畢業(yè)論文外文翻譯-標(biāo)準(zhǔn)分離方法

6.0標(biāo)準(zhǔn)分離方法6.1富集培養(yǎng)6.2用微量移液管進(jìn)行單細(xì)胞分離6.3用瓊脂進(jìn)行單細(xì)胞分離6.3.1細(xì)胞在瓊脂板上劃線分離6.3.2瓊脂傾注平板6.3.3霧化細(xì)胞噴淋技術(shù)6.3.4通過瓊脂劃線后分離6.4稀釋技術(shù)6.5重力分離：離心、沉淀6.6生物趨光性分離技術(shù)

6.0標(biāo)準(zhǔn)分離方法6.1富集培養(yǎng)Enrichmentcultureshavelongbeenusedasapreliminarysteptowardsingle-cellisolations.Theyareestablishedbyaddingnutrientstothenaturalsample,whichenrichthesamplesothatalgalgrowthoccurs.Commonenrichingsubstancesincludeculturemedium,soil-waterextract,ormacronutrients(i.e.,nitrate,ammonium,andphosphate),butinsomecasesthelimitingfactorisatracemetal(e.g.,BoothbayHarbor,Maine,seawaterinMay2003).Soil-waterextractisperhapsthesimplestandmostsuccessful,providedthattheoriginalsoilisofgoodquality(Pringsheim1912,1950;seeChapter2).Peatmosscanbesubstitutedforsoil-waterextractwhenenrichingfordesmidsandsomeotheralgaefromacidhabitats.Organicsubstances,suchasyeastextract(primarilyforvitamins),casein(foraminoacids),orurea(fornitrogen),canbeaddedwhentryingtoisolateosmotrophicalgae,buttheamountshouldbesmall,becausetheseorganiccompoundsalmostalwaysresultinrapidbacterialgrowth.Ifbacterialgrowthistoohigh,thentheenrichmentculturecanbecomeanoxicortoxic,andthealgawilldie.Forspeciesnotpreviouslybroughtintoculture,itsometimespaystobeinventive.Droop(1959)purchasedvariousfruitsandvegetablesandaddedtinyamountswhentryingtoisolateOxyrrhisintoculturewithphytoplanktonforprey.Hediscoveredthatunfilteredlemonjuice,andsubsequentlyextractsfromlemonrind,ledtosuccess.Ultimately,fromanalysisofthelemonrind,hewasabletodeterminethattherequirementwassatisfiedwithubiquinoneorplastiquinone(Droop1966,1971).Conversely,this“grocerycart”approachfailedwhentryingtoisolateDinophysisaccuminataClaparèdeetLachmann(Andersen,unpublishedobservations).

富集培養(yǎng)一直是作為單細(xì)胞分離的一個初步的步驟。通過在天然樣品中添加豐富的營養(yǎng)來完成藻類的生長。常見的富集培養(yǎng)物包括培養(yǎng)基、土-水提取液，或大量營養(yǎng)素（如硝酸鹽、銨、和磷酸鹽），但在某些情況下，限制因素是一種微量金屬（如布斯貝港，緬因州，海水在5月2003）。土壤水提取物可能是其中最簡單的、最成功的，只要原來的土壤質(zhì)量是好的就可以（Pringsheim1912、1950；見2章）。泥炭蘚可以代替土壤水提取當(dāng)從酸性環(huán)境中增殖鼓藻和其他藻.

有機(jī)物質(zhì)，如酵母提取物（主要是維生素），酪蛋白（氨基酸），或尿素（氮），可以添加來試圖分離滲透營養(yǎng)的藻類，但是量要小，因為這些有機(jī)化合物幾乎總是導(dǎo)致細(xì)菌快速生長。如果細(xì)菌的生長太快，那么富集培養(yǎng)就可能變成缺氧或有毒，而藻類就會死亡。對于以前沒有被培養(yǎng)的個體，它有時是有創(chuàng)造力的。德魯普（1959）購買各種水果和蔬菜，并添加少量當(dāng)試圖分離培養(yǎng)與浮游植物獵物的尖尾。他發(fā)現(xiàn)了未經(jīng)過濾的青檸汁，隨后提取檸檬皮導(dǎo)致成功。最終，從檸檬皮的分析，他能夠確定的要求是輔酶Q和質(zhì)體醌是滿意的（德魯普1966，1971）。反過來，這種“購物車”的方法試圖分離漸尖鰭藻是失敗的（安徒生，未發(fā)表的觀察）。Formixotrophsrequiringbacteria,adrygrainofricemaybeadded,whichreleasesorganicmatterveryslowlyControlledbacterialgrowth,inturn,provides88TraditionalMicroalgaeIsolationTechniquesTraditionalMicroalgaeIsolationTechniques89nutritionforthephagotrophicalga(e.g.,Ochromonas,Chrysophyceae).Thericegrainshouldnotbeautoclavedintheculturemedium,becausetoomuchdissolvedorganicmatterisreleasedastheheatcooksthericegrain,makingitsoftanddiffuse.Oncethecultureisestablished,someorganisms,especiallyheterotrophicalgae,growwellonautoclavedrice(LeeandSoldo1992).Dissolvedorganicmattercanalsobeadded(e.g.,glucose,acetate,oryeastextract);however,asPringsheim(1950)states,theamountofdissolvedorganicmatter(e.g.,acetateorglucose)shouldbelowwhenculturingchrysophyteflagellatesandotherslow-growingmixotrophs.Otherseeds(e.g.,1/4peaseed)aresometimesaddedtosoil-watermediumwhentryingtoisolateeuglenoids,butinthiscase,theadditionisforammoniaororganiccompounds,notforbacterialgrowth(Nichols1973).

對需要的細(xì)菌的類生物體，添加干燥的大米顆?？赡軙褂袡C(jī)質(zhì)緩慢釋放出來，轉(zhuǎn)而控制細(xì)菌的生長，提供了88個傳統(tǒng)的微藻的分離技術(shù)傳統(tǒng)的微藻的分離技術(shù)89個營養(yǎng)的吞噬藻類（如棕鞭藻、金藻）。稻谷不應(yīng)壓在培養(yǎng)基中，因為太多的溶解有機(jī)物和熱烹飪稻米釋放，會使其柔軟和擴(kuò)散。一旦培養(yǎng)成功，一些生物，特別是異養(yǎng)藻，蒸壓稻生長良好（李和索爾多1992）。溶解的有機(jī)物也可以被添加（例如，葡萄糖、乙酸或酵母提取物）；然而，作為普林斯海姆（1950）的規(guī)定，溶解性有機(jī)物的數(shù)量（如醋酸或葡萄糖）應(yīng)該低當(dāng)培養(yǎng)金藻鞭毛蟲等生長緩慢的類生物體。其他的種子（例如，1/4豌豆種子）有時被添加到土壤-水介質(zhì)時試圖分離裸藻，但在這種情況下，是氨或有機(jī)化合物的添加，而不是細(xì)菌的生長（尼克爾斯1973）。Naturalsamplesareoftendeficientinoneormorenutrients(i.e.,alimitingfactor),butinnaturethealgaesurvive,becausebacterialaction,grazing,anddeathoforganismsrecyclethosenutrients.Oncethesampleiscollected,recyclingmaybereducedoraltered,andnutrientstresscancausedeathtothetargetspecies.Thus,forsomespecies,weakenrichmentoffieldsamplescanextendthelifeofhealthyalgalcellsneededforisolation.Enrichmentoffieldsamplesmaybeeffectiveforoceanicspecieswherecollectionsaremadefromships,especiallyonlongcruiseswhereisolationisnotattempted.Inthiscase,minuteamounts(e.g.,1–10mL·L-1ofculturemediumoraspecificnutrientsolution)areaddedtothesamplestoavoidpoisoning,andthesamplesareincubatedinculturechambersaboardtheship.However,enrichmentscanalsobedetrimental,regardlessofthecollectionsite.Forexample,ifthetargetspeciesisrareandunabletocompetewithweedyspecies,thenenrichmentofsamplescandiminishthetargetorganisms,andinthatcase,unenrichedsamplesmaybemoreappropriate.Theenrichmentcultureisincubatedinaculturechamber,andthecultureisexaminedeveryfewdaysforgrowth

ofthetargetspecies.Oncehealthytargetcellsareabundant,individualcellsareisolated.Whenthetargetspeciesrespondsfavorablytoenrichment,theeaseandsuccessofisolationisgreatlyimproved.天然樣品往往缺乏一個或多個營養(yǎng)物（即，一個限制因素），但在自然界中幸存的藻類，因為細(xì)菌的活動，受傷和死亡的生物回收這些營養(yǎng)物質(zhì)。一旦收集到樣品，回收可以減少或改變因為營養(yǎng)的壓力而導(dǎo)致死亡的目標(biāo)物種。因此，對于某些物種，低富集的樣本可以延長生命健康的海藻細(xì)胞所需要的分離。實地樣品的有效的富集可能是由船舶完成，尤其是長的的郵輪，巡航的郵輪不需要分離。在這種情況下，微量（例如，1-10毫升/升的培養(yǎng)基或一個特定的營養(yǎng)液）添加到樣品中，以避免中毒，并在培養(yǎng)箱中培養(yǎng)的樣品在船上。然而，富集，也可以是有害的，無論收集的地點(diǎn)。例如，如果目標(biāo)物種是罕見的，無法與雜草物種競爭，然后濃縮樣品可以減少目標(biāo)的生物，在這種情況下，未濃縮的樣品可能更合適。富集培養(yǎng)是培養(yǎng)室培養(yǎng)，每幾天檢查培養(yǎng)的目標(biāo)品種。一旦健康的靶細(xì)胞是大量的，單個細(xì)胞就被分離。當(dāng)目標(biāo)樣品的反應(yīng)有利富集，就大大提高分離的容易度和成功率。Theenrichingsubstance,althoughoftennitrate,phosphate,orsoil-waterextract,canbevaried.If10mLofthecollectionsampleisplacedintoeachof10dif-ferenttesttubes,then10differentenrichmentscanbeattempted(i.e.,nitratemaybeaddedtothefirst,phosphatetothesecond,silicatothethird,ammoniatothefourth,irontothefifth,andsoon).Also,combinationsofnutrients(e.g.,nitrateandsilicafordiatomgrowth)orcompleteculturemediumadditionscanbeattempted.Adifferentorganismoftendominateseachtubewhenvariousenrichmentsareestablished.Furthermore,organismsnotobservedintheinitialsampleoccasionallyappear7to10dayslater,becausetheadditionfavorstheirrapidgrowth;theyestablishacompetitiveadvantageoverotherspecies,evenifinitiallyrare.

雖然富集的物質(zhì)如硝酸鹽，磷酸鹽，或土壤-水提取物通?？梢愿淖?。如果采集10毫升的樣品放入10個不同的不同的試管，然后嘗試10個不同的富集（例如，硝酸鹽會被添加到第一、磷酸第二，硅酸第三，氨第四，鐵第五等等）。此外，組合的營養(yǎng)物質(zhì)（例如，硝酸鹽和二氧化硅的硅藻增長）或可以嘗試完全培養(yǎng)基的添加。往往不同的生物體占主導(dǎo)地位時，就各自進(jìn)行富集了。此外，在初始試樣偶爾出現(xiàn)7至10天后沒有觀察到生物體，因為添加營養(yǎng)物質(zhì)有利于它們的快速成長；他們建立了相比其他物種競爭優(yōu)勢，即使最初罕見。Howmuchenrichingmaterialshouldbeadded?Althoughtheanswervarieswiththesampleandthesubstance,thegeneralansweris“notverymuch.”Atmost,theadditionequalsthatfoundinacommonculturemedium;ataminimum,onlyone-thousandthofthatinaculturemediumisadded.Forexample,approximately800mMnitrateisequivalenttof/2medium(Guillard1975),whichisappropriateforSkeletonemagrowth,butforisolatingsinglecellsofoceanicspeciessuchascoccolithophores,800nMnitrateismoreappropriate.Second,theenrichmentmaybestaged(e.g.,800nMnitrateonday1,anadditional800nMonday10,1.6mMonday20,3.2mMonday25,andsoon).Notethatasthebiomassdoubles,thenutrientadditionmustbedoubled.Forr-selectedspecies(e.g.,Skeletonema),greateramountsofenrichmentarenotonlybeneficialbutoftennecessary.Conversely,k-selectedspecies(e.g.,oceaniccoccolithophores)growveryslowlyandrequirelittleadditionofnutrients;undertheseconditionsther-selectedspeciesdie,andovertimethek-selectedspeciesgraduallydominate.

應(yīng)該添加多少物質(zhì)？雖然答案隨樣本和物質(zhì)的不同而不同，一般的回答是“不太多”，在大多數(shù)情況下，添加量等于在普通培養(yǎng)基中找到的，至少在培養(yǎng)基中的一千分之一。例如，約800毫米硝酸等價于f/2培養(yǎng)基（姬拉德1975），這是適合它生長的，但分離單細(xì)胞海洋物種如顆石藻，800nm的硝酸鹽更合適。其次，富集可能會出現(xiàn)（例如，800納米的硝酸鹽1天，添加800納米的10天，1.6毫米20天，3.2毫米25天等）。注意，隨著生物量的增加，營養(yǎng)鹽量必須增加一倍。比如r-選擇物種（例如，肋骨），更大量的富集不僅是有益的但常常是必要的。相反，k-選擇物種（例如，海洋顆石藻）生長非常緩慢，不需要添加營養(yǎng)素；在這些條件下的r-選擇物種死亡，并隨著時間的推移，k-選擇物種逐漸占據(jù)主導(dǎo)地位。Theadditionofammoniumisparticularlyusefulinenrichingforspecieswithanabsoluteammoniumrequirement(e.g.,Aureoumbra).Understandably,theywillnotflourishwithnitrateaddition,buttheyareadaptedforrapidgrowthwhenammoniumisavailable.Insomecases(e.g.,Aureococcus),ammoniumislethalatconcentrationsaboveabout20mM.Thus,thesetwobrown-tideorganisms,sosimilarinmanyrespects,requiredifferentenrichmentstrategies.在富集對銨要求高的物種時銨的加入特別有用（例如，Aureoumbra）?？梢岳斫獾氖牵鼈儾粫S著硝酸鹽的添加而生長，但是它們可以快速生長在銨可利用的時候。在某些情況下（例如，Aureococcus），銨的致死濃度高于約20毫米。因此，這兩種褐潮生物，在許多方面都很相似，需要不同的富集方法Selectiveculturingisatypeofenrichmentculturingwithaspecialpurpose.IfthegoalistoisolatemicroalgaethathastheabilitytogrowatahighCO2concentration,thenanenrichmentculturethatisaeratedwith1–5%CO2isaneffectivewaytoselectforspecieswithhighCO2tolerance.Similarly,manydifferenttypesofselectiveculturescanbedesigned(e.g.,highorlowtemperature,light,salinity,orpH).Theadditionofspecificphysicalconditionscanhaveadramaticeffectonspeciesselectionandgrowthofenrichmentcultures.Forexample,certainbenthicraphiddiatoms,especiallylargePinnularia,mayrequireasedimentenvironmentthroughwhichtheyarefreetomigrateandgrow.選擇性培養(yǎng)是一種特殊用途的富集培養(yǎng)。如果目標(biāo)是分離有能力生長在一個高CO2濃度的微藻，那么然后曝氣于1-5%CO2富集培養(yǎng)是一種選擇具有高CO2耐受性的物種的有的方式。同樣，許多不同類型的選擇性培養(yǎng)，可以設(shè)計（例如，高或低溫，光，鹽度，或PH值）。此外，特定的物理條件下，可以有一個戲劇性的影響物種的篩選和增殖的富集培養(yǎng)。例如，某些底棲針晶米粒硅藻，特別是大型羽紋藻屬，可能需要一個通過它們的遷徙自由和成長的沉積環(huán)境。

6.2用微量移液管進(jìn)行單細(xì)胞分離Perhapsthemostcommonmethodissingle-cellisolationbymicropipette,althoughautomationmayreplacethistechniqueinpopularityinthefuture(seeChapter7).MicropipetteisolationisusuallyperformedwithaPasteurpipetteoraglasscapillary.APasteurpipettecanbeheatedinaflame,extended,andbroken(Fig.6.3).Withminimalpractice,thistechniquebecomesquickandeasy,butthebeginnermustspendsometimeprac-ticingbeforereliableproductionofmicropipettesisachieved.Thepipetteisheldinonehand,andaforcepsheldintheotherhandsupportsthetip.Thepipetteisrotatedtoprovideevensofteningasthepipettewarmstothemeltingpoint(seeFig.6.3a).Whentheheatedareaissufficientlysoft,thepipetteisremovedfromtheflameandsimultaneouslypulledtoproduceathintube(seeFig6.3b).If

drawnouttooquickly,orifdrawnoutintheflame,thenthethinextensionbreaksorburnsthrough,andtheresultingproductisunsatisfactory.Somepeoplelikeaverystraightmicropipettetipandothersproduceabentorcurvedtip.Thecurvedtipisadvantageouswhenpickingcellsfromadeepdish,butthestraighttipiseasiertousewhendischargingthecapturedcellintoasterilerinsingdroplet.Oncethetipisdrawn,theglassisallowedtocoolforacoupleofseconds.Next,theforcepsisrepositionedtothethinarea,approximatelywheretheweightofthetipbendsdownward(seeFig.6.3c).Withaslighttuggingandbendingmotion,theendisremovedanddiscarded(seeFig.6.3d).Properlydone,thebrokenendofthepipetteissmoothandround(seeFig.6.3f).Iftheendisjaggedorbroken(seeFig.6.3e),thenthepipetteshouldbediscarded,becauseitwillnotdrawupthetargetcellproperly.Bothdiscardedendsandusedmicropipettesshouldbecarefullydiscarded,becausetheyareextremelysharp.Thesefinetipscanbepushedintoaflame,wheretheywillquicklymeltintoaratherirregular,dullpieceofglass.Cansorbottlesmakegoodwastecontainers,butaboatpreparedfromaluminumfoilisbetter,becauseattheendoftheisolationsession,thefoilcanbefoldedtoenclosethesmallglassremains.

也許，微管分離是最常用的單細(xì)胞分離的方法，盡管自動化技術(shù)在未來可能取代這種技術(shù)（見7章）。微管分離通常是用巴斯德移液管或玻璃毛細(xì)管。巴斯德移液器可以在火焰上延長加熱、破碎（圖6.3）。這項技術(shù)用最少的練習(xí)是可以快速和容易掌握的，但初學(xué)者必須花一些時間在實踐設(shè)計上實現(xiàn)微量可靠的生產(chǎn)。移液管握在手中，另一只手支撐著鉗子的尖端。將移液管旋轉(zhuǎn)，甚至加熱軟化吸管到熔點(diǎn)（參見圖6.3a）。當(dāng)受熱面足夠軟時，吸管從火焰中移除，同時牽拉產(chǎn)生一個薄壁管（見圖6.3B）。如果抽得太快，或者如果在火焰中抽出，則薄的延伸管會斷裂或燃燒，所產(chǎn)生的產(chǎn)品是不能令人滿意的。有些人喜歡直的微管末梢與其產(chǎn)生彎曲制成的尖端。當(dāng)從深皿中取細(xì)胞時，彎曲的尖端有優(yōu)勢，但在將捕獲的細(xì)胞放到無菌沖洗液滴時，直尖更容易使用。一旦尖端被拉出，允許玻璃冷卻幾秒鐘。接下來，鉗子回到薄壁區(qū)，大約在尖端的重量下向下彎曲（圖6.3c）。隨著輕輕一拉和彎曲運(yùn)動，最終被取下并丟棄（見圖6.3D）。做得好，移液管的斷裂處就是圓滑的（見圖6.3f）。如果端部鋸齒狀或破損（參照圖6.3e），然后吸移管應(yīng)該被丟棄，因為它不會制定適當(dāng)?shù)陌屑?xì)胞。都拋棄兩端，用微量移液器應(yīng)小心丟棄，因為他們非常鋒利。這些細(xì)小的尖端可以被推入火焰，在那里他們將迅速融化成一個相當(dāng)不規(guī)則的，鈍的玻璃片。罐子或瓶子可以制成好的廢物容器，但是從鋁箔制成的小船更好，因為在分離結(jié)束時鋁箔可以折疊起來封閉小玻璃。

FIGURE6.3.Preparationofamicropipettefroma

Pasteurpipette.(a)ThePasteurpipetteisheldinthehottestregionoftheflame,supportedontheleftbyahandandontherightbyforceps.Thepipetteshouldberotatedastheglassisheatedtoasoft,pliablecondition.(b)Whentheglassissoft,thepipetteisquicklyremovedfromtheflamewithagentlepulltoproduceathintube.(c)Theforcepsisthenrelocatedtotheappropriateregionofthethintube.(d)Theforcepsisusedtogentlybendthethinareasothatitbreaks,formingamicropipette.(e)Anenlargedtipofamicropipette,showingajaggedbreak;thistipisnotsuitableforuse.(f)Anenlargedtipwithaverysmoothbreak;thistipissuitableforuse.Notethatthediameterofthetipislargerth

圖6.3。巴斯德吸管微量制備。（一）巴斯德移液管在火焰最熱的區(qū)域進(jìn)行，可以用左手或右手。移液管應(yīng)旋轉(zhuǎn)，將玻璃加熱至軟而且柔韌。（b）當(dāng)玻璃是軟的，吸管很快可以輕拉以產(chǎn)生一個薄管從火焰去除。（C）然后鉗子移動到薄管的適當(dāng)區(qū)域。（d）鉗子是用來輕輕彎曲薄管的區(qū)域?qū)⑺蚱?，形成微管。（E）微量放大的尖端呈現(xiàn)出鋸齒狀斷裂；這提示不適合使用。（f）一個非常光滑的放大的尖端，這是適合使用的。請注意，尖端的直徑較大

Somepeopleprepareseveralmicropipettesinadvance,whereasothersprepareamicropipetteimmediatelybeforeuse.Apreviouslyusedmicropipettecanberedrawntoformanewtip,andtheheatrequiredtomelttheglassissufficienttosterilizeit,assumingthatnocontaminatingliquidisfurtherupinthepipette.Redrawnpipettesusedinseawateroftenformasmallsaltcrustwhenanyremainingseawaterisevaporated有些人提前準(zhǔn)備幾個微管，而其他人在馬上使用前準(zhǔn)備一個微管。以前使用的微管可以重新形成一個新的尖端，并且以熔化玻璃的熱足以將其消毒，假設(shè)沒有進(jìn)一步的污染液體在吸液管。在海水中使用重繪移液器往往在形成一個小鹽殼時蒸發(fā)任何剩余的海水Thegoalofmicropipetteisolationistopickupacellfromthesample,depositthecellwithoutdamageintoasteriledroplet,pickupthecellagain,andtransferittoasecondsteriledroplet(Fig.6.4).Thisprocessisrepeateduntilasinglealgalcell,freeofallotherprotists,canbeconfidentlyplacedintoculturemedium.Theprocessbalancestwofactors:celldamagebyexcessivehandling,whichisbad,andcleanisolationofasinglecell,whichisgood.Forrobustorganisms,repeatedhandlingcanbeachievedwithoutdamage;however,fordelicateorganisms,celldamageisanimportantconcern.微量分離的目的是從樣品中提取單細(xì)胞，將細(xì)胞沉積在無菌液滴中，再提取細(xì)胞，再轉(zhuǎn)移到一個無菌的液滴（圖6.4）。重復(fù)該過程，直到單個藻細(xì)胞，不含所有其他原生生

物，可放心地放置到培養(yǎng)基中。該過程平衡兩個因素：過度處理損傷細(xì)胞這是不好的，和徹底分離單個細(xì)胞是好的。對于生命力旺盛的生物，可以在不損害細(xì)胞下實現(xiàn)反復(fù)操作;然而，對于精巧的生物體，細(xì)胞損傷是一個重要的問題。Amicroscopeisnecessaryforobservingandisolatingthecell(seeSection4.1).Thesamplecontainingthetargetspeciescanbeplacedinaglassorplasticdish,inamultiwellplate,onamicroscopeslide,orinsimilarcontainers.Second,steriledropletsshouldbepreparedbeforeisolationbegins.Lewin(1959)recommendsplacingthedropletsonagartoreduceevaporation,butinourexperiencethisisn’tnecessaryiftheisolationpro-ceedswithoutdelays.Also,theagarisnotastransparentasglassorplastic,andforsmallcellsitismoredifficulttoseethemonagar.Thedropletscanbesterileseawater,sterilepondwater,culturemedium(dilutedornot),etc.Cellsmustbeabletosurviveinthedroplet,preferablywithoutstress.Forexample,afreshwateralgawilllikelydieifplacedinseawater,andasensitivespecieswilllikelydieifplacedinfull-strengthculturemedium.Typically,onepreparesseveralsteriledroplets,coveringthesewithapetridishcoverorsimilarcoverwhennotinuse.顯微鏡是觀察和分離細(xì)胞所必要的（見4.1節(jié)）。含有目標(biāo)物種的樣品可以被放置在玻璃或塑料盤，在多孔板，在顯微鏡載玻片上，或在類似的容器。第二，分離開始之前應(yīng)制備無菌液滴。列文（1959）建議液滴放置在瓊脂上以減少蒸發(fā)，但在我們的經(jīng)驗，這是沒有必要的，如果分離進(jìn)行而且沒有延遲。此外，瓊脂不如玻璃或塑料透明的，并且對于小細(xì)胞中，這是更難以看到它們在瓊脂上。液滴可以是無菌海水，無菌池塘水，培養(yǎng)基（稀釋或不稀釋）等，細(xì)胞必須能夠在液滴中生存，最好是沒有生存壓力的。例如，如果放在海水淡水藻類很可能會死，如果放在全強(qiáng)度培養(yǎng)基中，敏感的物種很可能會死亡。通常準(zhǔn)備幾個無菌水滴，在不使用時覆蓋這些培養(yǎng)皿蓋或類似的覆蓋。

FIGURE6.4.Apipetteisusedtoremoveothersmallcells(left,middle),leavingthetargetorganismfreeofcontamination(right).Thisprocedurelimitsthehandlingofthetargetorganism.

圖6.4移液管是用來排除其他小細(xì)胞（左，中），使目標(biāo)生物無污染（右）。這個程序限制在目標(biāo)生物處理。

Therearetwocommonmethodsforpickingupsinglecellswithamicropipette.Foronemethod,aflexible,latextubeisattachedtoamouthpieceononeendandamicropipetteorcapillarytubeattheotherend(seeFig.6.1i–k).Smalltubingisadvantageous,becauseitislightweightandeasytostore,butitmaybenecessarytouseareducingconnectortoconnectthetubingtothemicropipetteorcapillarytube,dependingonitssize.Reducingconnectorscanbepurchased,butsectionscutfromplasticpipettetips(seeFig.6.1l,rightside)orglassPasteur

pipettesworkquitewell.Ashortpieceoftubingattachedtothereducingconnectorprovidesaquickandsimpleseatforthemicropipette.Largertubingcanbeused(seeFig.6.1k),butitsweightmakesitmorecumbersometouse.Largetubingisusuallycuttoalongerlength,sothatonecanpassthetubingovertheshouldersandaroundtheneck,providingsupportforcomfortableuse(seeFig.6.2b)Althoughvariousmouthpiecesandmicropipetteconnectorscanbefittedtothelargertubing,1000-mLplasticpipettetipsworkwellatbothends.Regardlessoftubingsize,theoperatorplacesasmallamountofsterilewaterintothemicropipettetoactasacushion.Theoperatorplacesthetongueoverthemouthpiece,placesthemicropipettetipnearthetargetorganismandthenremovesthetonguetogentlyallowthecapillaryactiontodrawthecellupandintothemicropipettetiporcapillarytube.Aftersuccessfulcapturingofthecell,themicropipettetipisremovedfromthesampleordroplet;thetipisimmersedintothenextdroplet,testtube,ormultiwell;andthenbymeansofgentleblowingintothemouthpiece,thecapturedcellisdischargedintoasecond,steriledroplet.Thedrawingorexpellingpressureshouldbeslight,becauseexcessivepressureorrapidmovementcandamagethecell.有對于用微管拾取單個細(xì)胞兩種常用的方法。對于一種方法，柔軟的乳膠管連接到管口一端和在另一端的微管或毛細(xì)管（見圖6.1i-K）。小管是有利的，因為它重量輕，易于保存，但它可能有必要使用一個減少連接器連接到管路、微量或毛細(xì)管，這取決于它的尺寸。減少連接器可以購買，但是切段的塑料槍頭（見圖6.1升，右側(cè)）或玻璃巴斯德吸液管也能工作得非常好。附著在減少連接器的短管件提供了一個微量快速和簡單的地方。較大的管子可用（見圖6.1k），但它的重量使得它使用起來更笨重。大管子通常被切割成一個較長的長度，這樣就可以將管在肩上和圍繞頸部舒適的使用（參照圖6.2B）雖然各種吹嘴和微量的連接器可安裝到較大的管，1000毫升的塑料槍頭兩端工作。無論管道尺寸，操作員將少量無菌水注入作為緩沖。操作者將管的一端放在嘴上，則將微量移液器尖端放在目標(biāo)生物體附近，然后舌頭輕輕吸住靠毛細(xì)管作用吸取細(xì)胞并進(jìn)入微管尖端或毛細(xì)管。成功捕獲細(xì)胞后，微量移液器尖端從樣品中拿起放入試管，或多孔板；然后通過溫和吹入吸嘴裝置，所捕獲的細(xì)胞被排入第二個無菌液滴。拉出或排出的壓力應(yīng)該是很小的，因為壓力過大或快速運(yùn)動可能會損壞細(xì)胞。

Alternatively,themicropipettetipcanbetouchedintoasteriledropletsothatcapillaryactionpullswaterupintothemicropipette.Ifadrymicropipetteisimmersedinthesample(i.e.,withoutfirsttouchingthetiptothesteriledroplet),thenviolentcapillaryactionresults,drawingsubstantialunwantedmaterialintothemicropipette.Aftercapillaryloadingwithsterileliquid,themicropipetteisthendirectedtotheselectedcell,andresidualcapillaryactiongentlydrawsthecellintothemicropipette.Themicropipettetipcontainingthecapturedcellisthenmovedandsubmersedinasecondsteriledroplet,andthecellisdischargedbymeansofgentleblowingonthemicropipette.

另外，使得毛細(xì)管作用拉水成微量微量移液器尖端可觸及到無菌液滴。如果干微量浸入樣本中（即，不先接觸尖端到無菌液滴），那么劇烈毛細(xì)作用的結(jié)果，沾上大量不必要的物質(zhì)進(jìn)入微管。毛細(xì)管吸入無菌液體后，微管然后將選定的單元細(xì)胞，和殘留毛細(xì)作用的細(xì)胞輕輕吸取到微管。然后將含有捕獲的細(xì)胞的微量移液器尖端移動，并且在第二無菌液滴浸沒，細(xì)胞是通過在微量溫和鼓風(fēng)裝置排出。Thesteriledropletcontainingthetargetcell,andpossiblyothercells,isthenexaminedmicroscopically.Withthesametechnique,acleanmicropipetteisthenusedtopickupthecell

andtransferittoathirdsteriledroplet.Thisprocedureisrepeatedonlyuntilthesinglecellisisolatedfromothercells;unnecessaryadditionalisolationoftenleadstocelldamage.Therefore,afterthefinalcapture,thecellisdischargedintothefinalisolationvessel(testtube,multiwellplate,etc.).

無菌液滴含有靶細(xì)胞和其他可能的細(xì)胞，然后檢查顯微鏡。用同樣的方法，一個干凈的微管是用來將細(xì)胞轉(zhuǎn)移到三分之一消毒液滴。此過程是重復(fù)的，直到單細(xì)胞從其他細(xì)胞中分離；不必要的額外分離往往導(dǎo)致細(xì)胞損傷。因此，獲得單細(xì)胞后，細(xì)胞被釋放到最終的分離容器中（試管，多孔板等）。Thediameterofthemicropipetteopeningshouldbeatleasttwicethatofthecell,andoftenseveraltimesthecellsize.Iftheopeningistoosmall,thenfluidshearingforcescandamagethecellasitpassesintothemicropipette,especiallyifitisanaked,scaled,orflagellatecell.Iftheopeningistoolarge,thenitbecomesmoredifficulttopickupthecell,andthereisalsoanincreaseintheamountofunwantedmaterialthatiscaptured.Whenisolatingfilaments,chainsofcells,orlongsinglecells(e.g.,certainpennatediatoms),themicropipetteshouldbedirectedtooneendofthefilament,chain,orcell;themicropipetteshouldbeheldatananglesothatthefilamentorcellslidesupintothemicropipettetipwithoutseverebending.微管的開口直徑應(yīng)至少是細(xì)胞的兩倍，而且經(jīng)常是幾個細(xì)胞的大小。如果開口太小，則進(jìn)入微管時流體剪切力可以破壞細(xì)胞，尤其它是一個裸體、脫落、或生長晚期的細(xì)胞。如果開得太大，那么拿起細(xì)胞就會更難，也會有其他的增加數(shù)量和不需要的東西被提取。當(dāng)分離絲狀體、鏈狀體的細(xì)胞，或長的單細(xì)胞（例如，某些底棲硅藻）、微管應(yīng)該指向絲狀體，狀體鏈或細(xì)胞的一端；微管應(yīng)該在一個角度舉行以便絲狀體或細(xì)胞滑入到微管尖端而且沒有嚴(yán)重彎曲。Somecellsadheretothebottomofthedishormultiwellplate.Effortstopryacelllooseoftenresultindamageordeath.Rapidcell-handlingcancircumventthisproblem(i.e.,immediatelyafterthesampleisaddedtothedish,thecellshouldbepickedupbeforeitcansinktothebottomandadhere).Quickly,thecellshouldbedischargedintothesterilerinsingdroplet;cellscanalsoadheretotheinsideofthemicropipette.Immediatelyafterthecellisdischargedintothesterilerinsingdropletandbeforethecellcansettle,itshouldbepickedupagainandtransferred.Byquickaction,theisolationcanproceedbeforethecelladherestoasurface.

一些細(xì)胞粘附在培養(yǎng)皿或多孔板的底部。用力將細(xì)胞弄松散往往會造成損傷或死亡。細(xì)胞快速裝卸可以解決這個問題（即，將樣品添加到培養(yǎng)皿后立即將細(xì)胞應(yīng)被拾起之前，可以沉底和粘附）。很快，細(xì)胞應(yīng)該被釋放到無菌漂洗液滴；細(xì)胞也可以附著在微管的內(nèi)部。很快該細(xì)胞被排出到無菌沖洗液滴，在細(xì)胞沉淀之前，應(yīng)該再次提取并轉(zhuǎn)移。通過快速進(jìn)行，細(xì)胞附著在表面之前的分離可以繼續(xù)進(jìn)行。

Althoughoneapproachistofocusonasinglecell—fromoriginalsampletofinalisolationvessel—othertechniquescanbeemployed.Askilledtechniciancanpickseveraltargetcellsfromtheoriginalsample,andwitheachrinseonlyviablecellsaremovedtothenextstage.Withexperience,onecanassesstheviabilityofcells.Forflagellates,cessationofswimmingsometimesindicatesdamage.Anotherapproachistoisolateseveraltargetcellsintolargersteriledropletsofweaklynutrifiedliquid,andwithattentiongiventoavoidingevaporation(e.g.,

sealingwithParafilm),thesecellscanbeleftforminutestodays.Subsequently,viablecellscanbeprocessed,leavingdamagedordeadcells.Finally,inmanycases,itiseasiertoremovecontaminatingcellsfromaroundthetargetcell.Thismethodreducesthehandlingofthetargetcell,becauseeffortisdirectedtothenontargetcontaminatingmaterial.Whenmostorallcontaminationisremoved,thenthetargetcellispickedupandplacedintotheisolationvessel.雖然一種方法是集中在原始樣品中的一個單細(xì)胞至最終的分離容器，但其它技術(shù)也可以使用。一個熟練的技術(shù)人員可以從原始樣品選擇幾個靶細(xì)胞，并用每次漂洗僅有可行的細(xì)胞轉(zhuǎn)移到下一階段?？拷?jīng)驗，人們可以評估細(xì)胞的生存力。對于鞭毛蟲，游泳停止有時意味著傷害。另一種方法是把幾個目標(biāo)細(xì)胞分離成弱營養(yǎng)液體的較大的無菌液滴，并注意避免其蒸發(fā)（例如，用石蠟密封），這些細(xì)胞可以被放置數(shù)分鐘到數(shù)天。接著，活細(xì)胞可以被處理，留下受損或死亡的細(xì)胞。最后，在許多情況下，更容易從目的細(xì)胞周圍除去污染細(xì)胞。這種方法減少了靶細(xì)胞的處理，因為主要是針對非目標(biāo)污染材料。當(dāng)大部分或所有的污染被除去，則該目的細(xì)胞被提取并放置到分離容器中。

Anothermicropipettetechniquemayalsobeemployedtoinducecellwallruptureinsomediatoms,especiallylargerones,andmaybedesirableforexistingclonalstrainsthatarepresumablydioeciousandneartheendoftheircellsizeminimum.Thispurposefuldamageservestoremovethephysicalconstraintsofsizeregeneration.Rogersonetal.(1986)employedrepeatedintroductionandejectionofcells,suspendedina1%crudepapainsolution,intoandfromamicropipettetogenerateca.10%nakedcellsofCoscinodiscusasteromphalus.DavidCzarnecki(withandwithoutthepapaintreatment)andAnne-MarieSchmid(withoutthepapaintreatment)havebothhadsomesuccessusingthistechniquetogeneratenakedcellsofCampylodiscusclypeus;thesenakedcellsarereisolatedviamicropipetteintofreshmedium,andsomesuccessfullyregeneratenormallargercells(D.Czarnecki,personalcommunication).

另一個微管技術(shù)也可以用來誘導(dǎo)某些硅藻細(xì)胞壁破裂，特別是較大的，可能需要對現(xiàn)有的克隆菌株大概是雌雄異株和鄰近其最小單元尺寸的末端。這種有目的的損害是為了去除大小再生的物理約束。羅杰森等人（1986）采用重復(fù)引進(jìn)射血細(xì)胞懸浮在1%粗木瓜蛋白酶溶液，并微量生成星臍圓篩藻約10%原生質(zhì)體的方法。大衛(wèi)·查內(nèi)茨基談到（有和沒有的木瓜蛋白酶處理）和安妮·瑪麗·施密德（不含木瓜蛋白酶處理）也都有過使用這種技術(shù)來生成馬鞍藻唇基的裸細(xì)胞取得一定的成功，這些赤裸裸的細(xì)胞通過微管再分離到新鮮的培養(yǎng)基，有的成功地再生正常較大的細(xì)胞（大衛(wèi)·查內(nèi)茨基，個人通信）。

6.3用瓊脂進(jìn)行單細(xì)胞分離6.3.1細(xì)胞在瓊脂板上劃線分離Isolationofcellsonagarplatesisalsoanoldandcommonmethod.(Forpreparationofagarplates,seeChapter2.)Itisthepreferredisolationmethodformanycoccoidalgaeandmostsoilalgae,notonlyforeaseofusebutalsobecauseaxenicculturescanoftenbedirectlyestablishedwithoutfurthertreatment(seeChapter8).Forsuccessfulisolationontoagar,the

algamustbeabletogrowonagar.Someflagellates(e.g.,Heterosigma,Pelagomonas,andPeridinium)donotgrowonagar,butothers(e.g.,Chlamydomonas,Pavlova,Synura,andTetraselmis)growverywellonagar.Coccoidcellsfrequentlygrowwellonorinagar,butsome(e.g.,Aureococcus,Aureoumbra)donot.Mostdiatomsandchlorarachniophytesgrowverywellonagar;somecryptophytesdo,whereasothersdonot,anddinoflagellatesrarelygrowonagar.

瓊脂平板上的細(xì)胞分離，也是一個古老的和常用的方法。（用于制備瓊脂板，見2章。）這是許多球形藻類和大多數(shù)土壤藻類的首選分離的方法，不僅方便使用，而且也因為純性培養(yǎng)物通?？梢灾苯硬唤?jīng)進(jìn)一步處理得到（見8章）。在瓊脂上成功地分離，藻類必須能夠在瓊脂上生長。有些鞭毛蟲（如赤潮，浮葉藻，和多甲藻）不生長在瓊脂，但其他（如衣藻、巴夫藻、黃群藻和周氏扁藻）在瓊脂培養(yǎng)基上生長的很好。球菌細(xì)胞上或瓊脂普遍生長良好，但是（如Aureococcus，aureoumbra）有的并不是。大多數(shù)硅藻在瓊脂上生長很好；一些隱芽植物可以做到，而別的沒有，甲藻很少生長在瓊脂。Inmostcases,theconcentrationofagarisnotanimportantfactor,assumingtheagarisbetween0.8%and1.5to2.0%(seeChapter2).Afewalgaegrowon“sloppy”agar(i.e.,preparationswithbetween0.3and0.6%agar),butthealgaeareprobablygrowinginliquidpocketsratherthanonthe“solid”substrate.Agaralsoisagoodmediumforfungalandbacterialgrowth.Fieldsampleswithsubstantialfungalcontaminationcanprovefrustrating,becausethefungusoftengrowsquickly,producingsporangiaandsporesthatcontaminateeffortstoisolatethealga.Filtersandorganicsubstratescanbeusedtoremovefilamentousfungi(seeprevioustext).WhenfungalgrowthappearsonParafilm-sealedagarplates,itisalmostalwaysbettertodiscardtheplatewithoutopeningit.Conversely,bacteriausuallyproducesmall,limitedcolonies,andunialgalculturescanbeobtainediftheplatehasbeenproperlystreaked.Oneexceptionisthebacteriafrombenthictropicalsamples,becausetheyoftencontainagar-digestingbacteriathatwill“dissolve”regionsoftheagarplate.

在大多數(shù)情況下，瓊脂的濃度是不是一個重要因素，假設(shè)瓊脂是在0.8%和1.5至2%（見2章）。一些藻類生長在“草率”的瓊脂（即，在0.3和0.6%瓊脂的制劑），但藻類可能在液體口袋里增殖，而不是在“固體”基板。瓊脂培養(yǎng)基也是真菌和細(xì)菌生長的良好培養(yǎng)基。有大量的真菌污染的樣品的菌落可以證明令人沮喪，因為真菌通常生長迅速，產(chǎn)生孢子囊和污染來分離藻類孢子。過濾器和