單個微生物在適宜的固體培養基表麵或內部生長、繁殖到一定程度可以形成肉眼可見的

、有(you)一(yi)定(ding)形(xing)態(tai)結(jie)構(gou)的(de)子(zi)細(xi)胞(bao)生(sheng)長(chang)群(qun)體(ti)，稱(cheng)為(wei)菌(jun)落(luo)。當(dang)固(gu)體(ti)培(pei)養(yang)基(ji)表(biao)麵(mian)眾(zhong)多(duo)菌(jun)落(luo)連(lian)成(cheng)一(yi)片(pian)時(shi)，便(bian)成(cheng)為(wei)菌(jun)苔(tai)。不(bu)同(tong)微(wei)生(sheng)物(wu)在(zai)特(te)定(ding)培(pei)養(yang)基(ji)上(shang)生(sheng)長(chang)形(xing)成(cheng)的(de)菌(jun)落(luo)或(huo)菌(jun)苔(tai)一(yi)般(ban)都(dou)具(ju)有(you)穩(wen)定(ding)的(de)特(te)征(zheng)，可(ke)以(yi)成(cheng)為(wei)對(dui)該(gai)微(wei)生(sheng)物(wu)進(jin)行(xing)分(fen)類(lei)、鑒定的重要依據。大多數細菌、酵母菌、以yi及ji許xu多duo真zhen菌jun和he單dan細xi胞bao藻zao類lei能neng在zai固gu體ti培pei養yang基ji上shang形xing成cheng孤gu立li的de菌jun落luo，采cai用yong適shi宜yi的de平ping板ban分fen離li法fa很hen容rong易yi得de到dao純chun培pei養yang。所suo謂wei平ping板ban，即ji培pei養yang平ping板ban的de簡jian稱cheng，它ta是shi指zhi固gu體ti培pei養yang基ji倒dao入ru無wu菌jun平ping皿min，冷leng卻que凝ning固gu後hou
，盛sheng固gu體ti培pei養yang基ji的de平ping皿min。這zhe方fang法fa包bao括kuo將jiang單dan個ge微wei生sheng物wu分fen離li和he固gu定ding在zai固gu體ti培pei養yang基ji表biao麵mian或huo裏li麵mian。固gu體ti培pei養yang基ji用yong瓊qiong脂zhi或huo其qi它ta凝ning膠jiao物wu質zhi固gu化hua的de培pei養yang基ji，每mei個ge孤gu立li的de活huo微wei生sheng物wu體ti生sheng長chang、繁殖形成菌落，形成的菌落便於移植。最常用的分離、培養微生物的固體培養基是瓊脂固體培養基平板。這種由Kock建立的采用平板分離微生物純培養的技術簡便易行

，100多年來一直是各種菌種分離的最常用手段。

首先把微生物懸液作一係列的稀釋（如1:10
、1:100
、1:1000、1:10000），然後分別取不同稀釋液少許
，與已熔化並冷卻至50℃左zuo右you的de瓊qiong脂zhi培pei養yang基ji混hun合he，搖yao勻yun後hou，傾qing入ru滅mie過guo菌jun的de培pei養yang皿min中zhong
，待dai瓊qiong脂zhi凝ning固gu後hou
，製zhi成cheng可ke能neng含han菌jun的de瓊qiong脂zhi平ping板ban，保bao溫wen培pei養yang一yi定ding時shi間jian即ji可ke出chu現xian菌jun落luo。如ru果guo稀xi釋shi得de當dang
，在zai平ping板ban表biao麵mian或huo瓊qiong脂zhi培pei養yang基ji中zhong就jiu可ke出chu現xian分fen散san的de單dan個ge菌jun落luo，這zhe個ge菌jun落luo可ke能neng就jiu是shi由you一yi個ge細xi菌jun細xi胞bao繁fan殖zhi形xing成cheng的de。隨sui後hou挑tiao取qu該gai單dan個ge菌jun落luo

，或huo重zhong複fu以yi上shang操cao作zuo數shu次ci
，便bian可ke得de到dao純chun培pei養yang。

yinweijiangweishengwuxuanyexianjiadaojiaotangdepeiyangjizhongzaidaopingbanyizaochengmouxiereminganjundesiwang，qiecaiyongxishidaopingbanfayehuishiyixieyangehaoyangjunyinbeigudingzaiqiongzhizhongjianquefayangqieryingxiangqishengchang，yincizaiweishengwuxueyanjiuzhongchangyongdechunzhongfenlifangfashitubupingbanfa。qizuofashixianjiangyironghuadepeiyangjidaoruwujunpingmin，zhichengwujunpingban，lengqueningguhou，jiangyidingliangdeweishengwuxuanyedijiazaipingbanbiaomian
，zaiyongwujunbolitubangjiangjunyejunyunfensanzhizhenggepingbanbiaomian
，jingpeiyanghoutiaoqudangejunluo（圖1）。

最簡單的分離微生物的方法是平板劃線法
，即用接種環以無菌操作沾取少許待分離的材料，在無菌平板表麵進行連續劃線（圖2），微wei生sheng物wu細xi胞bao數shu量liang將jiang隨sui著zhe劃hua線xian次ci數shu的de增zeng加jia而er減jian少shao，並bing逐zhu步bu分fen散san開kai來lai，如ru果guo劃hua線xian適shi宜yi的de話hua，微wei生sheng物wu能neng一yi一yi分fen散san
，經jing培pei養yang後hou，可ke在zai平ping板ban表biao麵mian得de到dao單dan菌jun落luo。有you時shi這zhe種zhong單dan菌jun落luo並bing非fei都dou由you單dan個ge細xi胞bao繁fan殖zhi而er來lai的de
，故gu必bi須xu反fan複fu分fen離li多duo次ci才cai可ke得de到dao純chun種zhong。其qi原yuan理li是shi將jiang微wei生sheng物wu樣yang品pin在zai固gu體ti培pei養yang基ji表biao麵mian多duo次ci作zuo“由點到線”稀釋而達到分離目的的。劃線的方法很多，常見的比較容易出現單個菌落的劃線方法有斜線法、曲線法、方格法、放射法、四格法等。

yonggutipeiyangjifenliyangeyanyangjunyouteshuxing，ruguogaiweishengwubaoluyukongqizhongbulijisiwang，keyicaiyongtongchangdefangfazhibeipingban，ranhouzhifangzaifengbiderongqizhongpeiyang，rongqizhongdeyangqikecaiyonghuaxue、物(wu)理(li)或(huo)生(sheng)物(wu)的(de)方(fang)法(fa)清(qing)除(chu)。對(dui)於(yu)那(na)些(xie)對(dui)氧(yang)氣(qi)更(geng)為(wei)敏(min)感(gan)的(de)厭(yan)氧(yang)性(xing)微(wei)生(sheng)物(wu)，純(chun)培(pei)養(yang)的(de)分(fen)離(li)則(ze)可(ke)采(cai)用(yong)稀(xi)釋(shi)搖(yao)管(guan)培(pei)養(yang)法(fa)進(jin)行(xing)
，它(ta)是(shi)稀(xi)釋(shi)倒(dao)平(ping)板(ban)法(fa)的(de)一(yi)種(zhong)變(bian)通(tong)形(xing)式(shi)。先(xian)將(jiang)一(yi)係(xi)列(lie)盛(sheng)無(wu)菌(jun)瓊(qiong)脂(zhi)培(pei)養(yang)基(ji)的(de)試(shi)管(guan)加(jia)熱(re)使(shi)瓊(qiong)脂(zhi)熔(rong)化(hua)後(hou)冷(leng)卻(que)並(bing)保(bao)持(chi)在(zai)50℃左zuo右you，將jiang待dai分fen離li的de材cai料liao用yong這zhe些xie試shi管guan進jin行xing梯ti度du稀xi釋shi，試shi管guan迅xun速su搖yao動dong均jun勻yun，冷leng凝ning後hou，在zai瓊qiong脂zhi柱zhu表biao麵mian傾qing倒dao一yi層ceng滅mie菌jun液ye體ti石shi蠟la和he固gu體ti石shi蠟la的de混hun合he物wu，將jiang培pei養yang基ji和he空kong氣qi隔ge開kai。培pei養yang後hou
，菌jun落luo形xing成cheng在zai瓊qiong脂zhi柱zhu的de中zhong間jian。進jin行xing單dan菌jun落luo的de挑tiao取qu和he移yi植zhi，需xu先xian用yong一yi隻zhi滅mie菌jun針zhen將jiang液ye體ti石shi蠟la--shilagaiquchu，zaiyongyizhimaoxiguancharuqiongzhiheguanbizhijian，chuiruwujunwuyangqiti，jiangqiongzhizhuxichu，zhifangzaipeiyangminzhong，yongwujundaojiangqiongzhizhuqiechengbopianjinxingguanchahejunluodeyizhi。

 

 

大(da)多(duo)數(shu)細(xi)菌(jun)和(he)真(zhen)菌(jun)
，用(yong)平(ping)板(ban)法(fa)分(fen)離(li)通(tong)常(chang)是(shi)滿(man)意(yi)的(de)，因(yin)為(wei)它(ta)們(men)的(de)大(da)多(duo)數(shu)種(zhong)類(lei)在(zai)固(gu)體(ti)培(pei)養(yang)基(ji)上(shang)長(chang)得(de)很(hen)好(hao)。然(ran)而(er)迄(qi)今(jin)為(wei)止(zhi)並(bing)不(bu)是(shi)所(suo)有(you)的(de)微(wei)生(sheng)物(wu)都(dou)能(neng)在(zai)固(gu)體(ti)培(pei)養(yang)基(ji)上(shang)生(sheng)長(chang)，例(li)如(ru)一(yi)些(xie)細(xi)胞(bao)大(da)的(de)細(xi)菌(jun)、許多原生動物和藻類等

，這些微生物仍需要用液體培養基分離來獲得純培養。

稀(xi)釋(shi)法(fa)是(shi)液(ye)體(ti)培(pei)養(yang)基(ji)分(fen)離(li)純(chun)化(hua)常(chang)用(yong)的(de)方(fang)法(fa)。接(jie)種(zhong)物(wu)在(zai)液(ye)體(ti)培(pei)養(yang)基(ji)中(zhong)進(jin)行(xing)順(shun)序(xu)稀(xi)釋(shi)
，以(yi)得(de)到(dao)高(gao)度(du)稀(xi)釋(shi)的(de)效(xiao)果(guo)，使(shi)一(yi)支(zhi)試(shi)管(guan)中(zhong)分(fen)配(pei)不(bu)到(dao)一(yi)個(ge)微(wei)生(sheng)物(wu)。如(ru)果(guo)經(jing)稀(xi)釋(shi)後(hou)的(de)大(da)多(duo)數(shu)試(shi)管(guan)中(zhong)沒(mei)有(you)微(wei)生(sheng)物(wu)生(sheng)長(chang)

，那(na)麼(me)有(you)微(wei)生(sheng)物(wu)生(sheng)長(chang)的(de)試(shi)管(guan)得(de)到(dao)的(de)培(pei)養(yang)物(wu)可(ke)能(neng)就(jiu)是(shi)純(chun)培(pei)養(yang)物(wu)。如(ru)果(guo)經(jing)稀(xi)釋(shi)後(hou)的(de)試(shi)管(guan)中(zhong)有(you)微(wei)生(sheng)物(wu)生(sheng)長(chang)的(de)比(bi)例(li)提(ti)高(gao)了(le)，得(de)到(dao)純(chun)培(pei)養(yang)物(wu)的(de)機(ji)率(lv)就(jiu)會(hui)急(ji)劇(ju)下(xia)降(jiang)。因(yin)此(ci)，采(cai)用(yong)稀(xi)釋(shi)法(fa)進(jin)行(xing)液(ye)體(ti)分(fen)離(li)，必(bi)須(xu)在(zai)同(tong)一(yi)個(ge)稀(xi)釋(shi)度(du)的(de)許(xu)多(duo)平(ping)行(xing)試(shi)管(guan)中(zhong)，大(da)多(duo)數(shu)（一般應超過95%）表現為不生長。

 

 

zhinengfenlichuhunzaweishengwuquntizhongzhanshuliangyoushidezhongleishixishifadeyigezhongyaoquedian。zaiziranjie，henduoweishengwuzaihunzaquntizhongdoushishaoshu。zheshi
，keyicaiquxianweifenlifaconghunzaquntizhongzhijiefenlidangexibaohuodangegetijinxingpeiyangyihuodechunpeiyang，chengweidanxibao（或單孢子）分離法。單細胞分離法的難度與細胞或個體的大小成反比，較大的微生物如藻類、原生動物較容易，個體很小的細菌則較難。

較jiao大da的de微wei生sheng物wu，可ke采cai用yong毛mao細xi管guan提ti取qu單dan個ge個ge體ti，並bing在zai大da量liang的de滅mie菌jun培pei養yang基ji中zhong轉zhuan移yi清qing洗xi幾ji次ci
，除chu去qu較jiao小xiao微wei生sheng物wu的de汙wu染ran。這zhe項xiang操cao作zuo可ke在zai低di倍bei顯xian微wei鏡jing，如ru解jie剖pou顯xian微wei鏡jing下xia進jin行xing。對dui於yu個ge體ti相xiang對dui較jiao小xiao的de微wei生sheng物wu，需xu采cai用yong顯xian微wei操cao作zuo儀yi，在zai顯xian微wei鏡jing下xia用yong毛mao細xi管guan或huo顯xian微wei針zhen
、鉤、環(huan)等(deng)挑(tiao)取(qu)單(dan)個(ge)微(wei)生(sheng)物(wu)細(xi)胞(bao)或(huo)孢(bao)子(zi)以(yi)獲(huo)得(de)純(chun)培(pei)養(yang)。在(zai)沒(mei)有(you)顯(xian)微(wei)操(cao)作(zuo)儀(yi)時(shi)
，也(ye)可(ke)采(cai)用(yong)一(yi)些(xie)變(bian)通(tong)的(de)方(fang)法(fa)在(zai)顯(xian)微(wei)鏡(jing)下(xia)進(jin)行(xing)單(dan)細(xi)胞(bao)分(fen)離(li)，例(li)如(ru)將(jiang)經(jing)適(shi)當(dang)稀(xi)釋(shi)後(hou)的(de)樣(yang)品(pin)製(zhi)備(bei)成(cheng)小(xiao)液(ye)滴(di)在(zai)顯(xian)微(wei)鏡(jing)下(xia)觀(guan)察(cha)
，選(xuan)取(qu)隻(zhi)含(han)一(yi)個(ge)細(xi)胞(bao)的(de)液(ye)體(ti)來(lai)進(jin)行(xing)純(chun)培(pei)養(yang)物(wu)的(de)分(fen)離(li)。單(dan)細(xi)胞(bao)分(fen)離(li)法(fa)對(dui)操(cao)作(zuo)技(ji)術(shu)有(you)比(bi)較(jiao)高(gao)的(de)要(yao)求(qiu)，多(duo)限(xian)於(yu)高(gao)度(du)專(zhuan)業(ye)化(hua)的(de)科(ke)學(xue)研(yan)究(jiu)中(zhong)采(cai)用(yong)。

 

 

meiyouyizhongpeiyangjihuoyizhongpeiyangtiaojiannenggoumanzuyiqieweishengwushengchangdexuyao
，zaiyidingchengdushangsuoyoudepeiyangjidoushixuanzexingde。ruguomouzhongweishengwudeshengchangxuyaoshiyizhide，yekeyishejitedinghuanjingshizhishihezhezhongweishengwudeshengchang，yinernenggouconghunzadeweishengwuquntizhongbazhezhongweishengwuxuanzepeiyangchulai，jinguanzaihunzadeweishengwuquntizhongzhezhongweishengwukenengzhizhanshaoshu。zhezhongtongguoxuanzepeiyangjinxingweishengwuchunpeiyangfenlidejishuchengweixuanzepeiyangfenli，tebieshiyongyucongziranjiezhongfenli、xunzhaoyouyongdeweishengwu。ziranjiezhong，zaidaduoshuchangheweishengwuqunluoshiyouduozhongweishengwuzuchengde，congzhongfenlichusuoxudetedingweishengwushishifenkunnande，youqidangmouyizhongweishengwusuocunzaideshuliangyuqitaweishengwuxiangbifeichangshaoshi，dancaiyongyibandepingbanxishifajihushibukenengde。yaofenlizhezhongweishengwu，bixugenjugaiweishengwudetedian，baokuoyingyang、生理、生sheng長chang條tiao件jian等deng，采cai用yong選xuan擇ze培pei養yang分fen離li的de方fang法fa。或huo抑yi製zhi使shi大da多duo數shu微wei生sheng物wu不bu能neng生sheng長chang，或huo造zao成cheng有you利li於yu該gai菌jun生sheng長chang的de環huan境jing
，經jing過guo一yi定ding時shi間jian培pei養yang後hou使shi該gai菌jun在zai群qun落luo中zhong的de數shu量liang上shang升sheng，再zai通tong過guo平ping板ban稀xi釋shi等deng方fang法fa對dui它ta進jin行xing純chun培pei養yang分fen離li。

根據待分離微生物的特點選擇不同的培養條件，有多種方法可以采用。例如要分離高溫菌，可在高溫條件下進行培養；要分離某種抗菌素抗性菌株
，可在加有抗菌素的平板上進行分離；有些微生物如螺旋體
、粘細菌

、藍lan細xi菌jun等deng能neng在zai瓊qiong脂zhi平ping板ban表biao麵mian或huo裏li麵mian滑hua行xing
，可ke以yi利li用yong它ta們men的de滑hua動dong特te點dian進jin行xing分fen離li純chun化hua
，因yin為wei滑hua行xing能neng使shi它ta們men自zi己ji和he其qi它ta不bu能neng移yi動dong的de微wei生sheng物wu分fen開kai。可ke將jiang微wei生sheng物wu群qun落luo點dian種zhong到dao平ping板ban上shang，讓rang微wei生sheng物wu滑hua行xing，從cong滑hua行xing前qian沿yan挑tiao取qu接jie種zhong物wu接jie種zhong，反fan複fu進jin行xing，得de到dao純chun培pei養yang物wu。

富fu集ji培pei養yang法fa原yuan理li和he方fang法fa非fei常chang簡jian單dan
，利li用yong不bu同tong微wei生sheng物wu間jian生sheng命ming活huo動dong特te點dian的de不bu同tong
，製zhi定ding特te定ding的de環huan境jing條tiao件jian
，使shi僅jin適shi應ying於yu該gai條tiao件jian的de微wei生sheng物wu旺wang盛sheng生sheng長chang，從cong而er使shi其qi在zai群qun落luo中zhong的de數shu量liang大da大da增zeng加jia，很hen容rong易yi地di分fen離li到dao所suo需xu的de特te定ding微wei生sheng物wu。富fu集ji條tiao件jian可ke根gen據ju所suo需xu分fen離li的de微wei生sheng物wu的de特te點dian從cong物wu理li、化學、生物、及綜合多個方麵進行選擇，如溫度、pH
、紫外線、高壓、光照、氧氣、營(ying)養(yang)等(deng)等(deng)許(xu)多(duo)方(fang)麵(mian)。在(zai)相(xiang)同(tong)的(de)培(pei)養(yang)基(ji)和(he)培(pei)養(yang)條(tiao)件(jian)下(xia)
，經(jing)過(guo)多(duo)次(ci)重(zhong)複(fu)移(yi)種(zhong)，最(zui)後(hou)富(fu)集(ji)的(de)菌(jun)株(zhu)很(hen)容(rong)易(yi)在(zai)固(gu)體(ti)培(pei)養(yang)基(ji)上(shang)長(chang)出(chu)單(dan)菌(jun)落(luo)。如(ru)果(guo)要(yao)分(fen)離(li)一(yi)些(xie)專(zhuan)性(xing)寄(ji)生(sheng)菌(jun)，就(jiu)必(bi)須(xu)把(ba)樣(yang)品(pin)接(jie)種(zhong)到(dao)相(xiang)應(ying)敏(min)感(gan)宿(xiu)主(zhu)細(xi)胞(bao)群(qun)體(ti)中(zhong)，使(shi)其(qi)大(da)量(liang)生(sheng)長(chang)。通(tong)過(guo)多(duo)次(ci)重(zhong)複(fu)移(yi)種(zhong)便(bian)可(ke)以(yi)得(de)到(dao)純(chun)的(de)寄(ji)生(sheng)菌(jun)。

 

 

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