～25℃培養(yǎng)箱中培養(yǎng)，時間不少于5天。每批培養(yǎng)基應有對照試驗，檢驗培養(yǎng)基本身是否污染?？擅颗x定3只培養(yǎng)皿作對照培養(yǎng)。

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

II.Determinationofsettlingmicrobe（五）測試步驟

(V)Teststeps04菌落計數(shù)Colonycount用肉眼對培養(yǎng)皿上所有的菌落直接計數(shù)、標記或在菌落計數(shù)器上點計，然后用5～10倍放大鏡檢查，有否遺漏。若平板上有2個或2個以上的菌落重疊，可分辨時仍以2個或2個以上菌落計數(shù)。

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

II.Determinationofsettlingmicrobe（五）測試步驟

(V)Teststeps05結(jié)果計算Calculationofresults平均菌落數(shù)按下列公式計算。

Theaveragecolonyformingunitsiscalculatedbythefollowingformula.為平均菌落數(shù)；

istheaveragecolonyformingunits;m1為1號培養(yǎng)皿菌落數(shù)；m2為2號培養(yǎng)皿菌落數(shù)；mn為n號培養(yǎng)皿菌落數(shù)；n為培養(yǎng)皿總數(shù)。

m1isthecolonyformingunitsinPetridish1;m2isthecolonyformingunitsinPetridish2;mnisthecolonyformingunitsinPetridishn;nisthetotalnumberofpetridishes.二、沉降菌的測定

II.Determinationofsettlingmicrobe（五）測試步驟

(V)Teststeps06結(jié)果評定Resultsevaluation用平均菌落數(shù)判斷潔凈室（區(qū)）空氣中的微生物。每個測試點的平均菌落數(shù)必須低于所選定的評定標準。在靜態(tài)測試時，若某測點的沉降菌平均菌落數(shù)超過評定標準，則應重新采樣兩次，兩次測試結(jié)果均合格才能判為符合。

Determinethemicroorganismsintheairofacleanroom(zone)byusingtheaveragecolonyformingunits.Theaveragecolonyformingunitsforeachtestpointmustbebelowtheselectedevaluationstandard.Inthestatictest,iftheaveragecolonyformingunitsofsettlingmicrobeatatestingpointexceedstheevaluationstandard,thenitshouldbere-sampledtwice,andtheresultsofthetwotestsshallbequalifiedinordertobejudgedascompliant.潔凈度級別

Cleanlinessclass浮游菌

AirbornemicrobeCFU/m3沉降菌90mm

Settlingmicrobe90mmCFU/4h表面微生物

Surfacemicroorganisms接觸55mm

Contact55mmCFU/碟

CFU/dish5指手套

5-fingerglovesCFU/手套

CFU/glovesA級

LevelA1111B級

LevelB10555（六）注意事項

(VI)Notes二、沉降菌的測定

II.Determinationofsettlingmicrobe測試用具要做滅菌處理。

Sterilizetestinstruments.防止人為對樣本的污染。

Preventhumancontaminationofsamples.計數(shù)時不要漏計培養(yǎng)皿邊緣生長的菌落，并注意細菌菌落與培養(yǎng)基沉淀物的區(qū)別，必要時用顯微鏡鑒別。Whencounting,donotmissthecoloniesgrowingattheedgeofthepetridishandnotethedifferencebetweenthebacterialcoloniesandthemediumprecipitate,usingthemicroscopetoidentifythemifnecessary.采樣。

Sampling.對照試驗。

controlledtrial.二、沉降菌的測定

II.Determinationofsettlingmicrobe（七）日常監(jiān)測

(VII)Routinemonitoring（八）沉降菌測試報告

(VIII)Settlingmicrobetestreport測試單位

Testunit環(huán)境溫度（