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

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.

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.

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?

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

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

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

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?

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.

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

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

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

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

Tartrazine Effect on E. coli and Staph Survivorship (Liquid Pulse) S. epidermidis p-value: 0.06437* E. coli p-value: 0.03746*

Tartrazine Effect on E. coli and Staph Survivorship (Agar Infusion) S. epidermidis p-value: 0.89673* E. coli p-value: 0.95134*

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 0.03746 Not Significant 1% 2.5131 Significant

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

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

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

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%

E. coli Liquid Pulse Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control 8 3244 405.5 8505.142857 0.001 3185 398.125 6432.125 0.01 2480 310 2635.428571 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 45175.08333 2 22587.54167 3.856131316 0.03746232329 3.466800112 Within Groups 123008.875 21 5857.565476 Total 168183.9583 23

S. epidermidis Liquid Pulse Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control 8 2199 274.875 915.5535714 0.001 2424 303 1521.714286 0.01 2587 323.375 2103.410714 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 9489.083333 2 4744.541667 3.134691165 0.06437421326 3.466800112 Within Groups 31784.75 21 1513.559524 Total 41273.83333 23

E. coli Infusion Anova: Single Factor SUMMARY Groups Count Sum Average Variance 0.01 8 2917 364.625 9663.410714 0.1 2894 361.75 7474.5 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 33.0625 1 0.003858404977 0.9513485106 4.600109908 Within Groups 119965.375 14 8568.955357 Total 119998.4375 15

S. epidermidis Infusion Anova: Single Factor SUMMARY Groups Count Sum Average Variance 0.01 8 1910 238.75 2461.642857 0.1 1936 242 2376 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 42.25 1 0.01746718443 0.8967356586 4.600109908 Within Groups 33863.5 14 2418.821429 Total 33905.75 15

References More, Daniel. “The Potential Risks of Tartrazine.” Verywell Health, Verywellhealth, www.verywellhealth.com/tartrazine-free-diet-83227. “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, www.cdc.gov/ecoli/index.html. B.anthracis, web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.html.