BIOL 2325L - Section 1

Bacillusmegaterium
GraceDauer
July,222014

Purpose/Introduction

Thissemester,studentsinMicrobiologylab(BIOL2525L)learneddifferenttechniquestoidentify
givenorganisms.Thisisaveryimportantaspectofmicrobiology,becauseknowingwhatorganismis
givenwillallowforinformationofwhatdiseasesorbenefitsitmightcause.Anotherimportantaspectof
microbiologyisobtainingapureculture,whichwillbringaboutcorrectidentificationanddrugsensitivity
patterns.Stainingprocedurescanhelpdeterminemorphology,arrangement,size,andcellwall
characteristicsofanorganism.Forexample,aGramStainwilldetermineifthecellwallsareGram
negativeorGrampositive,whichwillhelpruleoutmanyorganisms.Manipulationoftheculturemedium
canalsoassistinobtainingapurecultureandidentificationofanunknownmicrobe.Examplesofdifferent
culturemediaareselectiveanddifferentialmedia,suchasPhenylethylalcohol(PEA),MacConkeyagar,or
Eosinmethyleneblue(EMB),whichwillselectforGramnegativeorpositiveandwilldifferentiate
betweencertainfermenters.Additionaltestsforidentificationcanberun(calleddiagnostictests)to
determineifthemicrobecontainscertainenzymes.AfinaltestontheorganismsDNAinvolving
extractionofDNAviafreezingandthawingcellsandsequencingbasepairscanbedonetobecertainof
whatmicrobeiscontainedinthegivenculturetube.Thepurposeofthislabistousetheseteststoidentify
theunknownmicroorganism,#4,giveninclass.
Materials/Methods:The following procedures (Part A-D) for this experiment are in Biology 2325:
Microbiology Lab Manual, Erin White, 2013, in the procedures sections of experiments: 2, 3, 4, 6 , 7 & 8.
PartAProcedure(StreakingforIsolation)(Exp.2)

ANutrientAgarplatewaslabeledwiththeunknownnumber,studentinitials,andthedate.Next,
theplatewasdividedintoquadrantsandlabeledI,II,III,andIV.Usingproperaseptictechnique(to
preventcontamination)theunknownorganismwasobtainedfromculturebrothandstreakedforisolation
usinganinoculatingloop.Next,theplatewasincubatedupsidedown(topreventcontaminationfrom
condensationonlid)for48hoursat37C.Atthenextclassperiod,theresultswereobservedandrecorded.

PartBProcedure(GramStainandMicroscope)(Exp.3,4)

Acleanglassslidewaslabeledwiththeunknownnumber,studentinitials,andthedate.Oneloop
fullofsuspendedcellswasobtainedfromthebrothandsmearedonacleanglassslide.Next,aGramstain
wasperformedonthesmearandviewedunderthemicroscope.Whenusingthe100xobjective,immersion
oilwasplacedontheslidetodecreasetherefractiveindex(lesslightwaslost).Theentireprocedurewas
repeatedusingtheNutrientAgarplatefrompartAinsteadoftheculturebroth.
PartCProcedure(StarchHydrolysisTest)(Exp.6,8)

APotatoDextroseAgar(PDA)platewaslabeledwithstudentinitials,unknownnumber,date,and
thetestbeingperformed(starchhydrolysis).Usingproperaseptictechnique,asampleoftheunknown
specimenwasobtainedfromtheNutrientAgarplatefrompartAandstreakedonthePDAplate.Theplate
wasincubatedupsidedownfor24hoursat37C.Atthenextclassperiod,thePDAplate(containing
growth)wasfloodedwithiodinetolookforstarchhydrolysis.
PartDProcedure(HemolysinsTest)(Exp.8)

Thebottomofabloodagarplatewaslabeledwithstudentinitials,unknownnumber,date,andthe
procedurebeingperformed(hemolysinstest).TheplatewasdividedintofourquadrantsandlabeledI,II,
III,andIV.Next,usingproperaseptictechnique,asampleofthespecimenfromtheNutrientAgarplate
frompartAwasobtainedandstreakedforisolationonthebloodagarplate.Theplatewasthenincubated
upsidedownfor24hoursat37C.Observationswererecordedatthenextclassperiod.
PartEProcedure(CarbohydrateFermentation)(Exp.7)

Atubeofnutrientbroth(containinglactose,anindicatorcalledphenolred,andaDurhamtube)
waslabeledwithstudentinitials,unknownnumber,thedate,andthesugarbeingtested(lactose).Using
properaseptictechnique,oneloopfullofsuspendedcellswasobtainedfromtheunknowntubeanddipped
intothenutrientbrothtubetoinoculate.Thetubewasincubatedat37Cfor24hours.Observationswere

recordedatthenextclassperiod.
PartFProcedure(DNAExtraction)*

Amicrocentrifugetubewaslabeledwithstudentinitials,unknownnumber,andthedate.Five
hundredmicrolitersoftheunknownculturebrothwaspipettedintothelabeledmicrocentifugetube.Next,
thesamplewascentrifugedfor5minutesatmaximumspeed.Then,theliquidatthetopofthesampletube
(supernatant)wasdiscardedwithoutdislodgingthepelletatthebottomofthetube.Fivehundred
microlitersofdiH2Owaspipettedintothetubewiththepellet.Next,thetubewasvortexeduntilthe
solutionwashomogenized.Then,thetubewasplaceinadryiceethanolbathforthreeminutestofreeze.
Next,thetubewasputinahotwaterbathforthreeminutestothaw.Thissameprocessoffreezingand
thawingwasrepeatedtwice.Thesamplewasagaincentrifugedfor5minutesatmaximumspeed.Finally,
usingamicropipettethesupernatantwastransferredintoanewclean,sterilemicrocentrifugetubeandthe
pelletwasdiscarded.
PartGProcedure(PolymeraseChainReaction)*

APCRtubewaslabeledwithstudentinitials,unknownnumber,andthedate.Twentytwo
microlitersofthePCRbufferand8microliterseachoftheforwardandreverseprimerswerepipettedinto
thePCRtube.Next,10microlitersofthepurifiedDNAfrompartEwasaddedtothereactiontube.Then,2
microlitersofTaqPolymerasewaspipettedintothereactiontubeandvortexedtomix.Thesampleswere
storediniceuntiltheinstructorperformedPCRreactionsinthethermocycler.Theinstructoremailedthe
16SrRNAsequencesthatwererunonBLAST.
*Theinstructor,Ms.Simms,gavetheMaterialsandProceduresfrompartFandpartGtothestudents
throughtheclasspageonMoodle.
Results/Charts
PartA:

Figure1:Diagramofstreakedplate

TableA

Observationsofgrowthonnutrientagar

Whatisthecolorofthecolony?

Offwhite

Doesthecolonyhaveroughorsmoothedges?

smooth

Isthecolonysizesmallorlarge?Shape?

Small,circular

Isthecolonyflatorraised?

Raised(convex)

Isthecolonytransparentoropaque?

Opaque

Otherobservations

Lookswaxy

Inferences:Onlyonequadrantcontainedgrowthofcoloniesandthegrowthwasminimum.This
minimumgrowthmayhavebeenduetoerrorsmadeduringstreakingsuchasnotlettingtheloopcoolafter
sterilizing(whichwouldkilltheculturebeinggrown)orbreakingtheagar.Thegrowthontheplatehelped
determinewhichbacteriaisgivenbyobservingthesecharacteristicslistedabovesuchascolonyshape
(circular)andcolor(offwhite).
PartB:
Figure2:GramStainedDiagram

TableB
Shape

Arrangement

Color

Gram(+/)

bacillus

strepto

purple

Gram+

Inferences:ThepurplecolorofthebacteriashowedthatthecellwallisGrampositivewithathick
peptidoglycanlayer.Becauseofthewaythatthisorganismstained,itwasdifficulttodistinguishbetween
coccusorbacillusmorphology.Thecrystalvioletstainedsplotchesontotherodsmakingthemlooklike
spheres.Itwasdetermined,though,thatthismicrobeisGrampositiveandstreptobacillus.Thisobservation
concludesdthattheGramPosRodsflowchartprovidedbyMs.Simmsshouldbeusedtodetermine
whichtesttoperformnext.
PartC
Figure3:StarchHydrolysisPlateDiagram

TableC
PositiveorNegative

Appearance/Observations

Positive

Clearingoftheblue/blackmediaaroundthegrowth

Inferences:Afterincubation,theplatewasfloodedwithiodine,whichturnedthemediumblue/black
becauseofpresenceofstarchonthePDAplate.Afterafewseconds,theblue/blackcolorturnedclear
whichindicatedthatthebacteriahadhydrolyzedthestarch.Theprocedurewasnotproperlyperformed
becausestreakingforisolationwasnotperformed.Instead,onezigzagstreakwasdoneonthePDAplate.
Ifstreakingforisolationhadbeenperformed,theresultsmayhavebeenclearer.Becausethestarchwas
hydrolyzed,theorganismsontheflowchartthatdonothydrolyzestarchwereruledoutandthehemolysins
testwasperformednext.

PartD
Figure4:HemolysinsResultonBloodAgar

TableD
Results(alpha,beta,gamma)

Appearance/Observations

gamma

Growthwithnochangeinagarappearance

Inferences:Thehemolysinstestwasperformedtwice,becausethefirsttrydidnotresultingrowthonthe
bloodagarplate(thismostlikelyhappenedduetonotcoolingtheloopenoughbeforeobtainingthe
specimen,whichkilledthespecimen).Theplatefromthesecondtryispicturedabove(Figure4).The
bacteriumgrewabundantly,buttherewasnochangeinthebloodagarsurroundingthegrowth.(This
appearanceconcludedthatthismicrobeisgammahemolyticwhichischaracteristicfornodestructionof
theredbloodcellmembranes.Ontheflowchart,theorganismthatisbetahemolyticwaseliminatedandit
wasconcludedthatthenextstepwastotestforlactosefermentation.

PartE

Figure5:LactoseFermentationResult(Diagram)

TableE
Color

yellow

GasProduction

none

Inferences:Thenutrientbrothstartedoutasredandchangedtoyellowafterincubation.Thisinfersthat
thebacteriafermentedthelactosebecausethephenolredindicatorpickeduptheacidbeingformedand
changedtheliquidtoyellow.TherewerenoairbubblesintheDurhamtube,whichmeansthatthis
organismdidnotproducegas.Sincethisorganismispositiveforlactosefermentation,itwasconcluded
thattheorganismpresentwasBacillusmegaterium(basedontheflowchart).
PartF:BLASTResults:Bacillusmegaterium
TableF
SpeciesName

Bacillusmegaterium

AccessionNumber

DQ408589

Authors

Lian,B., Hou,W.G., Wang,J.J., Xiao,Z.J., Hu,Q.N.,

B.megateriumcomesfromtheGreekmegameaninglargeandtheGreekteratismeaningmonsterorbeast.
ItisthebiggestofallBacilliandisthereforeusefulinstudyingcellmorphologyandsporulation.Itsnon
pathogenicpotentialmakesitoneofthepreferredorganismsforindustrialuse.B.megateriumhas made it
a workhorse in food and pharmaceutical production processes for decades (i.e., - and -amylases used for

starch modification in the baking industry and penicillin acylases (Eppinger, 2011). It is also used for the
industrial production of vitamin B12. B. megaterium was also one of the first bacterias genome that has
been fully coded and was used in the studies by Lwoff and Guttman who discovered lysogeny (Glogowki,
2010).
Figure 6: Electron Micrograph of B. Megaterium

Fig.6PhotoCredit:GaryE.Kaiser(1998)
Conclusion:
Throughthedifferentidentificationtests,theunknownbrothculturegiven(#4)isdeterminedtobe
Bacillusmegaterium.TheGramStainrevealedthattheunknownisstreptococcusGrampositive.The
Gramstainfromthebrothwasnotassuccessfulasthestainfromtheplatedgrowth.Thisismostlikely
becausetheplatedgrowthwasmoreabundantthanthebroth.ThegrowthontheNutrientAgar(NA)
revealedthattheunknownscolonymorphologywassomewhatcircular,offwhite,andraised.Performing
anotherstreakplateontheNAwouldhavebeenhelpfulbecausetheinitialplatedidnotcontainmany
colonies.Thevariousidentificationtestsalsoconcludedthattheunknownhydrolyzesstarch,ferments
lactose,andisnonhemolytic.Overall,thisexperimentwassuccessfulindeterminingtheunknown

specimenbuttheminorerrorscouldhavebeenavoidedsuchasnotlettingtheloopcoolbeforeinoculation
andoverstaining.
References:

Eppinger, M. (2011, June). Genome Sequences of the Biotechnologically Important Bacillus megaterium
Strains QM B1551 and DSM319. Retrieved from http://jb.asm.org/content/193/16/4199.full#xrefref-43-1
Glogowski, M. (2010, December). Bacillus Megatarium. Retrieved from
https://microbewiki.kenyon.edu/index.php/Bacillus_megaterium
White, E. Biology 2325: Microbiology Lab Manual. 2013. Print. (in the experimental procedures sections
of the following experiments: 1, 2, 3, 4, 6, 7 & 8)
Nucleotide BLAST (2014). Basic Local Alignment Search Tool. Retrieved from
http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome
Images:
Fig.4
Figure4wastakenbyDawnSimmsinclassonJuly,102014
Fig.6
Kaiser,G.E.(1998).ElectronMicrographofB.Megatarium.[Photograph].Retrievedfrom
http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/u1fig1.html

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