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
Cl2 + 2NaOH →NaClO + NaCl + H2O
NaClO (s) + H2O (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
- 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

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
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.
mL aliquots of the samples were plated synchronously in LB media plates. 7 replicates were performed of each tube.
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

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
P- 2.2E-13
Significant
Bleach variation 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
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

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 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 Web. 9 November 2014.
-“Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008.” 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 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, Web.

21. 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

22. 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
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.

23. 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

24. Analysis and Stats