Coffee Effects on Escherichia coli

David McFallGrade 9Central Catholic High School

Coffee

•Americans consume 400 million cups of coffee per year.

•Coffee has a high caffeine content due to its high concentration in its endosperm.

•Coffee can be decaffeinated by a treatment of methylene chloride.

The Effects of Coffee

Coffee’s side effects are drawn from the release of the hormone epinephrine, shown here.

The most notable effects include:

high heart beat

high blood pressure

higher blood sugar

content

tense muscles

Epinephrine

How caffeine interacts with the body

The binding of the hormone adenosine to an adenosine receptor in the brain brings about sleep.

Caffeine blocks adenosine from being connected to the adenosine receptor.

The hormone epinephrine is then produced. This creates the notable effects of caffeine.

Brands Tested

20 mg of Caffeine per ounce 0.6 to 1.4 mg of Caffeine per ounce

Starbucks X-bold Sumatra Roast (CAFF) Starbucks Decaf House Blend (DCAFF)

Rationale for Experiment:

Usually, when an ingested material is used as a variable, its effects on the human that ingested it are recorded and observed.

However, this experiment deals with the ingested material’s effect on the flora of the human body.

Escherichia coli

Escherichia coli is a large and diverse group of gram (-) bacteria.

It is found in the intestinal tract of most animals, including humans.

Estimated to cause infection in more than 70,000 patients a year in the United States.

E. coli has been tested in conjunction with caffeine before in previous studies.

Previous Studies

A study from the Lithuanian State Science and Studies Foundation concluded that when in high concentration, caffeine lowers the population of E. coli.

Another study from the Indiana University School of Medicine supported the previous study.

Purpose:

To determine the effect of coffee on human coliform populations.

Hypotheses:

Null Hypothesis: Neither caffeinated nor decaffeinated

coffee will have an effect on the survivorship

of E. coli.

Alternative Hypothesis: Caffeinated and decaffeinated

coffee will significantly affect

survivorship.

Materials:

E. coliLatex GlovesMicropipettesMicro tubesStarbucks X-bold Sumatra RoastStarbucks Decaf House Blend Sterile Dilution Fluid (SDF) (per 1 liter) (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)58 LB agar platesLB media (Per Liter:1% Tryptone, 0.5% Yeast Extract, and 1% NaCl)2 syringe Sterile FiltersBunsen BurnersSpread BarIncubatorEthanolMatchesVortexKlett Spectrophotometer

Procedure:1. E. coli was grown overnight in sterile LB agar.2. A sample of the overnight culture was added to fresh media in a sterile

sidearm flask.3. The culture was placed in an incubator (37°C) until a density of 50

Klett spectrophotometer units was reached. This represents a cell density of approximately 108 cells/mL.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.

5. Caffeinated and decaffeinated coffee was mixed with the appropriate amount Sterile Distillation Fluid to create coffee concentrations of 10%, 1%, and .1%.

6. 100 µL of cell culture was then added to the solutions, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.

7. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.

8. 100 µL aliquots were removed from the tubes and spread on LB plates.9. The plates were incubated at 37 degrees for 24 hours.10.The resulting colonies were counted visually. Each colony was

assumed to have arisen from one cell.11.The appropriate statistical analyses were performed to adequately

assess the hypothesis.

Procedure: (Infused Plates)

1. Repeat steps 1-6 in the Preliminary Procedure.2. Both brands of coffee were brewed as directed.3. The coffee was then sterile filtered.4. 200 µL of sterile coffee were removed from the

tubes and spread on LB plates.5. 100 µL of E. coli (control tube) was removed

from the tubes and spread on LB plates.6. The plates were incubated at 37 degrees for 24

hours.7. The resulting colonies were counted visually.

Each colony was assumed to have arisen from one cell.

8. The appropriate statistical analyses were performed to adequately assess the hypothesis.

Control .01x .1x x

E. coli 0.1 ml 0.1 ml 0.1 ml 0.1 ml

Sterile Dilution Fluid (SDF) 9.9 ml 9.89 ml 9.8 ml 8.9 ml

Coffee 0 ml 0.01 ml 0.1 ml 1 ml

Total Volume 10 ml 10 ml 10 ml 10 ml

Chart of Concentration

Contro

l

0.01

x0.

1x x

Caffei

nate

d In

fuse

d

Decaff

eina

ted

Infu

sed

0

100

200

300

400

500

600

700

Coffee Effects on E. coli Survivorship

Control

Caffeinated

Decaf-feinated

Infused Plates

Concentrations

Colo

nie

s o

f E.

coli

Dunnett’s Test Analysis

T Critical= 3.49 (Significant)

Variable Concentration T Value Interpretation .01x CAFF 2.2 Insignificant .1x CAFF 3.46 Insignificant x CAFF 4.78 Significant .01x DCAFF 7.63 Significant .1x DCAFF 4.54 Significant x DCAFF 3.69 Significant

Results- Key Questions

Did liquid exposure to coffee significantly effect survivorship? P-Value 7.29E-8 Significant

Which coffee exposures significantly effected survivorship? 10% Caffeinated Exposure T-Value 4.78 Significant 0.1% Decaffeinated Exposure T-Value 7.63 Significant 1% Decaffeinated Exposure T-Value 4.54 Significant 10% Decaffeinated Exposure T-Value 3.69 Significant

Did caffeinated coffee vary from decaffeinated in survivorship? 0.1% CAFF-P-Value 1.41E-3 Significant 1% CAFF-P-Value 0.28 Insignificant 10% CAFF-P-Value 0.62 Insignificant

Did continuous exposure to coffee (Infused Plates) alter survivorship?

CAFF-P-Value 0.12 Insignificant DCAFF-P-Value 0.3 Insignificant

Conclusions:

It seems as if caffeinated coffee increased the population of E. coli, but only in the 10% concentration.

Decaffeinated coffee, on the other hand, seemed to increase the population of E. coli in all concentrations.

Difficulties Encountered:

Plating was not exactly synchronized(10 minute lag), which could have resulted in extra time for bacterial replication. A team of students could remedy this technical problem.

Future Changes:

Test more brands of coffee

Use a different model

Test coffee in conjunction with other caffeinated beverages, such as tea or energy drinks

Refrences

http://www.espressoforums.com/viewtopic.php?t=137http://www.miyerbamate.com/content/Yerba%2BMate%2Bharmful%2Bingredients%2Bin%2Bcoffee%2Band%2Btea.htmhttp://www.starbucks.comhttp://www.coffeeresearch.org/science/decaffeination.htmhttp://www.about-ecoli.com/http://www.nlm.nih.gov/medlineplus/ecoliinfections.html