cultural characteristics physiological...
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MiramarCollegeBiology205Microbiology
LabExercise18:APhysiological&CulturalExaminationoftheMinorUnknownBackgroundInorder to identifyabacterial species,microbiologists employmany techniques thatwill allow them togatherbothphysicalandphysiologicaldataaboutaparticularisolate.Initially,anisolateismicroscopicallyexaminedforGramstainreactionandthepresenceofsub‐cellular/cellularcharacteristics (e.g.,capsules,spores,acid‐fastness and flagella). Inaddition, the approximate size, morphology and arrangement of the cells are also determined. Once thesemorphological characteristics have been determined, the cultural characteristics of the isolate are determined: theoxygenrequirement; itsgrowthpatternsonvarioussolidandliquidmediaareobservedandtheoptimaltemperatureoftheorganismisdetermined.Finally,theisolate’sphysiologicalcharacteristicsareexamined.Bacteriausecatabolicchemicalreactions inordertoharnesstheenergyreleasedfromthedecompositionoforganicmoleculesasATP. Thesereactions, likeallmetabolic reactions,aregovernedbyenzymescatalysts. Enzymescanbedividedintotwogroups,endoenzymeswhichfunctioninsidethebacterialcellandexoenzymeswhicharesecretedbythe cell in order to accomplish digestion in the exterior environment, (Figure 1). Becausemany bacteria share themorphological and cultural characteristicsmentioned above, the identification of the physiological capabilities of anunknown bacterium is paramount to its identification. The collection of these physiological data is themost time‐consumingandcomplicatedaspect, andemploystheuseofmultipleselectiveanddifferentialmedia. Eachof thesemedia infers the presence of a specific enzyme and/or enzymes by measuring the absence of a substrate or thepresenceof aproduct fromagivenenzyme‐regulated reaction. Becauseeachenzyme, as aprotein, is coded in thegenesofanorganism,theresultsofthesephysiologicaltestsindicatessomethingaboutthegenomeoftheorganismyouarestudying.
Figure1:Actionofenzymesinside(endoenzymes)andoutside(exoenzymes)thebacterialcellonaparticularsubstrate.TheseenzymescatalyzethereactionsandresultsinATPgeneration.
Physiological characteristics can be subdivided into categories: carbohydrate metabolism; fermentation; proteincatabolism; and respiration. Carbohydrate metabolism includes those reactions which break down moleculescontaining the subunit ratio of (CH2O)n: polysaccharides, oligosaccharides, disaccharides and monosaccharides.Fermentation reactions, you will remember from lecture, are catabolic redox reactions whose terminal electronacceptorisanorganicmolecule,typicallyanacidicbyproductofpyruvateand/orcarbondioxide(CO2)gasoracetoin.Protein catabolism includes those reactions whose substrate is a protein or amino acid which is converted into aproduct which can enter the glycolytic pathways of the cell. When the amido functional group (‐NH2) which isdeaminated as ammonia (NH3) during the catabolism of proteins is liberated, it causes an increase in the pH of themedium.Thecarboxylgroup(‐COOH)canalsobecleavedbyadecarboxylase(Figure2).Additionally,hydrogensulfide(H2S)isliberatedfromsulfur‐containingaminoacidspriortotheirentryintotheglycolyticpathwaysandoneimportantproduct,indole,canbeproducedfromtheaminoacidtryptophan.Enzymesinvolvedinrespirationareusedduringtheredoxreactions leadinguptotheuseofan inorganicelectronacceptor intheelectrontransportchain (e.g.,O2,SO4,CO3 or NO3). Because aerobic respiration (using O2) creates toxic oxygen species, the presence of enzymes whichprotectthecellfromthesecanalsobeusedtoinferthistypeofmetabolism.
Figure2:Themolecularstructureoftheaminoacid.Notethatthedefiningcharacteristics,theamide(NH2)andcarboxyl(COOH)groupswhichareliberatedduringthecatabolismofanaminoacidduringdeaminationsanddecarboxylationsrespectivelyAminoacidsdifferonlyintheirRgroup.
ThetestsperformedinlaboratoriestoinferthepresenceoftheenzymesdescribedaboveareoftenbasedonthepHofthemediachangingoverthecourseofincubation.ThepresenceoffermentationproductswilllowerthepH,whilethepresenceofammonia(NH3)willraisethepH. Inthesecases,differentialmediawhichincludepHindicatorsareused
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(Table 1). Other, more sophisticated tests are used to detect the absence of media components or their resultingproductswhenpHcannotelucidatethecharacteristicsofagivenmicrobe.Table1:variouspHindicatorsandtheirresultingcolorsinsolutionsasthepHofthesolutionchanges.pHindicator acidiccolor neutralcolor alkalinecolor
methylred yellow red red
phenolred yellow red pink
bromthymolblue green green blue
bromcresolgreen yellow green green
bromcresolpurple yellow yellow purple
In addition to the single tests researchers use to physiologically characterize and identify bacteria, manymulti‐testmediaareavailableforuse.Thesemediacombineingredients,suchaspHindicatorsandspecialsubstratesandcanbeused to show more than one physiological characteristic. These media are useful because often just a fewcharacteristicscanbeusedtoidentifya likelyorganismonceGramstaininghasbeendone.OnesuchexampleisSIMmedium,which tests for H2S production, indole production andmotility. These three tests are useful in identifyingpathogenicmembersoftheentericbacteriaoncelactosefermentationhasbeenruledout.Because certain characteristics can be used to identify organisms from one another, inoculations are sometimesgrouped together to identify closely‐relatedorganisms. Anexampleof this is the IMViCgroup of tests (i.e., indole,methyl‐red,Voges‐Proskauerandcitrate)whichareusefulinidentifyingabacterialspeciesamongtheentericbacteria(Table2).Table2:IMViCresultsforEscherichiacoliandEnterobacteraerogenes.IMViCisanexampleofmultiplemediainoculationsbeingusedtoidentifyaparticularbacterialspeciesfromclosely‐relatedspecies.organism indolereaction(rxn) methylredrxn Voges‐Proskauerrxn citraterxn
E.coli + + ‐ ‐
E.aerogenes ‐ ‐ + +
Becausethesemultipletestsaresouseful,particularlyinidentifyingcloselyrelatedspecies,severalcommercialkitsareavailabletocompilethesedatawithasingleinoculation.OnesuchkitistheBBLEnterotubeII,whichcombinesseveralof thetestsuseful in identifyingentericbacteria (Figure 3). Intheclinical setting, suchas in ahospitalmicrobiologylaboratory, even theEnterotube is too time‐consumingamethod for the identificationof entericpathogens. In thiscase, the Vitek® Gram Negative identification (GNI) card is used as a miniaturized test within which 30biochemical/physiologicaltestsfortheidentificationofentericbacteriaaredone.Thesesystemsaresosensitivethatacomputerisrequiredtointerprettheresults,andcandosoinaslittleasfourhours(Figure4).AnimportantdistinctionbetweenthisViteksystemandtheothermethodsdiscussedinthishandoutisthefactthatthecardisinoculatedwithorganismsalreadygrowninliquidculture.
Figure3:DiagramoftheBBLEnterotubeII,whichincludesmanyoftheusefulmediainoculationstoidentifymembersoftheEnterobiaceae.
Figure4:AVitekEPSentericpathogenscreencardispictured,afterinteractionwithmediacomponentsinwells(left).ThiscardwillthenbereadintheVitekinstrumentandanidentificationwillbeprintedoutontheattachedprinter(right).
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IntroductionDuringthenextfewlaboratoryperiods,youwillbeperformingvariousmorphological,culturalandphysiologicaltestsonanunknownorganismandwillbecollectingthesedataintheDescriptiveChartfoundattheendofthislabexercise.Thegoalofthisexerciseisnottoidentifytheunknownorganism,buttofamiliarizeyouwiththepositiveandnegativeresults of each of the tests you will perform. This knowledge will be helpful when you begin your SSE andMajorUnknownslaterinthesemester.Itisrecommendedthatyoukeepphotographsofbothpositiveandnegativeresultsinyourlaboratorynotebookforeachofthetestsperformed.BecauseyouhaveperformedeachofthestainingandinoculationtechniquesoutlinedintheDescriptiveChart,therewillbe littletonoexplanationfortheseprocedures. Youmayinsteadrefertotherelevant labsinbothyourLabManualandLaboratoryNotebookforguidance. Inaddition,detaileddescriptionsofeachofthephysiological inoculationstobe performed using your Minor Unknown organism is provided in yourMicrobiology: A Photographic Atlas for theLaboratory inChapter5: BiochemicalTestsforthe IdentificationofBacteria. Pleasenotethatwewillnotperformthedecarboxylasetestsorthedenitrificationtest.In addition to the physiological tests outlined in yourMicrobiology: A Photographic Atlas for the Laboratory, will beperforming fathydrolysis andnitrate reduction assays. Fathydrolysis is achieved when the glycerolmolecule in atriglyceride iscleavedfromtheadjoiningfatty acids. This reaction iscatalyzedbytheexoenzyme lipase. Spirit blueagarcontainstributyrinasasubstrateforlipaseandapHindicatorthatbecomescolorlessasthepHofthemediumisloweredinresponsetothebuild‐upoffattyacidsduetothefat‐hydrolyzingactionoflipase.Itisimportanttonotethatnotall lipasepositivemicroorganismscancleaveenoughtriglyceridesto lowerthepHsufficientlytoproduceacolorchange (Figure5). Youmustconfirmthenegativeresultbyconfirmingtheshinyappearanceof theagar(dueto thepresenceoftriglycerides).Adull,matteappearanceoraclearingofthebluecolorinthemediaisconsideredapositiveresultforfathydrolysis. Nitratereductionassaystesttheabilityofmicroorganismstousenitrate(NO3)asaterminalelectronacceptorduringanaerobicrespiration.Oneofthekeyenzymesinnitraterespirationisnitratereductasewhichtransfers electrons from the electron transport chain carrier cytochrome b to nitrate, reducing it to nitrite (NO2).Nitratebroth,whichcontains0.1%potassiumnitrate,isusedtodeterminewhetherornotabacterialspeciescanreducenitrate. After incubation, sulfanilic acid anddimethyl‐α‐napthylamine are added to themediumwith positive resultsindicatedbyacolorchangetodeepred. Negativeresultsareconfirmedbyaddingzincdust. It is importanttonotethatacolorchangetoredaftertheadditionofzincdustdoesnotindicateapositiveresult;itmerelyindicatesthatthereagentsareworkingproperly(Figure6).
Figure5:Actionofthebacterialexoenzymelipaseontriglycerides,resultinginthecleavageofglycerolfromthefattyacids,whichlowersthepHoftheSpiritblueagarandcausesaclearingofthepHindicatorinthemediumsurroundingtheinoculum(top),thiszoneofhydrolysisindicatesapositiveresult(bottom,left).
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Figure6: Reactioncatalyzedbynitratereductase,reducingnitrate(NO3)tonitrite(NO2)withtheoxidationofcytochromeb(top).InoculationoforganismintoNitrateBrothwiththesubsequentadditionofsulfanilicacidanddimethyl‐α‐napthylamineproducesanegativeandpositiveresult(bottom).
Someofthedatayoucollectwillberedundant.Youwillfindallofthephysicalandphysiologicalcharacteristicsweareinterestedinforthislab,alongwiththeirredundantmediainoculationsinTable3.Youshouldpoolyourdatafromeachofthese inoculationsandrecordtheconsensus inyourDescriptiveChartasonesingleentryunderthecorrespondingheadings. For instance,glucosefermentationcanbedeterminedfromDurhamglucosebroth,Kligler’s ironagarandtheoil‐containingO/Fglucosetube.Table3:AlistofthemicrobialcharacteristicstestedinthisLabExerciseandthemediainoculationswhichareusedtodeterminetheresultsforeachofthecharacteristicsshown.Physical/PhysiologicalCharacteristic MediaInoculation(s)
ColonyCharacteristics NutrientAgarPlate,NutrientAgarSlant&GelatinDeep
SurfaceGrowth NutrientBroth
OptimalTemperature NutrientAgarSlant
OxygenRequirement FTMMedia
GelatinHydrolysis GelatinDeep
GlucoseFermentationtoGas&Acid DurhamGlucoseMedia,Kligler’sIronAgar&O/FGlucoseMedia
LactoseFermentationtoGas&Acid DurhamLactoseMedia&Kligler’sIronAgar
MannitolFermentationtoGas&Acid DurhamMannitolMedia
AerobicGlucoseMetabolism O/FGlucoseMedia
StarchHydrolysis StarchAgar
CaseinHydrolysis SkimMilkAgar
FatHydrolysis SpiritBlueAgar
TryptophanHydrolysis/IndoleProduction TryptoneBroth&SIMMedia
MixedAcidProductionfromGlucose MR‐VPMedia
2‐3,butanediolProductionfromGlucose MR‐VPMedia
CitrateUtilization Simmon’sCitrateAgar
NitrateReduction NitrateBroth
H2SProduction SIMMedia&Kligler’sIronAgar
UreaseProduction UreaBroth
CatalaseProduction Substantialamountofgrowthonsolidmedia(NAplateaftercharacteristicshavebeendetermined)
CytochromeOxidaseProduction TSAplate
PhenylalanineDeaminaseProduction PhenylalanineAgarSlant
LitmusMilkReactions LitmusMilkMedia
Youshouldbefamiliarwithallofthetestsperformedinthislabexerciseintermsofsubstrates,enzymes,productsandreagentsusedfortesting.Asummaryofthemedia,characteristics,enzymes,reagentsandpositive&negativeresultsforalloftheinoculationsperformedinthislabcanbefoundattheendofthelabexerciseinTable5.
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Objectives1. Becomefamiliarwitheachofthemedia,positiveandnegativeresults,andphysiologicalcharacteristicstestedfor
ineachoftheinoculationsperformedinthislabexercise.
ProtocolPerformeachoftheinoculationsoutlinedbelowforyourMinorUnknownOrganism.Somemediawillrequirethatyouadd reagents or manipulate your inoculation in someway after it has incubated. Thesemedia are indicated in thecolumnentitled“Reagents,ifnecessaryforresults”below.LabPeriodOne:MorphologicalTests
IndividualSuppliesbrothcultureofyourunknownorganism
DayOneRefer to your notes and previous lab exercises in order to perform the following inoculations/stainingtechniques:1. Gramstaining2. Capsulestaining3. Sporestaining4. Acid‐faststaining5. MotilitymediainoculationUsethesedatatocompletetheMorphologicalCharacteristicsportionofyourDescriptiveChart.
LabPeriodOne:CulturalCharacteristics
IndividualSupplies Reagents,ifnecessaryforresultsbrothcultureofyourunknownorganism FTMmedia GelatinDeep 4°Cincubator2Nutrientagarslants(orsimilarcomplexmedium) NutrientBroth(orsimilarcomplexmedium) 2agarplates
DayOnePerformthefollowingmediainoculations,usingthetechniquesoutlinedinpreviouslylabexercises:1. Needle inoculation into gelatin deep for gelatin growth & gelatin hydrolysis (this second result will be
recordedintheHydrolysisportionoftheDescriptiveChart)2. Yourinoculationonto2nutrientagarslantsforreserve&workingstockandoptimumtemperatureshould
onlybestreaked,notstabbed.Theseslantsshouldbeplacedseparatelyinthe25°Cand37°Cincubators.3. Inoculationintonutrientbrothtoidentifypossiblesurfacegrowth(ifapplicable).4. Streakeachunknownonaagarplateforisolation.DayTwo1. EvaluatethegrowthoftheseinoculationsaccordingtoFigure7,Figure8andFigure9.2. Identifyyouroptimaltemperatureastheslantwiththemostgrowth.ThisslantwillbeyourWorkingStock.
Ifamplegrowthexistsonthesecondslant,placeitinthe4°Cincubator(refrigerator)asyourReserveStock.IfthegrowthonyourReserveStockislight,placeitbackintotheincubator(atitsoptimaltemperature)for48hours.
3. UsethesedatatocompletetheCulturalCharacteristicsportionofyourDescriptiveChart.4. Onceyourresultshavebeencollected,youmaydiscardofallbutyourworkingandreservestocks.
Figure7:growthonagarslants.RecordthenameofthegrowthwhichmostcloselyresemblesthegrowthofyourorganismintheGrowthinAgarSlantsectionoftheDescriptiveChart.
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Figure8:surfacegrowthinnutrientbroth,surfacegrowthmaynotbeapparent.RecordthenameofthegrowthwhichmostcloselyresemblesthegrowthofyourorganismintheGrowthinNutrientBrothsectionoftheDescriptiveChart.
Figure9:colonyconfiguration,marginationandelevationonnutrientagar.RecordthenameofthegrowthwhichmostcloselyresemblesthegrowthofyourorganismintheNutrientAgarGrowthsectionsoftheDescriptiveChart.For the followingbiochemical/physiological tests, youwill followperformyour inoculations according to thedirectionsbelow.YouwillinoculateeachofthetestsusingyourWorkingStock.WiththeexceptionofthefathydrolysisandnitratereductiontestsexplainedintheBackground,youwillreadthecorrespondingportionsofyour Photographic Atlas for the biochemical pathways and enzymes involved as well as for positive andnegativeresults.Allmedia,unlessotherwisenoted,shouldbeinoculatedaspreviouslydescribedinyourLabManual:
• Platesshouldbestreakedforisolation;• Agarslantsshouldbestreakedandthenstabbedwithaninoculatingloop;• Agardeepsshouldbestabbedwithaninoculatingneedle;• Brothsshouldbeinoculatedwithaninoculatingloop.
Where noted in the supply tables below, our laboratory has commercially prepared kits for your use indeterminingtheresultsofeachofyourinoculations.Ifthisisthecase,pleaserefertotheenclosedinstructionsineachofthekits.Ifnoreagentsarelisted,resultscanbereaddirectlyfromthemedia.
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LabPeriodTwo:CarbohydrateMetabolismIndividualSupplies Reagents,ifnecessaryforresultsworkingstock ½Starchagarplate Gram’siodine(Gramstainingkit)(2)O/Fglucosemedia&mineraloil
DayOne1. Inoculateallmediawithyourworkingstock.
a. CoverthesurfaceofoneofyourO/Fmediawithmineraloil.b. Yourinoculationontothestarchagarneedonlybealineinoculation.
2. Incubateallmediaatyouroptimaltemperature.DayTwo1. AddGram’siodinetothestarchplate.RecordresultsasindicatedinyourPhotographicAtlas.2. ReadresultsandrecordtheminyourDescriptiveChart.3. Discardallmediainoculations.
LabPeriodTwo:FermentationIndividualSupplies Reagents,ifnecessaryforresultsworkingstock Durhamglucosebroth Durhamlactosebroth Durhammannitolbroth MR‐VPmedia(oneforMR&VPtest) emptytesttube;methylredfor½;reagentkitforVP½Simmon’scitrateagarslant ½spiritblueagarplate
DayOne1. Inoculateallmediawithyourworkingstock.
a. BoththemethylredandVoges‐ProskauertestswillbedonefromasingleMR‐VPinoculation.b. Yourinoculationontothespiritblueagarneedonlybealineinoculation.
2. Incubateallmediaatyouroptimaltemperature.DayTwo1. Obtainanemptytesttube,labelit.2. Transfer1.0mlofyourMR‐VPinoculumintothistube.Add5dropsofmethylredandrecordyourresults.3. TotheotherhalfofyourMR‐VPinoculum,addtheVPreagentsaccordingtothepackageinsert.4. ReadresultsandrecordtheminyourDescriptiveChart.5. Discardallmediainoculations.
LabPeriodThree:ProteinCatabolism
IndividualSupplies Reagents,ifnecessaryforresultsworkingstock GelatinDeep 4°Cincubatorureabroth tryptonebroth reagentkitforindoleproduction½skimmilkagarplate phenylalanineagarslant reagentkit
DayOne1. Inoculateallmediawithyourworkingstock.
a. Yourinoculationontotheskimmilkagarneedonlybealineinoculation.2. Incubateallmediaatyouroptimaltemperature.DayTwo1. Placeyourgelatindeepintothe4°Cincubatorforatleast30minutespriortoreadingtheresultsforgelatin
hydrolysis. Additionally,recordtheculturalcharacteristicsofthegelatindeepgrowthaccordingtoFigure10.
2. Addreagents,ifnecessary.ReadresultsandrecordtheminyourDescriptiveChart.3. Discardallmediainoculations.
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Figure10:growthingelatindeep,inorganismsthatarepositiveforgelatinhydrolysis(left)andnegativeforgelatinhydrolysis(right).
LabPeriodThree:RespirationIndividualSupplies Reagents,ifnecessaryforresultsworkingstock ½TSAplate Oxidropsreagentkit,hydrogenperoxide(H2O2)nitratebroth nitratereagentsA,B&Ckit
DayOne1. Inoculateallmediawithyourworkingstock.
a. StreakyourinoculationontotheTSAplateforisolation.2. Incubateallmediaatyouroptimaltemperature.DayTwo1. Inordertotestforcytochromeoxidaseproduction,transferaclumpofyourorganismfromtheTSAplate
ontoasterilecottonswab.AddOxidropsaccordingtopackageinsert.2. Inordertotest forcatalaseproduction, transfer a large clumpof yourorganismfromtheTSAplateto a
cleanmicroscopeslide.Add3–4dropsofH2O2ontotheclump.Vigorousbubblingindicatestheproductionofcatalase.
3. ReadresultsandrecordtheminyourDescriptiveChart.4. Discardallmediainoculations.
LabPeriodThree:Multi‐TestMedia
IndividualSupplies Reagents,ifnecessaryforresultsworkingstock SIMmedia reagentkitforindoletestKligler’sironagarslant Russelldoublesugaragarslant litmusmilk
DayOne1. Inoculateallmediawithyourworkingstock.2. Incubateallmediaatyouroptimaltemperature.DayTwo1. HydrogensulfideproductioncanberecordedfromboththeSIMandKligler’sironagarslant.2. Addreagentsforindoletestaccordingtopackageinsert.3. ReadresultsandrecordtheminyourDescriptiveChart.4. Discardallmediainoculations.
VariousLabPeriods:PositiveControlInoculation
DayOne1. PerformthePositiveControlInoculationlistedin2. Table4accordingtoyourstudentnumber.DayTwo1. RefertotheappropriatesectionintheProtocolinordertocompletetheinoculations.2. PlaceyourcompletedmediatestsontheInstructorBenchatthefrontofthelaboratory.
Table4:Positivecontrolinoculations,listedaccordingtostudentnumber.Pleasesharemediawhenappropriate.Notethattherearenotpositivecontrolinoculationsforallofthemediatestedinthislab.
AtlasBiochemicalTestName DescriptiveChartCharacteristic Medium Organism Stu.No.carbohydrateutilization fermentation(acidand/orgas)bysugar Durhamglucosetube E.coli 1
catalaseproduction catalase anysolidmedia S.aureus 4
citrateutilization citrate Simmonscitrateagar E.aerogenes 9
hydrogensulfideproduction H2Sproduction Kligler'sironagar P.vulgaris 17,22
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AtlasBiochemicalTestName DescriptiveChartCharacteristic Medium Organism Stu.No.indoleproduction(tryptophanhydrolysis)
indole SIM E.coli 20
indoleproduction(tryptophanhydrolysis)
indole tryptonebroth E.coli 14
mixedacidfermentation methylred MR‐VP E.coli 2
motilitytest motility motilitymedium E.coli 21
(cytochrome)oxidaseproduction
oxidase TSAplate P.aeruginosa 5
O/Fglucosetest glucose(oxidation/fermentation) O/Fglucosemedia E.coli 7,23
O/Fglucosetest glucose(oxidation/fermentation) O/Fglucosemedia&oil E.coli 8,24
phenylalaninedeamination phenylalaninedeamination phenylalanineagarslant P.vulgaris 15,19
skimmilkutilization caseinhydrolysis skimmilkplate B.subtilis 11
starchutilization starchhydrolysis starchagarplate B.subtilis 10
Kligler’stest lactose&glucosefermentation Kligler’sironagar E.coli 18
urea ureahydrolysis ureabroth P.vulgaris 16
Voges‐Proskauertest VP MR‐VP E.aerogenes 3
notinatlas,seeIntroduction nitratereduction nitratebroth P.aeruginosa 6
notinatlas,seeIntroduction fathydrolysis spiritblueagarplate S.aureus 12,13
DataCollection&Analysis1. Make sure that you have collected the results for all of your unknown inoculations and recorded them in your
DescriptiveChart.
DiscussionYouwill be required to recognizeand identify themedia, characteristics, enzymes, reagents andpositive&negativeresultsforalloftheinoculationsdoneinthislabexerciseforyourLaboratoryMidterm,useTable5asaresource.Table5:Asummaryofthemedia,characteristics,enzymes,reagentsandpositive&negativeresultsusedinthislabexercise.
mediumbiochemical/
physiologicalcharacteristicenzyme reagent(s) positive&negativeresults
anysolidmediadeterminecatalaseproduction
catalase H2O2 bubblesindicateapositiveresult
anysolidmedia(TSA)
determineiforganismisoxidase‐positive
cytochromeoxidase Oxidrops colorchangetoblackindicatespositiveresult
Durhamglucosetube
glucosefermentationtoacidwithgas
multiple phenolred(inmedium)yellowbrothindicatesacidproduction,bubblestrappedinDurhamtubeindicatesgasproduction
Durhamlactosetube
lactosefermentationtoacidwithgas
multiple phenolred(inmedium)yellowbrothindicatesacidproduction,bubblestrappedinDurhamtubeindicatesgasproduction
Durhammannitoltube
mannitolfermentationtoacidwithgas
multiple phenolred(inmedium)yellowbrothindicatesacidproduction,bubblestrappedinDurhamtubeindicatesgasproduction
fluidthioglycollatemedia(FTM)
determineoxygenrequirements
multiplethioglycollate,cysteine,agar,sodiumresazurin(inmedium)
placementofgrowthindicativeofoxygenrequirement
gelatindeepdeterminewhetherorganismcanhydrolyzegelatin
gelatinase 4°Cincubationliquidmediumwhencoolindicatesgelatinhydrolyzed
Kliglerironagar
multi‐mediumtotestforsugarfermentationandH2Sproduction
cataboliclactoseand/orglucoseenzymes,cysteinedesulfurase
phenolred(inmedium),ironsalts(inmedium)
yellowbuttindicateslactosefermentation,yellowneckindicatesglucosefermentation,blackprecipitateindicatesH2Sproduction
litmusmilkmilkmetabolism:proteins,sugarandfathydrolysis
multiple litmus(inmedium)pink=acid,blue/purple=alkaline,curds=coagulation=curdsform,clear=peptonization,white=litmusreduction
motilitymedium
determinemotilitymultipleandstructuralproteinflagellin
2,3,5‐triphenyl‐tetrazolium,5%agar(inmedium)
burgundycolorshowsgrowth,burgundythroughoutindicatesmotility,burgundyinoculationlineindicatesnomotility
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mediumbiochemical/
physiologicalcharacteristicenzyme reagent(s) positive&negativeresults
MR‐VPmediumdetermineiforganismisamixedacidfermenter
formichydrogenlyaseandotheracidproducingenzymes
methylredifbrothturnred,itispositiveforacidicfermentationproducts
MR‐VPmedium acetoinproduction acetoin(notanenzyme) α‐napthol&40%KOHburgundycolorchangeispositiveforbutanediolproduction&presenceofacetoin
nitratebrothproductionofnitrogenreductase,convertsNO3NO2
nitratereductasesulfanilicacid,N,N‐dimethyl‐I‐napthylamine,zincdust
Ifcultureinbrothturnsred,itispositivefornitratereductiontonitrite.Ifnegative,addapinchofzincdust(willturnredafteraddingzinctoconfirmitisnegative).
O/Fglucosemedia
determineoxidative&fermentativeglucosemetabolism
multiplemineraloilforFtube,pHindicator(inmedium)
yellowcolorindicatesmetabolism,whetherfermentativeoroxidativedependsontube
phenylalaninebroth
productionofphenylalaninedeaminase
phenylalaninedeaminase ferricchloridegreencolorindicatesphenylalaninedeaminaseproduction
rabbitplasma productionofcoagulase coagulase n/acoagulationofmediumwithin0.5–24hoursindicatescoagulaseproduction
Russelldoublesugar
sugarfermentation multiple phenolred(inmedium)yellowbuttindicateslactosefermentation,yellowneckindicatesglucosefermentation
SIMH2SProduction;indoleproduction;motility
cysteinedesulfuraseironsalts(inmedium);Kovac’sreagent
blackprecipitateindicatesH2Sproduction;redcolorchangewithKovac’sreagentindicatesindoleproduction;cloudythroughoutindicatesmotility
Simmoncitrateagar
citrateasasolesourceofcarbon
citrasebromthymolblue(inmedium)
bluecolorchangeindicatespresenceofcitrase
skimmilkagar caseinhydrolysis caseinase N/Aclearzoneinmediumindicatesproductionofcaseinase
spiritblueagar fathydrolysis lipase N/Amatte(depletionofoil)and/orclearingofbluecolorindicatesproductionoflipase
starchagar starchhydrolysis amylase iodineclearzoneinagarafteradditionofiodineindicatespresenceofamylase
tryptonebroth tryptophanhydrolysis tryptophanase Kovacs'reagentredlayerontopofmediumafteradditionofreagentindicateproductionoftryptophanase(indole‐positive)
ureabroth productionofurease urease phenolredcolorchangetohotpink/cerisecolorindicatespresenceofurease
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DESCRIPTIVECHART
StudentName:
StudentNumber:
Unknown:MINORSSEMAJOR
Organism:
MORPHOLOGICALCHARACTERISTICSPHYSIOLOGICALCHARACTERISTICS
(Indicateaspositiveornegativeforeach.)
CellShape: CARBOHYDRATEMETABOLISM(acidand/orgas)
Arrangement: O/Fglucose,aerobic
Size: O/Fglucose,anaerobic
Spores:POSITIVEorNEGATIVE starchhydrolysis
Gramreaction:POSITIVEorNEGATIVE FERMENTATION
Motility:MOTILEorNON‐MOTILE glucose
Capsules:POSITIVEorNEGATIVE mannitol
Acid‐fast:POSITIVEorNEGATIVE lactose
CULTURALCHARACTERISTICS fathydrolysis
PROTEINCATABOLISM
caseinhydrolysis
gelatinhydrolysis
phenylalaninedeamination
ureahydrolysis
hydrogensulfideproduction
RESPIRATION
oxidase
catalase
nitratereduction
IMViC
indole citrate
NutrientAgarGrowth:configuration:margination:elevation:GrowthonAgarSlant:GrowthinNutrientBroth:GrowthinGelatinStab:OxygenRequirement:OptimumTemperature:37°Cor25°C
MR VP
LITMUSMILK(PositiveorNegative&IncubationTime)
REACTIONTIME
REACTIONTIME
Acid Alkaline Coagulation Reduction
Peptonization NoChange