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Effect of Bacterial Growth Stage on Chlorine DisinfectionProject By: Warren SipeGrade 9Central 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 + H2ONaClO (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 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
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
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
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
Data
InteractionP- 2.2E-13Significant
Interaction P= 2.2E-13
No bleach variation P= 8.54E-6
Bleach variation P=2.78E-8
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
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 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
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
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
Analysis and Stats