THE EFFECTS OF OLIVE OIL AGAINST CIGARETTE SMOKE ON YEAST

Peter Wagstaff

PROBLEM

Cigarette smoking is the leading cause of preventable death in the United States. Quitting is ideal but not always easy or expedient.

CIGARETTE SMOKE

Cigarette smoke contains thousands of chemicals, many of them toxic or cancer causing, such as arsenic and tar

An estimated 480,000 deaths are caused by cigarettes every year in the United States.

Cigarettes are the leading cause of preventable death in the United States

OLIVE OIL

Contains Oleic Acid Unsaturated fat Unsaturated fats are cited as a lung

supporting nutrient by the National Lung Association

Previous studies have reported that diets high in unsaturated fats can shield against some effects of smoking

However, other studies have indicated that the burning of cooking oils can increase risk for lung cancer

WHY YEAST (SACCHAROMYCES CERERISAE)

Yeast is used as a model because: Eukaryotic, just like human cells Most studied cell Many characteristics like nutrition and cell

growth are similar to human cells Aerobic

PURPOSE

Can unsaturated fats (olive oil) reduce the negative effects of cigarette smoke on yeast?

Will oil alone affect yeast survivorship? Will smoke alone affect yeast survivorship?

HYPOTHESIS

Null: The olive oil and cigarette smoke will NOT have a significant effect on yeast survivorship, individually or synergistically.

Alternative 1: The presence of olive oil or cigarette smoke will have a significant individual effect on yeast survivorship.

Alternative 2: Olive oil will significantly increase the survivorship of smoke stressed yeast.

MATERIALS Saccharomyces cerevisiae (Yeast) YEPD agar plates (1% yeast extract, 1.5% agar, 2% glucose) Aldi brand virgin olive oil Sterile micropipettes and tips (200ml) Sharpie Sterile dilution fluid (100mM KH2PO4. 100mM K2HPO4, 10mM

MgSO4, 1mM NaCl) Sterile spreader bars Ethyl alcohol Burner Marlboro cigarettes Stop watch Smoke Chamber Incubator Spectrophotometer

PROCEDURE

1. A culture Saccharomyces cerevisiae was grown.

2. The culture was incubated at 30 degrees Celsius until a density of 50 Klett spectrophotometer units were reached. This represents a density of approximately 10⁷ cells/mL.

3. The culture was diluted with sterile dilution fluid to a concentration of 10^3 cells/mL

PROCEDURE

4. 100μl of the cell suspension was pipetted into olive oil infused YEPD agar plates

Agar infusion: After autoclaving YEPD agar, sterile olive oil

was mixed with the molten agar to create the following infused plates; 0%, 0.1%, 1%, and 10%

5. Six plates of each concentration were taped to the top of the smoke chamber and exposed to 0, 45, 90, and 130 seconds of smoke

PROCEDURE

7. A fresh cigarette was lit and fitted into the socket for each exposure, and the air pump was pumped every three seconds for consistent airflow.

8. The plates were covered and incubated at 30 degrees Celsius for 72 hours and the resulting colonies were quantified.

DOCUMENTATION OF SMOKE CHAMBER

DOCUMENTATION OF SMOKE CHAMBER

DOCUMENTATION OF SMOKE CHAMBER

ANOVA

Single Factor Stat test comparing the means of multiple

groups Alpha of 0.05 is selected

Two Factor Can reveal synergistic affects of two variables

SMOKE EFFECTS ON YEAST SURVIVORSHIP

0 45 90 1300

10

20

30

40

50

60

70

80

90

a

Yeast without oil

Number of colonies

Overall P-value:0.00171

Average Yeast Survivorship

Smoke Exposure [seconds]

OIL EFFECT ON YEAST SURVIVORSHIP

0.00% 0.10% 1.00% 10.00%0

20

40

60

80

100

120

Yeast with out smoke

Number of colonies

Average Yeast Survivorship

Overall P-value:4.24E-06

Concentration[Oil]

DUNNETT’S TEST

The Dunnett's test is a follow up to the ANOVA that quantifies the difference between a variable and control group to find significant variation.

DUNNETT'S TEST

Group T Value Significance

0.1% Oil 2.432 Not Significant

1% Oil 0.024 Not Significant

10% Oil -4.936 Significant

Oil did not begin to have a significant effect on yeast until the concentration reached 10%

T-Crit = 3.098391

SMOKE AND OIL EFFECTS ON STRESSED YEAST

0 Seconds Exposure

45 90 1300

20

40

60

80

100

120

0% Oil0.10%1%10%

Number of colonies Average Yeast

Survivorship

Overall P-value:0.017711

Did olive oil have an effect by itself?P-Value 4.24E-6 Significant

Did cigarette smoke reduce yeast survivorship? P-Value 0.00171 Significant

Did oil increase survivorship in smoke stressed yeast?P-Value 0.017711 Significant

CONCLUSION

The null hypothesis was rejected. Both variables had individual effects as well as a synergistic effect. Both oil and smoke harmed the yeast, however oil created a shielding affect at high concentrations on smoke stressed yeast.

LIMITATIONS/EXTENSIONS

Limitations Lag time between smoke exposures could

have created unwanted variance Yeast should have been exposed more

immediately after platingExtensions More replicates More exposure times and concentrations of

oil A more sophisticated smoke chamber

REFERENCES

[http://atvb.ahajournals.org/content/21/6/1029.long]

[http://www.ncbi.nlm.nih.gov/pubmed/10618003]

[http://cancergrace.org/lung/2007/11/27/cooking-oil-fumes-and-lcins/]

SINGLE FACTOR ANOVA FOR SMOKE EFFECTS

Groups Count Sum Average Variance

Column 1 6 481 80.16667 47.76667

Column 2 6 455 75.83333 52.56667

Column 3 6 425 70.83333 42.56667

Column 4 6 381 63.5 25.5

ANOVASource of Variation SS df MS F P-value F critBetween Groups

921.8333 3 307.2778 7.29876 0.00171

3.098391

Within Groups 842 20 42.1

Total1763.83

3 23

SINGLE FACTOR ANOVA FOR OLIVE OIL EFFECTS

Groups Count Sum Average Variance

Column 1 6 481 80.16667 47.76667

Column 2 6 581 96.83333 282.5667

Column 3 6 482 80.33333 223.4667

Column 4 6 278 46.33333 9.866667

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

Between Groups 8101.5 3 2700.5 19.16381 4.24E-06 3.098391

Within Groups 2818.333 20 140.9167

Total 10919.83 23

2 FACTOR ANOVA SUMMARY

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

Sample 14810.86 3 4936.955 63.92777 3.46E-21 2.718785

Columns 1598.615 3 532.8715 6.90006 0.000343 2.718785

Interaction 1677.594 9 186.3993 2.413652 0.017711 1.999115

Within 6178.167 80 77.22708