The Effects of Chlorinated Water on Microbial Life

The Effects of Chlorinated Water on Microbial Life Jeff Van Kooten 9 th Grade Central Catholic High School

Chlorine’s Use in Sanitation l l l The most common substance used in water sanitation Solid, liquid, and gaseous form An important reaction occurs that allows sanitization l When chlorine (Cl 2) is added to water (H 20), a chemical reaction occurs that produces hypochlorous acid (HOCl) and hypochloric acid (HCI) l CI 2+H 20 HOCI+ HCI l Hypochlorous acid is the active, killing form of chlorine l When Hypochlorous encounters a nitrogen or ammonia, it becomes a chloramine (which is not capable of sanitation)

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

Chartiers Valley Pool Water l l l p. H level is between 7. 8 - 8. 2 Higher p. H reduces the sanitizing power of the chlorine due to reduced oxidation-reduction potential (ORP), Lower p. H 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 l l l Single-celled fungi Eukaryotes even though they have some traits of prokaryotes Most commonly studied organism- similar biochemistry and genes as other Eukaryotes

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

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

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

Materials l l l l l YEPD agar plates (YEPD media + 1. 5% agar) YEPD media (1% yeast extract, 2% peptone, 2% glucose) LB agar plates LB media (0. 5% yeast extract, 1% tryptone, 1% sodium chloride) Sterile Dilution Fluid [SDF] (100 m. M KH 2 PO 4, 100 m. M K 2 HPO 4, 10 m. M Mg. SO 4, 1 m. M Na. CI) Sterile pipette tips Micropipettes Vortex Incubator § § § § § Sidearm flask Spreading turntable Spreader bar Ethyl alcohol Sterile capped test tubes with Sterile distilled water. Saccharomyces cerevisiae (Yeast) DH 5 -Alpha (E. coli) 0. 22 micron syringe filters + 10 m. L 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 7 of 50 Klett spectrophotometer was reached. This is a density of about 107 cells/m. L 5 4) The cultures were then diluted in Sterile Dilution Fluid to a concentration of 105 cells/m. L 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 103 cells/ml.

Procedure (Continued) Concentration Chart 0% 10% 50% 99% Sterile Dilution Fluid 9. 9 m. L 8. 9 m. L 4. 9 m. L 0 m. L Microbe 0. 1 m. L Chlorinated Pool Water 0 m. L . 1 m. L 5 m. L 9. 9 m. L Total Volume 10 m. L

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

[colonies] Pool waters effect on Yeast P-value: . 098755 130. 33 113. 66 109. 16 89. 33 [Pool water]

Pool waters effect on E. coli P-value: 5. 66 x 10 -6 [Colonies] 299. 83 214 161. 5 101 [Pool water]

Dunnett’s Test Table T crit. =3. 10 Alpha=. 05 E. Coli T value Interpretation 10% 50% 99% . 789 2. 934 4. 74 not significant Significant

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

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

Limitations and Extensions l l l Limitations Synergizing the spreading of plates more precisely Water might have had chloramines in it, making the water less effective Extensions Increase trial size to 20 or 30 for more accurate results exposure time to the water Conduct test on different micro organisms Determine the solute concentrations in water, providing evidence for or against osmotic shock

References l l l www. poolcenter. com/chlor. htm www. pested. msu. edu/Resources/Bulletins/pdf/2621/ e 2621 chap 7. pdf http: //water. epa. gov/drink/contaminats/basienformati on/ecoli. cfm#four http: //ict-science-tosociety. org/pathogenomics/Ecoliand. Salmonella. htm http: //answer. yahoo. com/question/index? quid=2007 0606205537 AAygisg

Yeast 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 Between Groups 3296. 833 3 1098. 944 Within Groups 9185. 667 20 459. 2833 12482. 5 23 Total F P-value 2. 392737 F crit 0. 098755 3. 098391

E. Coli 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 Between Groups SS df MS F 18. 41934 61055. 5 3 20351. 83 Within Groups 22098. 33 20 1104. 917 Total 83153. 83 23 P-value F crit 5. 66 E-06 3. 098391