The University of Akron
IdeaExchange@UAkron
Honors Research Projects
The Dr. Gary B. and Pamela S. Williams Honors
College
Spring 2017
Measuring Bacterial Growth Using a 3D-Printable
Spectrometer
Samuel R. Bunting
The University of Akron, [email protected]
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Recommended Citation
Bunting, Samuel R., "Measuring Bacterial Growth Using a 3D-Printable Spectrometer" (2017). Honors Research
Projects. 433.
http://ideaexchange.uakron.edu/honors_research_projects/433
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Bunting1
MeasuringBacterialGrowthUsinga3D-PrintableSpectrometer
1.Introduction
Spectroscopyisabroadlyapplicablelaboratorytechniqueandanessentialpartof
qualityeducationinthebiologicalandphysicalsciencesatearlyeducationlevels.However,
traditionallyhighcostsandsophisticationhaverelegatedthenecessaryequipmenttoindustry
andhighereducation.Commercialspectrometersare‘blackboxes,’withtheirinnerworkings
andphysicshiddenfromthestudent.
Wehavepreviouslyreportedonaninexpensive,
3D-printablespectrometer,calledtheSpecPhone(Grasse,
Torcasio&Smith,2016).ThisapparatususesaniPhoneas
thecamera,alongwithseveralotherinexpensiveadditions
tomakeafullyfunctioningspectrometerforafractionof
thecostofacommercialinstrument.Theoverall
Figure1.TheSpecPhoneapparatusispictured.The
housingis3D-printed,andadditionalcomponents
areaddedintocompletetheapparatus(Grasse,
Torcasio&Smith,2016).
purposeoftheSpecPhoneprojectistoprovidelaboratoryexperienceswithspectroscopyin
low-resourceeducationalenvironments.Thesimplicityofthedesign,andwidespread
availabilityofthecomponentsmaketheSpecPhoneparticularlyaptforthisgoal.
PreviousprotocolshaveonlyappliedtheSpecPhonetocolorimetricassays(Smith,
2015),andsimpleproteinquantificationexperiments(Smith,2017).Inpractice,spectroscopy
extendsfarbeyondthesetwoapplications.Here,twoprotocolsusingtheSpecPhoneto
measurebacterialgrowtharediscussed.Bothprotocolsprovideinstructionsforthecreationof
abacterialgrowthcurve.Oneoutlinesaconventionalprocedure:inoculatingaflaskofblank
mediawithastockEscherichiacoliculture,andtakinggrowthmeasurementsatregular
Bunting2
intervals.Theseconddescribesasmaller-scaleversionofthisconcept,withtheentireculture
beinggrownina4.5-mLcuvette.Designingandtestingtheseprotocolsbroadensthe
applicationsoftheSpecPhonetoanewrealm:measurementoflivingsystems,thusextending
itseducationalpotential.
Bacterialgrowthcurvesareawidely-usedlaboratorymeasurement.Theirbasic
underlyingprincipledependsontherelationshipbetweenconcentrationandtheamountof
lightabsorbedbyasample,asrepresentedbytheBeer-LambertLaw:A=εℓC.Inthisequation,A
representsthelightabsorbedbythesample,ℓisthepathlength(1cm.intheseexperiments),
andCisthesampleconcentration.Themolarabsorptivity(ε)isaconstantthatdependsonthe
solution.Followinginoculationofsterilemedia,theincreaseinbacterialcellswillproportionally
increasethedegreeoflightscattercausedbytheturbidityoftheculture.Regularabsorbance
measurementsareusedtomonitorthegrowthofbacteriaintheliquidculture.These
absorbancemeasurementscanbecorrelatedwithdiluted,bacterialplatesmadeatthesame
timepoints.Fromthiscorrelation,therelationshipbetweencultureabsorbance(turbidity)and
cellnumber,determinedbythenumberofcoloniesgrowingontheplates,canbeestablished.
Thislinearrelationshiprepresentsthestandardcurve,anditcanbeusedtodeterminethecell
numberinthatculture,aswellasinunknownculturesofthesameorganism.
Bacterialgrowthcurvesarebroadlyapplicableineducation,researchandindustry(Hall,
Acar&Nadipati,2014).Growthofbacteriaundervaryingconditionscanbemonitoredand
comparedusinggrowthcurves,aprocedurewhichisusedextensivelyindevelopmentoffood
safetyguidelines(Zwietering,et.al.,1990).Effectsofenvironmentalfactors(Hall,Acar&
Nadipati,2014),andantibiotics(Yourassowsky,etal.,1985)arealsomeasurableusinggrowth
Bunting3
curves.Growthratesofvaryingspecies,includingdifferentphenotypesofthesamespeciescan
becomparedusingthiskindofmeasurementaswell.
2.Materials&Methods
2.1GrowthofLiquidBacterialCultures
Tobegin,astockculturecontainingastandardlaboratorystrain(K12)ofE.coliwith
Kanamycinresistancewasgrown.Steriletrypticasesoybroth(TSB)containingKanamycinwas
inoculatedwithasingleE.colicolony.Theculturewasincubatedat37°Cfor24hoursina
shakingincubator(VWR,RadnorPA)at265rpm.ThesameprocedureforcreatingthestockE.
coliculturewasfollowedforalltrials,regardlessofthevesselusedforgrowth.
Fortheflaskcultures,a125-mLErlenmeyerflask,containing95mLofsterileTSBwas
inoculatedwith5mLofthestockE.coliculture.Thisflaskwasplacedintheshakingincubator
atthesamespecificationsasbefore.Forthecuvettegrowthprotocols,twoplastic,4.5-mL
cuvetteswerefilledwith3mLofsterileTSBcontainingKanamycin.Bothwereinoculatedwith
100μLofthestockE.coliculture.Onecuvettewassecuredintheshakingincubatoratthe
samesettingsastheprevioustrial(mechanically-shakencuvette).Thesecondcuvettewas
placedinastationaryincubatorat37°C.Thiscuvettewasremovedfromtheincubatorevery5
minutes,covered,vigorouslyshakenbyhand,andthenreturnedtotheincubator(hand-shaken
cuvette).Theflaskculturetrialswerereplicatedfourtimes,andtheeachofthecuvettecultures
wasreplicatedthreetimes.
2.2AssemblingtheSpecPhoneApparatus
BeforemeasurementsweretakenwiththeSpecPhone,thecomponentsofthedevice
wereassembled.Asmentioned,thehousingoftheSpecPhoneisa3D-printablefiledesigned
Bunting4
usingSolidWorks(DassaultSystèmes
SOLIDWORKSCorp.,WalthamMA).
Afterprinting,theremaining
componentsareadded.A1in.2
aluminummirrorisinsertedtoreflect
lightcominginthroughtheopeningin
therearofthehousing.Lightpasses
Figure2.AschematicofthecompletedSpecPhoneapparatus,including
the3D-printedhousing,lightsource,slitinsert,mirroranddiffraction
grating(Grasse,Torcasio&Smith,2016).
througharemovable,3D-printedslitinsert,withaslitwidthof1mm.Thefinalcomponentisa
smallpieceofdiffractiongrating,containing1,000lines/mm.Lightpassesthroughtheslitand
thenthroughthesamplesolutioninthecuvette,whichisinsertedinthehousingofthe
SpecPhone.Thislightisreflectedoffthemirror,andthenpassedthroughthediffractiongrating
beforereachingtheiPhonecamera,whereanimageofthespectrumiscaptured.Aschematic
ofthefully-assembledSpecPhoneapparatusisshownaboveinFigure2.
2.3RecordingAbsorbanceMeasurementswiththeGeneSys20andtheSpecPhone
AbsorbancemeasurementsweretakenwithaGeneSys20VisibleSpectrophotometer
(ThermoFisherScientific,WalthamMA),andcomparedtoresultsobtainedwiththeSpecPhone
outfittedwithaniPhone5.Beforeanyabsorbancemeasurementsweretaken,acuvette
containingsterileTSBwasusedtoblanktheGeneSys20.ToblanktheSpecPhone,threeimages
ofthespectrumfortheblankTSBcuvettewerecapturedv.
Thewavelengthwassetat600nmfortheGeneSys20forallmeasurements,andthe
samewavelengthwaswhenanalyzingtheSpecPhoneresultsaswell.FortheSpecPhone,the
lightsourcewasadesklampwitha40Wlightbulb.Fortheflaskcultures,absorbance
Bunting5
measurementsweretakenevery20minuteswithbothdevicesoveraperiodof4hours,
followingtheinitial(T0)measurement.A3-mLaliquotoftheculturewastransferredtothe
samplecuvetteformeasurements.Betweenmeasurements,thecultureflaskwaskeptinthe
shakingincubatorunderthepreviouslyspecifiedconditions.Spectrumimagesweretakenin
triplicatewiththeSpecPhoneateachtimepoint.
Inthecuvetteculturegrowthprotocols,measurementsweretakenwiththesametwo
devices,howevertherewasnotransferofasampleoftheculturefromalargervessel.Cultures
weregrownoveraperiodof2.5hours,withabsorbancemeasurementstakenevery10minutes
forboththemechanically-shakenandhand-shakencuvettes,removingthemfromtheir
respectiveincubators.Imagesofeachbacterialculturespectrumwerecapturedintriplicate
usingtheSpecPhone.
2.4PlatingfromBacterialCulture
Bacterialplateswerealsocreatedfromthreeofthefourflaskgrowthcultures.The
initialflaskculturetrialwasonlyperformedtodeterminethefeasibilityofmeasuringculture
turbidity,andplateswerenotconstructed.Fortheremainingflaskcultures,dilutiontubesand
platesweremadebeginningatT20.Samplesoftheflaskculturewereplatedat40-minute
intervals.Thefirstthreetimepoints(T20,T60,T100)weredilutedbyfactorsof10-2,10-3,and10-4.
Thelatterthreetimepoints(T140,T180,T240)werediluteduptoafactorof10-5.
Afterabsorbancemeasurementshadbeenrecorded,a100-μLaliquotoftheculture
samplewastransferredfromthesamplecuvettetothefirstdilutiontube,whichcontained9.9
mLofsterileTSB.Fromthisinitialtube,threesubsequenttransfersof1mLofculturewere
madetoperformserialdilutions.Eachsubsequentdilutiontubecontained9.0mLofsterileTSB.
Bunting6
Thesetubesofblankmediawerecreatedbeforetheexperimentwasbegun.Aftereach
transfer,thetubeswereadequatelyshakentoensurepropersuspensionofthebacteria.
Fromeachdilutiontube,a100-μLaliquotwasplated.Twoplateswerecreatedfrom
eachdilutiontube.Theplateswereincubatedat37°Cfor24hours.Aschematicofthisdilution
processisbelowinFigure3.
Cuvette
100μL
1mL
9.9
mL
TSB
1
10-2
Plate
100μL
1mL
9.0
mL
TSB
2
10-3
Plate
100μL
1mL
9.0
mL
TSB
3
10-4
Plate
100μL
9.0
mL
TSB
4
10-5
Plate
100μL
Figure3.Thisdilution
schemewasfollowed
foreachtimepointat
whichplateswere
created.Thecuvette
representsthesample
takenfromthelarger
culturefor
measurementatthat
period.Thefourpurple
tubeswerefilledwith
sterile,warmTSB
beforetheexperiment
wasbegunandwere
thoroughlymixed
betweentransfers.
Twoplateswere
createdforeach
dilutionfactor,and
wereincubatedfor24
hoursat37°C.
Onlythoseplateswithcolonycountsbetween30-300werecounted,asthisisthe
establishedrangeforstatisticalrobustness(Sutton,2011).Confluentplateswerenotcounted,
andtheircolonycountdatawasnotincludedinanyanalyses.Thecolonycountsfromeachtime
pointwerethencorrelatedwiththeabsorbancemeasurementsfromeachdeviceatthesame
timepointtocreateastandardcurve.
Bunting7
2.5ImageandDataAnalysis
SpectralimagescapturedbytheSpecPhonewereanalyzedusingImageJ,thefreeimage
analysissoftwareavailablefromtheU.S.NationalInstitutesofHealth(Rasband,2016).A
protocoldetailingthestepsforimageanalysiswithImageJhasbeenpreviouslypublished
(Smith,2015).Thesestepswerefollowedforanalysisofallspectrumimagescollectedfromthe
SpecPhone.AllsubsequentdataanalysisandrepresentationweredoneusingMicrosoftExcel
andMiniTabstatisticalsoftware(MinitabInc.,StateCollegePA).
Tocorrelatetheabsorbanceandtimedata,timewasplottedontheX-axis,and
absorbanceontheY-axis,andtheequationofalineofbestfitwascalculated.Thiswas
adequatefordescriptionofthegrowthcurvedatafromboththeflaskandcuvetteculture
protocols.Asmentioned,multiplerepetitionswereperformed,andtheabsorbancevaluesfor
eachtimepointwereaveragedtogethertoyieldthevaluesusedtoconstructthegrowthcurves
forboththecuvetteandflaskcultures,plottedwiththestandarderrorofthemean(SEM).
Forthestandardcurvedata,whichinvolvedabsorbanceandcolonyformingunitsper
milliliter(CFU/mL)datapoints,moreadvancedanalysiswasdone.Eachtimepointthatincluded
platingproducedtwoplatesforeachdilutionfactor,andassuchthenumberofcoloniesfrom
eachwereaveragedtogether.Usingtheaveragenumberofcolonies,thedilutionfactor,and
theamountplated,thenumberofCFU/mLwerecalculatedusingtheformula:
!"#
!"
=
!"#$%&# !"#"$% !"#$%&
!" !"#$%& × !"#$%"&' !"#$%&
.CorrelatingtheaveragenumberofCFU/mLforeachtime
pointwiththeaverageabsorbanceatthesametimepointsplottedthecreatedcurve.The
significanceofthecorrelationwasdetermined,bycalculatinga95%confidenceintervaland
predictionintervalforthedatafromboththeGeneSys20andSpecPhone.Finally,thestatistical
Bunting8
differencebetweentheequationsofthelinesofbestfitfortheGeneSys20andtheSpecPhone
datawascalculated.
TheGeneSys20reportedpercenttransmissionvalues(%T),whichwereconvertedto
absorbancevaluesusingtheformulaA=-log(%T).AsamplespectrumofE.coliisshownin
Figure4B,comparedtoahighlyconcentratedsampleofBSAinFigure4A,withanoverall
comparisonofpixelintensityinFigure4C.Thiscomparisonshowsthedifferenceinspectrum
appearancewithaturbidculture,comparedtomeasurementofasimple,coloredsolution.
A BSA(1mg/mL)
B
E.coliatT240
Intensity(GrayValue)
C
70
BSA
60
E.coli@T240
50
40
30
20
10
0
0
100 200 300 400 500 600 700 800 900 1000
PixelPosiKon
Figure4.Image4Arepresentsa
spectrumofBovineSerumAlbumin
(BSA)ataconcentrationof1mg/mL
withtheSpecPhoneusingtheiPhone5
camera.Thehorizontaledgesareclearly
definedandtherearesomewhatsharp,
verticaldivisionsbetweentheindividual
colorsofthespectrum.Image4Bisa
spectralimageofthebacterialculture
attheT240timepoint.Thebordersare
lessdefinedandtheoverallcolor
intensityisreduced.TheplotinImage
4Cshowstheoverallpixelintensityfor
theentirespectrumforbothimages.
Theintroductionofaturbidculture
causedmorelightscatter,andthis
causeslesslighttoreachthecamerafor
measurement.However,inthecolored
solution(BSA),thereisareductionin
theamountoflightrecordeddueto
morelightbeingabsorbedbythe
sample,ratherthanbeingscattered.
Bunting9
Fullprotocolsareattachedtothisreport,withtheFlaskCultureProtocolinAppendix2
andtheCuvetteCultureProtocolinAppendix3.Bothprotocolsincludedetailedlaboratory
procedures,aswellasdataanalysisinstructions,andsafetyguidelines.Theseappearinthe
sameformataswillbedistributedwiththeSpecPhoneforeducationalpurposes.
3.Results
Twoprotocolscenteredonthesamebasicprinciple,usingspectroscopytomeasure
bacterialgrowth,weredesignedandtestedinthisproject.Multipletrialsofeachexperimental
designwereconducted,andthedatawascombined.Thefirstflaskculturegrowthtrialwas
conductedonlytodetermineiftheSpecPhonewascapableofmeasuringcultureturbidity(data
notshown).Basedonsatisfactoryresultsfromthisinitialexperiment,theremainingtrialswere
conducted.Threeadditionalflaskculturesweregrownandmeasured,andaveragedwiththe
datafromthefirst.Platecountswerealsoaveragedtocreatethestandardcurve.Similarly,
threehand-shakencuvetteculturesandthreemechanically-shakencuvettecultureswere
grown,andtheresultsaveraged.
3.1FlaskCultureGrowthCurves
AbsorbancevaluesfromallfourflaskgrowthculturesrecordedbytheGeneSys20and
theSpecPhonewereaveragedandplottedwithrelationtotime.Anexponentialtrendlinewas
fitted,whichshowsapositivecorrelationbetweenincreasingabsorbanceandturbidity.The
GeneSys20andSpecPhoneabsorbancevalueswereverysimilarattheearlytimepoints.
However,asturbidityincreasedtheGeneSys20valuesbegantotrendhigherthanthose
recordedbytheSpecPhone,beginningaroundT120.Thereisstrongcorrelationbetweenthe
datapointsforbothdevices,withR2valuesof0.979and0.953fortheGeneSys20andthe
Bunting10
SpecPhone,respectively.TheGeneSys20yieldedanequationofy=0.0378e0.0119x,andthe
SpecPhoneyieldedtheequationy=0.0385e0.0104x.Bothequationshavesimilarcoefficients,
whichisanimportantcharacteristicforverifyingtheaccuracyoftheSpecPhoneascomparedto
acommercialdevice.TheAbsorbance±SEMvaluesarepresentedinFigure5,alongwiththe
linesofbestfitandthecorrelationvalues.
Absorbance(600nm)
0.7
0.6
y=0.0378e0.0119x
R²=0.979
0.5
0.4
0.3
0.2
y=0.0385e0.0104x
R²=0.953
0.1
0
0
20
GeneSys20
40
60
80
SpecPhone
100 120 140 160 180 200 220 240
Time(min)
Expon.(GeneSys20)
Expon.(SpecPhone)
Figure5.Thecombinedresultsfromtheflaskgrowthcultures.Thereisastrongtrendupwardcorrespondingtoincreased
cultureturbidity,asrepresentedbythelinesofbestfitandtheirequations.ValuesarepresentasAbsorbance±SEM.
3.2CuvetteCultureGrowthCurves
Followingtheinitialflaskculture,whichdemonstratedthattheSpecPhonewascapable
ofmeasuringturbidity,thesmaller-scalecuvettecultureprotocolsweretested.Theresults
fromthemechanically-shakencuvetteculturesarepresentedfirst.Overall,thereissimilarity
betweenthevaluesrecordedbytheSpecPhoneandtheGeneSys20,withminordisparities,
particularlybetweenT30-T80,wheretheGeneSys20recordedslightlyhighervalues,and
betweenT120-T150wheretheSpecPhonerecordedslightlyhighervalues.Generally,thereisa
muchmorepronouncedmarginoferrorinthesevalues,asrepresentedbythemoredramatic
Bunting11
errorbars.Whenlookingattheshapeofthecurves,theSpecPhonedatafollowsasmoother
upwardtrajectory,eventhoughthecorrelationisslightlylesspronouncedthanthatofthe
GeneSys20,withR2valuesof0.886and0.897,respectively.Thegrowthcurvefromthe
mechanically-shakencuvettecultureisbelowasFigure6.
Absorbance(600nm)
0.3
0.25
y=0.0263e0.0137x
R²=0.886
0.2
0.15
0.1
y=0.0396e0.0099x
R²=0.897
0.05
0
0
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time(min)
GeneSys20
SpecPhone
Expon.(GeneSys20)
Expon.(SpecPhone)
Figure6.Growthcurvefromthemechanically-shakencuvettecultures.ValuesarepresentasAbsorbance±SEM.
Thenexttrialswerethehand-shakencuvettecultures,whichwereremovedfromthe
incubatorandvigorouslyshakenbyhandat5-minuteintervals.Betweenshaking,these
cuvetteswerekeptat37°C.However,theabsorbancevaluesfromthisculturearemarkedly
differentfromthoseofthemechanically-shakenculture.
Thefinal,averageabsorbancevaluesatT150forthemechanically-shakencultureswere
0.163±0.0456and0.214±0.0252fortheGenesys20andSpecPhone,respectively.Atthatsame
timepointinthehand-shakencultures,theabsorbancevalueswere0.0576±0.0214and
0.0626±0.00434fortheGenesys20andSpecPhone,respectively,representingasubstantial
Bunting12
reductioninthetotalabsorbanceofthehand-shakencultures.Thisdataisalsomuchnoisier
thanthatofthemechanically-shakencuvettes,particularlyforthedatafromtheGeneSys20,
whichhasanR2valueof0.727.TheSpecPhonedatahasastrongercorrelation,withanR2of
0.902,howevertheupwardtrendinabsorbanceasafunctionoftimeismuchlessrobustfor
bothdevices.Thegrowthcurvefromthehand-shakencuvettecultureisbelowasFigure7.
0.09
y=0.0312e0.0038x
R²=0.727
Absorbance(600nm)
0.08
0.07
0.06
0.05
0.04
0.03
y=0.0241e0.006x
R²=0.902
0.02
0.01
0
0
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time(min)
GeneSys20
SpecPhone
Expon.(GeneSys20)
Expon.(SpecPhone)
Figure7.Growthcurvefromthehand-shakencuvettecultures.ValuesarepresentasAbsorbance±SEM.
3.3BacterialPlatingandStandardCurveConstruction
Threeofthefourflaskculturetrialsinvolvedbacterialplating.Fromthebacterialplates,
astandardcurverelatingabsorbancetothenumberofCFU/mLwasconstructed.Eachcolony
growingonaplatewasdeemedtohavebegunfromasinglebacterium,andthuswascounted
asaCFU(Sutton,2011).Correlatingthenumberofcolonieswiththeabsorbanceatthesame
timepoint,therelationshipbetweenabsorbanceandthenumberofcellspresentintheculture
wasdetermined.Usingtheequationofthelineofbestfitofthestandardcurve,thebacterial
Bunting13
concentrationofanunknownK12E.coliculturecanbecalculated,providedanabsorbance
valueisknown.
Theequationofthelineofbestfitforthestandardcurvecreatedfromtheabsorbance
valuesrecordedfromtheSpecPhoneisy=6.39E+07x+4.53E+05,withanR2valueof0.984.
Thislinerepresentsthedatawithstatisticalsignificancewhensubjectedtoanalysisbylinear
regression(p<0.05).Similarly,thesameanalysiswasperformedonthelineofbestfitdescribing
thedatarecordedfromtheGeneSys20,yieldinganequationofy=4.85E+07x+1.01E+06,with
anR2valueof0.970(p<0.05).Furthermore,thecorrelationsarenotstatisticallydifferentfrom
eachother(p=0.741).ThestandardcurveispresentedasFigure8.
4.00E+07
y=6.39E+07x+4.53E+05
R²=0.984
3.50E+07
CFU/mL
3.00E+07
2.50E+07
2.00E+07
y=4.85E+07x+1.01E+06
R²=0.970
1.50E+07
1.00E+07
5.00E+06
0.00E+00
0
GeneSys20
0.1
0.2
0.3
0.4
Absorbance(600nm)
SpecPhone
Linear(GeneSys20)
0.5
0.6
0.7
Linear(SpecPhone)
Figure8.ThestandardcurverelatingabsorbanceandthenumberofCFU/mL.ValuesarepresentasAbsorbance±SEMintheXdirectionandCFU/mL±SEMintheY-direction.
ThelinesofbestfitforthestandardcurveinFigure8weresubjectedtostatistical
analysisbylinearregression,followedbyplottingwithina95%confidenceinterval.These
annotatedgraphsappearbelow,withthedatafromtheGeneSys20representedinFigure9A
andthatfromtheSpecPhoneinFigure9B.
Bunting14
Figure9.9AShowstheannotated
standardcurvecreatedfromthedataof
theGeneSys20.Alldatapointsfallwithina
95%confidenceinterval,aswellasthe
95%predictioninterval.Theresulting
equationofthelineofbestfitisalso
shown.9Bshowsthesamemetricsforthe
datacollectedfromtheSpecPhone.The
coefficientsoftheequationsaredifferent,
2
howeverbothhavegoodR values.
Additionalanalysiswasperformedonthedatafromtheflaskculturetrialsasan
alternativetomeasuringabsorbanceusingtheSpecPhone.Theoverallintensityofthe
spectrumimagesfromtheblankmediaandthecultureswerecomparedasaratiotermedthe
intensity/arearatio(IR).Foreachblankandeachsample,theaveragepixelintensityperunitof
area(PIA)wasmeasuredusingImageJandthefollowingformula:PIA =
!"#$"%&#' (!"#$ !"#$%)
!"#$%&'( !"#$
.
Bunting15
ThePIAvaluewascalculatedforeachblankandeachsample,andthesevalueswereusedto
calculatetheIRusingthefollowingformula:IR =
!"#!"#$%&
!"#!"#$%
.Thisratioisintendedtoshowthe
differenceintheoverallintensityofthelighttransmittedthroughthesampleandtheblank
media.Astheculturegrewmoreturbid,theamountoflightthatpassedthroughthesample
decreasedastheamountscatteredbythebacteriaincreased.Whencorrelatedwiththetime
pointsofmeasurement,theIRservesasanalternativemeasurementtoabsorbance.Thisdata
Intensity/AreaRaKo
ispresentedbelowasFigure10.
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
y=0.923e-0.007x
R²=0.977
0
20
40
60
80
100 120 140 160 180 200 220 240
Time(min)
Figure10.Thisrelationshiprepresentsthedecreaseintheintensity/arearatiosbetweentheblankmediaandthebacterial
cultureat20-minuteintervalsofmeasurement.Asthenumberofbacteriainthecultureincreased,theamountoflightscattered
bytheirturbidityproportionallyincreased,decreasingamountoflightthatreachedthecamera.ValuesarepresentasIR±SEM.
4.Discussion
TheresultsofthisprojectsupportthehypothesisthattheSpecPhonecanbeusedto
measurebacterialgrowth.BroadeningthescopeoftheSpecPhonetomeasurementofliving
bacterialsystemsopensneweducationalopportunities,thusadvancingtheoverallgoalofthe
SpecPhoneproject.Further,twodistinctbacterialgrowthprotocolshavebeendesigned,which
Bunting16
outlinedetailedinstructionsforuseinavarietyofsettings.Thedistribution-readyversionsof
theseprotocolsareincludedinAppendices2and3ofthisreport,representingtheflask
culturesandcuvettecultures,respectively.
AmajorconcernwhendevelopingprotocolsfortheSpecPhoneistheaccuracyand
reproducibilityoftheresults.Thiswasespeciallypertinentforthisproject,whichfocusedon
determiningtheplausibilityandpracticalityofusingtheSpecPhonetoquantifyactivelydividing
bacteria.Regardingthegrowthcurvesthatresultedfromtheflaskcultureexperiments,the
absorbancevaluesrecordedfromtheSpecPhoneandGeneSys20arecloselycorrelatedwith
eachother,asshownbythesimilarityofthecoefficientsoftherespectivelinesofbestfit.
SpecPhonevaluesarerepresentedbytheequationy=0.0385e0.0104x,andtheGeneSys20values
bytheequationy=0.0378e0.0119x.Theerrorbarsforbothsetsofdataarealsosmall,indicating
thestandarderroroftheaverageabsorbancevalueswassmall.Again,thispointstothe
reproducibilityofthedataobtainedfromtheseprotocols.
InanefforttosimplifyuseoftheSpecPhonetomeasurebacterialgrowth,thecuvette
culturegrowthprotocolsweredesignedandtested.Thegoaloftheseprotocolswasto
decreasethetimenecessaryforcompletion.Resultsfromthecuvetteculturetrialswerenotas
robustasthosefromtheculturesgrownintheflasks.Giventhesmallervesselandthe
shorteneddurationofmeasurement,cuvettecultureabsorbancemeasurementsaremuch
lowerthanthoserecordedintheflaskculturetrials.Atthelastpointofmeasurement,T150,the
absorbancevaluerecordedbytheSpecPhonefromtheflaskgrowthprotocolswas
0.480±0.0281.Conversely,themaximumabsorbancevaluesrecordedbytheSpecPhonewere
0.214±0.0252and0.0626±0.00434forthemechanically-shakenandhand-shakencuvette
Bunting17
cultures,respectively.But,whencomparingthemaximumvaluesrecordedbytheSpecPhoneto
thoserecordedwiththeGeneSys20,theyaresimilar.Absorbancevaluesrecordedfromthe
GeneSys20were0.163±0.0456and0.0576±0.0214forthemechanically-shakenandhandshakencuvettecultures,respectively,and0.601±0.0274fortheflaskcultures.
Thereisadramaticdifferenceinthemaximumabsorbancevalueoftheculturegrownin
thehand-shakencuvettewhencomparedtothemechanically-shakencuvette.Thisislikelydue
toinadequateaerationoftheE.coli,whichisnecessarytoobservegrowthinashortperiodof
time(Juergensmeyer,Nelson,&Juergensmeyer,2007).Withoutadequateaccesstooxygen,
thegrowthrateofthebacteriaisslowed,andthustherewaslikelynotenoughcelldivisionto
produceaculturewithnoticeablyincreasedturbidity.Thisrepresentsthetrade-offofusingthe
hand-shakingprotocol.Costandequipmentrequirementsarereduced,andtheprotocolcanbe
completedinashorterperiodoftime.But,theresultsarenaturallynotasstrongasthose
completedusingmoreadvancedequipment,largerculturevessels,andoveralongertime
period.
Thecuvettecultureprotocolsalsodonotresultinthecreationofastandardcurve
relatingabsorbanceandcolonynumber,asthisrequiresremovalofasampleoftheculturefor
plating.Giventhesmallvolumeofthecuvettesandthelimitedamountofcultureavailablefor
analysis,periodicremovalofcultureforplatingwouldinterferewithgrowth.However,ifa
standardcurvefromanotherexperimentisavailable,theequationofthelineofbestfitforthis
plotcanbeusedtocalculateCFU/mLofthecuvettecultures.Ultimately,thepurposeofthe
cuvettegrowthprotocolsistointroducethetopicofmeasuringbacterialgrowth,andtodoso
inashorterdurationoftime.Despitetheselimitations,thisgoalwasaccomplishedoverall.
Bunting18
Platingwasalsodoneinthisexperimentseriestoestablishtherelationshipbetween
absorbanceandbacterialcolonynumber,andtocreateastandardcurveofbacterialgrowth
(Figure8).Thestandardcurvecorrelatesabsorbancevalueswiththenumberofbacterial
coloniesthatgrewontheplatesfromculturesamplesdilutedatthesametimepoint.The
equationsofthelinesofbestfitfromthestandardcurveconstructedinthisexperimentcanbe
usedtodeterminethenumberofCFU/mLofanunknownK12E.coliculture,iftheabsorbance
valueisknown.Rewritingtheseequationstoincludethevariablesofthisexperiment,the
GeneSys20equationbecomes:CFU/mL=4.85E+07(Absorbance)+1.01E+06,andthe
SpecPhoneequationbecomes:CFU/mL=6.39E+07(Absorbance)+4.53E+05.Asmentioned,
theselinesarestatisticallysignificantintheirrepresentationofthedataforboththeGeneSys20
(p<0.05)andtheSpecPhone(p<0.05)whenanalyzedbylinearregression,andarenot
statisticallydifferentfromeachother(p=0.741).And,asshowninFigures9Aand9B,thedata
pointsarealignedwithina95%confidenceinterval.Again,thispointstothereproducibilityand
accuracyoftheSpecPhoneresults,whenjudgedagainstacommercialdevice.
Littlevariationexistsintheabsorbancevalues(X-axiserrorbars)recordedfromeach
instrument,however;thereissignificantvariationinthecalculatedCFU/mLvalues(Y-axiserror
bars)ateachdatapointofthestandardcurve.Thereareanumberofpotentialexplanationsfor
thisvariation,thefirstofwhichhastodowiththeuniquegrowthpatternsofeachindividual
culture.AllstockcultureswerecreatedwiththesameK12strainofE.coli,howevertherewere
stilldifferencesingrowth,andsubsequentlyabsorbancemeasurements,andultimatelyfewer
viablecellsavailabletoestablishcoloniesuponplating.Thiscouldhavetodowiththeageof
thecellsusedtocreatethestockculture.Insomeofthetrials,theplatesofK12E.coliusedto
chemical properties, a limited nutrient status, and consor- for growth and environmental parameters are optimal, the
tia of different microorganisms all competing for the same increase in numbers or bacterial mass can be measured as
limited nutrient supply (see Chapter 4). Thus, the current a function of time to obtain a growth curve. Several dischallenge facing environmental microbiologists is to under- tinct growth phases can be observed within a growth curve Bunting19
stand microbial growth in natural environments. Such an (Fig. 3.3). These include the lag phase, the exponential or
understanding would facilitate our ability to predict rates of log phase, the stationary phase, and the death phase. Each
nutrient cycling (Chapter 14), microbial response to anthro- of these phases represents a distinct period of growth that
growthestockculturewereseveraldaysold.Itispossiblethatthenumberofviablecells
pogenic perturbation
of the environment (Chapter 17), is associated with typical physiological changes in the cell
microbial interaction with organic and metal contaminants culture. As will be seen in the following sections, the rates
(Chapters 20 and 21),
and survival and growth of pathogens of growth associated with each phase are quite different.
containedineachcolonyontheplatehaddecreasedbythatpoint,andthusfewerviablecells
in the environment (Chapters 22 and 27). In this chapter,
we begin with a review of growth under pure culture condi- 3.1.1 The Lag Phase
wereavailablewhentheblankmediawasinoculated.
tions and then discuss
how this is related to growth in the
The first phase observed under batch conditions is the lag
environment.
phase in which the growth rate is essentially zero. When
Correlatedwiththiscouldbeasecondpossibleexplanation:anincreaseinthetime
an inoculum is placed into fresh medium, growth begins
after a period of time called the lag phase. The lag phase is
necessaryforsynthesisofgrowthenzymesandsubstrates.AsshowninFigure11,thereisa
defined to transition to the exponential phase after the initial population has doubled (Yates and Smotzer, 2007). The
lag phase is thought to be due to the physiological adaptadistinctphaseatthebeginningofthebacterialgrowthtrajectoryinwhichthecellsarenot
tion of the cell to the culture conditions. This may involve
a time requirement for induction of specific messenger
rapidlydividing,andinsteadaresynthesizingtheneededsubstratesandmolecularmachinery
vs.
RNA (mRNA) and protein synthesis to meet new culture
requirements. The lag phase may also be due to low initial
densities of organisms that result in dilution of exoenzymes
requiredfordivision.Dependingonthenumberofviablecellsatthebeginningofthetrial,this
(enzymes released from the cell) and of nutrients that leak
from growing cells. Normally, such materials are shared by
periodmayhavebeenlongerforsomeculturesthanothersasthebacteriapreparedfor
cells in close proximity. But when cell density is low, these
materials are diluted and not as easily taken up. As a result,
FIGURE 3.2 Compare the complexity of growth in a flask and growth
initiation of cell growth and division and the transition to
growth.Beginningwithfewercellswouldnaturallyleadtoalongertimenecessarytovisualize
n a soil environment. Although we understand growth in a flask quite
well, we still cannot always predict growth in the environment.
exponential phase may be slowed.
d 38
anincreaseinturbidityasincreasedabsorbance.
Turbidity (optical density)
9.0
0.75
8.0
7.0
0.50
entia
l
De
ath
6.0
0.25
5.0
4.0
Optical density
Stationary
Exp
on
Log10 CFU/ml
1.0
0.1
Lag
Figure11.Anexampleofa
standardbacterialgrowth
curveisrepresented,with
labeledphasesofgrowth.In
theseexperiments,onlythe
initialphaseswerevisualized
intheresults.However,
allowingthetrialstogrowfor
alongerperiodoftimemay
allowforvisualizationoflater
phases(Maier,Pepper,&
Gerba,2009).
Time
FIGURE 3.3 A typical growth curve for a bacterial population. Compare the difference in the shape of the
curves in the death phase (colony-forming units versus optical density).
Finally,whenlookingatthenumberofcoloniespresentattheT240timepointinthe
standardcurves,thereisalargeincreaseinAbsorbanceaswellasCFU/mL.Thisdifferencemay
7/21/2008 3:38:38 PM
beexplainedbythefactthatthefinalplateswerecreatedfromtheT240timepoint,whichis
actually60minutesfollowingtheT180point,insteadofthe40-minuteintervalsatwhichthe
previousplateswerecreated.Thiswasdonetoensuretherewereplatesavailableforthefinal
Bunting20
pointoftheculturegrowth,ratherthanplatingfromthecultureatT220andneglectingthe
growththatoccurredinthefinal20minutes.Inafuturerevision,itmaybebeneficialtoextend
thetrialto260minutes,allowingforplatestobeconstructedattheT220andT260timepoints,
andaddinganadditionalabsorbancemeasurementatT260.
Withintheseprotocols,thereiseducationalvalueandpotentialforextendingthat
value.Onepotentialareawouldbeextensionoftheprotocolstoincludemoreplating,withthe
hopeofvisualizingmorephasesofbacterialgrowth.Ahallmarkcharacteristicofbacterial
growthcurvesistheirshape,showingfour,distinctphasesofgrowth:lag,exponential,
stationaryandcelldeath(Figure11).Thesephasesofgrowthwillnotberepresentedas
changesinturbidity,butinsteadcanbeseenwhenplottingCFU/mL.Aftertheplateauphase
hasbeenreached,turbiditywillremainconstantaslongasthecultureiscontinuallyshakento
keepthecellsinsuspension.However,activecelldivisionwillnolongerbeoccurring.Ifsamples
aretakenfromthecultureatregularintervalsandplated,thecalculatednumberofCFU/mL
willremainconstantthroughoutthestationaryphase,andwilleventuallybegintodeclineas
thecellsdepletethenutrientsofthemediaandbegintodie.Ifabsorbancemeasurementsare
takeninconcurrencewiththeseplates,agrowthtrajectorysimilartothatofFigure11will
result,withabsorbanceremainingconstantandtheCFU/mLeventuallydeclining.Thiscould
representaninterestingconcepttobroadentheeducationalprospectsoftheseprotocols.
Asecondoptiontovisualizethesesameeffectswouldbetogrowacontrolflaskculture
alongwiththeonetobemeasured(sampleflask).Asecondflaskofmediacouldbeinoculated
withthestockcultureatthesametimeasthesampleflask.Thecontrolflaskculturewould
growalongwiththesampleculture,andabsorbancemeasurementswouldbetakenatthe
Bunting21
sametimepoints.However,aftertheT240measurement,absorbancemeasurementofthe
controlflaskwouldcontinueforapproximatelyfouradditionalhours.Eventually,the
absorbancemeasurementswouldbeexpectedtoreachaplateau,pointingtocessationof
bacterialgrowth.However,thenumberofcellsisnotdecreasing,astheyarestillinsuspension
becauseofshakingintheincubator.But,thispresentsatradeoffinthatthisextended
measurementwouldincreasethetimenecessaryforcompletion.Todecreasetheamountof
activelaboratorytimerequired,absorbancemeasurementsforthecontrolculturecouldbe
takenat40-minuteintervalsinsteadof20-minutes.Thepointofthismodificationwouldbeto
showthegeneralshapeofastandardbacterialgrowthcurve,ratherthanclosemonitoringof
cultureturbidity.
Finally,furthereducationalopportunitiesareimplicitwithintheseprotocolsbythe
additionofvariousotheragentstothecultures.Forexample,antibioticscouldbeaddedtoone
culture,grownalongsideonewithoutthemtovisualizeandteachtheeffectsofthesedrugs.
Forafurthernuancedcomparison,abactericidalantibiotic,onethatkillsthecellscouldbe
comparedwithabacteriostaticdrug,onethatdoesnotkilltheexistingcellsbutprevents
furtherdivision.Thehands-onnatureoftheSpecPhone,coupledwiththeabilitytogrowand
visualizebacterialculturesinarelativelyshortamountoftimepresentsapowerfulteaching
opportunity.Similarly,oneculturecouldbesupplementedwithaglucosesolution,and
comparedtoaculturegrowninun-supplementedmedia.Theculturewithglucose
supplementationwouldbeexpectedtogrowatafasterrate,asglucoseisthepreferredcarbon
sourceforE.coliandcouldenterdirectlyintocentralmetabolism.
Bunting22
Theseprotocolscouldalsobeextendedtocomparethegrowthofdifferentbacterial
species.Forexample,thegrowthrateofaGram-positivespeciescouldbecomparedtothatof
aGram-negativespecies.Importantly,thesemodificationswouldinvolvebacterialstrainsthat
mayhavedifferentsafetyrecommendationsthantheonespresentedhere.Thisshouldbe
carefullyconsideredbeforeanyadditionaltrialsareperformed,andbeforeanyprotocol
modificationsaremade.Importantly,thesemodificationscouldbeappliedtoeithertheflaskor
cuvettecultureprotocols,dependingontheresourcesandtimerestrainsoftheindividual
settinginwhichtheywillbeimplemented.
DespitetheworkpresentedheretoincreasetheapplicationsoftheSpecPhone,several
limitationspersistregardingtheseprotocols.Thelargestoftheseissuesrevolvearoundthe
conditionsthatarenecessaryforE.coligrowthinthelaboratory.Whileagitationand37°C
incubationarenotabsolutelyrequiredforE.coli,therateofcellulargrowthisdramatically
increasedundertheseconditions.Withoutagitation,thebacteriawillgrow;however,therate
willbedramaticallyslowerbecausetheywillbeforcedintoanaerobicconditionsandrelyon
fermentationratherthanoxidativemetabolism(Juergensmeyer,Nelson,&Juergensmeyer,
2007).Inorderfortheseprotocolstobeperformedinareasonablybriefperiodoftime,
instrumentstoensureagitationandheatwerereliedupon.Availabilityofthiskindof
equipmentmaynotbearealityforeducationalsettingsthatwouldbenefitfromteaching
spectroscopyusingtheSpecPhone,thuspresentingamajorlimitationfortheseprotocolsthat
didnotaffecttheformerlydesignedones.Aspreviouslystated,apotentialextensionofthe
protocolswouldbethesupplementationofthemediawithaglucosesolution.Thisispredicted
toincreasethegrowthrate,howeveritdoesnotsolvetheissueoftheneedforconstant
Bunting23
agitationtoaerate,andtokeepthecellsinsuspensionformeasurement.Effortstoshakethe
cultureandsuspendthecellswithoutadvancedequipmentproducedsubparresults,asshown
bythehand-shakencuvettecultures.
Onepotentialavenuethatcouldsolvetheissueofagitation,whichwasnotexploredin
thepresentexperiment,wouldbetheuseofamagneticstirbar.Fortheflaskcultures,apillsizedstirbarplacedatopamagneticstirplatecouldproducesufficientagitationforbacterial
aeration.Forthecuvettecultures,aricegrain-sizedstirbarcouldbeemployed,withthe
cuvettesplacedatopthestirplate.Toprovideheat,theflaskorcuvettecouldbeplacedina
waterbaththatwouldbeheatedandkeptintherangeof30-37°Ctopreventcelldamageand
death.Thebenefitsherearetwofoldinthattheneedforanexpensiveshakingincubatorwould
beeliminated,anditwouldalsoallowtheseprotocolstobeperformedinamorepractical
durationoftime.Whilethisoptionwasnotpursuedinthisexperiment,itdoespresentan
interestingfutureoption,andwarrantsfurtherinvestigation.
Anotherissuesurroundingtheseprotocols,specificallythosethatwereconductedinthe
flasks,istheamountoftimerequired.Currently,theflaskcultureprotocolsaredesignedfor
completionin4hours,andthecuvettecultureprotocolsin2.5hours.Neitherestimateincludes
thenecessarypreparationtimetocreatethestockculture,northeadditionaltimerequiredfor
platecounting.Whilethisisarelativelyshortperiodoftimeoverall,itdoeslimitthepotential
forvisualizationofallfourphasesofbacterialgrowth.Fullphasevisualizationisnotanabsolute
necessityforusingtheseprotocolstoteachtheprinciplesofbacterialgrowth,butitwouldadd
anadditionallayerofdepthtotheexperience.Asmentioned,thismaybecompensatedforby
theadditionofacontrolculturethatisgrownoveralongerperiodoftime.Extendingthe
Bunting24
amountoftimegiventocompleteeachisapotentialsolution,yetthisfurthersthechallengeof
usingtheseprotocolsinsettingswithinflexibleschedulesorlimitedlaboratorytime.Evenwith
visualizationoftheexponentialgrowthphaseonly,therearestillvaluableteaching
opportunitiesforlearningaboutbacterialgrowth.
Turningnowtoissueswithimageanalysis,theImageJanalysiswasmorecomplicated
fortheseexperimentsthaninthepreviousSpecPhoneprotocols,whichdealtwithsimpler,
coloredsolutions.Bacterialculturesareturbid,andthespectrabecameincreasinglydispersed
anddimastheculturegrew.ThisisshowninthecomparisonbetweenBSAandE.coliinFigure
4Aand4B.Overalllightintensityisdiminishedasthenumberofindividualphotonsreaching
thecameraisreducedbecauseoflightscatteringfromthebacteria.Thisoveralldecreasein
intensityisrepresentedinthecomparisonplotofFigure4C.Thespectraareaalsobecame
largerastheculturegrew,andanalysiswithImageJreliesoncomparingintensityatspecific
pixelpositions.Ensuringtheareasofanalysisbetweenthecultureandblankswerealigned
becamemorecomplicatedwithincreasingcultureturbidity.
Apotentialalternativeforthismethodofmeasurementsistouseoverallspectrum
intensity,ratherthanintensityataparticularpixelposition,whichcorrespondstoaspecific
wavelength.ImageJallowsformeasurementofintensity(Grayvalue)overaunitofarea,orPIA.
ThePIAofasampleandblankcanbecomparedtocalculatetheIR.Atthebeginning,the
sampleandblankspectrumswillbenearlyidenticalbecausetherehasbeenlimitedbacterial
growth,meaningIR≈1.However,astheculturegrowsandbecomesincreasinglyturbid,the
intensityoflightpassingthroughtheculturesampleswillbegintogloballydecrease,rather
thansolelyataspecificwavelength.IRvalueswillcontinuallydecreasebelow1,resultingina
Bunting25
downwardtrend.Thisdecreaseinoverallintensitycanbeplottedasafunctionoftime,which
wasexploredandispresentedinFigure10.Thiscorrelationwouldnotbeverifiablebya
commercialinstrument,suchastheGeneSys20,whichonlyreportstheabsorbancevalue,not
overallintensity.Further,whenattemptingtousetheIRvaluestocreateanalternativegrowth
curveforthedata,thecorrelationwassubpar,withanR2valueof0.676(datanotshown).
WhileIRmaynotbeausefulforquantifyinganunknownK12E.coliculture,itdoeshave
potentialforuseasateachingtooltodemonstratethephysicsoflightscatteringwith
increasingcultureturbidity.
Finally,thereareseveralissuesthatarerelatedtothelargerSpecPhoneproject,that
arenotuniquetotheseprotocols.ThefirstistheimageanalysisprocessusingImageJ.While
theaforementionedprotocolisavailable(Smith,2015),analysisisstillnotanovertly
streamlinedprocess.Forbeginners,ImageJmaynotbeaninherentlyuser-friendlyprogram,
anditpresentsasignificantlearningcurveforsomeoneusingitforthefirsttime.Thatbeing
said,ImageJiscommonlyusedsoftwareinmanyareasofresearch,andearlyintroductionmay
beanadvantage.Anareaoffurtherresearchcouldbecenteredarounddevelopmentofa
mobileapplicationthatcouldperformthedataanalysisonthephone,eliminatingtheneedfor
importandanalysiswithImageJ.AsecondissuecentersonthedesignoftheSpecPhone,which
currentlyonlyfitstheiPhone5/5s.Thisplacesasignificantlimitationontheapplicationofthe
apparatus,andeffortstowardaredesignareongoing.
5.Conclusion
TheworkpresentedhereshowsthattheSpecPhonecanbeconsistentlyandaccurately
usedtoquantifyanactivelydividingK12E.coliculture.Fromthisproject,threeprotocolswere
Bunting26
designedandtested.OneprotocolprovidesstepsforgrowthofE.coliinanErlenmeyerflask
withroutinemeasurementofabsorbanceandCFU/mLviaplating.Theothersgiveinstructions
forsmaller-scaleculturesgrownentirelyinplasticcuvettes,witheithermechanicalorhandshaking.Theresultsobtainedfromthecuvettecultureprotocolswerenotasrobustasthose
fromtheflaskcultures,howevertheystillpresentvaluable,educationalopportunities.When
comparingtheresultsobtainedfromtheSpecPhonetothoseoftheGeneSys20,thereissome
deviationbutoverallthetrendsareconsistentforallprotocols.Thisreproducibilityand
consistencymaketheSpecPhoneacompellingandusefultoolforteachingtheuseof
spectroscopytomeasureactivelydividingbacterialcultures.Combinedwiththerelative
inexpensivenessoftheSpecPhonecomponents,thisbodyofworkmakestheubiquitousand
powerfultechnologyofspectroscopymorehands-onandaccessible.
Bunting27
References:
CentersforDiseaseControlandPrevention.(2009).BiosafetyinMicrobiologicalandBiomedical
Laboratories(5thed.).(C.Chosewood,&D.Wilson,Eds.)Atlanta,Georgia.
Grasse,E.K.,Torcasio,M.H.,&Smith,A.W.(2016).TeachingUV-VisSpectroscopywitha3DprintableSmartphoneSpectrophotometer.JournalofChemicalEducation,93(1),146151.
Hall,B.G.,Acar,H.,Nandipati,A.,&Barlow,M.(2014).GrowthRatesMadeEasy.Molecular
Biology&Evolution,31(1),232-238.
Juergensmeyer,M.,Nelson,E.,&Juergensmeyer,E.(2007).Shakingalone,withoutconcurrent
aeration,affectsthegrowthcharacteristcsofEscherichiacoli.LettersinApplied
Microbiology,45,179-183.
Maier,R.M.,Pepper,I.L.,&Gerba,C.P.(2009).EnvironmentalMicrobiology.AcademicPress.
Rasband,W.S.,ImageJ,U.S.NationalInstitutesofHealth,Bethesda,Maryland,USA,
https://imagej.nih.gov/ij/,1997-2016.
Smith,A.W.(2017,January5).CreationofaProteinStandardCurveUsingtheSpecPhone.
RetrievedFebruary15,2017,fromSpecPhoneProtocols:
https://smithlab.uakron.edu/files/2017/01/BSA-SpecPhone-Protocol-V1.3-1517.pdf
Smith,A.W.(2015,August28).ProcessingSpecPhoneImagesandWavelengthCalibration.
RetrievedFebruary16,2017,fromSpecPhoneProtocols:
https://smithlab.uakron.edu/files/2015/09/Protocol-1-Image-Processing-andCalibration.pdf
Smith,A.W.(2015,August28).SpectroscopyofCherryKool-AidUsingtheSpecPhone.Retrieved
February15,2017,fromSpecPhoneProtocols:
https://smithlab.uakron.edu/files/2015/09/Protocol-2-The-Beer-Lambert-Law.pdf
Sutton,S.(2011).AccuracyofPlateCounts.JournalofValidationTechnology,17(3),42-46.
Yourassowsky,E.,VanderLinden,L.P.,Lismont,M.J.,Crokaert,F.,&Glupczynski,Y.(1985).
Effectongrowthcurvepatternsofbriefexposreofbacteriatodifferentconcentrations
ofB-lactamantibiotics.JournalofAntimicrobialChemotherapy,15(Suppl.A.),27-36.
Zwietering,M.H.,Jongenburger,I.,Rombouts,F.M.,&Van'tRiet,K.(1990).Modelingofthe
BacterialGrowthCurve.AppliedandEnvironmentalMicrobiology,56(6),1875-1881.
Appendix1.1
SafetyPrecautions&Guidelines
Giventhatthisprotocoldealswithbacteria,safelaboratorypracticeisofamore
concerncomparedtothepreviousSpecPhoneprotocols.Allworkwascompletedinaccordance
withtheCDC’sguidelinesforaBSL-1laboratory(CentersforDiseaseControlandPrevention,
2009).Gloves,eyeprotectionandalabcoatwerewornthroughouttheentiretyofalltrials,
noneofwhichleftthelaboratory.
Appendix2.1
CreatingaStandardBacterialGrowthCurve
Materials:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Laboratorystrain(K-12)ofEscherichiacoli
ErlenmeyerFlask,125mLcapacity
TrypticaseSoyBroth(TSB)(~400mL)
TrypticaseSoyAgar(TSA)plates(48)
Ethanol
Culturetubes,sterile(25)
p5000&p1000Pipettesandpipettetips
Bunsenburnerorotherflame
Shakingincubator,capableofreaching37°Cand265rpm
Bacteriaspreader
Plasticcuvettes(2),4.5mL1cm.x1cm.
LensCloth(KimWipes)
SpecPhoneapparatuswithnecessaryadditions
LightSource(desklamp)
iPhoneoranothercamera-containingmobiledevice
Laboratorynotebook
Commercialspectrometer(ifavailable)
Introduction:
Thisprotocolprovidesthestepsforquantifyingbacterialgrowthusingthe
SpecPhone.AstockcultureofE.coliiscreatedandthenusedtoinoculatesterilemediato
growanewculture.Atregularintervalsalongtheprocess,asampleofthegrowingculture
isremovedandanabsorbancemeasurementistaken.Platingisperformedatregular
intervals,andtheseplatesareusedtodeterminethenumberofbacteriaintheculture
alongthegrowthprocess.Whenallofthedatahasbeencollected,astandardcurvecanbe
plottedrelatingabsorbancetothenumberofbacteria,whichcanbeusedtoassessthe
numberofcellsinanunknownculture.
Astheculturegrows,itbecomesincreasinglyturbid.Thisincreasingturbidity
causesmorelighttobescatteredbytheculture,resultinginadecreaseintheintensityof
thelightrecordedbytheSpecPhone.Lightscatterisdirectlydependentonthedensityof
bacterialcellspresentintheculture.Theamountoflightallowedtopassthroughthe
cultureisthepercenttransmission(%T),whichcanbeconvertedtoanabsorbancevalue
usingtheequation:A=-log(%T).Theconventionistomeasurebacterialculturesat600nm.
Aspecialnoteshouldbemadethatthisprotocolrequiresuptothreedaysfor
completion.Onedayisneededtocreatethestockbacterialcultureandallowittoincubate.
Aseconddaywillbespenttakingtheactualmeasurementsandthethirdwillbedevotedto
countingbacterialplates.
Appendix2.2
Procedure:
Day1:
CreationofStockE.coliCulture:
1. Fillaculturetubewith10mLofsterileTSB,andbringtoatemperatureof37°C.
2. IftheE.coliareinitiallyonabacterialplate,selectasinglecolonyfromtheplate
withtheloopandinoculatetheTSB.Flametheloopbeforetouchingtheplateto
ensuresterility.IftheE.coliareinitiallyinbroth,pipette100μLofthecultureinto
9.9mLofwarmTSB.
3. Incubatethisculturefor24hoursintheshakingincubator,shakingataspeedof
265rpm,andatatemperatureof37°C.
PreparationforMeasurements:
1. Platinganddilutionswillbedoneinthisexperimenttocreatethestandardcurve
relatingcolonynumbertoabsorbance.BlanktubesofTSBwillberequiredforthis,
andshouldbefilledaheadoftime.Createsixsetsofthefollowingtubevolumes:3
filledwith9.0mLofTSB,and1filledwith9.9mLofTSB.Ultimately,therewillbea
totalof24culturetubes;sixsetsoffourtubes.
2. FilltheErlenmeyerflaskwith95mLofTSB.Placetheflaskalongwiththeculture
tubesinanincubatorat37°Candallowthemtocometotemperature.
3. PreparetheSpecPhoneapparatus.Ensurethesurroundingsarefreefromoutside
lightaspossible,andthatthelightsourcebeingusedisbrightenough.Placethe
mobiledeviceintheSpecPhoneandlaunchthecameraapp.
4. Noneofthecamerapixelsshouldbesaturatedbythelightsource,andthespectrum
shouldhaveevenintensityacrossthecolorrange.Onceadequateandappropriate
lightingisfound,tapethedeviceinplaceormakemarksonthesurfacetodenote
theproperpositioning.Takegreatcaretokeeptheapparatusstationary
throughoutthetrialstoensureaccuracyacrossallmeasurements.
5. FillonecuvettewithTSB,thiswillserveastheblank.
Day2:
TakingAbsorbanceMeasurements:
1. InoculatetheErlenmeyerflaskofwarmTSBwith5mLofthestockE.coliculture,
transferringusingthep5000pipette.
2. Vortextoensuremixingandimmediatelytransfer2.5mLofthefreshlyinoculated
culturetoacuvetteusingthep5000pipette.
3. PlacetheblankcuvetteintheSpecPhoneandcapturethreespectrumimages,and
thenreplacetheblankwiththeculturecuvetteandcapturethreemorespectrum
images.Recordtheexacttimeofmeasurementinthelabnotebook,andrecord
whichpicturesinthecamerarollcorrespondtotheblankorculturesamples.Thisis
theT0measurement.
Appendix2.3
Tip:Itmaybeusefultophotographsomeobjectatthebeginningandendofthe
seriesofphotostodenotethesequenceinthephone’scameraroll.
4. PlacetheErlenmeyerflaskintheshakingincubatorat37°Cand265rpm,andseta
timerfor20minutes.
5. At20-minuteintervals,pipette2.5mLofthecultureintheErlenmeyerflasktothe
samplecuvetteandrepeatthemeasurementprocess,rememberingtoblankthe
SpecPhonebeforetakingeachmeasurement.Keepdetailedrecordsinthe
laboratorynotebook,orusingthetableprovidedintheAppendix.
6. Thisprocesswillcontinueoveraperiodof4hours,withabsorbancemeasurements
beingtakenatthefollowingtimepoints:T0,T20,T40,T60,T80,T100,T120,T140,T160,
T180,T200,T220,T240.
7. Ifacommercialspectrometerisavailable,takemeasurementsforcomparison.
BacterialPlating:
1. At40-minuteintervals,bacterialplateswillbemadefromthegrowingculture.
Platesshouldbemadeatthefollowingtimepoints:T20,T60,T100,T140,T180,T240.
2. ThepreparedculturetubesofsterileTSBwillbeneededhere.Aftertaking
absorbancemeasurements,pipette100μLofthecultureinthesamplecuvetteto
thefirstculturetubecontaining9.9mLofsterileTSB.
3. Vortexthefirsttubetoensureacellsuspensioniscreated.
4. Transfer1mLofthesolutioninthefirsttubetothesecond,andvortexagain.
Transfer1mLofthesecondtubetothethirdtube,vortex,andthentransfer1mLof
thethirdtothefourthtube.Thiscreatestheserialdilutions,uptoafactorof10-5.
5. Fromeachtube,pipette100μLontotwoTSAplates.Therewillbeatotalofeight
platesforeachtimepoint.Usethebacterialspreadertoevenlydistributetheculture
ontheplate.Beforespreading,dipthespreaderintheethanolandthenintheflame
tosterilize.Allowthespreadertocoolbeforetouchingtheplatetopreventkilling
thecells.Aschematicofthedilutionprocessisbelow.
100μL
Cuvette
1mL
9.9
mL
TSB
1
10-2
Plate
100μL
1mL
9.0
mL
TSB
2
10-3
Plate
100μL
1mL
9.0
mL
TSB
3
10-4
Plate
100μL
9.0
mL
TSB
4
10-5
Plate
100μL
Appendix2.4
6. Labeltheplatesusingthefollowingconvention:tXXX,dilutionfactor,plate#(eg.
T100,10-3,#1).Thedateshouldalsobeincluded.
Tip:Onlywriteontheplatenotthelidtosavetheinformationincasethelids
getseparated.
7. Placetheplatesintheincubatorat37°Covernight.
Day3:
PlateCounting&DataAnalysis:
1. Followingincubation,countthenumberofcoloniesoneachplate.Onlythoseplates
thathavebetween30-300coloniesshouldbecounted,asthisisthebestwindowfor
statisticalaccuracy.
Tip:Ifaplatehasmorethan300colonies,labelitas‘TooManyToCount.’
2. Countthecolonynumberforbothplatesfromeachdilutionfactorforthesixtime
points.Averagethetwocolonycountsforeachdilutionfactor.
3. CalculatethenumberofcolonyformingunitspermL(CFU/mL)usingthefollowing
formula:
𝐶𝐹𝑈
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐶𝑜𝑙𝑜𝑛𝑦 𝑁𝑢𝑚𝑏𝑒𝑟
=
𝑚𝐿
(𝑚𝐿 𝑃𝑙𝑎𝑡𝑒𝑑)×(𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟)
Tip:ThemLPlatedforallis100μLor0.1mL.
4. ImagestakenwiththeSpecPhonecanbeimportedtoacomputercontainingImageJ
(Rasband,2016)andaphotobrowser.Followtheinstructionsforimageanalysis
providedintheImageProcessingandCalibrationprotocol(Smith,2015).Atableis
includedintheAppendixtoorganizethedataandcalculations.
5. Createthestandardcurvebyplottingtheabsorbancevalueforeachofthesixtime
pointsontheX-axis,andthecalculatedCFU/mLvalueontheY-axis.Anexample
standardcurveappearsbelow:
4.00E+07
3.50E+07
y=6.39E+07x+4.53E+05
R²=0.984
CFU/mL
3.00E+07
2.50E+07
y=4.85E+07x+1.01E+06
R²=0.970
2.00E+07
1.50E+07
1.00E+07
5.00E+06
0.00E+00
0
GeneSys20
0.1
0.2
SpecPhone
0.3
0.4
0.5
0.6
0.7
Absorbance(600nm)
Linear(GeneSys20)
Linear(SpecPhone)
Appendix2.5
6. IfanunknownK12E.colicultureisgiven,thenumberofCFU/mLcanbecalculated
usingtheequationofthelineofbestfitforthestandardcurve,substitutingthe
knownabsorbancevalueinforXandsolvingforY(CFU/mL).
Important:Thedataobtainedfromthesetrialscanonlybeusedtoquantify
unknownculturesofK12E.coli.Ifanotherbacterialspeciesistobeanalyzed,a
standardgrowthcurveforthatspeciesmustbeconstructedaseachbacterium
hasitsowngrowthcharacteristics.
SafetyGuidelines:
Giventhatthisprotocoldealswithbacteria,thesafetyguidelinesaresomewhat
stricterthanwhencompletingtheotherSpecPhoneprotocols.Whenchoosingabacterial
straintoworkwith,werecommendK-12E.coli,asthisisastandardlaboratorystrainthat
hasbeendeterminedtobenon-pathogenicforhumansandothermammals(U.S.
EnvironmentalProtectionAgency,1997).
WorkingwithnonpathogenicorganismsfallsundertheBiosafetyLevel1(BSL-1)
guidelinesfromtheCDC.ExtensiveguidelinescanbefoundontheCDCwebsite(U.S.
CentersforDiseaseControlandPrevention,2009).Briefly,allpersonnelshouldwashtheir
handsbeforeenteringandleavingthelab,andwearglovesthroughouttheentire
procedure.Glovesshouldnotbewornoutsideofthelab,andshouldbeimmediately
replacedifanydamageoccurs.Ifpossible,wearinglabcoatsispreferabletoprotect
personalclothing.Nofoodordrinkshouldbekeptorconsumedintheareawherethe
experimentisbeingconducted.Attheendofthelabperiod,disinfectallsurfacesandany
equipmentthatcameincontactwiththebacterialcultures.Allstudentscompletingthe
protocolshouldbeinstructedtoreportanymistakes,spills,injuriesetc.tothelaboratory
coordinatorwhowilldecidetheappropriateaction.Anyindividualinstitutionpractices
thatextendbeyondtheseshouldbefollowedaswell.
ProtocolExpansions:
Thereareanumberofmodificationsthatcanbemadetothisprotocoltofurther
increasetheeducationalpotential.Onesuchmodificationwouldbetheadditionofan
antibiotictooneculture,andtothencomparethegrowthrateofthisculturetoonethatdid
notcontainthedrug.Ifamorenuancedcomparisonisdesired,differingclassesof
antibioticcouldbecompared.Forexample,oneculturecouldbesupplementedwitha
bactericidalantibiotic,whichwillkillthecellsandanothercouldbesupplementedwitha
bacteriostaticantibiotic,whichdoesnotkillthebacteriabutratherpreventsfurther
division.Twodifferentantibioticsofthesameclasscouldalsobecomparedtodetermineif
oneismoreeffectivethananother.
Anotherpotentialexpansionwouldbetosupplementoneculturewithaglucose
solutionandcompareitsgrowthtothatofanon-supplementedculture.Glucoseisthe
preferredcarbonsourceforE.coli,anditisabletoenterdirectlyintocentralmetabolism.
Thisadditionisexpectedtoincreasethegrowthrateofthesupplementedculture.Finally,
thegrowthratesofdifferingspeciesofbacteriacouldalsobecompared.Apotentially
interestingcomparisonwouldbebetweenaGram-negativespeciesandaGram-positive
Appendix2.6
species.However,thiswouldinvolvedifferentstrainsofbacteriathatcarrydifferingsafety
recommendations,whichshouldbecarefullyconsideredbeforeanyadditionaltrialsare
performedoranyprotocolmodificationsaremade.
References:
Rasband,W.S.,ImageJ,U.S.NationalInstitutesofHealth,Bethesda,Maryland,USA,https://imagej.nih.gov/ij/,
1997-2016.
Smith,A.W.(2015,August28).ProcessingSpecPhoneImagesandWavelengthCalibration.Retrieved
February16,2017,fromSpecPhoneProtocols:https://smithlab.uakron.edu/files/2015/09/Protocol-1Image-Processing-and-Calibration.pdf
U.S.CentersforDiseaseControlandPrevention.(2009).BiosafetyinMicrobiologicalandBiomedical
Laboratories(5thed.).(C.Chosewood,&D.Wilson,Eds.)Atlanta,Georgia.
U.S.EnvironmentalProtectionAgency.(1997,February).AttachmentI-FinalRiskAssessmentofEscherichia
coliK-12Derivatives.RetrievedFebruary24,2017,fromEPAUnitedStatesEnvironmentalProtectionAgency:
https://www.epa.gov/sites/production/files/2015-09/documents/fra004.pdf
Appendix2.7
Appendix:
Usethefollowingtablestoorganizetheplatecountingandabsorbancedata.Remember,onlycountthose
platesthathave30-300colonies.UsetheprovidedformulatocalculatetheCFU/mL.
T20
AbsorbanceValue:
DilutionFactor:
10-2
10-3
10-4
10-5
10-4
10-5
10-4
10-5
Plate1
Plate2
Average
CFU/mL
T60
AbsorbanceValue:
DilutionFactor:
10-2
10-3
Plate1
Plate2
Average
CFU/mL
T100
AbsorbanceValue:
DilutionFactor:
Plate1
Plate2
Average
CFU/mL
10-2
10-3
Appendix2.8
T140
AbsorbanceValue:
DilutionFactor:
10-2
10-3
10-4
10-5
10-4
10-5
10-4
10-5
Plate1
Plate2
Average
CFU/mL
T180
AbsorbanceValue:
DilutionFactor:
10-2
10-3
Plate1
Plate2
Average
CFU/mL
T240
AbsorbanceValue:
DilutionFactor:
Plate1
Plate2
Average
CFU/mL
10-2
10-3
Appendix3.1
BacterialGrowthCurves-CuvetteCultures
Materials:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Laboratorystrain(K-12)ofEscherichiacoli
TrypticaseSoyBroth(TSB)(~20mL)
Culturetube,sterile(1)
p5000&p1000Pipettesandpipettetips
Bunsenburnerorotherflame
Ethanol
Incubator,capableofreaching37°Cand265rpm
Bacteriaspreader
Plasticcuvettes(2),4.5mL1cm.x1cm.
LensCloth(KimWipes)
SpecPhoneapparatuswithnecessaryadditions
LightSource(desklamp)
iPhoneoranothercamera-containingmobiledevice
Laboratorynotebook
Commercialspectrometer(ifavailable)
Introduction:
Thisprotocolprovidesstepsforcreatingabacterialcultureandmeasuringgrowth
withinaplasticcuvette.AstockcultureofE.coliiscreatedandthenusedtoinoculate
sterilemediatogrowanewculture.Atregularintervalsalongtheprocess,thecultureis
placedintheSpecPhoneandanabsorbancemeasurementistaken.Whenallthedatahas
beencollected,theabsorbancemeasurementsacrossthetimespancanbeusedtosolvefor
thenumberofbacteriausingtheequationofthetrendlineofastandardcurve.The
advantageofthisprotocolisareducedamountoftimeandresourcesneededfor
completion.
Astheculturegrows,itbecomesincreasinglyturbid.Thisincreasingturbidity
causesmorelighttobescatteredbytheculture,resultinginadecreaseintheintensityof
thelightrecordedbytheSpecPhoneLightscatterisdirectlydependentonthedensityof
bacterialcellspresentintheculture.Theamountoflightallowedtopassthroughthe
cultureisthepercenttransmission(%T),whichcanbeconvertedtoanabsorbancevalue
usingtheequation:A=-log(%T).Theconventionistomeasurebacterialculturesat600nm.
Aspecialnoteshouldbemadethatthisprotocolrequirestwodaysforcompletion.
Onedayisneededtocreatethestockbacterialcultureandallowittoincubateaswellas
preparations.Aseconddaywillbespenttakingtheactualmeasurements.
Appendix3.2
Procedure:
Day1:
CreationofStockBacterialCulture:
1. PrepareaculturetubefullofsterileTSB,andbringittoatemperatureof37°C.
2. IftheE.coliareinitiallyonabacterialplate,selectasinglecolonyfromtheplate
withaloopandinoculatetheTSB.Flametheloopbeforetouchingtheplateto
ensuresterility.IftheE.coliareinitiallyinbroth,pipette100μLofthecultureinto
9.9mLofwarmTSB.
3. Incubatethisculturefor24hours,shakingataspeedof265rpm,andata
temperatureof37°C.
Day2:
PreparationforExperiment:
1. Thisprotocolinvolvesgrowingabacterialcultureinacuvette.Fillaplasticcuvette
with3mLofwarmTSB,whichwillbelaterinoculatedtocreatetheculture.This
willbethesamplecuvette.
2. PreparetheSpecPhoneapparatus.Ensurethesurroundingsareasfreeofoutside
lightaspossible,andthatthelightsourcebeingusedisbrightenough.Placethe
mobiledeviceintheSpecPhoneandlaunchthecameraapp.
3. Noneofthecamerapixelsshouldbesaturatedbythelightsource,andthespectrum
shouldhaveevenintensityacrossthecolors.Onceadequateandappropriate
lightingisfound,tapethedeviceinplaceormakemarksonthesurfacetorecord
theproperpositioning.Takegreatcaretokeeptheapparatusstationary
throughoutthetrialstoensureaccuracyacrossallmeasurements.
4. Fillasecondcuvettewith3mLofTSB,thiswillserveastheblank.
TakingAbsorbanceMeasurements:
1. InoculatethesamplecuvetteofwarmTSBwith100μLofthepreviouslycreated
stockbacterialculture,transferringusingthep1000pipette.
2. Vortextoensuremixingandimmediatelybegintakingspectrumimageswiththe
SpecPhone.
3. PlacetheblankcuvetteintheSpecPhoneandcaptureafewspectrumimages.
Replacetheblankwiththeculturecuvette,andcaptureseveralmorespectrum
images.Recordtheexacttimeofmeasurementinthelabnotebook,aswellas
denotewhichpicturesinthecamerarollcorrespondtoblanksorculturesamples.
ThisistheT0measurement.
Tip:Itmaybeusefultophotographsomeobjectatthebeginningandendofthe
seriesofphotostodenotethesequenceinthephone’scameraroll.
4. Dependingontheavailabilityoflaboratoryequipment,therearetwooptionsfor
completingthisstep:
a. Ifashakingincubatorisavailable:Securethesamplecuvettewithtapeinthe
incubatorat37°Candaspeedof200rpm.
Appendix3.3
b. Ifastationaryincubatoristheonlyoption:Placethesamplecuvetteinthe
incubator,uncovered.At5-minuteintervals,removetheculturecuvetteand
vigorouslyshakebyhand,placingalidonthecuvettetopreventspilling.
Replacethecuvetteintheincubatoruntilitistimeforabsorbance
measurement.
5. At10-minuteintervals,removetheculturecuvettefromtheincubatorandtakea
seriesofabsorbancespectrumimages.Aseriesofcapturesoftheblankcuvette
shouldbedonebeforetheculturecuvetteisplacedinthedevice.Keepdetailed
recordsinthelaboratorynotebook,orusingthetableintheAppendix.
6. Thisprocesswillcontinueoveraperiodof2.5hours,withabsorbance
measurementsbeingtakenatthefollowingtimepoints:T0,T10,T20,T30,T40,T50,T60,
T70,T80,T90,T100,T110,T120,T130,T140,T150.
ImageAnalysis:
1. ImagescanbeimportedtoacomputercontainingImageJ(Rasband,2016)anda
photobrowser.FollowtheinstructionsforimageanalysisprovidedintheImage
ProcessingandCalibrationprotocol(Smith,2015).
2. Bycomparingtheratioofintensitiesfortheblankcuvetteandtheculturecuvette,
absorbancevaluesarecalculatedforthecultureat10-minuteintervals.Atableis
availableintheAppendixtoorganizethedata.
3. Fromtheseabsorbancemeasurements,thenumberofbacteriapresentinthe
cultureatthattimepointcanbedeterminedbyusingtheequationofalineofbest
fitforastandardcurve.Asamplestandardcurveappearsbelow.
Constructionofastandardcurverequiresdilutionsofthecultureatregular
timepointsandbacterialplating.Aseparateprotocol(CreatingaStandard
BacterialGrowthCurve)isavailablewhichoutlinestheprocedureforcreation
ofthestandardcurve.
4.00E+07
3.50E+07
y=6.39E+07x+4.53E+05
R²=0.984
3.00E+07
2.50E+07
y=4.85E+07x+1.01E+06
2.00E+07
R²=0.970
1.50E+07
1.00E+07
5.00E+06
CFU/mL
0.00E+00
0
GeneSys20
0.1
0.2
SpecPhone
0.3
0.4
0.5
0.6
0.7
Absorbance(600nm)
Linear(GeneSys20)
Linear(SpecPhone)
Appendix3.4
SafetyGuidelines:
Giventhatthisprotocoldealswithbacteria,thesafetyguidelinesaresomewhat
stricterthanwhencompletingtheotherSpecPhoneprotocols.Whenchoosingabacterial
straintoworkwith,werecommendK-12E.coli,asthisisastandardlaboratorystrainthat
hasbeendeterminedtobenon-pathogenicforhumansandothermammals(U.S.
EnvironmentalProtectionAgency,1997).
WorkingwithnonpathogenicorganismsfallsundertheBiosafetyLevel1(BSL-1)
guidelinesfromtheCDC.ExtensiveguidelinescanbefoundontheCDCwebsite(U.S.
CentersforDiseaseControlandPrevention,2009).Briefly,allpersonnelshouldwashtheir
handsbeforeenteringandleavingthelab,andwearglovesthroughouttheentire
procedure.Glovesshouldnotbewornoutsideofthelab,andshouldbeimmediately
replacedifanydamageoccurs.Ifpossible,wearinglabcoatsispreferabletoprotect
personalclothing.Nofoodordrinkshouldbekeptorconsumedintheareawherethe
experimentisbeingconducted.Attheendofthelabperiod,disinfectallsurfacesandany
equipmentthatcameincontactwiththebacterialcultures.Allstudentscompletingthe
protocolshouldbeinstructedtoreportanymistakes,spills,injuriesetc.tothelaboratory
coordinatorwhowilldecidetheappropriateaction.Anyindividualinstitutionpractices
thatextendbeyondtheseshouldbefollowedaswell.
ProtocolExpansions:
Thereareanumberofmodificationsthatcanbemadetothisprotocoltofurther
increasetheeducationalpotential.Onesuchmodificationwouldbetheadditionofan
antibiotictooneculture,andtothencomparethegrowthrateofthisculturetoonethatdid
notcontainthedrug.Ifamorenuancedcomparisonisdesired,differingclassesof
antibioticcouldbecompared.Forexample,oneculturecouldbesupplementedwitha
bactericidalantibiotic,whichwillkillthecellsandanothercouldbesupplementedwitha
bacteriostaticantibiotic,whichdoesnotkillthebacteriabutratherpreventsfurther
division.Twodifferentantibioticsofthesameclasscouldalsobecomparedtodetermineif
oneismoreeffectivethananother.
Anotherpotentialexpansionwouldbetosupplementoneculturewithaglucose
solutionandcompareitsgrowthtothatofanon-supplementedculture.Glucoseisthe
preferredcarbonsourceforE.coli,anditisabletoenterdirectlyintocentralmetabolism.
Thisadditionisexpectedtoincreasethegrowthrateofthesupplementedculture.Finally,
thegrowthratesofdifferingspeciesofbacteriacouldalsobecompared.Apotentially
interestingcomparisonwouldbebetweenaGram-negativespeciesandaGram-positive
species.However,thiswouldinvolvedifferentstrainsofbacteriathatcarrydifferingsafety
recommendations,whichshouldbecarefullyconsideredbeforeanyadditionaltrialsare
performedoranyprotocolmodificationsaremade.
Appendix3.5
References:
Rasband,W.S.,ImageJ,U.S.NationalInstitutesofHealth,Bethesda,Maryland,USA,https://imagej.nih.gov/ij/,
1997-2016.
Smith,A.W.(2015,August28).ProcessingSpecPhoneImagesandWavelengthCalibration.Retrieved
February16,2017,fromSpecPhoneProtocols:https://smithlab.uakron.edu/files/2015/09/Protocol-1Image-Processing-and-Calibration.pdf
U.S.CentersforDiseaseControlandPrevention.(2009).BiosafetyinMicrobiologicalandBiomedical
Laboratories(5thed.).(C.Chosewood,&D.Wilson,Eds.)Atlanta,Georgia.
U.S.EnvironmentalProtectionAgency.(1997,February).AttachmentI-FinalRiskAssessmentofEscherichia
coliK-12Derivatives.RetrievedFebruary24,2017,fromEPAUnitedStatesEnvironmentalProtectionAgency:
https://www.epa.gov/sites/production/files/2015-09/documents/fra004.pdf
Appendix3.6
Appendix:
Usethefollowingtablestoorganizethespectrumdataandtocreateagrowthcurve.The
actualtimeofmeasurementwillbethetimeontheclockwhentheabsorbance
measurementwastakenusingtheSpecPhone.
ActualTimeof
SpecPhone
TimePoint Measurement AbsorbanceValue
T0
T10
T20
T30
T40
T50
T60
T70
T80
T90
T100
T110
T120
T130
T140
T150