UVandAppleCiderVinegarInteractionEffectsonFlora

OscarShaverGrade11

CentralCatholicHighSchool

ElectromagneticSpectrum• Rangeofalltypesoffrequenciesofelectromagneticradiation• Thetypesofradiationareradiowaves,microwaves,infrared,visible,ultraviolet,x-rays,andgammarays

UVLightRays• Ultraviolet(UV)raysarelightraysthathaveshorterwavelengthsthanvisiblelight• Rangefrom150nm–300nm• Theyarenaturallygivenoffbythesun,butmostareabsorbedbytheozonelayer• Thewavelengthusedinthisexperimentis254nmlight

AppleCiderVinegar• Bragg‘sAppleCiderVinegar(unfiltered)Ø FermentedrawapplejuiceØ UsesfromcookerytohealthbenefitsØ Internetphenomenon- manyclaimsØ Contains:vitaminB,vitaminC,folicacid,magnesium,potassium,iron,calcium,andbiotin

ØpH:3.075

VitaminCandUVlight• FreeradicalsinducedbyUVexposureØ createsoxidativestressØ chainreactionofdamagingcells‘sDNA• VitaminCisanantioxidantØ inhibitsoxidationØ reducesoxidativestress

Staphylococcusepidermidis

• Grampositivebacteria• Partofnormalhumanflora• Foundonskin• Coccalbacteria• Whenincubated,formswhitecolonies

Purpose• TodeterminetheinteractioneffectsofapplecidervinegarandUVlightonStaphsurvivorship

Hypotheses

Null:ApplecidervinegarwillnothelptoincreasesurvivorshipofUVstressedStaph

Alternate:ApplecidervinegarwillhelptoincreasesurvivorshipofUVstressedStaph

Materials(DirectExposure)• YEPDagarplates(YEPDmedia+1.5%agar)

• YEPDmedia(1%yeastextract,2%peptone,2%glucose)

• Incubator• Ethanol

• Spreaderbars• 0.2MicronSterileSyringeFilter

• Staphylococcusepidermidis

• Bragg‘sAppleCiderVinegar(unfiltered)• UVlamp(254nmlight)

• Testtubes• SterileDilutionFluid[SDF](100mMKH2PO4,100mMK2HPO4,10mMMgSO4,1mMNaCl)

• Sterilepipettetips

• Micropipettes• Sterilecappedtesttubeswithsteriledistilledwater

• Sidearmflask

Procedures(DirectExposure)1. Bacteria(Staph)wasgrownovernightinsterileYEPDMedia2. Asampleoftheovernightculturewasaddedtofreshmediaina

sterilesidearmflask3. Theculturewasplacedinanincubator(37°C)untiladensityof50

Klett spectrophotometerunitswasreached.Thisrepresentsacelldensityofapproximately10⁸cells/mL

4. Theculturewasdilutedinsteriledilutionfluidtoaconcentrationofapproximately10⁵cells/mL

5. Applecidervinegarwassterilefilteredusing0.2micronsterilesyringefilter

6. ApplecidervinegarwasaddedtotesttubestocreatethefollowingpH’s:4and5

ConcentrationChartFinalConcentrationinTubes

0%(pH6.5) 2%(pH5) 5%(pH4)

SterileWater 9.9ml 9.7ml 9.4ml

Bacteria 0.1ml 0.1ml 0.1ml

AppleCiderVinegar 0ml 200microliters 500microliters

TotalVolume 10ml 10ml 10ml

Procedurescontinued7. 0.1mlofcellculturewasthenaddedtothetesttubes,yieldingafinal

volumeof10mL.8. Thesolutionsweremixedbyvortexing andallowedtositatroom

temperaturefor10minutes9. Aftervortexing toevenlysuspendcells,0.1mlwasremovedfromthe

tubesandspreadonYEPDplates10. PlateswerethenplacedunderaUVlampforcertaintimes(0,4,and8

seconds)11. Theplateswereincubatedat37°Cfor24hours12. Theresultingcolonieswerecounted.Eachcolonyisassumedtohave

arisenfromonecell

pHandRadiationExposure

00

L0

H0

0L

LL

HL

0H

LH

HH

6.5pH(0) 5pH(L) 4pH(H)

0sec

4sec(L)

8sec(H)

P-valueInteraction:0.035003

P-value:0.040385

P-value:1.47E-05

Procedures(LiquidExposure)1. Bacteria(Staph)wasgrownovernightinsterileYEPDMedia2. Asampleoftheovernightculturewasaddedtofreshmediaina

sterilesidearmflask3. Theculturewasplacedinanincubator(37°C)untiladensityof50

Klett spectrophotometerunitswasreached.Thisrepresentsacelldensityofapproximately10⁸cells/mL

4. Theculturewasdilutedinsteriledilutionfluidtoaconcentrationofapproximately10⁵cells/mL

5. Applecidervinegarwassterilefilteredusing0.2micronsterilesyringefilter

6. ApplecidervinegarwasaddedtotesttubestocreatethefollowingpH’s:4and5

Procedurescontinued7. 0.1mlofcellculturewasthenaddedtothetesttubes,yieldinga

finalvolumeof10mL8. Thesolutionsweremixedbyvortexing andallowedtositatroom

temperaturefor10minutes9. Aftervortexing toevenlysuspendcells,800microliters were

removedandaddedtomicrotubes10. Openedmicrotubes werethenplacedunderaUVlampforcertain

times(0,4,and8seconds)11. Afterexposure,0.1mlwasremovedfromthemicrotubes and

spreadonYEPDplates12. Theplateswereincubatedat37°Cfor24hours13. Theresultingcolonieswerecounted.Eachcolonyisassumedto

havearisenfromonecell

P-valueInteraction:5.27e-12

P-value:1.66E-05

P-value:7.5E-09

Conclusions

• Thenullhypothesiscanberejectedforeverygroupinbothdirectandliquidexposureexperiments• ItcanbeinferredthatapplecidervinegarhadaprotectioneffectagainsttheUVstressedStaph

ØApplecidervinegarandUVlightappearedtoreducestaphsurvivorshipindividually

Limitations

• Spreadplatingwasnotperformedinperfectsynchronization,thereforesomebacteriahadaslightlyhigherexposuretime• Onlyonespeciesused• Onlysurvivorshipassessed• Platingwasdoneinroomwithpartialsunlight

Extensions

• Testandcompareotherliquidscontainingvitaminc• Morespeciesofbacteriawillbeused• Spreadplatingwillbeperformedmoresynchronously• UVexposuretimewillbeatsmallerincrements

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LibraryofMedicine,Feb.2000.Web.24Jan.2018.Lucas,Jim."WhatIsUltravioletLight?"LiveScience.TechMediaNetwork,31.Mar.2015.Web.23Nov.

2017."StaphylococcusEpidermidis."- Wikipedia,theFreeEncyclopedia.N.p.,n.d.Web.23Nov.2017.“Sunburn." MayoClinic.MayoFoundationforMedicalEducationandResearch,01June2017.Web.

Nov.2017."TypesOfUVLight- UVC,UVB,UVA ." AmericanAir&WaterUVLightAirCleanersand Ultraviolet

WaterPurifiers.N.p.,n.d.Web.24Jan.2018.“UV&FreeRadicals." Albus&Flora.N.p.,n.d.Web.24Jan.2018."VitaminCandSkinHealth." LinusPaulingInstitute.N.p.,01Jan.2018.Web.24Jan. 2018."WhatIsGermicidalUltraviolet?|Ultraviolet.com."Ultraviolet.com.N.p.,n.d.Web.23Nov.2017.

2FactorANOVA(DirectExposure)

2FactorANOVA(LiquidExposure)