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

Transcription

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

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

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

4 Bacterial Growth Curve

5 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

6 Gram Positive vs. Gram Negative 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 Gram positive - simple, thick membrane - most human pathogens - antibiotics such as penicillin prevent linking of peptidoglycan and formation of cell wall

7 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

8 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

9 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

10 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

11 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

12 Procedures 1. Parallel cultures were grown to different growth stages (50 KU log phase, 100 KU early stationary phase, 150 KU late stationary phase). 2. The cultures were synchronized to a KU reading of 50 on a klett spectrophotometer by dilution in LB media. 3. Tubes were prepared according to the chart below and incubated for 3 minutes at room temperature. Bleach Tube Sterile Fluid 9.8 ml 9.8 ml Bleach stock (10-2 %) 0.1 ml 0 ml Diluted E. Coli culture 0.1 ml 0.1 ml Total 10 ml 10 ml No Bleach Tube 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.

13 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

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

15 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 -Accept alternate hypothesis 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 -Accept alternate hypothesis

16 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

17 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

18 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

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

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

21 Bleach Kill Curve Concentration of Bleach Avg. Number of Survivors

22 Data 50 KU Bleach No Bleach Difference in Survivorship Percentage 100 KU Bleach No Bleach Difference in Survivorship Percentage % Total Avg % Total Avg

23 Data (cont.) 150 KU Bleach No Bleach Difference in Survivorship Percentage % Total Average

24 Analysis and Stats