Physiologicalunknown#1–Spring2020CellandColonyMorphologyMyphysiologicalunknownwas#25andwasobtainedastwoplateculturesdesignated“A”and“B”.AGramstainandanigrosinindirectstainwereusedtodeterminethecellmorphologyforthetwocultures.Thecellsofculture“A”werepink,soweredeterminedtobeGram-negative.Theirshapewasbacilli,theirarrangementwassinglecells,andtheirsizewas0.7µmindiameterand3-4µminlength.AKOHtestwasrunonthisculture,andcellsmixedwith3%potassiumhydroxideformedslimethatextendedintothreadswhenliftedfromtheslidesurface.Iconcludedmy“A”cultureformedcellswiththinpeptidoglycanwallsplusanoutermembrane.Thecellsofculture“B”werepurple,soweredeterminedtobeGram-positive.Theirshapewascocci,theirarrangementwaschains(streptococci),andtheirsizewas1-1.5µmindiameter.AKOHtestwasrunonthisculture,andcellsmixedwith3%potassiumhydroxidedidnotformslime.Iconcludedmy“B”cultureformedcellswiththickpeptidoglycanwallsandnooutermembrane.Thecolonymorphologyformy“A”cultureonTrypticSoyagar(TSA)wascircular,entire,low-convex,smooth-shiny,semi-translucent,paletan-grayincolorand3-5mmindiameter.MycoloniesonBrainHeartagar(BHA)werecircular,entire,raisedtolow-convex,opaque,whiteand2-3mmindiameter.NewmediausedandbeginningofenzymatictestsIrestreakedmy“A”cultureontoMacConkey’sagar(MAC)andTergitol-7agar(T-7)platesandmy“B”cultureontoagarcontaining5%sheepblood(bloodagar).The“A”culturewasalsorestreakedontoTSAandthe“B”culturewasrestreakedontoBHAplates.After24hoursofincubationmyMACplatecontainedtan-coloredcoloniesthatweresimilarinmorphologytothosegrowingonTSA.ThepHindicator(neutral-red)causedthemediumtoappearmoretanthanpinkbehindthecolonies.OnT-7thecolonieswerepalegray-blue,butthepHindicatorinthemedium(bromothymolblue)hadturnedfromgreentoblue.Theseresultsindicatedthatmy“A”culturecouldnotfermentlactose,becauselactose-positivecoloniesonMACarepink,andonT-7areyellow.Onbloodagarmy“B”cultureformedcoloniessimilartothoseonBHA,butsurroundedbyclearzones(theredcolorofthebloodagarwasgone).Thisindicatedthe“B”cultureisβ-hemolytic(producedhemolysins).

Cellsamplesfromculture“A”wereusedtoinoculateaseriesofmedia(enzymatictestmedia)inordertodeterminemoremetaboliccapabilitiesoftheculture.Themediaused,howtheywork,testresultsandconclusionsaredescribedbelow.Oxidation/Fermentation(O/F)testTheO/Fmediumwasintwotubes,onewithaDurhamtubeandonewithout.ThepHindicatorwasbromothymolblueandthemediumwasinitiallygreen(pHneutral).Inoculationinvolvedstabbingtheculturetothetubebottomswithaloop,andthenthetubewithouttheDurhamtubewassealedwithvaspar(50%vasolinemixedwith50%paraffin).Bothtubeswereincubatedatroomtemperaturefor2weeks(normallywouldbe48hoursat37oC).Bothtubesturnedyellowindicatingpositiveforfermentation,andtherewasagapunderthevasparseal.SincetheculturegrewandfermentedunderthevasparIknewthebacteriawerefacultativeanaerobes(couldgrowwithorwithoutoxygen).Theyellowcolorindicatedacidformation,andthegapindicatedgasformation(fermentationofglucoseyieldedacidandgas).Ifthemediumunderthevasparhadremainedgreentheculturewouldhavebeenrecordedasnegativeforfermentation(positiveforrespiration)andnotcapableoffermentingglucose.

CarbohydratedeepsEightdifferenttubescontainingdifferentcarbohydrateswereinoculatedbystabbingcellstoeachtubebottom.Thecarbohydrateswerelactose,sucrose,raffinose,rhamnose,arabinose,mannitol,sorbitolandinositol.ThepHindicatorinallofthesewasphenolred,anditwasred(neutral)tostart.Ifthecarbohydrateswerefermentedbythecultureadded(acidwasformed)thepHindicatorwouldturnyellow.Iftheculturecouldnotfermentthecarbohydratesthemediawouldstayred.Afterinoculationandtwoweeksofincubationatroomtemperature(normally24hoursat37oC)themediumwasyellowinthemannitol,inositol,sorbitol,arabinoseandrhamnosetubes,butredinthelactose,sucroseandraffinosetubes.Thisindicatedmy“A”culturewascapableoffermentingandformingacidfrommannitol,inositol,sorbitol,arabinoseandrhamnose.Butitcouldnotfermentlactose,sucroseorraffinose.Therewassomegasformationindicatedbybubbles,cracksand/orsplitagarinthetubescontainingmannitol,inositol,sorbitol,arabinoseandrhamnose.Gasformationinthesewasnotcriticaltotheidentification.

Resultsformycarbohydratedeeps

Methylred–Voges-Proskauer(MR-VP)testAtubeofmethylred-Voges-Proskauerbroth(MR-VP)wasinoculatedbyaddingavisibleblobofcellsfromculture“A”tothemedium.Afterincubationforoneweekatroomtemperature(normally48hoursat37oC)theculturewascloudyindicatingtheculturehadgrown.TocompletetheVouges-Proskauertest(VP)onemLofculturewastransferredintoacleanscrew-toptubeusinga1000µLpipetteandsterilebluetip.Then18dropseachofBarrit’sreagentsAandBwereaddedtothetubeandthecontentsweremixedusingavortexmixerforoneminute.Thetubewasallowedtorestfor15minutesandthenthecontentsweremixedagainfor10seconds.ThemixtureintheVPstayedasortofuglybrownishyellowcolorindicatinganegativeresult.Theculturecannotformacetoin(acetylmethylcarbinol)fromglucosefermentation.Apositivetestresultwouldhavebeenpink.

ThecultureremainingintheoriginalMR-VPtubewasusedtoconductthemethylredtest(testingfortheformationoflargequantitiesofacidfromglucosefermentation).Tocompletethistest5-6dropsofmethylredpHindicatorwasaddedtothetubecontents.Redcolorformationindicatesapositiveresult,andthecontentsofmytubeturnedred.Anegativeresultwouldhavebeenyellow.My“A”culturecanformalargequantityofacidthroughthefermentationofglucose.CitrateUtilizationandUreaHydrolysisAtubeofSimmon’scitrateagarwasstreakedandstabbedwithasampleofculture“A”.Theculturewasalsousedtostreakuptheslantofatubeofureaagar.Bothtubeswereincubatedatroomtemperatureforoneweek(normally48hoursat37oC).ThecitrateslantwasgreenincolorduetothepHindicatorbromothymolblue,andtheureaslantwaspalepeach-coloredduetothepHindicatorphenolred.Bothmediawereclosetoneutral.Iftheculturecouldmakecitratepermease(anenzyme)andtakeincitrate,thenammoniawouldbeformedinthetubeandthepHindicatorwouldturnblue(alkaline).Iftheculturecouldhydrolyzeureaandformammonia(usingtheenzymeurease),thepHindicatorwouldturnhotpink(alkaline).

Themediuminmycitrateslantturnedblueindicatingapositiveresultforcitrateutilizationandmyureaslantstayedpalepeach-coloredindicatinganegativeresultforureahydrolysis.Culture“A”wasabletoformbothenzymescitratepermeaseandurease.Hydrogensulfide(H2S)productioninTSIAlargeslantcontainingtriplesugarironmedium(TSI)wasstreakedandstabbedwithasampleofcellsfromculture“A”.Themediumwasincubatedatroomtemperatureforoneweek(normallyfor24hoursat37oC).ThepHindicatorpresentwasphenolredandthemediumwasinitiallyred-colored(itwasalittlebrownish-red).ThepHwasneutral.Iftheculturecouldfermentlactoseandformacid,theslantwouldturnyellow.Ifnot,theslantwouldstayred.Iftheculturecouldfermentglucoseandsucroseandformacid,thetubebuttwouldturnyellow.Ifnotthebuttwouldstayred.Ifhydrogensulfidewasformed(duetocatabolismofsulfur-containingaminoacids),therewouldbeablackprecipitate(ironsulfide)formedinthebottomofthetube.IfnoH2Swasformed,therewouldbenoblackcolorpresent.ThecolorofthemediumintheTSI(redvs.yellow)waslessaccuratethantheindividualcarbohydratedeepsbecausetheresultsforglucoseandsucrosecouldnotbeseparated,lactoseutilizationofteninvolvesinducibleenzymes,andifacetoin(aneutralproduct)wasformedfromglucosefermentation,lessacidwouldbemade.

TheTSImediumwasblackthroughoutthebottomofthetube,andonlytheslantremainedred.Thisindicatedmy“A”culturewaspositiveforH2Sformation,andnegativeforlactosefermentation.Itcouldnotfermentlactoseandformacid,butitcouldcatabolizesulfur-containingamino-acidmoleculesandformhydrogensulfide.Theresultsforglucoseandsucrosefermentationwerenotvisibleduetotheironsulfide(blackprecipitate).ResultsobtainedforTSIandSIMmedia

SulfurIndoleMotilityMedium(SIM)My“A”culturewasstabbedtothebottomofalargetubecontainingSulfurIndoleMotility(SIM)mediumandincubatedforoneweekatroomtemperature(normally24hoursat37oC).Thismediumallowsculturesabletocatabolizetheaminoacidtryptophantoformindole,italsoallowsthemtoformhydrogensulfideandtoshowifornottheyaremotile(canswim).IndoleistestedforbyaddingKovac’sreagenttothemediumsurface;ifitturnsredtheculturecanformindole,andifitstaysyellow,theculturecannotformindole.Motilityisdeterminedbytheappearanceofthestabline.Ifitisclearlyvisible,thecultureisnotmotile,butifitisdifficultorimpossibletosee,thecultureismotile.Hydrogensulfideformation(throughthecatabolismofsulfur-containingaminoacids)isindicatedbytheblackprecipitate(ironsulfide)atthetubebottom.MySIMmedium(likemyTSImedium)wasmostlyblacktowardthebottom,sotheculturewasH2S-positive.Thestabtimewasnotvisiblebecausetheblackwasallovertheplaceandaboveitthemediumwascloudy.Thismeanttheculturewasmotile.TheKovac’sreagentdidnotchangecolorwhenaddedtothemediumsotheindoletestwasnegative.Culture“A”formedH2S,wasmotilebutcouldnotcatabolizetryptophanandformindole.AminoaciddecarboxylationandamineformationTwotubeswereusedfortheaminoaciddecarboxylationtest.Onecontainedglucose,lysine(theaminoacid)andbromocresolpurple(thepHindicator).Theothertube(thecontrol)containedonlyglucoseandthepHindicator(itdidnotcontaintheaminoacidlysine).Bothtubesappearedpurpletostartwith.Afterinoculationbyaddingavisibleblobofcellstoeachtube,bothwere

sealedwithvaspar.Vasparkeepsoxygenout,somakesthemediumanaerobic,itkeepsanycarbondioxideformedinside,andkeepsanyvolatileaminesformedfromlysineinthetube.Thelysinetubeandtheaminoacidcontroltubewereincubatedatroomtemperatureforoneweek(normally48hoursat37oC).Iftheglucoseineithertubewasfermentedandacidwasformed,thepHindicatorwouldturnyellow.Iflysinewasdecarboxylated,andtheaminecadaverinewasformed,thelysinemediumwouldappearpurple.Thisisbecauseaminesarealkaline(theyarealsovolatileandhaveanastyodor).Iflysinewasnotdecarboxylated,themediuminthelysinetubewouldturnyellowduetotheacidfromglucosefermentation.

Thecontroltubehadtobeyellowtovalidatethistest,becauseifbothtubescontainedpurplemedium,afterincubation,theorganismsusedmighthavebeendead,orwereobligateaerobes.Themediumwaspurpletobeginwith.

EsculinHydrolysisTestEsculin(alsospelledaesculin)isaglycoside(acompoundthatcontainsglucose).Inthemediumweusedtheesculinwascombinedwithferriccitrate.Themediumwasasortofgray-tantobeginthetest,andiforganismscouldhydrolyzetheesculin(breakitapart)theywouldformglucoseandesculitin(aesculitin).Thiswouldcombinewithferricsaltstoformablackprecipitateinthemediumanditwouldturndarkbrown(nearlyblack).Onetubeofesculinagarwasinoculatedbystreakingthecultureuptheslantina“fishtail”pattern.

My“A”cultureturnedthemediumintheaminoacidcontroltubeyellow,socouldfermentglucoseandformacid.Therewasalsogasunderthevasparseal.Myculturedidnotturnthelysinemediumyellowbecauseitwasstillpurple.Thismeantmyculturecoulddecarboxylatelysineandformtheaminecadaverine.Thegasunderthevasparsealwouldhavebeenmostlycarbondioxide.Myconclusionwasthatmy“A”culturecoulddecarboxylatetheaminoacidlysine.Thetestwasvalidbecausethecontroltubeturnedyellow.

My“A”cultureformedGram-negativebacilliarrangedassinglecells.Itwasnegativefortheoxidasetestsocouldnotformcytochromecoxidase.Theculturewasfermentative(notrespiratory),wasnegativeforindoleformation,acetoinformation,ureahydrolysis,esculinhydrolysis,andthefermentationoflactose,sucroseandraffinose.Itwaspositiveforcitrateutilization,hydrogensulfideformation(inbothTSIandSIMmedia),andlysinedecarboxylation.Itwasabletofermentglucoseandformlotsofacid,italsofermentedmannitol,inositol,sorbitol,arabinoseandrhamnoseformingbothacidandgas.WhenIcomparedmyresultstotheBacterialIdentificationchartprovidedinclass,IdeterminedthattheidentityofmyPUNK1-AwasSalmonellacholeraesuis.ActuallythenameforthisculturehasbeenchangedanditisSalmonellaenterica,withthenameCholeraesuisasaserotype.Testsrunonmyculture“B”organismsCatalaseandOxidaseTestsTwotestsrunonmy“B”typeculturewerethecatalasetestandtheoxidasetest.Thecatalasetestinvolvedputtingavisibleblobofcellmaterialonaglassslideandaddingadropof3%hydrogenperoxide(H2O2).CatalaseenzymeswouldconverttheH2O2intowaterandfreeoxygengas.Ifbubblesformed,thetestresultwouldbepositiveandifnobubblesformed,thetestresultwouldbenegative.Toconductanoxidasetest,Iusedacleantoothpicktopickupavisibleblobofcellsfrommy“B”cultureandrubthemintofilterpapersaturatedwiththeoxidasetestreagent.Ifthecellscouldformcytochromecoxidase,apurplespotwouldappearandiftheycouldn’t,therewouldbenocolorchange.

Theesculinmediumwasincubatedforoneweekatroomtemperature(normally24hoursat37oC).Afterincubationmyesculintubelookedjustlikeitdidtobeginwith;itwasasortofgray-tancolor.Thiswasanegativetestresult.Therewasnoblackcolorformed,somy“A”culturewasnotabletocarryoutesculinhydrolysis.TheesculinhydrolysistestwasthelasttestIwasrequiredtoperformwithmyPUNK1“A”culture.Iwasnotrequiredtodoanoxidasetestwiththisculture,butIdidandtherewasnocolorchangewhenmy“A”culturewasrubbedonthefilterpaper.

My“B”culturedidnotformbubblesinthecatalasetest,sowascatalase-negative.Whenrubbedintothefilterpaperfortheoxidasetest,my“B”culturedidnotformapurplespotsowasoxidase-negative.My“B”culturecouldnotformcatalaseenzymesanditcouldnotformcytochromecoxidaseenzymes.EsculinHydrolysisMy“B”culturewasstreakedontoaslantofesculinagar(asdescribedabove).Theculturedidn’tgrowwellonthismediumandtherewasnotcolorchangeintheagar.My“B”cultureappearedtobenegativeforesculinhydrolysis.HemolysisReactiononBloodAgarAsstatedearlier,my“B”cultureshowedbeta-hemolysisonsheepbloodagar(shownabove)becausethebloodagarbecameclearbehindthecolonies(Icouldseerightthroughtheagar).Thismeantmy“B”culturecouldcausecompletehemolysisoftheredbloodcells(RBCs)present.My“B”culturewasGram-positivecocciarrangedinchains(streptococci)andbothcatalaseandoxidase-negative.Theculturecouldnotcausethehydrolysisofesculin,butwasabletocausethecompletehemolysisofRBCs.Bylookingatthepresentation“PUNK#1Tests2”IwasabletoidentifymyGram-positive“B”typecultureasStreptococcuspyogenes.

Physiological Unknown #1 Identification Chart

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Indole - - - + - + - + - - d + + - -Methyl Red + - - + + d + + + d + + + + +Voges Proskauer - + + - - + - - - + - - - + -Citrate + + + - + + d - + + + - + + +H2S (TSI) + - - - - - + + + - - - - - +Urease + - - - + + + + - - - + + - -Lysine Decarboxylase - + - + + + - - + + + - - - +Esculin Hydrolysis - + + - + + - + - + d - - - -Motility (SIM) + + + + - - + + + + + - + - +Glucose (acid) + + + + + + + + + + + + + + +Glucose (gas) + + + + + + + + + + - - - + +Lactose (acid) + + + + + + - - - - + - - + -Sucrose (acid) + + + - + + - + - + d - d - -Mannitol (acid) + + + + + + - - + + + - - + +Inositol (acid) - + - - + + - - + + + - d - +Sorbitol (acid) + + + + + + - - + + + - - + +Arabinose (acid) + + + + + + - - + - + - - + +Raffinose (acid) - + + - + + - - - - d - - - -Rhamnose (acid) + + + + + + - - + - + - - + +

"d" = variable results