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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 • Cell wall contains an extra are gram (+) organisms layer of lipopolysaccharides for extra protection • Simple cell wall • Outer membrane protects • Some antibiotics work against the against several antibiotics cell wall 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 • 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 0.1 ml 0.1 ml 0.1 ml Total Volume 10 ml 10 ml 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) Number of Colonies Staph p-value: 0.002974* E. Coli p-value: 0.157312 Cutoff: 0.05 Concentration of Dye Tartrazine Effect on E. coli and Staph (Infusion) Number of Colonies Staph p-value: 0.720708 E. Coli p-value: 0.176972 Cutoff: 0.05 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 3.6823 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 Control 0.1% 1% E. coli 394 352 380 312 382 456 334 600 316 346 298 300 352 266 346 408 308 364 334 346 336 248 320 362 Staph 288 246 286 296 238 226 298 248 332 242 342 Contaminated 300 364 336 302 Contaminated 324 302 400 282 316 390 302 1% infused 10% infused E. coli 416 314 230 302 478 422 450 340 332 432 352 454 302 358 328 362 Staph 250 242 292 242 250 188 284 246 236 260 310 266 236 220 210 334 E. Coli ANOVA (Liquid Pulse) Outliers were eliminated before each ANOVA was calculated. Staph ANOVA (Liquid Pulse) E. Coli ANOVA (Infusion) Staph ANOVA (Infusion)