1. Azo Dye Effects on Human Microflora
By Alex Bilyak
Pittsburgh Central Catholic High School
Grade 9

2. Tartrazine A chemical used in azo yellow dye and cosmetic products
Is both ingested and applied to skin
Has been shown to cause health problems in humans, specifically in children.
Migraines, fatigue, blurred vision, hyperactivity, an inability to focus, etc.

3. Yellow Azo Food Dye Commonly sold in stores
Exposed to human microflora such as E. coli in the intestines
Used in products such as breakfast cereals, Kool-Aid, pasta, ramen noodles, and many makeup products.

4. Human Microflora
Bacteria that primarily thrive on the surface of the skin and in the respiratory, urinary, and digestive systems.
Most are completely harmless and many are even beneficial, although some can be harmful.
Human body is comprised of a collection of cells, cell products, and symbionts
Do ingested or surface products have an effect on the microbiome?

5. Gram (+) Gram (-) The majority of pathogenic bacteria are gram (+)
Antibiotics work against cell wall
Simple cell wall
Cell wall contains layers of lipopolysaccharides for protection
Outer membrane protects against many antibiotics

6. Staphylococcus epidermidis
Bacteria found on healthy human skin
Gram (+)
Used as a model for human microbial flora
Form clusters and biofilms

7. Escherichia coli Mammalian intestinal prokaryotic symbionts
A few strains are pathogenic and carry powerful toxins but the majority are harmless
Gram (-)
Used as a model for human microbial flora

8. Rationale
Tartrazine is often exposed to human microbial flora on the skin and in the intestines in various food and cosmetic products
Could azo yellow dye affect human microbial flora on the skin and in the intestines?

9. Hypotheses
Null : Tartrazine will not have a significant toxic effect on human microbial flora survivorship.
Alternative : Tartrazine will have a toxic effect on human microbial flora survivorship.

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

11. Procedure - Liquid Pulse Exposure
The bacteria were grown to a density of approximately 10^8 cells/mL
The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL
The yellow food dye was diluted to a 10% stock solution.
The following ingredients were mixed in sterile test tubes
The solutions were vortexed and allowed to sit for 15 minutes.
0.1mL of the aliquots were spread onto the agar plates
The plates were incubated at 37C overnight and the resulting colonies were counted

12. Liquid Pulse Exposure Concentration of Dye 0% 0.1% 1% Sterile fluid
9.9
9.8
8.9
Dye stock
0.1 1
E.Coli / Staph .1
Total
10ml

13. Procedure - Agar Infusion
100 ul of the dye stock solution were spread and infused onto eight agar plates to create approximately a 0.1% exposure. These plates were incubated for an hour.
10 ul of the dye solution and 90 ul of the sterile fluid were also spread and infused onto eight agar plates, creating approximately 0.01% exposure. These plates were also incubated for an hour.
100 ul from the bacteria control tubes were spread on these infused plates. The plates were incubated and the colonies were counted

14. Tartrazine Effect on E. coli and Staph Survivorship (Liquid Pulse)
S. epidermidis p-value: *
E. coli p-value: *

15. Tartrazine Effect on E. coli and Staph Survivorship (Agar Infusion)
S. epidermidis p-value: *
E. coli p-value: *

16. Significance Chart
The dunnett's test was performed for the E. coli liquid pulse test because the f-value in the ANOVA was greater than the f-critical.
T-value
P-value
Significance
0.1%
0.1940
Not Significant 1%
2.5131
Significant

17. Conclusions
•Fail to reject null for all tests except E. coli liquid pulse test at 1% concentration
•E. coli liquid pulse test at 1% concentration showed that the tartrazine had a significant toxic effect on E. coli colonies

18. Limitations Only tested survivorship of microflora
Spread plating may not have been completely synchronized (human error)
Data collecting was not completely synchronized (Counting of colonies did not occur at the same time.)
Limited number of replicates

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

20. Rough Data Liquid Pulse E. coli 306 459 562 425 322 382 478 310 494
402
410
398
272
368
515
326
408
246
308
288
356
318
282
274
Staph
260
284
318
292
249
282
221
293
329
219
312
348
288
321
315
294
360
367
378
281
340
Control
Infusion
E. coli
518
382
243
302
281
435
458
298
312
289
350
421
486
396
422
218
Staph
168
304
298
232
248
190
204
266
308
226
188
244
208
210
324
228
0.01%
0.1%
0.1% 1%

21. E. coli Liquid Pulse Anova: Single Factor SUMMARY Groups Count Sum
Average
Variance
Control 8
3244
405.5
0.001
3185
0.01
2480
310
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 2
Within Groups 21
Total 23

22. S. epidermidis Liquid Pulse
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Control 8
2199
0.001
2424
303
0.01
2587
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 2
Within Groups 21
Total 23

23. E. coli Infusion Anova: Single Factor SUMMARY Groups Count Sum Average
Variance
0.01 8
2917
0.1
2894
361.75
7474.5
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 1
Within Groups 14
Total 15

24. S. epidermidis Infusion
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
0.01 8
1910
238.75
0.1
1936
242
2376
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
42.25 1
Within Groups 14
Total 15

25. References
More, Daniel. “The Potential Risks of Tartrazine.” Verywell Health, Verywellhealth,
“Tartrazine.” National Center for Biotechnology Information. PubChem Compound Database, U.S. National Library of Medicine, pubchem.ncbi.nlm.nih.gov/compound/Tartrazine#section=Top.
“E.coli (Escherichia Coli).” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 20 Nov. 2018,
B.anthracis, web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.html.