Effect of Bacterial Growth Stage on Chlorine Disinfection Project By: Warren Sipe Grade 9 Central Catholic High School

Chlorine Bleach -Common household disinfectant -Active ingredients -Sodium hypochlorite -Sodium chlorate -Sodium hydroxide -Sodium polyacrylate -Sodium carbonate

Sodium Hypochlorite -NaClO -Active disinfectant in chlorine bleach -Results when chlorine gas is mixed with sodium hydroxide Cl2 + 2NaOH →NaClO + NaCl + H2O NaClO (s) + H2O (l) → HOCl (aq) + NaOH- (aq) -Kills bacteria by denaturing their proteins

Bacterial Growth Curve

Model Bacteria (E. coli) -Extremely common bacteria -Frequently used for scientific research -Rod-shaped microflora -Hundreds of species -Measure of water quality -Some strains can be extremely harmful

Gram Positive vs. Gram Negative - simple, thick membrane - most human pathogens - antibiotics such as penicillin prevent linking of peptidoglycan and formation of cell wall Gram Negative (E. coli) - cell membrane of peptidoglycan and lipid membrane - Outer membrane is a thin extra layer of lipopolysaccharide - Outer membrane protects the bacteria from several antimicrobials

Past Studies - Resistance - Persister cells- slower growing cells that are resistant - Bacterial quorum- regulation of gene expression according to perceived surrounding population density - Discovered other factors in resistance -Chemical reasons -Biological reasons

Interaction of Components - Chlorine is very effective against E. coli - Sodium hypochlorite- used in water treatment - Quorum sensing could cause a change that increases resistance to chlorine - SRT- how long activated sludge is kept in the treatment system - A long SRT could increase sewage’s resistance to disinfection -E. coli serves as a model for other gram negative bacteria

Purpose - To determine the effect of bacterial growth stage on percent survivorship of E. coli through chlorine disinfection - To determine whether bacterial growth stage has an effect on the number of viable cells in the population

Hypothesis -Null hypothesis- Bacterial growth stage will have no significant effect on bacterial survivorship, with chlorine disinfection or without it -Alternate hypothesis- Bacterial growth stage will have a significant effect on bacterial survivorship, both with chlorine disinfection and without it

Materials -E. coli -Klett Spectrophotometer (reading in KU) -Clorox bleach -LB agar plates -LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) -Sterile dilution fluid (100 mM KH2PO4, 100 mM K2HPO4, 10 mM MgSO4, 1mM NaCl) -Sterile pipette tips -Micropipettes -Vortex -Incubator (37 degrees C) -Sidearm flask -Sterile spreader bars -Ethanol -Sterile capped test tubes with sterile distilled water

Procedures Parallel cultures were grown to different growth stages (50 KU log phase, 100 KU early stationary phase, 150 KU late stationary phase). The cultures were synchronized to a KU reading of 50 on a klett spectrophotometer by dilution in LB media. Tubes were prepared according to the chart below and incubated for 3 minutes at room temperature. 4. 0.1 mL aliquots of the samples were plated synchronously in LB media plates. 7 replicates were performed of each tube. 5. Plates were incubated at 37 degrees C overnight and quantified. Bleach Tube No Bleach Tube Sterile Fluid 9.8 mL Bleach stock (10-2 %) 0.1 mL 0 mL Diluted E. Coli culture Total 10 mL

ANOVA - Analysis of Variance- multiple mean comparison - Statistical test comparing variation between and within groups - P-Value is smaller than the Alpha Value (0.05)= variation is significant

Data No bleach variation P= 8.54E-6 Interaction P- 2.2E-13 Significant Bleach variation P=2.78E-8 Interaction P= 2.2E-13

Analysis and Stats Did the bacterial growth stage cause significant variation in percent survivorship through chlorine disinfection? -Two Factor Anova- Interaction- P of 2.2E-13/ Significant -Accept alternate hypothesis Did bacterial growth stage cause significant variation in numerical survivorship through chlorine disinfection? -Single Factor Anova for bleach column- P of 2.74E-8/ Significant Did bacterial growth stage have a significant effect on the fraction of viable cells in the population? -Single Factor Anova for no bleach column-P of 8.54E-6/ Significant

Conclusions - The null hypothesis was rejected - The alternate hypothesis was supported - E. Coli showed greater resistance to chlorine bleach at higher growth stages - Unstressed bacterial survivorship was lower at higher growth stages

Possible Limitations - Spread plating was not perfectly synchronized - Only survivorship, and not growth, was measured - Only E. coli was tested - Only chlorine bleach was tested - Only one concentration of bleach was used

Extensions - Efforts will be made to achieve more synchronous spread plating - Survivorship and growth will be measured - More species will be tested - Tests will be done at more stages of growth - Other disinfectants will be tested - More concentrations of bleach will be tested

Resources -“How to Count Bacteria.” disknet. n.p., n.d. Web. 5 October 2014. -“Culturing E. Coli.” newton.dep. US Department of Energy Office of Science, n.d. Web. 5 October 2014. -“Your Disinfection Team: Chlorine and pH.” cdc.gov. US Center for Disease Control and Prevention, 23 April 2013. Web. 9 November 2014. -“Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008.” cdc.gov. US Center for Disease Control and Prevention, 29 December 2013. Web. 9 November 2014.

Resources (cont.) -¨E. coli (Eschericia coli).¨ cdc.gov. US Center for Disease Control and Prevention, 29 December 2013. Web. 12 November 2014. - Krotec, Mark. Personal interview. 28 October, 2014. -Cherchi, C., and A.Z. Gu. Effect of Bacterial Growth Stage on Resistance to Chlorine Disinfection. Diss. Northwestern University, 2011. Web.

Bleach Kill Curve Concentration of Bleach Avg. Number of Survivors 489.25 10-5 611.5 10-4 537 10-3.5 6.3 10-3

Data 145% 28% 50 KU Bleach No Bleach Difference in Survivorship Percentage 185 598 145% 153 454 220 532 278 554 241 545 258 586 263 530 Total 1335 3269 Avg. 222.5 544.8333333 100 KU Bleach No Bleach Difference in Survivorship Percentage 374 517 28% 408 437 344 456 368 443 346 452 348 476 501 Total 2556 3282 Avg. 365.1428571 468.8571429

Data (cont.) 7% 150 KU Bleach No Bleach Difference in Survivorship Percentage 420 424 7% 379 432 414 440 386 402 415 355 412 392 Total 2748 5705 Average 392.5714286 422.4285714

Analysis and Stats