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
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
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
1.
2.
3.
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.
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
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.
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
Bleach
P-variation
2.2E-13
Significant
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
-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
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
0
489.25
10-5
611.5
10-4
537
10-3.5
6.3
10-3
0
Data
50 KU
Bleach
Difference in
Survivorship
Percentage
No Bleach
100 KU
145%
Bleach
No Bleach
374
517
185
598
153
454
408
437
220
532
344
456
278
554
368
443
241
545
346
452
258
586
348
476
263
530
368
501
Total
1335
3269
Total
2556
3282
Avg.
222.5
544.8333333
Avg.
365.1428571
468.8571429
Difference in
Survivorship
Percentage
28%
Data (cont.)
Difference in
Survivorship Percentage
150 KU
Bleach
No Bleach
420
424
379
432
414
440
386
414
402
415
355
412
392
420
Total
2748
5705
Average
392.5714286
422.4285714
7%
Analysis and Stats