Download CCHS Tom Maier Food Dye Toxicity Effects on Microbes

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)