國外微生物學(xué)基礎(chǔ)實(shí)驗(yàn)(三)-英文版課件

GettingReadyforToday’sLabGrabyourlabcheck-incardandthedirtycopiesofLab#2and#3thatwesavedfromlastweek.Grabyourplasticboxofsupplies.Putonyourlabcoatandhaveyourgoggles,labcardanddirtycopiesoflabexercises/reportsonthebenchnexttoyou.Pluginandturnonyourmicroincinerator.TURNIN:NolabreportorPre-labduethisweek.EndofToday’sLabAfteryouhavecleanedyourlabbench,copyyourLab#2and#3materialontoacleancopytotakehome.ImustseeyouthrowoutyourdirtycopyofLab#2.YoumayturninyourdirtycopyofLab#3tosaveforusenextweek.Lab

Report#2and#3duenextweek,asweek,aswellasPre-lab#4.GettingReadyforToday’sLab1Laboratory3DifferentialStainingsueGLaboratory3DifferentialStain2ThreeMainThingsWeareDoingTodayI.LookingatResultsfromLastWeeksCulturesII.LearningtouseanInoculationLoopforStreakPlateMethodIII.DifferentialStainsThreeMainThingsWeareDoing3I.LookingatResultsfromLastWeeksCulturesI.LookingatResultsfromLas4OtherTypesofMedia:

McConkey’s,BloodAgar&MannitolSaltLookattheplatesonyourlabbenchthatarered/pinkIncolor.BloodAgar=verydarkredMcConkeys=lighter,purplish-pinkMannitolSalt-=orangish-pinkMacConkey's

(alreadyprepared)MacConkey’sisbothaselective&differentialmedia.1.WhatDoesSelectiveMean?MacConkey’sisselectivemediabecauseitinhibitsthegrowthofsomeorganisms[Grampositivebacteria].2.WhatDoesDifferentialMean?Because“neutralred”hasbeenaddedtothemedia,itisalsodifferential.

-Bacteriathatuselactose(atypeofsugar)forfood,produceacidicmetaboliteswhichcausesthepHindicator neutralredtoturnred. -These“l(fā)actosefermenters”willgrowinredcolonieswhilenon-lactosefermenterswillbecolorlessandclear.

MembersofthefamilyEnterobacteriaceae(Gramnegativebacilli)arethemostfrequentlyencounteredbacteriaisolatedfrommanytypesofclinicalspecimens.Theyaremostcommonlylactosefermenters.OtherTypesofMedia:

McConke5Bloodagar

MostspecimensreceivedinaclinicalmicrobiologylabplatedontoBloodAgar,sinceitsupportsthegrowthofmostorganisms.Bloodagarcontains5%sheepblood.Thismediaisdifferentialbecause:Certainbacteriaproduceenzymes(hemolysins…h(huán)emo-lice-ins)thatactontheredcellstoproduceeither:

Betahemolysis:Enzymeslysethebloodcellscompletely,producinga clearareaaroundthecolony.

Alphahemolysis:Incompletehemolysisproducesagreenish discolorationaroundthecolony.

Gammahemolysis:Noeffectontheredcells.Bloodagarisusuallyinoculatedfromapatient’sthroatswab.MicrobiologisttrytodetectpresenceofGroupAbetahemolyticStreptococci(aGram-positivecoccithatcausesBetahemolysisonbloodagar.)MajorhumanpathogeninthisgroupisStreptococcuspyogenes,thecausativeagentofstrepthroat.Normalthroatflorawillexhibitalphaorgammahemolysis.Betameansstrep.Bloodagar6MannitolSaltIsaselectivemediawithahighconcentrationofNaCI(7.5%).Mostbacteriacannotsurviveinthisenvironment.ThegenusStaphylococcus,however,growswellinthismedia.MannitolSaltisalsodifferentialbecauseitcontainsadyethatidentifiesorganismsthatfermentmannitol.Theorganicacidsproducedchangethedyefromredtoyellow.Thismediumworkswellforidentifyingpathogenicstaphylococci,suchasStaphylococcusaureus,whichwillfermentmannitol.Mostnon-pathogenicstaphylococciwillnotfermentmannitol.MannitolSalt7RecordingResultsfromLastLab

CulturesRecordresultsofenvironmentalsamples,foreachquadrants1–4(TSYandMacConkeys).AreMacConkeycoloniespinkorcolorless?Whatdoesitmeaniftheyarepink?2.Describeresultsfromtouchplatesandarmplates(TSY).

a.Whatisthedifferencebetweenplates1,2and3?3.Recordresultsfromthroatswabplate(Bloodagar).

a.DoyouseeAlphahemolysis?Betahemolysis?Gammahemolysis?

b.Whichhemolysispatternisbadnews?4.Recordresultsfromnasalswabplates(TSYandMannitolSalt).

a.DidanyofthecoloniesturntheMannitolSaltmediumyellow?

b.Whatwoulditmeaniftheydid?RecordingResultsfromLastLa8國外微生物學(xué)基礎(chǔ)實(shí)驗(yàn)(三)-英文版課件9國外微生物學(xué)基礎(chǔ)實(shí)驗(yàn)(三)-英文版課件10II.InoculationLoop&StreakPlateMethodWewillcultureourunknownontoMacConkey’sandMannitolSalt.Todoastreakplatetechnique,wewillneedtouseaninnoculationloop.AKAsmearloop,inoculationwandormicrostreakerSimpletoolusedtoretrieveaninoculumfromacultureofmicroorganisms.Alwayssterilizeinaflameuntilitbecomesredhotbeforeandaftereachuse.Bydoingthis,thesametoolcanbereusedindifferentexperimentswithoutfearofcross-contamination.Besurethatyourinoculationloophascooledbeforeusingittoretrieveaninoculumorstreakaplate!Ifyouhearthemediumsizzle… theloop’stoohot!MicroincineratorII.InoculationLoop&Streak11StreakPlateTechniqueWhenwewanttoisolateaspecificspeciesofbacteria,weneedtouseatechniquethatwillspreadouttheoriginal“parentbacteria”inasparseenoughpatternsothataftergrowth,individualcolonieswillresult.Afterincubation,the4thquadrantofyourplateshouldhavedots.Thesesmall“dots”areindividualcolonies,andrepresentmillionsofbacteriaofthesamespeciesStreakPlateTechnique12III.DifferentialStainsIII.DifferentialStains13DifferentialStainsMoststainsusedinmicrobiologyaredifferentialstains.Usemorethanonedyesothatdifferentcells,chemicalsorstructurescanbedistinguished.DifferentialStainsMoststains14PreparingSpecimensforViewing–DifferentialStainsGramStainDistinguishesbetweentwolargegroupsofmicroorganisms: -purplestaining,Gram-positivecells -pinkstaining,Gram-negativecellsThesetwotypesofcellsdiffersignificantlyinthechemicalandphysicalstructureoftheircellwall.ThestructureofthethinnercellwallsofGramnegativebacteriacannotholdthedyespreviouslyused,oncethedecolorizerisapplied.Crystalviolet(1min):

rinse:Iodine(1min):

rinse:AcetoneAlcohol(10–15sec):rinse:Safrinin(1min)MordantisachemicalusedtofixthestainingreactionsueGPreparingSpecimensforViewin15PreparingSpecimensforViewing–DifferentialStainsAcid-FastStainThisstainsthecellsofthegeneraMycobacteriumandNocardia,whichcausemanydiseasesinhumans,includingtuberculosis,leprosy,andotherlungandskininfections.Cellsofthesebacteriahavelargeamountsofwaxylipidintheircellswalls,sotheGramstainandotherwater-basedstainsdon’tworkwellonthem.Cellsaredeterminedtobeacid-fastornot,basedonwhethertheyretaintheirprimarystainafterdecolorization.Createasmearoftheorganismthatyouaretesting.Coverthesmearwithastripofblottingpaper.SaturatepaperwithZiehl’scarbolfuchsinandheatfor3–5minutes.Removeblottingpaper.Washslidewithtapwaterandthendecolorizethesmearfor10-15secondswithacidalcohol.Rinseagainwithtapwater.Applycrystalvioletfor1minute,wash,blotdry.OBSERVATIONOFMICROORGANISMSBlottingpaper:Ziehlscarbolfuchsin(3–5minheat):rinse:AcidAlcohol(10–15sec):rinse: