微生物學(xué)與免疫學(xué)：普通微生物學(xué)實(shí)驗(yàn)

ExperimentandGuideforMicrobiology*1*2EXPIBright-FieldMicroscopeEXPIIMicrobialMorphologyEXPIIIInoculation,Growth,BiochemicalTestsofBacteriaEXPIVActinomycetes,FungiandSpirochetesEXPVDisinfection&Sterilization,HeredityandVariationEXPVIDeterminationof

AntimicrobialActivity,BiologicalAssayofAntibioticsEXPVIISterilityTestofdrugsandMicrobiallimitTestsEXPIBright-FieldMicroscopePurpose：1.Structureandprincipleofthebright-fieldmicroscope2.Howtousethemicroscope(*theoilimmersionlens)3.Observe:Stainedmicroorganismspecimens*3Principle

1.Thestructureofthebright-fieldmicroscopemachinesystemopticalsystem*4ArmTube

Nosepiece

StageclipsStageStagemotionknobBaseCoarseadjustmentknobFineadjustmentknob*5Lightsourcecontrol1:on/offLightsourcecontrol2OcularlensObjective

lensLink

computerCondenserTest*62.Thebasicprincipleofoil-immersionlensR=0.61λ/N.A.N.A.=n·sinθ/2resolvingpower:0.2μm.*71.25(0.65、0.25)100(40、10）160/0.17

數(shù)值孔徑放大倍數(shù)鏡筒長(zhǎng)/蓋玻片厚度

數(shù)值孔徑：N.A.=n﹒sinθ/2

n—玻片和物鏡之間的折射率。

θ—光線最大射入角。

分辨率：R＝0.61λ/N.A.

λ—波長(zhǎng)

物鏡上的標(biāo)識(shí)*8Specimens：1.BasicbacterialmorphologyStaphylococcusaureusStreptococcusEscherichiacoliBacillussubtilisVibriocholerae2.Specialbacterialstructurespore(Bacillussubtilis,Clostridiumtetani)flagella(Salmonellatyphosa)capsule(Streptococcuspneumoniae)*9Procedures：1.Gettingreadyforexperiments2.Gettingthelight3.Adjusttheirisdiaphragm,condenser,andlightsource4.ObservationLow-power→High-power→Oil-immersionobjectivelensAttention:Can’tturnthecoarse-adjustmentknobtomaketheobjectivelensimmersedintotheoilonlyifviewingfromtheside.Thefine-adjustmentknobcan’tbeturnedexcessively.5.AfterexperimentsMicroscopeslide*10ResultsandDiscussionDrawing:(3figures/line,9figures/page)StrainMagnificationStaphylococcusaureus100×10*11PreviewNext:MicrobialMorphology

Exp6,7,10,11OnDuty:No.?-?*12EXPIIMicrobialMorphologyPurpose1.TobefamiliarwithGram’sstaintechniques2.Tobefamiliarwiththeprocedureofthesporestain3.observethemotionoflivingbacteriavideo*13Bacteriacanbedividedintotwomajorgroups,calledgram-positiveandgram-negative.Theoriginaldistinctionbetweengram-positiveandgram-negativewasbasedonaspecialstainingprocedure,theGramstain.Principle-Gram’s

stain*14Specimens：1.Cultures:24hnutrientagarslantculturesofStaphylococcusaureusandEscherichiacoli.2.Reagents:Crystalviolet,95%ethylalcohol,Lugol’siodine,safranin,physiologicalsaline,cedarwoodoil,xylene.3.Apparatus:glassslides,water-adsorptionpaper,inoculatingloop,lamp,lenspaper,microscope.*15Procedures：

SimpleStain*16washingSmearsAir

dryFixationStaining(Methyleneblue)Examine(oilimmersion)DrawingProcedures：SimpleStain*17Procedures：GramStain①Preparationofbacterialsmears,airdry,andfixationrefertosimplestainingtechniques.(3-area)②Primarystain1-2min③Mordant1min④Decolorizing30seconds(10’’x3)⑤Counterstain1minObservinganddrawingthemixture*18CrystalvioletLugol’siodine95%ethylalcohol,safranin,PrimarystainMordantDecolorizingCounterstainwashingwashingwashingwashing3-areamixtureE.coliS.aureus*19Gram’s

stain*20E.coliS.aureusG+G-*21ResultsandDiscussion:①what’stheprincipleofGramstain?②Drawarepresentativefieldforgrampositiveandgramnegativebacteria.*22PrinciplesporestainCentralcoreCortexSporecoat/membraneexosporium*23MaterialsandApparatus:①Cultures:48hoursnutrientagarslantcultureofBacillussubtilis.②Reagents:0.5%malachitegreen,0.5%safranin,physiologicalsaline,cedarwoodoil,xylene.③Apparatus:glassslides,water-adsorptionpaper,inoculatingloop,lamp,lenspaper,microscope.*24Procedures:sporestain①Preparationofbacterialsmears,airdry,andfixation.②Heat-stain(0.5%malachitegreen,1minute)③CoolandWashthesmear④Stainthesmearwith0.5%safranin(30seconds)⑤Examinethestainedslideunderoilimmersion.Drawing:Vegetativecell:red,Spore:green.*25Bacillussubtilis*26ResultsandDiscussion:①Whyisheatnecessaryinsporestaining?②Drawarepresentativefieldforthevegetativecellandbacterialsporeandindicatethecolorofvegetativecellandthespore.*27Principle-HangingdropFlagellaaremotionorganellesofbacteria.Bacteriawithflagellacanmoveformoneplacetoanotherinculturesolution.Ontheotherhand,bacteriawithoutflagellawillvibrateduetowatercollision.ThatiscalledBrown’smotion.Herehangingdropmethodisusedtoobservethemotionoflivingbacteriawithbright-fieldmicroscope.Asshown,adropofbacterialiquidcultureissuspendedonthecenterofcoverslip.*28MaterialsandApparatus:①Cultures:8hoursculturemediaofStaphylococcusarueusandBacillussubtilis.②Apparatus:concavityslide,coverslip,toothpick,inoculatingloop,lamp,lenspaper,microscope.*29ProceduresPreparationofspecimen(1)PlaceadropofculturemediaofSaccharomyces

cerevisiae

andBacillussubtilisrespectivelyonaglassslideandmixitwithinoculatingloop.(2)Smearsomevaselinaroundtheedgesofthecoverslipwithtoothpick.(3)Placeaconcavityslideonthecoverslideandpressitsoftlyforsticking.(4)Reversethespecimenrapidly.Observationwithmicroscope(1)Placethespecimenonthestage.(2)Observeitwithlowpowerlens(10×)andfixtheedgeofdrop.(3)Observeitwithhighpowerlens(40×).*30*31concavityslidecoverslipbacteriasolutionvaselin*32*33ResultsandDiscussion①DescribethemotivecharacteristicsofSaccharomycescerevisiaeandBacillussubtilis.②Howtodecidewhetherbacteriacanmove?*34EXPIIIInoculation，Growth，BiochemicalTestsofBacteriaPurpose1.Tobefamiliarwiththetechniqueofasepticremovalandtransferofmicroorganismsforsubculturing.2.Toknowhowtoestablishpureculture.3.Todeterminetheculturalcharacteristicsofmicroorganismsasanaidinidentifyingandclassifyingorganismsintotaxonomicgroups.4.Tounderstandtheprincipleofbiochemicaltestsandthejudgmentofthesetests.*35Principle—InnoculationAndCulturalCharacteristicsOfBacteria

Themostusefulandpragmaticmethodforobtainingpureculturesisthestreakplateinwhichamixescultureisspreadorstreakedoverthemediumsurfacesuchthatindividualcellsbecomeseparatedfromoneanother.Eachisolatedcellgrowsintoacolony.agarslantBrothsemisolidmedia3-way

streak-plateSterilizationoftheinoculatingloop*36MaterialsandApparatusS．a(chǎn)ureusE．coliB．SubtilisagarslantBrothsemisolidmedia3-way

streak-plateMedia：1set/personCultures：1set/group*37Proceduresagarslant*38Proceduresbroth*390.5cmProceduressemisolidmedia*40Proceduresstreak-plate*41*42*43ResultsAndDiscussion1.Observethecultureonagarslant.2.Observethecultureinnutrientbrothandwritedowntheculturalcharacteristicsofdifferentbacteria.strains

culturalcharacteristicsS．a(chǎn)ureus

E．coli B．subtilis

*443.Observethecultureinsemisolidmediaandwritedowntheresultsinthefollowingtable.ResultsandDiscussionStrainculturalcharacteristicsmobilityS．a(chǎn)ureusB．subtilis*454.

Observethecharacteristicsofthecoloniesofdifferentbacteriaandwritedowntheresultinthetable.ResultsAndDiscussionStrainsSizeFormSurfaceMarginTransparencePigmentationS.aureusE.coliB.subtilisS.lutea*461.UtilizationofCarbohydrates2.IMViCSeries3.HydrolysisofGelatin4.HydrogensulfideProductionPrinciple—thebiochemicaltestsofbacteria*47*48Result—UtilizationofCarbohydrates*49CarbohydratesglucoselactosemaltosemannitolsucroseE.coliB.typhiResults—UtilizationofCarbohydrates*50E.coliE.aerogenesIndoleproductionRed:+*51E.coliE.Aerogenes

cotrol

TheMethylRedTestRed:+*52TheVoges-ProskauerTestE.coliE.aerogenesRed:+*53CitrateUtilization*54IMViCSeries*55Results—IMViCSeriesIMViCIndoleMethylRedVoges-ProskauerCitrateUtilizationE.coliE.aerogenes*56Results—HydrolysisofGelatinBacteriaBacillussubtilisEscherichiacoligelatincontrol*57Results—HydrogensulfideProductionBacteriaResultPrincipleE.coliP.vulgaris*58HydrogensulfideProduction*59EXPIVActinomycetesandFungiPurpose1.Tobefamiliarwiththeobservationmethodof

yeast,

fungiandantinomycetes.2.Toobservethemorphologyofantinomycetesandmolds.3.Toobservethecolonymorphologyofbacteria,antinomycetes,yeastandmolds.4.Tobefamiliarwiththespirochetesstainandtoobservethemorphologyofspirochetes.*60methylene

blue(oxidationtype）bluemethyleneblue(reductiontype）nocoloryeastcellsmetabolismpossibilityofreductionPrinciple—YeastMorphologyViableyeastcelldeadcell*61Procedures—YeastMorphology1.

Dropablobofmethyleneblueonthecenterofglassslide.2.Takesomeyeastandsuspendaloopfulofyeastscultureintoafewdropsofmethylenebluesolution.3.Coverthesuspensionslowlywithacoverslipandstayitfor3minutes.4.Examinetheslidewithmicroscopeunderlowandhighpower.*621.Dropablobofmethyleneblue2.Takesomeyeast3.Coverthesuspension4.Examinetheslide*63*64ResultsandDiscussion1.Drawarepresentativefieldforyeastunderhigh-powermagnificationsoroilimmersion.Saccharomyces

cerevisae*65Principle—MorphologyofMyceliaandSporeforAntinomycetesandMoldsAgarsurface氣生菌絲基內(nèi)菌絲*66Procedures—MorphologyofMyceliaandSporeforAntinomycetesandMolds1.Placeadropofmethyleneblue2.Pressacoversliponthecolonysurface3.Coverthesuspension4.Examinetheslide*67放線菌2809*68青霉菌*691.Describethemorphologicalcharacteristicsofthemyceliaandsporeofantimycetesandmolds.ResultsandDiscussionantinomycetesmolds*702.Describethecharacteristicsof

ColoniesofActinomycetes2809,Yeast(Sacchariomyces)andMolds(Penicillum)ResultsandDiscussion2809SacchariomycesPenicillumSizeShapeSurfaceEdgeColorDissolvedpigment*713.LawnofActinomycetes(1~5)AntinomycetesNo.1No.2No.3No.4No.5SizeShapeSurfaceShapeandcolorofaerialmyceliaColorofsporemyceliaColorofsubstratemyceliaDissolvedpigment*724.DescribethecharacteristicsofMolds(4Penicillum,Aspergillus,Mucor,Rhizopus)FungiPenicillumAspergillusMucorRhizopusSacchariomycesSizeShapeSurfaceShapeandcolorofaerialmyceliaColorofsporemyceliaColorofsubstratemyceliaDissolvedpigment*73Principle—SpirochetesMorphologySpirochetesareslender,soft,curved,movable,andhelicalprokaryoticcellswhichareabout5to10nminlengthand0.2μminwidth.Inadditiontodupilication,theprimarystructureofspirochetesissimilartobacteriawithcellwallandnucleoid.Spirochetesaresensitivetoantibioticsandcanmovecausedbyaspecialaxialfilamentotherthanflagellarrotation.*74Procedures—SpirochetesMorphology1.

Smears（tartar）→Air

dry→2.

Fixation→solutionA，1minute→wash→3.

Stain→solutionB，30seconds（Heat-stain）→cool→wash→

solutionC，30seconds（Heat-stain）→cool→wash→

4.Examine(oilimmersion)*75*761.Drawarepresentativefieldforspirochetes.ResultsandDiscussionSpirochetes*77EXPVDisinfection&Sterilization,HeredityandVariationPurpose1.Tobefamiliarwiththephenomenaofmicroorganism’svariationsofcolonialmorphologyanddrugresistancemutation.2.Tobefamiliarwithasimplemethodtodeterminepenicillinase.3.Tobecomefamiliarwiththeprincipleandmethodsofhightemperature.4.Tobecomefamiliarwiththeprincipleandmethodsofeffectofultravioletlight.5.Tobecomefamiliarwithspore’sthermotolerance.*78Principle--Disinfection、UltravioletLigh1.Pasteurization2.Actionofultravioletlight253.7nm*79Materials--Disinfection、UltravioletLighBacillussubtilis

Escherichiacoli*80Procedures--Disinfection、UltravioletLigh1.Bacillussubtilis、Escherichiacoli→

broth→63℃waterbath,30minutes→37℃,18-24hours

→

observe2.Streakplate→coverhalf→UVlight,30minutes

→37℃,upsidedown,18-24hours→observe*81Discussion--Disinfection、UltravioletLight1.Recordtheactionof60℃waterbathandUVlight.2.ComparetheresultoftheactionofUVlightontwostrains.Howdoyouaccountforthedifference?3.HowcanweacquiremasssporeinliquidofBacillus?*82Principle--HeredityandVariationofMicroorganismHeredityandvariationarecommonpropertiesofmicroorganisms.Therearemanyphenomenaofbacterialvariationincludingcolonialorcellularmorphologicalvariation,structuralvariationofcapsule,sporeandflagella,biochemicalvariationsuchasvirulenceanddrugresistance.*83Procedures--FlagellaVariationnutrientagarnutrientagar（0.1%phenol）Proteusvulgaris

*84Procedures--Drug-ResistantMutation(drugsensitivitytest)filterpaperdiscscontainingpenicillinresistantstrainsSensitive

strainsInhibitionzone*85Procedures--DeterminationofPenicillinase(I2-starchplatemethod)RpenicillinG20000U/mlStarchagar50℃SRⅠ2*86*87ResultsanddiscussionTheresultsofflagellavariation.StrainMediaDescriptionofgrowthProteusvulgarisStrainsMediaDescriptionofgrowthStaphylococcusaureus(penicillinsensitive)Staphylococcusaureus(penicillinresistant)Theresultsofdrug-resistantmutation*88EXPVIDeterminationof

AntimicrobialActivity，BiologicalAssayofAntibioticsPurposeTodeterminetheantimicrobialactivityofantibiotics.Tounderstandtheprincipleofbiologicalassayofantibiotics.Todeterminetheantibioticsconcentrationwithcylinderplatemethod.*89Principle--DeterminationofAntimicrobialActivityTheminimalinhibitoryconcentration(MIC)Theminimalbactericidalconcentration(MBC)MaterialsandApparatusCultures:Staphylococcusaureus,Staphylococcusepidermidis,Escherichiacoli,Klebsiella

pneumoniae,andstandardstrainsofStaphylococcusaureus(ATCC25925)

andEscherichiacoli(ATCC25922).Antibiotics:penicillinG,streptomycin,szithromycin,levofloxacin.Media:Mueller-Hintonagarincubatedat55℃inwaterbath.*90*91Procedures12345678media*92ResultsandDiscussionAntimicrobialactivityofdrugs

*+:growth.-:nogrowthStrainsDrugconcentration（μg/ml）MIC(μg/ml)12864321684210S.

aureusS.

aureus(ATCC25925)S.

epidermidisE.coliE.coli(ATCC25922)K.pneumoniae*93Principle—BiologicalAssayofAntibiotics*94Procedures--BiologicalAssayofAntibioticsSporesuspensionofBacillussubilis*95I=lg2D=1ResultsandDiscussion1.Calculatetheantibioticsconcentrationwiththemethoddescribedabove.W=(SH+UH)–(SL+UL)V=(UH+UL)–(SH+SL)θ=D×antilog(IV/W)*96EXPVII

SterilityTest