t he e ffects of o live o il a gainst c igarette s moke on y east peter wagstaff
TRANSCRIPT
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