The Effect of Alcohol on Prokaryotic Survivorship

Luke Nedrow9th GradeCentral Catholic High School

Chemistry of Alcohol

Alcohol -the hydroxyl functional group is bound to a saturated carbon atom.

Ethanol – 1.Very common alcohol 2.Very toxic substance 3.Two main metabolites,

acetaldehyde and acetic acid.

Alcohol Statistics

Alcoholic beverages are one of America’s favorite drinks.

57% of American adults over the age 18 said they consumed alcohol in the past month.

88 thousand people die annually from drinking related issues.

223.5 billion dollars are spent in the USA alone on alcohol misuse problems.

The 3 Types of Alcohol

Most Dangerous Second Most Dangerous

Least Dangerous

Takes a long time to digest and byproducts are very dangerous

Most are ethanol based-Byproducts are dangerous if consumed in large quantities

Tertiary and Secondary Alcohols

Vodka, Moonshine, Methanol (Wood alcohol)

Some beers and most wines

Quickly digested- Most Beers

Variables (Beer, Liquor, Wine)

The variables for this experiment were beer, liquor, and wine.

Beer-Coors Light Alcohol Level- 4.2%

Wine- Sutter Home Sauvignon Blanc Alcohol Level – 12.5%

Liquor- Smirnoff Vodka Alcohol Level- 37.5%

Human Micro Flora

About 10% of the biomass on the body are symbionts

E.coli is one of the most prominent and well known models

E.coli was chosen for this experiment because of it’s diversity, it lives in the intestinal tract of humans and many other mammals, and it serves as a common prokaryotic cell model.

E.coli

Large and diverse group of gram(-) bacteria

One of the most common forms of bacteria: free living, symbiotic, or pathogens

Most strains are not dangerous, but the strains that are can produce fatal diseases.

Common prokaryotic cell model

Rationale

The human micro flora performs many functions vital to human health.

Close to 60% of the legal population drink alcohol on a monthly basis.

Does alcohol effect the micro flora in the digestive system?

Which type of alcohol effects the microflora the most?

Hypothesis

Null- The alcohol will have no significant effect on E.coli survivorship.

Alternate- The liquor (Smirnoff Vodka) will have the most significant effect on E.coli survivorship.

Materials Coors Light Smirnoff Vodka Sutter Home

Sauvignon Blanc LB agar plates (1%

tryptone, .5% yeast extract, 1%NaCl)

E.coli Spreader plates Open flame and

ethanol (for disinfecting spreader bars)

Various sized pipettes

Sterile Fluid Test tubes Test tube rack Heat plate/stirrer Vortex Incubator Bunsen Burner

Procedure

1.Bacteria (E.coli) were grown overnight in sterile LB media.

2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask.

3. The cultures were placed in an incubator (37˚C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^8 cells/mL.

4. The cultures were diluted in sterile fluid to a concentration of approximately 10^5 cells/mL.

Procedure ( Liquid Pulse)

1.LB agar plates(1% tryptone, .5% yeast extract, 1%NaCl) were prepared

2. Alcohol was pipetted into tubes containing SDF, to create the following concentrations: 0%, 1%, 10%, and 50%, yielding a final volume of 10 mL.

3.The cells were vortexed for even suspension.

Concentration Chart

Control (0%)

1% 10% 50%

Variable (Type of Alcohol)

0 mL .1 mL 1 mL 5 mL

Sterile Fluid 9.9 mL 9.8 mL 8.9 mL 4.9 mL

E.coli .1 mL .1 mL .1 mL .1 mL

Final 10 mL 10 mL 10 mL 10 mL

Procedure (Cont.)

4. Removed test tubes from test tube rack and 0.1mL was pipetted into each plate.

5. Plates were incubated at 37°C overnight. The resulting colonies were counted.

Each colony is assumed to have risen from one cell.

Smirnoff Vodka Effects on E.Coli Survivorship

0% 1% 10% 50%0

100

200

300

400

500

600

700

P-Value =3.47 ^-05

Amount of Surviving Cells

Smirnoff Vodka Concentrations

Coors Light Effects on E.Coli Survivorship

0% 1% 10% 50%0

100

200

300

400

500

600

700

800

P- Value =2.45 ^ -08

Amount of Surviving Cells

Coors Light Concentrations

Sauvignon Blanc Effects on E.Coli Survivorship

0% 1% 10% 50%0

100

200

300

400

500

600

700

P-Value= 1.06 ^-10

Amount of Surviving Cells

Sauvignon Blanc Concentrations

Dunnett’s Test

Replicates

Avg. of Group – Avg. of Control

(2) (MS Within Group)

A Dunnett’s quantifies significant change in data between the control and experimental groups.

Dunnett’s Test Results

Beverages Concentrations

T-value T-crit Conclusions

Sauvignon Blanc

1%10%50%

13.718.124.5

3.49 SigSigSig

Coors Light 1%10%50%

2.212.715.1

3.58 Not SigSig Sig

Smirnoff Vodka

1%10%50%

3.26.17.5

3.56 Not SigSigSig

Conclusions

Reject Null Hypothesis for all the exposures except:

1% Coors Light 1% Smirnoff Accept Alternative Hypothesis for all

other trials except for the aforementioned

The alcohol appeared to have a negative effect on E.coli survivorship.

Limitations and Extensions

Only tested one type of microbe Only one exposure time Possible contamination from spread-

plating Only three concentrations of alcohol Only thee alcoholic drinks Only survivorship quantified, not

growth

References

“E. Coli Food Poisoning.” About. About E. Coli, n.d Web. < http://www.about-ecoli.com/>

Melton, Lisa. “Alcohol.” Wikipedia, Web. http://en.m.wikipedia.org/wiki/Alcohol.

Special Thanks to Mr. Krotec

Percent Survivorship of E.Coli when exposed to alcohol

1% 10% 50%0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Smirnoff VodkaCoors LightSauvignon Blanc

Percent Survivorship

Concentrations

Smirnoff Results (All)

0% 566 438 486 422

1% 370 424 378 296

10% 224 304 286 258

50% 192 194 202 206

Surviving Colonies

Coors Light (All)

0% 872 828 582 (Outlier)

738

1% 686 804 744 676

10% 394 318 346 322

50% 212 204 246 238

Surviving Colonies

Wine Results (All)

0% 606 654 618 722

1% 360 371 313 362

10% 264 268 258 240

50% 102 112 134 126

Surviving Colonies

Smirnoff Single Factor Anova

Anova: Single Factor

SUMMARYGroups Count Sum Average Variance

Column 1 4 1912 478 4181.333Column 2 4 1468 367 2806.667Column 3 4 1072 268 1218.667Column 4 4 850 212.5 958.3333

ANOVASource of VariationSS df MS F P-value F crit

Between Groups163662.8 3 54554.25 23.80982 2.43E-05 3.560839Within Groups27495 12 2291.25

Total 191157.8 15

Coors Light Single Factor Anova

Anova: Single Factor

SUMMARYGroups Count Sum Average Variance

Column 1 4 3248 812 23369Column 2 4 2910 727.5 3499.667Column 3 4 1380 345 1220Column 4 4 900 225 406.6667

ANOVASource of VariationSS df MS F P-value F crit

Between Groups837393 3 279131 39.1827 1.78E-06 3.580803Within Groups85486 12 7123.833

Total 922879 15

Wine Single Factor AnovaAnova: Single Factor

SUMMARYGroups Count Sum Average Variance

Column 1 4 2600 650 2720Column 2 4 1406 351.5 681.6667Column 3 4 1030 257.5 153Column 4 4 474 118.5 203.6667

ANOVASource of VariationSS df MS F P-value F crit

Between Groups608096.8 3 202698.9 215.7328 1.06E-10 3.490295Within Groups11275 12 939.5833

Total 619371.8 15