AmmoniumHydroxideandOrangeJuiceSynergisticEffects

onEscherichiacoliJohnLynch

PittsburghCentralCatholicHighSchoolGrade9

pHpHScale

• Measureshowacidicorbasicasubstanceis• Scalefrom0-14• pH<7acidic,pH>7basic,pH=7neutral

Neutralization

• Themixtureofanacidandabase.

• Mixtureofacidandbasecommonlyyieldswater

NavelOrangeJuice

• Verycommonlysoldorange

• MeasuredpHof3(raw)

• ContainslargeamountsofVitaminAandC• Mayhelpanimalsformconnectivetissue• AlsocontainsmanyotherVitamins

ComponentsofOrangeJuice

• Sugars• Proteinsandaminoacids• VitaminsA,C,D,E,K,B12,B6• Thiamin• Pantotheic Acid• Sodium• Potassium

• Water• Mono/Polysaturated fats• Omega-3fattyacids• Calcium• Iron• Magnesium• Phosphorus

AmmoniumHydroxide

• Basicliquid• MeasuredpHof10(2Molar)

• Verycorrosiveandantibacterial• Usedatverysmallmolaritiesincleaners

• ReleasesAmmoniavaporswhenmixedwithanacidorwater.• Toxictohumans athighconcentrations

Escherichiacoli(E.coli)

• Largeanddiversegroupofgram(-)bacteria• Freeliving,symbionts,orpathogens• Moststrainsarenotpathogenic• Serveasacommonprokaryoticcellmodel• Commonmammalian intestinalsymbiont• Environmentalprokaryoticmodel

Rationale

• IndividualOrangeJuice:• Orangefallingfromtree• Disposalofpeels

• IndividualAmmoniumHydroxide• DisposalofAmmonium-basedcleaners

• Mixtureofboth:• Possiblesynergisticeffects• PossibleremedyforAmmonium-basedcleanerspills

Purpose

• TodeterminetheindividualandsynergisticeffectsofNavelorangejuiceandAmmoniumHydroxideonE.Colisurvivorship.

Problem

• WilltherebeasignificanteffectwhenAmmoniumHydroxide,Navelorangejuice,oramixtureofbothisaddedtoEscherichiacoli?

Hypothesis

AlternativeHypothesis:

AmmoniumHydroxideandOrangejuicewillsignificantlyreduceE.colisurvivorship.

Incombination,asynergisticeffectwillimprovesurvivorshipcomparedtoindividualexposures.

Materials

• Borosilicatesteriletesttubes• LatexGloves• LaboratoryGoggles• Micropipettes• SidearmFlask• SterileFilters• Incubator(37o C)• VortexMachine• E.coli(DH5Alpha)• LBAgarPlates(LBMedia)(1%Tryptone,0.5%Yeastextract,1%NaCl)• Spreadbars

• Ethanol• pHstrips• Matches• SterileDilutionFluid(SDF)(100mMKH2PO4,100mMK2HPO4,10mMMgSO4,1mMNaCl)• NavelOrangeJuice• Juicer• Knife• 2MolarAmmoniumHydroxide• SterileDilutionFluid• BunsenBurner

Procedure

1. E.coliwasgrownuntiladensityof50klett spectrophotometerdensitywasreached.Thiswasapproximately10^8cells/mL

2. Theculturewasdilutedinsteriledilutionfluidtoaconcentrationofapproximately10^5cells/mL

3. Stocksofthevariableswerecreated;

1. 10mLoforangejuicewassterilefiltered

2. A10-2 stockofAmmoniumHydroxidewasmade

4. Thevariableswereaddedtothetubesasfollows:

ExperimentalExposuresAmmoniumHydroxide(10-2stock)Zero Low High

Zero 0mL 0mL 0mL Key:0mL 10uL 1mL OrangeJuice

OrangeJuice 100uL 100uL 100uL AmmoniumHydroxide9.9mL 9.89mL 8.8mL Escherichiacoli

Low 100uL 100uL 100uL SterileDilutionFluid0mL 10uL 1mL100uL 100uL 100uL Concentrationin%:9.8mL 9.79mL 8.8mL Low High

High 1mL 1mL 1mLAmmoniumHydroxide 0.00005% 0.005%

0mL 10uL 1mLOrangeJuice 1% 10%

100uL 100uL 100uL8.9mL 8.89mL 7.9mL

5. 0.1mLofE.colistockwasaddedtoeachtube,andallowedtoincubateatroomtemperaturefor15minutes

6. 0.1mLofthealiquotswerespreadontotheagarplates

7. Theplateswerethenincubatedfor24hoursandthecolonieswerecounted

179

153

136

179

138

115

100110120130140150160170180190

[Zero] [Low] [High]

Numbe

rofC

olon

ies

ConcentrationLevels

IndividualVariable’sEffects

OrangeJuice AmmoniumHydroxide

P- value:0.024806

P-value:0.008053

Alpha:0.05

SynergisticEffectsonE.Coli

020406080100120140160180200

ZeroOrangeJuice LowOrangeJuice HighOrangeJuice

Num

bero

fColon

ies

AveragesofAllSolutions

ZeroAmmoniumHydroxide LowAmmoniumHydroxide HighAmmoniumHydroxide

DataAnalysis:Two-factorANOVA

• Two-WayANOVAforbothVariables:• InteractionP-value:Yes,thepvaluewasat0.000262

• Interpretation:Rejectnull;therewasasignificanteffect

• Typeofeffect:Themixedvariablesappearedtoreducethenegativeeffects

Alpha:0.05

FurtherInterpretation

AVERAGESOFCOLONIES

AmmoniumHydroxide

Zero Low High

OrangeJuice

Zero 179 138 115

Low 153 151 151

High 136 145 179

pHChartandInterpretation

• pHsolelydoesn’tseemtobetheaffectingfactor

pHLevels

AmmoniumHydroxide

Zero Low High

Orange Zero 7 7 7

Juice Low 7 7 7High 6 6 6

KeyQuestions

• DidtheNavelOrangeJuicealonehaveasignificanteffectonE.coli?• Yes,p-valuewaslessthan0.05

• DidtheAmmoniumHydroxidealonehaveasignificanteffectonE.coli?• Yes,p-valuewaslessthan0.05

• DidthemixtureofbothhaveasignificanteffectontheE.coli?• Yes,p-valuewaslessthan0.05

OverallConclusions

• Therewerenegativeindividualeffectsofthevariables,butapositivesynergisticeffect.

LimitationsandExtensions

Limitations:

• Onlyoneexposuretime• Chanceofcontamination• UnevendistributionofE.coliinsolution• Smallexposuretolight• Narrowrangeofvariableconcentrations

Extensions:

• Varyexposuresandconcentrations• Morereplicates• Otherculturesofbacteria• Differenttypesoforangejuice• Othersynergisticeffects

Sources

• "NutritionFacts."AndAnalysisforOrangeJuice,Raw.N.p.,n.d.Web.11Jan.2016.

• "PHScale."PHScale.N.p.,n.d. Web.15Dec.2015.http://chemistry.elmhurst.edu/vchembook/184ph.html

• "#1DentistRecommendedToothpasteFor Sensitivity."Sensodyne ToothpasteforSensitiveTeeth.N.p.,n.d. Web.15Dec.2015.

• us.sensodyne.com

• "Molarity."Molarity.N.p.,n.d. Web.15Dec.2015.

• http://dl.clackamas.edu/ch105-04/molarity.htm

• "Aqion."Aqion.N.p.,n.d. Web.15Dec.2015.

• http://www.aqion.de/site/191

• "OrangeFruitNutritionFactsandHealthBenefits."NutritionAndYou.com.N.p.,n.d. Web.16Dec.2015.

• http://www.nutrition-and-you.com/orange-fruit.html

• OfficeofDietarySupplements- VitaminA."VitaminA— Consumer.N.p.,n.d. Web.16Dec.2015.

• https://ods.od.nih.gov/factsheets/VitaminA-Consumer/

• "AmmoniumHydroxide."AmmoniumHydroxide.N.p.,n.d. Web.16Dec.2015.

• http://pubchem.ncbi.nlm.nih.gov/compound/ammonium_hydroxide#section=Metabolism-Metabolites

• "BugsintheNews- WhattheHeckIsanE.Coli?"BugsintheNews- WhattheHeckIsanE.Coli? N.p.,n.d. Web.16Dec.2015.

• http://people.ku.edu/~igmdoc/ecoli.html

• "ResultFilters."NationalCenterforBiotechnologyInformation.U.S.NationalLibraryofMedicine,n.d. Web.16Dec.2015.

• http://www.ncbi.nlm.nih.gov/pubmed/21558695

• "QuestionsandAnswersaboutAmmoniumHydroxideUseinFoodProduction."FoodInsight.org.N.p.,n.d. Web.18Dec.2015.

• http://www.foodinsight.org/Questions_and_Answers_about_Ammonium_Hydroxide_Use_in_Food_Production

DataChartAmmoniumHydroxide

Zero Low High

Zero 193 179 88127 107 118195 130 115

Orange Juice 174 217 111148 134 116

Low 140 145 237240 163 178139 158 132169 136 144165 105 149

High 119 55 177130 157 171147 167 196165 151 170147 105 128

One-WayANOVA:AmmoniumHydroxideSUMMARYGroup Count Sum Average VarianceC1 4 710 177.5 476.333C2 4 550 137.5 907C3 4 460 115 8.66667

ANOVASource SS df MS Ftest P-value F-CriticalBetween 8016.67 2 4008.33 8.63865 0.008053 4.2565Within 4176 9 464Total 12192.7 11

ConclusionsHo: μ1=μ2=μ3=...Ha Atleast2μ'sdifferF-Test 8.63865F-Critical 4.2565Alpha 0.05P-Value 0.008053Decision: RejectHo

One-WayANOVA:Orange JuiceSUMMARYGroup Count Sum Average VarianceC6 4 710 177.5 476.333C7 4 613 153.25 254.917C8 4 543 135.75 188.917

ANOVASource SS df MS Ftest P-value F-CriticalBetween 3516.5 2 1758.25 5.73238 0.024806 4.2565Within 2760.5 9 306.722Total 6277 11

ConclusionsHo: μ1=μ2=μ3=...Ha Atleast2μ'sdifferF-Test 5.73238F-Critical 4.2565Alpha 0.05P-Value 0.024806Decision: RejectHo

TwoFactorANOVAWithReplicationa

Summary O L H Total

O

Count 4 4 4 12

Sum 710 550 460 1720

Average 177.5 137.5 115 143.333

Variance 476.333 907 8.66667 1108.42

L

Count 4 4 4 12

Sum 613 602 603 1818

Average 153.25 150.5 150.75 151.5

Variance 254.917 151 380.917 216.273

H

Count 4 4 4 12

Sum 543 580 714 1837

Average 135.75 145 178.5 153.083

Variance 188.917 754.667 145.667 664.992

ANOVA

Source SS df MS Ftest P-value F-Critical

Samples(rows) 657.056 2 328.528 0.904735 a 3.35413

Columns 775.056 2 387.528 1.06722 0.358032 3.35413

Interaction 11307.3 4 2826.82 7.7848 0.000262 2.72777

Within 9804.25 27 363.12

Total 22543.6 35

Conclusions

InteractionHo: NOInteractionHa InteractionExistsF-Test 7.7848F-Critical 2.72777Alpha 0.05P-Value 0.000262Decision: RejectHo

Samples(rows)Ho: μ1=μ2=μ3=...Ha Atleast2μ'sdifferF-Test 0.904735F-Critical 3.35413Alpha 0.05P-Value 0.416567Decision: FailtorejectHo

ColumnsHo: μ1=μ2=μ3=...Ha Atleast2μ's differF-Test 1.06722F-Critical 3.35413Alpha 0.05P-Value 0.358032Decision: FailtorejectHo