手機版
1、用固體培養基分離和純化
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、yijixuduozhenjunhedanxibaozaoleinengzaigutipeiyangjishangxingchenggulidejunluo，caiyongshiyidepingbanfenlifahenrongyidedaochunpeiyang。suoweipingban，jipeiyangpingbandejiancheng，tashizhigutipeiyangjidaoruwujunpingmin，lengqueningguhou，shenggutipeiyangjidepingmin。zhefangfabaokuojiangdangeweishengwufenlihegudingzaigutipeiyangjibiaomianhuolimian。gutipeiyangjiyongqiongzhihuoqitaningjiaowuzhiguhuadepeiyangji，meigegulidehuoweishengwutishengchang、繁殖形成菌落，形成的菌落便於移植。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常用手段。
1.1 稀釋倒平板法
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℃zuoyoudeqiongzhipeiyangjihunhe，yaoyunhou，qingrumieguojundepeiyangminzhong，daiqiongzhiningguhou，zhichengkenenghanjundeqiongzhipingban，baowenpeiyangyidingshijianjikechuxianjunluo。ruguoxishidedang，zaipingbanbiaomianhuoqiongzhipeiyangjizhongjiukechuxianfensandedangejunluo，zhegejunluokenengjiushiyouyigexijunxibaofanzhixingchengde。suihoutiaoqugaidangejunluo，huozhongfuyishangcaozuoshuci，biankededaochunpeiyang。
1.2 塗布平板法
因yin為wei將jiang微wei生sheng物wu懸xuan液ye先xian加jia到dao較jiao燙tang的de培pei養yang基ji中zhong再zai倒dao平ping板ban易yi造zao成cheng某mou些xie熱re敏min感gan菌jun的de死si亡wang，且qie采cai用yong稀xi釋shi倒dao平ping板ban法fa也ye會hui使shi一yi些xie嚴yan格ge好hao氧yang菌jun因yin被bei固gu定ding在zai瓊qiong脂zhi中zhong間jian缺que乏fa氧yang氣qi而er影ying響xiang其qi生sheng長chang，因yin此ci在zai微wei生sheng物wu學xue研yan究jiu中zhong常chang用yong的de純chun種zhong分fen離li方fang法fa是shi塗tu布bu平ping板ban法fa。其qi做zuo法fa是shi先xian將jiang已yi熔rong化hua的de培pei養yang基ji倒dao入ru無wu菌jun平ping皿min，製zhi成cheng無wu菌jun平ping板ban，冷leng卻que凝ning固gu後hou，將jiang一yi定ding量liang的de微wei生sheng物wu懸xuan液ye滴di加jia在zai平ping板ban表biao麵mian，再zai用yong無wu菌jun玻bo璃li塗tu棒bang將jiang菌jun液ye均jun勻yun分fen散san至zhi整zheng個ge平ping板ban表biao麵mian，經jing培pei養yang後hou挑tiao取qu單dan個ge菌jun落luo。
1.3 平板劃線法
簡jian單dan的de分fen離li微wei生sheng物wu的de方fang法fa是shi平ping板ban劃hua線xian法fa，即ji用yong接jie種zhong環huan以yi無wu菌jun操cao作zuo沾zhan取qu少shao許xu待dai分fen離li的de材cai料liao，在zai無wu菌jun平ping板ban表biao麵mian進jin行xing連lian續xu劃hua線xian，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。
1.4 稀釋搖管法
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zai50℃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)--石(shi)蠟(la)蓋(gai)取(qu)出(chu)，再(zai)用(yong)一(yi)隻(zhi)毛(mao)細(xi)管(guan)插(cha)入(ru)瓊(qiong)脂(zhi)和(he)管(guan)壁(bi)之(zhi)間(jian)，吹(chui)入(ru)無(wu)菌(jun)無(wu)氧(yang)氣(qi)體(ti)，將(jiang)瓊(qiong)脂(zhi)柱(zhu)吸(xi)出(chu)，置(zhi)放(fang)在(zai)培(pei)養(yang)皿(min)中(zhong)，用(yong)無(wu)菌(jun)刀(dao)將(jiang)瓊(qiong)脂(zhi)柱(zhu)切(qie)成(cheng)薄(bo)片(pian)進(jin)行(xing)觀(guan)察(cha)和(he)菌(jun)落(luo)的(de)移(yi)植(zhi)。
2、用液體培養基分離和純化
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。
3、單細胞(孢子)分離
隻zhi能neng分fen離li出chu混hun雜za微wei生sheng物wu群qun體ti中zhong占zhan數shu量liang優you勢shi的de種zhong類lei是shi稀xi釋shi法fa的de一yi個ge重zhong要yao缺que點dian。在zai自zi然ran界jie，很hen多duo微wei生sheng物wu在zai混hun雜za群qun體ti中zhong都dou是shi少shao數shu。這zhe時shi，可ke以yi采cai取qu顯xian微wei分fen離li法fa從cong混hun雜za群qun體ti中zhong直zhi接jie分fen離li單dan個ge細xi胞bao或huo單dan個ge個ge體ti進jin行xing培pei養yang以yi獲huo得de純chun培pei養yang，稱cheng為wei單dan細xi胞bao(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。
jiaodadeweishengwu，kecaiyongmaoxiguantiqudangegeti，bingzaidaliangdemiejunpeiyangjizhongzhuanyiqingxijici，chuqujiaoxiaoweishengwudewuran。zhexiangcaozuokezaidibeixianweijing，rujiepouxianweijingxiajinxing。duiyugetixiangduijiaoxiaodeweishengwu，xucaiyongxianweicaozuoyi，zaixianweijingxiayongmaoxiguanhuoxianweizhen、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。
4、選擇培養分離
沒mei有you一yi種zhong培pei養yang基ji或huo一yi種zhong培pei養yang條tiao件jian能neng夠gou滿man足zu一yi切qie微wei生sheng物wu生sheng長chang的de需xu要yao，在zai一yi定ding程cheng度du上shang所suo有you的de培pei養yang基ji都dou是shi選xuan擇ze性xing的de。如ru果guo某mou種zhong微wei生sheng物wu的de生sheng長chang需xu要yao是shi已yi知zhi的de，也ye可ke以yi設she計ji特te定ding環huan境jing使shi之zhi適shi合he這zhe種zhong微wei生sheng物wu的de生sheng長chang，因yin而er能neng夠gou從cong混hun雜za的de微wei生sheng物wu群qun體ti中zhong把ba這zhe種zhong微wei生sheng物wu選xuan擇ze培pei養yang出chu來lai，盡jin管guan在zai混hun雜za的de微wei生sheng物wu群qun體ti中zhong這zhe種zhong微wei生sheng物wu可ke能neng隻zhi占zhan少shao數shu。這zhe種zhong通tong過guo選xuan擇ze培pei養yang進jin行xing微wei生sheng物wu純chun培pei養yang分fen離li的de技ji術shu稱cheng為wei選xuan擇ze培pei養yang分fen離li，特te別bie適shi用yong於yu從cong自zi然ran界jie中zhong分fen離li、尋(xun)找(zhao)有(you)用(yong)的(de)微(wei)生(sheng)物(wu)。自(zi)然(ran)界(jie)中(zhong)，在(zai)大(da)多(duo)數(shu)場(chang)合(he)微(wei)生(sheng)物(wu)群(qun)落(luo)是(shi)由(you)多(duo)種(zhong)微(wei)生(sheng)物(wu)組(zu)成(cheng)的(de)，從(cong)中(zhong)分(fen)離(li)出(chu)所(suo)需(xu)的(de)特(te)定(ding)微(wei)生(sheng)物(wu)是(shi)十(shi)分(fen)困(kun)難(nan)的(de)，尤(you)其(qi)當(dang)某(mou)一(yi)種(zhong)微(wei)生(sheng)物(wu)所(suo)存(cun)在(zai)的(de)數(shu)量(liang)與(yu)其(qi)它(ta)微(wei)生(sheng)物(wu)相(xiang)比(bi)非(fei)常(chang)少(shao)時(shi)，單(dan)采(cai)用(yong)一(yi)般(ban)的(de)平(ping)板(ban)稀(xi)釋(shi)法(fa)幾(ji)乎(hu)是(shi)不(bu)可(ke)能(neng)的(de)。要(yao)分(fen)離(li)這(zhe)種(zhong)微(wei)生(sheng)物(wu)，必(bi)須(xu)根(gen)據(ju)該(gai)微(wei)生(sheng)物(wu)的(de)特(te)點(dian)，包(bao)括(kuo)營(ying)養(yang)、生理、shengchangtiaojiandeng，caiyongxuanzepeiyangfenlidefangfa。huoyizhishidaduoshuweishengwubunengshengchang，huozaochengyouliyugaijunshengchangdehuanjing，jingguoyidingshijianpeiyanghoushigaijunzaiqunluozhongdeshuliangshangsheng，zaitongguopingbanxishidengfangfaduitajinxingchunpeiyangfenli。
4.1 利用選擇平板進行直接分離
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、lanxijundengnengzaiqiongzhipingbanbiaomianhuolimianhuaxing，keyiliyongtamendehuadongtedianjinxingfenlichunhua，yinweihuaxingnengshitamenzijiheqitabunengyidongdeweishengwufenkai。kejiangweishengwuqunluodianzhongdaopingbanshang，rangweishengwuhuaxing，conghuaxingqianyantiaoqujiezhongwujiezhong，fanfujinxing，dedaochunpeiyangwu。
4.2 富集培養
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、yingyangdengdengxuduofangmian。zaixiangtongdepeiyangjihepeiyangtiaojianxia，jingguoduocizhongfuyizhong，houfujidejunzhuhenrongyizaigutipeiyangjishangchangchudanjunluo。ruguoyaofenliyixiezhuanxingjishengjun，jiubixubayangpinjiezhongdaoxiangyingminganxiuzhuxibaoquntizhong，shiqidaliangshengchang。tongguoduocizhongfuyizhongbiankeyidedaochundejishengjun。
5、二元培養物
fenlidemudetongchangshiyaodedaochunpeiyang。raner，zaiyouxieqingkuangxiazheshihennanzuodaode。dankeyongeryuanpeiyangwuzuoweichunhuapeiyangdetidaiwu。hanyouerzhongyishangweishengwudepeiyangwuchengweihunhepeiyangwu，erruguopeiyangwuzhongzhihanyouerzhongweishengwu，erqieshiyouyishidebaochierzhezhijiandetedingguanxidepeiyangwuchengweieryuanpeiyangwu。lirueryuanpeiyangwushibaocunbingdudeyouxiaotujing，yinweibingdushixibaoshengwudeyangedexibaoneijishengwu。youyixiejuyouxibaodeweishengwuyeshiyangedeqitashengwudexibaoneijishengwu，huoteshudegongshengguanxi。duiyuzhexieshengwu，eryuanpeiyangwushizaishiyanshikongzhitiaojianxiakenengdadaodejiejinyuchunpeiyangdepeiyangfangfa。lingwai，lieshixixiaoweishengwudeyuanshengdongwuyehenrongyiyongeryuanpeiyangfazaishiyanshipeiyang，peiyangwuyouyuanshengdongwuhetalieshideweishengwuerzhezucheng。liru，xianmaochong、變形蟲和粘菌。
在zai以yi上shang介jie紹shao的de幾ji種zhong方fang法fa中zhong，平ping板ban分fen離li法fa普pu遍bian用yong於yu實shi驗yan室shi微wei生sheng物wu的de分fen離li與yu純chun化hua。微wei生sheng物wu在zai固gu體ti培pei養yang基ji上shang生sheng長chang形xing成cheng的de單dan個ge菌jun落luo，通tong常chang是shi由you一yi個ge細xi胞bao繁fan殖zhi而er成cheng的de集ji合he體ti。因yin此ci可ke通tong過guo挑tiao取qu單dan菌jun落luo而er獲huo得de一yi種zhong純chun培pei養yang。獲huo取qu單dan個ge菌jun落luo的de方fang法fa可ke通tong過guo稀xi釋shi塗tu布bu平ping板ban或huo平ping板ban劃hua線xian等deng技ji術shu完wan成cheng。值zhi得de指zhi出chu的de是shi，從cong微wei生sheng物wu群qun體ti中zhong經jing分fen離li生sheng長chang在zai平ping板ban上shang的de單dan個ge菌jun落luo並bing不bu一yi定ding保bao證zheng是shi純chun培pei養yang。因yin此ci，純chun培pei養yang的de確que定ding除chu觀guan察cha其qi菌jun落luo特te征zheng外wai，還hai要yao結jie合he顯xian微wei鏡jing檢jian測ce個ge體ti形xing態tai特te征zheng後hou才cai能neng確que定ding，有you些xie微wei生sheng物wu的de純chun培pei養yang要yao經jing過guo一yi係xi列lie分fen離li與yu純chun化hua過guo程cheng和he多duo種zhong特te征zheng鑒jian定ding才cai能neng得de到dao。
