Ethanol Effects Upon Microbial Flora Survivorship

Siddarth NarayanGrade 9

North Allegheny Intermediate High School

What effect does ethanol have upon E Coli and Staph cultures?

Problem

More than 10% of your body mass is due to symbionts and pathogens

Mostly Prokaryotic cells Some may cause diseases Also protect the body from foreign invaders Reinforce immune system, prevent

allergies, and produce vitamins Lie within skin, saliva, and gastrointestinal

tracts Break down abnormally large nutrients

Human Symbionts, Pathogens, and Flora

Large and diverse group of gram (-) bacteria Free living, symbiontes, or pathogens Live in the intestinal tract of many

mammals. Most strains are not pathogenic Serve as a common prokaryotic cell model.

Escherichia coli (E.Coli)

Common surface symbiont in many mammals (Human).

Gram (+) bacteria Most strains considered non-pathogenic. Pathogenic strains can be life-threatening.

Staphylococcus epidermidis (Staph)

Often made through the process of fermentation

Fermentation is the process by which yeast breaks down sugar into carbon dioxide and alcohol.

Common uses include: Household chemicals, alcoholic beverages, and medical chemicals and disinfectants

Very toxic substance with numerous effects on body

Ethanol

To examine effects of ethanol upon endosymbiontes

Purpose

Null Hypothesis: Ethanol will not significantly affect microbial survivorship

Alternative Hypothesis: Ethanol will significantly reduce the survivorship of E coli and staph cells.

Hypotheses

LB agar plates (LB media + 1.5% agar)

LB media (0.5% yeast extract, 1% tyrptone, 1% NaCL)

Sterile pipette tips Micropipettes Vortex Incubator Sidearm flask Spreading turntable

Spreader bar Ethanol (Ethyl Alcohol) Sterile capped test

tubes with Sterile distilled water.

DH5 Alpha E coli Strain

Staphylococcus epidermidis (Ward’s)

0.22 micron syringe filters + 10mL syringe

Klett

Materials

1. The microbes were grown overnight in sterile LB media.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 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. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations of 0%, 1%, 10%, 25% and 50% to a total of 9.9 mL.

6. 0.1 mL of Staph/ E coli was then added to the test tubes, yielding a final volume of 10 mL and a cell density of 103 cells/mL per tube.

Procedure

Concentration Chart0% 1% 10% 25% 50%

Sterile Dilution Fluid

9.9 mL 9.8 mL 8.9 mL 7.4 mL 4.9 mL

Microbe 0.1 mL 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Ethanol 0 mL 0.1 mL 1 mL 2.5 mL 5 mL

Total Volume

10 mL 10 mL 10 mL 10 mL 10 mL

Procedure (Continued)

7. The solutions were mixed by vortexing and allowed to sit at room temperature

8. After vortexing to evenly suspend cells, 0.1mL aliquots were removed from the tubes and spread on LB agar plates (3 plates per concentration)

9. The plates were then incubated at 37 degrees for 24 hours.

10. The resulting colonies were counted.

Procedure (Continued)

E Coli Survivorship

5

P Value=1E-05

50 25 10 1 00

50

100

150

200

250

300

350

400

450

Ethanol Concentration (%)

Avera

ge N

um

ber

of

Cells

Data Analysis (ANOVA)

Anova: Single Factor (E Coli)

SUMMARYGroups Count Sum Average Variance

50% 3 2 0.667 0.33325% 3 1 0.333 0.33310% 3 1152 384 11788

1% 3 889 296.3 34900% 3 773 257.7 246.3

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 4E+05 4 93988 30.27 1E-05 3.478Within Groups 31051 10 3105

Total 4E+05 14

Concentration T- Value Interpretation

1% .81 Insignificant

10% 3.3 Insignificant

25% 7.6 Significant

50% 7.6 Significant

E Coli Dunnett’s Test Analysis(Alpha= .05) (T Crit=3.48)

Staph Survivorship P Value=1.46E-11

50 25 10 1 00

50

100

150

200

250

300

350

400

450

500

Series1

Data Analysis (ANOVA)Anova: Single Factor (Staph)

SUMMARYGroups Count Sum Average Variance

50% 3 0 0 025% 3 399 133 32710% 3 1422 474 468

1% 3 1141 380.3333 44.333330% 3 1092 364 268

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 462882.3 4 115720.6 522.5191 1.46E-11 3.47805

Within Groups 2214.667 10 221.4667

Total 465096.9 14

Concentration T-Value Interpretation

1% 1.7 Insignificant

10% 11.7 Significant

25% 24.5 Significant

50% 38.7 Significant

Staph Dunnett’s Test Analysis(Alpha=.05) (T Crit=3.48 )

Staph and E coli LD50

0 1 10 25 500

50

100

150

200

250

300

350

400

450

500

Ethanol Concentration s (%)

Nu

mb

er

of

Ce

lls

E coli~ 22% Staph~ 24%

The null hypothesis can be rejected for the following concentration of ethanol and species: E coli (25%, 50%), Staph (10%, 25%, 50%)

Both microbes display similar survivorship

Conclusions

Low sample sizes Only one type of exposure (Liquid Pulse

Exposure) Limited concentrations of variable Only one exposure time

Limitations

More replicates being used in same setup Other exposures (diffusion assay) Varied exposure time Synergistic Effects Can ethanol benefit survivorship in certain

quantities? Does ethanol only have an effect upon

pathogenic endosymbiontes?

Further Study

Data Results (E Coli)

Plate 1 Plate 2 Plate 3

0% Ethanol 263 270 240

1% Ethanol 364 255 270

10% Ethanol 420 262 470

25% Ethanol 0 1 0

50% Ethanol 0 1 1

Data Results (Staph)Plate 1 Plate 2 Plate 3

0% 360 350 382

1% 382 373 386

10% 480 492 450

25% 114 135 150

50% 0 0 0

E Coli ANOVA Data Analysis

Staph ANOVA Data Analysis

0 1 10 25 500%

200000%

400000%

600000%

800000%

1000000%

1200000%

1400000%

GroupsCountSumAverageVariance

References www.ncbi.nlm.nih.gov/pubmed/19609257 www.mayoclinic.com/health/staph

-infections/DS00973 www.mayoclinic.com/health/e-coli/DS01007 www.life.umd.edu/classroom/.../GramPosvs

GramNeg.htm www.ncbi.nlm.nih.gov/pubmed/15112929 http://

jb.asm.org/content/157/1/233.abstract