1. Toxicity Effect of Food Dye on Microbes
Tom Maier
Pittsburgh Central Catholic High School
Grade 9

2. Tartrazine Tartrazine is a yellow azo dye
Commonly used in foods, cosmetics, and medications
Placed on skin when in cosmetics
Ingested when in food
Known as F&DC Yellow 5

3. Yellow Azo Dye Easily bought in store Main ingredient is tartrazine
Normally exposed to E. coli in the intestines
Used to expose tartrazine on the different bacteria this experiment

4. Human Bacterial Flora
Microorganisms that reside in or on the surface of the body
Respiratory, urinary, digestive tracts, and skin
Consists of mostly bacteria, fungus, protists, and archea
Most are harmless, but some can cause disease
Most studies based on ingested materials study human cells, but human are a collection of cells and symbiotants
Estimated 10% body mass is symbiotants

5. Gram (+) vs. Gram (-) Bacteria
Gram (+) Gram (-)
Most pathogenic bacteria in humans are gram (+) organisms
Simple cell wall
Some antibiotics work against the cell wall
Cell wall contains an extra layer of lipopolysaccharides for extra protection
Outer membrane protects against several antibiotics

6. Staphylococcus epidermidis
Bacteria found on healthy human skin
Gram (+)
After incubation in agar, form 1/2 – 2 mm colonies that are easily counted
Used as a model for microbial flora in and on humans
Can produce biofilms
Form clusters

7. Escherichia coli Mammalian Intestinal Prokaryotic Symbiont
Some strains can be pathogenic and travel outside the intestines. These strains can be harmful.
Gram (-)
Used as a model for microbial flora in the body

8. Rationale
Tartrazine is exposed to the skin in cosmetics and to the digestive system in food and medications.
Could tartrazine affect the microbes on the skin and in the digestive system (modeled by Staph and E. coli)?
Purpose: Test the survivorship of E. Coli and Staph populations when exposed to Tartrazine

9. Hypotheses
Null Hypothesis: The Tartrazine will have no toxic effect on the microbes' survivorship.
Alternate Hypothesis: The Tartrazine will have a toxic effect on the microbes' survivorship.

10. Materials Incubator Ethanol Spreader bars
LB agar plates
LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)
Micropipettes
Sterile micropipette tips
Sterile dilution fluid
E. Coli
Staphylococcus epidermidis
Vortex
SDF (100mM KH2P0, 100mM K2PO4, 10mM MgSO4 1m NaCl)
Incubator
Ethanol
Spreader bars
F&DC Yellow food dye (Tartrazine)
Sidearm flask
Sterile test tubes
Burner
Klett spectrophotometer

11. Procedures
The bacterium were grown until a density of 50 klett spectrophotometer density 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
The yellow food dye was diluted into a 10% stock solution.
The solutions shown in the chart below were created in sterile test tubes.

12. Chart of Concentrations
Control
0.1x liquid pulse
1x liquid pulse
Sterile Fluid
9.9 ml
9.8 ml
8.9 ml
Dye Stock Solution
0 ml
0.1 ml
1 ml
E. coli/Staph Solution
Total Volume
10 ml

13. Procedures cont.
The solutions were vortexed and allowed to sit for 15 minutes.
0.1mL of the aliquots were spread onto the agar plates
A test using infusion was also performed in this experiment
100 ul of the dye stock solution were spread and infused onto eight agar plates to create a 10% infused plate. These plates were incubated for an hour.
10 ul of the the dye solution and 90 ul of the sterile fluid were also spread and infused onto eight agar plates, creating a 1% infused plate. These plates were also incubated for an hour.

14. Procedures cont.
The control solutions were used on the infused plates to test the effect of the infused dye on the microbes.
Each plate was placed in the incubator for 24 hours at a temperature of 37 degrees Celsius
After the incubation, the plates were taken out and the colonies of the bacteria were counted visually.

15. Tartrazine Effect on E. coli and Staph (Liquid Pulse)
Staph p-value: *
E. Coli p-value:
Cutoff: 0.05
Number of Colonies
Concentration of Dye

16. Tartrazine Effect on E. coli and Staph (Infusion)
Staph p-value:
E. Coli p-value:
Cutoff: 0.05
Number of Colonies
Concentration of Infusion

17. Dunnett's Test (Staph Liquid Pulse)
The Dunnett's test was performed for the Staph liquid pulse test because the f-value in the ANOVA was greater than the f-critical.
T-Value
F-Critical
0.1x
3.3607
3.6823
Not Sig 1x
3.8699
Sig

18. Conclusions Accept null for all tests except Staph liquid pulse
Staph liquid pulse at 1x was significant; stimulated growth rather than be toxic

19. Limitations Only tested survivorship
Spread plating may not have been synchronized (human error)
Data collecting was not completely synchronized (counted E. coli one day, Staph the next)
Limited number of replicates
Only one exposure time

20. Extensions Growth experiment Test different colored dyes
Other microbial models
Different concentrations
Different exposure times
Synergistic effects (with other dyes)

21. References Http://www.cdc.gov/ecoli/
0presentations/S%20epidermidis/sepidermidis.h tml 1

22. Rough Data E. coli Staph Control 394 288 352 246 380 286 312 296
0.1%
300 Contaminated
1% 308 Contaminated
E. coli Staph
1% infused
10% infused

23. E. Coli ANOVA (Liquid Pulse)
Outliers were eliminated before each ANOVA was calculated.

24. Staph ANOVA (Liquid Pulse)

25. E. Coli ANOVA (Infusion)

26. Staph ANOVA (Infusion)