The Effects of The Effects of Chlorinated Water Chlorinated Water on Microbial Life on Microbial Life

Jeff Van KootenJeff Van Kooten99thth Grade Grade Central Catholic High SchoolCentral Catholic High School

Chlorine’s Use in Sanitation The most common substance used in water sanitation Solid, liquid, and gaseous form An important reaction occurs that allows sanitization

When chlorine (Cl2) is added to water (H20), a chemical reaction occurs that produces hypochlorous acid (HOCl) and hypochloric acid (HCI)

CI2+H20 HOCI+ HCI Hypochlorous acid is the active, killing form of chlorine When Hypochlorous encounters a nitrogen or ammonia, it

becomes a chloramine (which is not capable of sanitation)

Chlorinated WaterChlorinated Water

Primary factors affecting the disinfection Primary factors affecting the disinfection of water using chlorine include:of water using chlorine include:1. Type of chlorine1. Type of chlorine2. pH of pool water2. pH of pool water3. Water concentration of dissolved 3. Water concentration of dissolved contaminants in the poolcontaminants in the pool4. Water temperature4. Water temperature5. Duration of exposure 5. Duration of exposure

Chartiers ValleyChartiers Valley Pool Water pH level is between 7.8 - 8.2 Higher pH reduces the sanitizing power of the

chlorine due to reduced oxidation-reduction potential (ORP),

Lower pH causes discomfort to swimmers Kept around 40 ppm Once a week the pool undergoes a shock

treatment, uses chlorine in the form of sodium hypochlorite (bleach)

YeastYeast

Single-celled fungiSingle-celled fungi Eukaryotes even though they have some traits Eukaryotes even though they have some traits

of prokaryotesof prokaryotes Most commonly studied organism- similar Most commonly studied organism- similar

biochemistry and genes as other Eukaryotes biochemistry and genes as other Eukaryotes

E. coli E. coli

Common symbiont found in the colon of Common symbiont found in the colon of animals (human) animals (human)

Gram negative bacteria Gram negative bacteria Most commonly studied Prokaryote Most commonly studied Prokaryote Most strains non pathogenicMost strains non pathogenic Easy to culture and work with Easy to culture and work with

Purpose Purpose

To examine the effects of chlorinated To examine the effects of chlorinated water on Yeast and E. coli survivorshipwater on Yeast and E. coli survivorship

Hypothesis Hypothesis

Null Hypothesis: Null Hypothesis: Chlorinated pool Chlorinated pool water will not have water will not have a significant effect a significant effect on Yeast and E. on Yeast and E. coli survivorship. coli survivorship.

Alternative Alternative Hypothesis: Hypothesis: Chlorinated pool Chlorinated pool water will water will significantly reduce significantly reduce Yeast and E. coli Yeast and E. coli survivorship.survivorship.

MaterialsMaterials YEPD agar plates YEPD agar plates (YEPD media + (YEPD media +

1.5% agar)1.5% agar) YEPD media YEPD media (1% yeast extract, 2% (1% yeast extract, 2%

peptone, 2% glucose)peptone, 2% glucose) LB agar platesLB agar plates LB media (0.5% yeast extract, 1% LB media (0.5% yeast extract, 1%

tryptone, 1% sodium chloride)tryptone, 1% sodium chloride) Sterile Dilution Fluid [SDF] (100mM Sterile Dilution Fluid [SDF] (100mM

KHKH22POPO44, 100mM K, 100mM K22HPOHPO44, 10mM , 10mM

MgSOMgSO44, 1mM NaCI), 1mM NaCI) Sterile pipette tipsSterile pipette tips Micropipettes Micropipettes VortexVortex IncubatorIncubator

Sidearm flaskSidearm flask Spreading turntable Spreading turntable Spreader barSpreader bar Ethyl alcoholEthyl alcohol Sterile capped test tubes with Sterile capped test tubes with

Sterile distilled water.Sterile distilled water. Saccharomyces cerevisiae Saccharomyces cerevisiae

(Yeast) (Yeast) DH5-Alpha (E. coli)DH5-Alpha (E. coli) 0.22 micron syringe filters + 0.22 micron syringe filters +

10mL syringe10mL syringe Klett SpectrophotometerKlett Spectrophotometer Chlorinated pool waterChlorinated pool water

Procedure Procedure

1)1) Pool water was obtained from Chartiers Valley Swim Pool water was obtained from Chartiers Valley Swim Pool and sterile filtered with 0.22 micron syringe filtersPool and sterile filtered with 0.22 micron syringe filters

2) 2) YeastYeast and and E. coliE. coli were grown overnight in sterile were grown overnight in sterile YEPDYEPD and and LBLB media media

3)3) The cultures were placed in an incubator until a density The cultures were placed in an incubator until a density of 50 Klett spectrophotometer was reached. This is a of 50 Klett spectrophotometer was reached. This is a density of about 10density of about 1077 cells/mL cells/mL

4) The cultures were then diluted in Sterile Dilution Fluid to 4) The cultures were then diluted in Sterile Dilution Fluid to a concentration of 10a concentration of 1055 cells/mL cells/mL

5) The chlorinated water was diluted with sterile dilution 5) The chlorinated water was diluted with sterile dilution fluid to concentrations of 0%, 10%, 50%, and 99% (to fluid to concentrations of 0%, 10%, 50%, and 99% (to total 9.9 ml) 0.1 ml. of cell culture was then added to total 9.9 ml) 0.1 ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml, and a cell the test tubes, yielding a final volume of 10 ml, and a cell density of approximately 10density of approximately 103 3 cells/ml.cells/ml.

7

5

Procedure (Continued)Procedure (Continued)

Concentration ChartConcentration Chart

  0% 10% 50% 99%

Sterile Dilution Fluid

9.9 mL 8.9 mL 4.9 mL 0 mL

Microbe 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Chlorinated Pool Water

0 mL .1 mL 5 mL 9.9 mL

Total Volume

10 mL 10 mL 10 mL 10 mL

Procedure Procedure Continued Continued

6) The tubes were allowed to incubate at room temperature 6) The tubes were allowed to incubate at room temperature for 20 minutes.for 20 minutes.

7) 7) After vortexing to evenly suspend cells, 0.1 ml. aliquots After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread either YEDP or were removed from the tubes and spread either YEDP or LB agar plates. LB agar plates.

7) The E. coli was incubated for 24 hours at 37C and the 7) The E. coli was incubated for 24 hours at 37C and the yeast was incubated for 48 hours at 30Cyeast was incubated for 48 hours at 30C8) The resulting colonies were counted. Each colony is 8) The resulting colonies were counted. Each colony is assumed to have arisen from one cell.assumed to have arisen from one cell.

0

20

40

60

80

100

120

140

0% 10% 50% 99%

Pool waters effect on Yeast[c

olon

ies]

[Pool water]

P-value: .098755

113.66130.33

89.33

109.16

0

50

100

150

200

250

300

350

0% 10% 50% 99%

Pool waters effect on E. coli

[Col

onie

s]

[Pool water]

P-value: 5.66x10-6

299.83

214

161.5

101

Dunnett’s Test Table Dunnett’s Test Table

T crit.=3.10 Alpha=.05T crit.=3.10 Alpha=.05E. Coli E. Coli T valueT value InterpretationInterpretation 10% 10% .789 .789 not significant not significant 50%50% 2.934 2.934 not significantnot significant99%99% 4.74 4.74 SignificantSignificant

Yeast Analysis Yeast Analysis

The Anova test did not reveal significant The Anova test did not reveal significant variation among the means, thus variation among the means, thus Dunnett’s test could not be performed Dunnett’s test could not be performed

The null hypothesis has to be accepted, The null hypothesis has to be accepted, there was no evidence that the pool water there was no evidence that the pool water reduced yeast survivorshipreduced yeast survivorship

E. coli Analysis

Anova revealed significant variation between the Anova revealed significant variation between the means means

The Dunnett’sThe Dunnett’s test revealed test revealed significant significant variation variation withwith the 99% groupthe 99% group

The null hypothesis rejected only for the 99% The null hypothesis rejected only for the 99% groupgroup

Limitations and Extensions Limitations and Extensions

LimitationsLimitations Synergizing the spreading of plates more precisely Synergizing the spreading of plates more precisely Water might have had chloramines in it, making the water less Water might have had chloramines in it, making the water less

effectiveeffective

ExtensionsExtensions Increase trial size to 20 or 30 for more accurate resultsIncrease trial size to 20 or 30 for more accurate results exposure time to the water exposure time to the water Conduct test on different micro organisms Conduct test on different micro organisms Determine the solute concentrations in water, providing evidence Determine the solute concentrations in water, providing evidence

for or against osmotic shock for or against osmotic shock

References References

www.poolcenter.com/chlor.htmwww.poolcenter.com/chlor.htm www.pested.msu.edu/Resources/Bulletins/pdf/www.pested.msu.edu/Resources/Bulletins/pdf/

2621/e2621chap7.pdf2621/e2621chap7.pdf http://water.epa.gov/drink/contaminats/http://water.epa.gov/drink/contaminats/

basienformation/ecoli.cfm#fourbasienformation/ecoli.cfm#four http://ict-science-to-society.org/pathogenomics/http://ict-science-to-society.org/pathogenomics/

EcoliandSalmonella.htmEcoliandSalmonella.htm http://answer.yahoo.com/question/index?http://answer.yahoo.com/question/index?

quid=20070606205537AAygisgquid=20070606205537AAygisg

Yeast Anova Yeast Anova Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 6 786 131 528.8

Column 2 6 785 130.8333 382.5667

Column 3 6 636 106 200.4

Column 4 6 655 109.1667 725.3667

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 3296.833 3 1098.944 2.392737 0.098755 3.098391

Within Groups 9185.667 20 459.2833

Total 12482.5 23        

Anova: Single Factor

SUMMARY

Groups Count Sum Average Variance

Column 1 6 1379 229.8333 1080.167

Column 2 6 1284 214 1146.8

Column 3 6 969 161.5 293.5

Column 4 6 606 101 1899.2

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 61055.5 3 20351.83 18.41934 5.66E-06 3.098391

Within Groups 22098.33 20 1104.917

Total 83153.83 23        

E. Coli AnovaE. Coli Anova