central catholic high school grade 9 project by: warren sipe science/pjas/pjas 15 pdf... · effect...
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Effect of Bacterial Growth Stage on Chlorine DisinfectionProject By: Warren SipeGrade 9Central Catholic High School
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Chlorine Bleach-Common household disinfectant
-Active ingredients-Sodium hypochlorite-Sodium chlorate-Sodium hydroxide-Sodium polyacrylate-Sodium carbonate
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Sodium Hypochlorite-NaClO-Active disinfectant in chlorine bleach-Results when chlorine gas is mixed with sodium hydroxide
Cl2 + 2NaOH →NaClO + NaCl + H2ONaClO (s) + H2O (l) → HOCl (aq) + NaOH- (aq)
-Kills bacteria by denaturing their proteins
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Bacterial Growth Curve
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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
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Gram Positive vs. Gram NegativeGram positive- 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
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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
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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
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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
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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
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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
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Procedures1. 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.
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 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
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ANOVA- Analysis of Variance- multiple mean comparison- Statistical test comparing variation between and withingroups- P-Value is smaller than the Alpha Value (0.05)= variation is significant
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Data
InteractionP- 2.2E-13Significant
Interaction P= 2.2E-13
No bleach variation P= 8.54E-6
Bleach variation P=2.78E-8
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Analysis and StatsDid 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 hypothesisDid 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 hypothesisDid 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
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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
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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
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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
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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.
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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.
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Bleach Kill CurveConcentration of Bleach Avg. Number of Survivors
0 489.25
10-5 611.5
10-4 537
10-3.5 6.3
10-3 0
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Data
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
368 501
Total 2556 3282
Avg. 365.1428571 468.8571429
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Data (cont.)
150 KU Bleach No Bleach
Difference in Survivorship Percentage
420 424 7%
379 432
414 440
386 414
402 415
355 412
392 420
Total 2748 5705
Average 392.5714286 422.4285714
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Analysis and Stats