The Effects of Chlorinated Water on Microbial Life Jeff Van Kooten 9 th Grade Central 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 (Cl 2 ) is added to water (H 2 0), a chemical reaction occurs that produces hypochlorous acid (HOCl) and hypochloric acid (HCI) CI 2 +H 2 0 HOCI+ HCI Hypochlorous acid is the active, killing form of chlorine When Hypochlorous encounters a nitrogen or ammonia, it b ecomes a chloramine (which is not capable of sanitation)

Chlorinated Water Primary factors affecting the disinfection of water using chlorine include: Primary factors affecting the disinfection of water using chlorine include: 1. Type of chlorine 2. pH of pool water 3. Water concentration of dissolved contaminants in the pool 4. Water temperature 5. Duration of exposure

Chartiers Valley Chartiers Valley Pool Water pH level is between 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)

Yeast Single-celled fungi Single-celled fungi Eukaryotes even though they have some traits of prokaryotes Eukaryotes even though they have some traits of prokaryotes Most commonly studied organism- similar biochemistry and genes as other Eukaryotes Most commonly studied organism- similar biochemistry and genes as other Eukaryotes

E. coli Common symbiont found in the colon of animals (human) Common symbiont found in the colon of animals (human) Gram negative bacteria Gram negative bacteria Most commonly studied Prokaryote Most commonly studied Prokaryote Most strains non pathogenic Most strains non pathogenic Easy to culture and work with Easy to culture and work with

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

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

Materials YEPD agar plates (YEPD media + 1.5% agar) YEPD agar plates (YEPD media + 1.5% agar) YEPD media (1% yeast extract, 2% peptone, 2% glucose) YEPD media (1% yeast extract, 2% peptone, 2% glucose) LB agar plates LB agar plates LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Sterile Dilution Fluid [SDF] (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCI) Sterile Dilution Fluid [SDF] (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCI) Sterile pipette tips Sterile pipette tips Micropipettes Micropipettes Vortex Vortex Incubator Incubator  Sidearm flask  Spreading turntable  Spreader bar  Ethyl alcohol  Sterile capped test tubes with Sterile distilled water.  Saccharomyces cerevisiae (Yeast)  DH5-Alpha (E. coli)  0.22 micron syringe filters + 10mL syringe  Klett Spectrophotometer  Chlorinated pool water

Procedure 1)Pool water was obtained from Chartiers Valley Swim Pool and sterile filtered with 0.22 micron syringe filters 2) Yeast and E. coli were grown overnight in sterile YEPD and LB media 3)The cultures were placed in an incubator until a density of 50 Klett spectrophotometer was reached. This is a density of about 10 7 cells/mL 4) The cultures were then diluted in Sterile Dilution Fluid to a concentration of 10 5 cells/mL 5) The chlorinated water was diluted with sterile dilution fluid to concentrations of 0%, 10%, 50%, and 99% (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 density of approximately 10 3 cells/ml. 7 5

Procedure (Continued) Concentration Chart 0%10%50%99% Sterile Dilution Fluid 9.9 mL8.9 mL4.9 mL0 mL Microbe0.1 mL Chlorinated Pool Water 0 mL.1 mL5 mL9.9 mL Total Volume10 mL

Procedure Continued 6) The tubes were allowed to incubate at room temperature for 20 minutes. 7) After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread either YEDP or LB agar plates. 7) The E. coli was incubated for 24 hours at 37C and the yeast was incubated for 48 hours at 30C 8) The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

Pool waters effect on Yeast [ colonies] [Pool water] P-value:

Pool waters effect on E. coli [ Colonies] [Pool water] P-value: 5.66x

Dunnett’s Test Table T crit.=3.10 Alpha=.05 E. Coli T valueInterpretation 10%.789not significant 50% 2.934not significant 99% 4.74Significant

Yeast Analysis The Anova test did not reveal significant variation among the means, thus Dunnett’s test could not be performed The Anova test did not reveal significant variation among the means, thus Dunnett’s test could not be performed The null hypothesis has to be accepted, there was no evidence that the pool water reduced yeast survivorship The null hypothesis has to be accepted, there was no evidence that the pool water reduced yeast survivorship

E. coli Analysis Anova revealed significant variation between the means Anova revealed significant variation between the means The Dunnett’s test revealed significant variation with the 99% group The Dunnett’s test revealed significant variation with the 99% group The null hypothesis rejected only for the 99% group The null hypothesis rejected only for the 99% group

Limitations and Extensions Limitations 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 effective Water might have had chloramines in it, making the water less effectiveExtensions Increase trial size to 20 or 30 for more accurate results Increase 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 for or against osmotic shock Determine the solute concentrations in water, providing evidence for or against osmotic shock

References e2621chap7.pdf e2621chap7.pdf e2621chap7.pdf e2621chap7.pdf on/ecoli.cfm#four on/ecoli.cfm#four on/ecoli.cfm#four on/ecoli.cfm#four society.org/pathogenomics/EcoliandSalmonella.htm society.org/pathogenomics/EcoliandSalmonella.htm society.org/pathogenomics/EcoliandSalmonella.htm society.org/pathogenomics/EcoliandSalmonella.htm AAygisg AAygisg AAygisg AAygisg

Yeast Anova Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups Within Groups Total

Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Column Column Column Column ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups E Within Groups Total E. Coli Anova