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