微生物实验1 细菌的形态与结构

实验室注意事项穿白大衣、严禁饮食无菌操作，实验完毕要洗手煤气水电随手关好若发生吸入菌液，割破手指，要及时报告实验前预习，课后认真、真实的完成报告1注意事项（一）除必需书籍、实验报告、文具外，学生书包一律不能带进教室。书包等存储于储物柜中：每次锁门需要1元硬币一枚，开门时自动退还。希望同学们好好保管钥匙，如有遗失须赔偿换锁芯成本费30元。（如以后找到交还，30元可退还）储物柜使用时间为学生本人病原学实验课时段，请勿超时存放，课后储物柜有实验室工作人员清理，并将存放物品移出，遗失概不负责。煤气灯使用结束，黄色旋钮必须关闭（垂直）学生抽屉中已放置擦镜纸、香柏油、火柴等物，使用完毕放置原处。2注意事项（二）显微镜请学生登记在显微镜记录表格上。靠窗一列学生实验台柜中可能储存2台显微镜，只有数字编号的为学生使用镜，其他一台写明“示*-*”为示教镜，严禁学生使用。（有些柜中另装一台解剖镜，也请勿动）白大衣借用：在213外室靠窗的一个柜中有备用白大褂，如需借用，请登记，并叠好归还。3MorphologyofBacteria,

Culture4ObjectiveMorphology&StructureGramstainGrowthphenomenonofbacteriaStreakplatemethodInoculationPart1MorphologyPart2Culture5BasicmorphologyofbacteriaCocciBacilliSpiralbacteria6StainStainsdifferentiatemicroorganismsfromtheirsurroundingenvironmentTheyallowdetailedobservationofmicrobialstructuresathighmagnificationCertainstainingprotocolscanhelptodifferentiatebetweendifferenttypesofmicroorganisms.7SpecialstructuresCapsuleCapsuleS.pneumoniae(Negativestain)8FlagellaProteusvulgaris(Peritrichous)Flagella9Spore：SporesporeC.tetani10GramstainTodifferentiate

bacterial

speciesintotwolargegroups(Gram-positive

and

Gram-negative)basedonthephysicalpropertiesofcellwallsofbacteria.Usage:IdentificationChooseantimicrobialagentsStudyonbacterialpathogenicityHansChristianGram,188411GramstainPreparationofasmearStainingTransfergrowthtotheslideDryFixcrystalviolet1minwaterIodine1min95%ethylalcoholSafranin30secDryObservationwaterwaterwater(untilthedyenolongerrunsfromthesample)(theoilimmersionobjective)12GramstainPreparationofasmearObtainacleanslidePlacealoopfulofnormalsalineintheslide.FlameloopAsepticallytransferaminuteamountofgrowthfromtheagarslanttothedropofwater.Flameloopbeforesettingitdownontable.Allowthesmeartoairdryorexpeditedbyholdingtheslidehighoveragasflame.Fixthefilmbyquicklypassingtheslide3timesthroughtheupperportionoftheflame.13StainprocedureCoverthesmearwithcrystalvioletandallowittoremainfor1min.PouroffthestainandrinsethewaterCoverthesmearwithiodineandallowittoremainfor1min.PouroffthestainandrinsethewaterApplyafewdropsfoethylalcoholtothesmear,allowthealcoholtodripofftheslide.Continuetorenewwithmorealcoholuntilthedyenolongerrunsfromthesample.WashtheslideinwaterCounterstainwithsafraninfor30sec.Washslideinwateragain.Blotslidedrywithbibulouspaperorpapertowel.ObservationExaminethesmearusingtheoilimmersionobjective.14ResultsofGramstain×1000Bytheoilimmersionobjective.PositiveNegative15PrerequisitesNutritionTemperaturepHGasenvironmentOsmolalityH2O

InorganicsaltCarbonsourceNitrogensourceGrowthFactor

CultureMedium16LiquidmediaSedimentgrowthamplificationSurfacegrowthTurbidity

17SolidmediaAgarplateAgarslantobtainingisolatedcoloniesobtainingpurecultures18Semi-solidmedia+-1.motilitytest2.short-timepreservation19IVII1/5I1/10Rotate90FlameloopRotate90Rotate90III1/4FlameloopSummaryofstreakplatemethod

IsolationofsinglecoloniesfrommixedbacteriaIncubateat37℃20IMViCtests

（E.coli、

E.aerogenes）Culturemedia：Glucosepeptonewaterbroth(4)peptonewaterbroth(2)citrateagarslant(2)E.coliE.aerogenes