實(shí)訓(xùn)項(xiàng)目二藥品生物檢定基本操作技術(shù)二PracticalTrainingProgramIIBasic

實(shí)訓(xùn)項(xiàng)目二藥品生物檢定基本操作技術(shù)（二）

PracticalTrainingProgramIIBasicOperationalTechniquesofBiologicalAssayofDrugs(Ⅱ)細(xì)菌接種技術(shù)）

BacterinationTechniques學(xué)習(xí)目標(biāo)

Learningobjective知識(shí)目標(biāo)

KnowledgeObjectives能力目標(biāo)

Abilityobjective1、掌握倒平板的方法。

1.Masterthemethodofpouringplate.2、熟悉平板劃線接種。

2.Familiarizewithplatestreakinoculation.3、了解細(xì)菌接種的原理。

3.Understandtheprincipleofbacterination.能正確制作培養(yǎng)細(xì)菌用的平板，能正確進(jìn)行平板劃線接種。

Beabletocorrectlymakeplatesforbacterialcultureandperformplatestreakinoculationcorrectly.素質(zhì)目標(biāo)

Qualityobjective培養(yǎng)學(xué)生樹(shù)立無(wú)菌觀念。

Trainstudentstoestablishtheasepticconcept實(shí)訓(xùn)內(nèi)容

Practicaltrainingcontent1.制作平板。

1.Makeaplate.2.練習(xí)劃線接種。

2.Practicestreakinoculation.連續(xù)劃線

Continuousstreaking分區(qū)劃線

Zonestreaking材料與儀器

Materialsandinstruments菌種葡萄球菌

Strainsstaphylococcus培養(yǎng)基固體培養(yǎng)基和液體培養(yǎng)基

2.CulturemediumSolidculturemediumandliquidculturemedium3.儀器接種環(huán)、酒精燈、恒溫箱、酒精棉球、打火機(jī)、記號(hào)筆、試管架等。

3.InstrumentsInoculatingrings,alcohollamps,thermostats,alcoholcottonballs,lighters,markers,testtuberacks,etc.一、培養(yǎng)基及制備方法

I.Culturemediumandpreparationmethods

培養(yǎng)基是人工配制的生物營(yíng)養(yǎng)物質(zhì)，即用人工方法將多種物質(zhì)按各種微生物生長(zhǎng)繁殖的需要配制成的一類(lèi)混合營(yíng)養(yǎng)物質(zhì)，用于細(xì)菌的培養(yǎng)、分離、鑒定、研究和保存。

Culturemediumisartificiallyformulatedbiologicalnutrients,thatis,artificialmethodscanbeusedtoformulateavarietyofsubstancesintothemixednutrientsaccordingtotheneedsofthegrowthandreproductionofvariousmicroorganisms,forbacteriacultivation,isolation,identification,researchandpreservation.（一）培養(yǎng)基分類(lèi)(I)Classificationofculturemedium1．按物理性狀劃分為液體、流體、固體、半固體；

Dividedintoliquids,fluids,solids,semi-solidsaccordingtophysicalproperties;LiquidculturemediumSolidculturemediumSlantculturemediumPlateculturemedium2．按用途劃分為：Dividedbyuse:基礎(chǔ)培養(yǎng)基：普通瓊脂培養(yǎng)基

Basalculturemedium:commonagarculturemedium增菌培養(yǎng)基：專(zhuān)一性強(qiáng)，使目的菌擴(kuò)大生長(zhǎng)。

Enrichmentculturemedium:strongspecialization,sothatthetargetbacteriatoexpandthegrowth.鑒別培養(yǎng)基、選擇培養(yǎng)基：根據(jù)各種細(xì)菌的生化特性鑒別種屬的培養(yǎng)基

Indicatorculturemedium,selectiveculturemedium:identifytheculturemediumofspeciesaccordingtothebiochemicalcharacteristicsofvariousbacteria第二節(jié)藥品生物檢定技術(shù)的基本操作

SectionIIBasicoperationofbiologicalassaytechnologyofdrugs（二）培養(yǎng)基制備

(II)Preparationofculturemedium培養(yǎng)基的制備可按處方配置，也可使用按處方生產(chǎn)的符合規(guī)定的脫水培養(yǎng)基。試劑要求使用化學(xué)純（CP）以上的規(guī)格。Preparationofculturemediummaybeconfiguredbyprescriptionorbyusingdehydratedculturemediummanufacturedtoprescriptionincompliancewiththeregulations.Reagentsarerequiredtobechemicallypure(CP)orabovespecifications.PreparationofculturemediumContainersGlasswareorenamelwarePHcalibrationAcidoralkalicalibrationSubpackageLiquidculturemedium:1/3ofthetesttubevolumeSolidculturemedium:Subpackedwithconicalflask,2/3ofthecontainer.Semi-solidculturemedium:1/3ofthetesttubevolumeSterilizewithin2hafterpreparationPHcalibrationStorageCoolanddryplace案例

Case配制LB培養(yǎng)基——液體

PreparationofLBculturemedium-liquid按照LB培養(yǎng)基的配方，分別稱(chēng)量10g胰蛋白胨，5g酵母提取物，10g置于1000ml的燒杯中，加入少于所需要的水量，用玻棒或磁力棒攪拌溶解。待藥品完全溶解后，轉(zhuǎn)移至1L的量筒，定容至1000ml

AccordingtotheformulaofLBculturemedium,weigh10goftryptone,5gofyeastextract,and10gina1,000mlbeakerrespectively,addthewaterlessthanrequired,andstirtodissolvewithaglassrodoramagneticbar.

Aftercompletedissolutionofthedrugs,transfertoa1Lmeasuringcylinderanddeterminethevolumeto1,000ml用1mol/L的NaOH調(diào)節(jié)溶液pH值為7.2左右。

AdjustthepHofthesolutiontoabout7.2with1mol/LNaOH.案例

Case配制培養(yǎng)基——固體

Preparationofculturemedium-Solid稱(chēng)量4.5g瓊脂粉末，置于錐形瓶中，量取300ml液體培養(yǎng)基，倒入錐形瓶，充分搖勻。微波爐加熱煮沸融化瓊脂（制備斜面時(shí)需操作）。

Weigh4.5gofagarpowderandplaceitinaconicalflask,measure300mlofliquidculturemedium,pouritintotheconicalflaskandshakeitthoroughly.Melttheagarbyheatingandboilinginmicrowaveoven(needtobeoperatedwhenpreparingbevel).制作平板

Makeaplate倒平板：將融化的滅菌后的培養(yǎng)基倒入平板

Pouringplate:Pourmeltedsterilizedculturemediumontotheplate.冷卻，凝固，倒置

Cool,solidifyandinvert防止培養(yǎng)液蒸發(fā)的附在上板的水蒸氣倒流到培養(yǎng)液中

Preventthewatervaporattachedtotheupperplatethatevaporatesfromtheculturesolutionfromflowingbackintotheculturesolution.二、微生物接種技術(shù)

Ⅱ.Microbialinoculationtechnology（一）接種工具

(I)Inoculatingtools1．接種環(huán)和接種針

Inoculatingringandinoculatingneedle環(huán)或針

Ringorneedle金屬柄

Metalhandle絕緣柄

Insulatedhandle用于固體、液體培養(yǎng)基的接種

Forinoculationofsolidandliquidculturemedium用于半固體培養(yǎng)基的接種

Forinoculationofsemi-solidculturemedium環(huán)（針）部分材質(zhì)為鉑金絲或鎳鉻絲

Thematerialofthering(needle)partisplatinumwireornickel-chromewire.接種是指將微生物移接到另一個(gè)滅菌的新鮮培養(yǎng)基中，使其生長(zhǎng)繁殖的過(guò)程。Inoculationistheprocessoftransferringmicroorganismstoanothersterilizedfreshculturemediumforgrowthandreproduction.2．玻璃涂棒

2.Glasscoatingbar在固體培養(yǎng)基表面將菌液均勻涂布時(shí)。主要用于液體標(biāo)本的涂布接種。

Whencoatingthebacterialliquidevenlyonthesurfaceofsolidculturemedium.Mainlyusedforliquidspecimenspreadinoculation.涂布棒是由直徑為2~3mm的玻璃棒彎曲成L形制成。

Spreadingrodismadeofglassrodswithadiameterof2-3mmbentintoL-shape.3．滴管或吸管

3.Dropperorpipette用于液體接種

Forliquidinoculation（二）無(wú)菌操作

(II)Asepticoperation無(wú)菌操作技術(shù)：指在微生物實(shí)驗(yàn)工作中，控制或防止各類(lèi)微生物的污染及其干擾的一系列操作方法和有關(guān)措施。

Asepticoperationtechnique:referstoaseriesofmethodsandmeasurestocontrolorpreventthecontaminationandinterferenceofvariousmicroorganismsinmicrobiologicalexperiments.目的：

Purpose:保證待測(cè)檢品不被環(huán)境中微生物污染；

Toensurethatthetestproducttobetestedisnotcontaminatedbymicroorganismsintheenvironment;防止被檢微生物在操作中污染環(huán)境或感染操作人員。

Topreventthemicroorganismstobetestedfromcontaminatingtheenvironmentorinfectingtheoperatorsduringtheoperation.接種環(huán)境

Inoculationenvironment接種室Inoculatingroom接種箱Inoculatingbox超凈工作臺(tái)Cleanbench接種場(chǎng)所必須進(jìn)行嚴(yán)格的消毒處理

Inoculationplacesmustbestrictlysterilized于接種之前打開(kāi)接種室和超凈工作臺(tái)紫外燈消毒30min，同時(shí)打開(kāi)風(fēng)機(jī)；

Beforeinoculation,turnontheUVlampsintheinoculatingroomandthecleanbenchtodisinfectfor30minutes,andturnonthefanatthesametime;接種時(shí)必須關(guān)閉紫外燈

TheUVlampsmustbeturnedoffduringinoculation要點(diǎn)：防止污染，嚴(yán)格的無(wú)菌操作

Keypoints:preventcontamination,strictasepticoperation接種者的雙手要進(jìn)行消毒處理，一般用75%酒精或0.01%新潔爾滅擦洗

Thehandsoftheinoculators

shouldbesterilized,usuallywith75%

alcohol

or0.01%bromogeramine

scrubbing整個(gè)操作過(guò)程都要靠近酒精燈火焰

Thewholeoperationshouldbeclosetotheflameofthealcohollamp接種工具、管、瓶口接種前后要通過(guò)灼燒滅菌

Inoculatingtools,tubesandvialsshouldbesterilizedbycauterizationbeforeandafterinoculation.棉塞不得亂放，操作中只能夾在手上

Cottonplugsshouldnotbemisplacedandshouldonlybeheldinthehandduringoperation.不能用嘴直接吸吹吸管。

Nodirectsuckingofthepipettewiththemouth.不能有跑、跳等力度大的動(dòng)作，以免引起空氣大振動(dòng)而增加染菌機(jī)會(huì)。

Cannothaverunning,jumpingandotherstrongmovements,soasnottocauseairvibrationandincreasethechanceofbacterialinfection.（三）接種的基本程序

(III)Thebasicprocedureofinoculation滅菌接種環(huán)

Sterilizetheinoculatingring沾取標(biāo)本

Dipthespecimen接種

Inoculation滅菌接種環(huán)

Sterilizetheinoculatingring殺滅接種環(huán)沾染的微生物，以免污染標(biāo)本

Killthemicroorganismscontaminatedbytheinoculatingringsoasnottocontaminatethespecimen.殺滅接種環(huán)沾染的微生物，以免污染環(huán)境

Killthemicroorganismscontaminatedbytheinoculatingringsoasnottocontaminatetheenvironment.操作需在無(wú)菌室或超凈工作臺(tái)內(nèi)進(jìn)行

Theoperationshouldbecarriedoutintheasepticroomorcleanbench.無(wú)菌室、超凈工作臺(tái)使用前后需進(jìn)行消毒。

Sterilizetheasepticroomandbechtopbeforeandafteruse.實(shí)驗(yàn)使用的物品使用前后需嚴(yán)格滅菌

Strictsterilizationbeforeandafteruseofexperimentalitems

1．斜面接種

Inoculationofanagarslant2．穿刺接種Punctureinoculation3.平板接種Plateinoculation4.液體接種Liquidinoculation斜面接種

Inoculationofanagarslant（四）常用的接種方法

(IV)Commonlyusedinoculatingmethods穿刺接種（水平法）

Punctureinoculation(horizontalmethod)穿刺接種（垂直法）Punctureinoculation(verticalmethod)劃線接種Streakinoculation涂布接種Spreadinoculation四、平板劃線分離法

IV.Platestreakingisolationmethod目的：使混合的細(xì)菌呈單個(gè)分散生長(zhǎng)，形成單個(gè)菌落，以便獲得純菌、活菌計(jì)數(shù)。

Purpose:tomakethemixedbacteriaareindividualdispersedgrowth,formingasinglecolonysoastoobtainpure,viablebacteriaforcounting.方法：

Method:分區(qū)劃線法：適用于含菌量較多的標(biāo)本

Zonestreaking:applicabletospecimenscontainingalargeamountofbacteria.連續(xù)劃線法：適用于含菌量較少的標(biāo)本。

Continuousstreaking:applicabletospecimenscontainingasmallamountofbacteria.連續(xù)劃線分離法：最常使用的、最基本的方法，其中圖B常用于含菌量較少的標(biāo)本。

Continuousstreakingisolationmethod:themostcommonlyusedandbasicmethod,ofwhichFigureBiscommonlyusedforspecimenscontaininglessbacteria.連續(xù)劃線法是從平板邊緣一點(diǎn)開(kāi)始，連續(xù)作波浪式劃線直到平板的另一端為止，當(dāng)中不需灼燒接種環(huán)上的菌。

Thecontinuousstreakingstartsfromtheedgeoftheplate,andmakesacontinuouswavystreakinguntiltheotherendoftheplate,inwhichthereisnoneedtocauterizethebacteriaontheinoculatingring.分區(qū)劃線法示意圖

Schematicdiagramofzonest