1. Joe Stern Pittsburgh Central Catholic Grade 9
Sodium Hypochlorite Effects on Escherichia coli and Staphylococcus epidermidis
Joe Stern
Pittsburgh Central Catholic
Grade 9

2. Research Questions
Does an environmentally friendly cleaning agent perform better, similar, or worse than a chemically based cleaning agent?
How do different concentrations of cleaning agents compare to each other in reducing microbial growth?

3. Cleaning Agents
LA’s Totally Awesome Bleach Javellisant: Chemical Based
pH = 12
Contains active ingredients:
Sodium hypochlorite %
Sodium chlorate
Sodium hydroxide
Sodium polyacrylate
Sodium carbonate

4. Cleaning Agents Cont.
Simple Green All-purpose Cleaner: Environmentally friendly
Biodegradable
U.S. EPA Safer choice product
pH = 9
Contains active ingredients
Sodium hypochlorite %
Sodium citrate
Sodium carbonate

5. Sodium Hypochlorite NaOCl Sodium cation and hypochlorite anion
Forms a hypochlorous acid when with water
Found in cleaning agents
Strong oxidizer
Proficient in killing bacteria
Base

6. Gram (-) vs. Gram (+) Bacteria
Gram Positive Bacteria (Staph)
Most pathogenic bacteria in humans are Gram (+) organisms
Thick layer of peptidoglycan in the cell wall
Antibiotics can impede the ability of the peptidoglycan to link
Gram Negative Bacteria (E. Coli)
Thin layer of peptidoglycan
The outer membrane is an extra thin layer of lipopolysaccharide, further adding protection to the cell
Outer membrane provides protection from some antibiotics

7. Model Bacteria: Escherichia coli
Gram negative bacteria
Rod-shaped
Intestinal symbiont
Lives in our intestinal tract
Commonly used as a model for microbial life in the human body
Some strains are pathogens

8. Model Bacteria: Staphylococcus epidermidis
Gram positive bacteria
Skin symbiont
Found on human skin
Commonly used as a model for microbial life in the human body
Strains are non-pathogenic

9. Purpose
Determine effects of two common cleaners on E. coli and Staph (each containing sodium hypochlorite)
Investigate the reliability of an environmentally friendly cleaning agent when compared to a chemical based cleaning agent

10. Hypotheses Null Hypotheses:
Various concentrations of sodium hypochlorite will have no toxic effect on the survivorship of the microbes
There will be no significant difference in microbial survivorship between an environmentally friendly cleaner and a chemical based cleaner
Alternative Hypotheses:
Various concentrations of sodium hypochlorite will have a toxic effect on the survivorship of the microbes
There will be a significant difference in microbial survivorship between an environmentally friendly cleaner and a chemical based cleaner

11. Materials Sidearm flask Spreading turntable LB agar plates
Spreader bars
Ethanol
Burner
Sterile capped test tubes with sterile distilled water
LA’s Totally Awesome Bleach Javellisant
Simple Green All-purpose Cleaner
LB agar plates
LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)
Sterile Dilution Fluid
Micropipettes
Sterile micropipette tips
E. coli C strain (Obtained from CMU)
Staphylococcus epidermidis (Obtained from Ward’s Science)
Vortex
Incubator

12. Procedure (Liquid Exposure)
Both bacteria were grown until a density of 50 klett spectrophotometer was reached. This was approximately 10^8 cells/mL
The cultures diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL
Cleaners were diluted to a concentration of 1% in SDF.
The solutions shown in the table of concentrations were created in sterile test tubes
Solutions were vortexed

13. Table of Concentrations: Liquid Exposure
0% (Control)
0.001%
0.01%
0.1%
Microbe
0.1 ml
Sodium Hypochlorite
Solution
0 ml
0.01 ml
1 ml
SDF
9.9 ml
9.89 ml
9.8 ml
8.9 ml
Total volume
10 ml

14. Procedure cont. (Liquid Exposure)
0.1 ml of each tube was spread onto agar plates
Plates were grown overnight at 37 C and the resulting colonies produced were counted

15. Procedure (Infused Agar)
Aliquots of cleaners were spread onto agar plates
Plates were then incubated for 1 hour to absorb
100 μL samples from the control tube were then added to the infused plates
The table below shows the concentrations of the infused agar plates
Plates were grown overnight at 37 C and the resulting colonies produced were counted

16. Table of Concentrations: Infused Agar (1%)
200 uL
20 uL
Percentage of Sodium Hypochlorite 1%
0.1%
SDF
0 uL
180 uL
Total volume
0.2 ml

17. Hypochlorite Effects on E. coli Survivorship (Liquid Exposure)
P-value: 8.66E-06 < 0.05
Individual p-values:
Chemical: 5.3E-05 < 0.05
Environmental: < 0.05

18. Hypochlorite Effects on Staph Survivorship (Liquid Exposure)
P-value: 3.29E-14 < 0.05
Individual p-values:
Chemical: 5.91E-10 < 0.05
Environmental: 1.18E-07 < 0.05

19. Dunnett’s Test (E. coli Liquid Exposure)
Chemical Based Bleach
T- Value
T- critical
Significance?
0.1%
2.56
3.49
Not sig. 1%
4.62
Significant
10%
5.50

20. Dunnett’s Test (E. coli Liquid Exposure) Cont.
Environmentally Friendly Bleach
T-value
T-critical
Significance?
0.1%
0.26
3.49
Not sig. 1%
2.20
10%
3.67
Significant

21. Dunnett’s Test (Staph Liquid Exposure)
Chemical Based Bleach
T-value
T-critical
Significance?
0.1%
0.75
3.49
Not sig. 1%
2.13
10%
7.22
Significant

22. Dunnett’s Test (Staph Liquid Exposure) Cont.
Environmentally Friendly Bleach
T-value
T-critical
Significance?
0.1%
0.07
3.49
Not sig. 1%
0.10
10%
4.42
Significant

23. Hypochlorite Effects on E. coli Survivorship (Infused Agar)
P-value: 1.59E-5 < 0.05

24. Hypochlorite Effects on Staph Survivorship (Infused Agar)
P-value: 1.0E-5 < 0.05

25. Dunnett’s Test (E. Coli Infused Agar)
Chemical Based
Environmentally Friendly
T-value
T-critical
Significance?
0.1%
0.46
3.48
Not sig. 1%
3.53
Significant
T-value
T-critical
Significance?
0.1%
6.45
3.48
Significant 1%
7.53

26. Dunnett’s Test (Staph Infused Agar)
Chemical Based
Environmentally Friendly
T-value
T-critical
Significance?
0.1%
40.7
3.48
Significant 1%
42.59
T-value
T-critical
Significance?
0.1%
42.98
3.48
Significant 1%
43.77

27. Conclusions
Reject null hypothesis for various concentrations of sodium hypochlorite having a toxic effect on microbial survivorship
Microbial survivorship decreased as concentration of sodium hypochlorite increased
Reject null hypothesis for the common cleaning agents not having a significant difference in reduction of microbial survivorship
The environmentally friendly bleach allowed for more microbial survivorship than the chemical based bleach in liquid exposure tests
In infused agar tests, the chemical based bleach allowed for more microbial survivorship than the environmentally friendly bleach

28. Explanations
The different pH levels in the two cleaners could have caused different microbial growth. This could be a result from the environmentally friendly bleach using more organic material instead of synthetic
Possible ingredient in one of the variables caused it to reduce or allow microbial growth
When active ingredients are applied in higher concentrations, they permit for less microbial survivorship

29. Limitations
Spread plating could have possibly not been synchronized due to human error
The timespan of collecting data may have not been completely synchronized due to time constraints
Only two species used
Only survivorship was tested
Only 4 concentrations were tested

30. Extensions Other cleaning agents and bacteria will be tested
Active ingredients of cleaning agents will be compared
A more diverse pool of microbes will be tested
Synchronized spread plating
Perform experiment over longer periods of time to test cleaning agent duration
A variety of concentrations will be tested

31. Works Cited

32. Chemical Bleach ANOVA: E. Coli (Liquid Exposure)

33. Chemical Bleach ANOVA: Staph (Liquid Exposure)

34. Natural Bleach Anova: Staph (Liquid Exposure)

35. Cleaning Agents Staph Microbial Survivorship ANOVA (Liquid Exposure)

36. Cleaning Agents E. coli Microbial Survivorship ANOVA (Liquid Exposure)