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

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

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

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

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

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

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

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

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.

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

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.

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

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.

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.

Tartrazine Effect on E. coli and Staph (Liquid Pulse) Staph p-value: 0.002974* E. Coli p-value: 0.157312 Cutoff: 0.05 Number of Colonies Concentration of Dye

Tartrazine Effect on E. coli and Staph (Infusion) Staph p-value: 0.720708 E. Coli p-value: 0.176972 Cutoff: 0.05 Number of Colonies Concentration of Infusion

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

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

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

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

References Http://www.cdc.gov/ecoli/ Http://web.uconn.edu/mcbstaff/graf/Student%2 0presentations/S%20epidermidis/sepidermidis.h tml http://www.ncbi.nlm.nih.gov/pubmed/1750576 1

Rough Data E. coli Staph Control 394 288 352 246 380 286 312 296 352 246 380 286 312 296 382 238 456 226 334 298 600 248 0.1% 316 332 346 242 298 342 300 Contaminated 352 300 266 364 346 336 408 302 1% 308 Contaminated 364 324 334 302 346 400 336 282 248 316 320 390 362 302 E. coli Staph 1% infused 416 250 314 242 230 292 302 242 478 250 422 188 450 284 340 246 10% infused 332 236 432 260 352 310 454 266 302 236 358 220 328 210 362 334

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

Staph ANOVA (Liquid Pulse)

E. Coli ANOVA (Infusion)

Staph ANOVA (Infusion)