1. Mineral Oil Effects on Yeast Mutagenesis
Luke Giannetta
Second Year in PJAS
Central Catholic High School
10th Grade

2. Mineral Oil
Unrefined mineral oil is a known carcinogen (American Cancer Society)
Studies by: International Agency for Research on Cancer National Toxicology Program
Principal Ingredients
D-alpha tocopherol (Vitamin E)
Liquid Paraffin

3. ?
Question
Does commercially sold mineral oil affect the mutagenic rate of yeast significantly?

4. Liquid Paraffin
General formula CnC2n+2H
Paraffin isomers

5. Cell Model Saccharomyces cerevisiae Commonly used model
Tolerant and safe to culture
Has similar reproduction, metabolism, and chemistry as other more advanced eukaryotic cells
(-) Lys Special strain unable to produce lysine

6. Lysine Lysine codons AAA, AAG
(-) lysine yeast mutants used in research
Lys 2 mutants are missing an enzyme function within the lysine biosynthesis pathway
Result: Cells require lysine supplementation

7. Ames Test Developed by Bruce Ames (c. 1970)
Tests for mutagenic/anti-mutagenic properties of various chemicals
Used a (-)-histidine mutant Salmonella (single-point substitution) Bacteria cannot synthesize histidine due to mutation
Exposure to suspected mutagen correlated with increased reversion (mutation) rate
Visible colonies appearing on complete (-)Histidine media  evidence of mutation through reversion
Obviously, a lower limit on mutation
Assayed only 1 DNA site in genome

8. Ames Test
-Lys yeast
Lys+

9. Modified Ames Test
The number of reverted colonies of yeast can be correlated with the rate of mutation.
A reversion at that point
can result in a reversion
back to wild type yeast (lys +)

10. Ultraviolet (UV) Rays Short-wavelength electromagnetic waves
greater energy than visible light
wavelengths range from 400nm to 10nm
Sun’s UV rays absorbed by ozone layer
Causes direct DNA damage (mutagen)

11. Purpose
To determine the effects of mineral oil (suspected mutagen) on the mutagenesis rate of (-) Lys yeast
To determine the effects of UV light (known mutagen) on the mutagenesis of (-) Lys and the survivorship of wild-type yeast

12. Hypotheses
Null Hypothesis Mineral oil will not have a significant effect on the mutagenesis rate of yeast.
Alternate Hypothesis Mineral oil will have a significant effect on the mutagenesis rate of yeast.

13. Materials
(-) Lysine agar plates 1% yeast nitrogen base w/o amino acids 2% glucose 1 mM amino acid mix 1.5% agar
YEPD plates
UV light hood (LD-50 on yeast is 30 s)
Sterile dilution fluid (SDF) 10 mM KH2PO4, 1 mM MgSO4 1 mM CaCl2, 100 mM NaCl
SDF Test Tubes
Sterile pipette tips, microplates
Vortex
Side-arm flask
Spreader bar
Ethanol
Micro burner
(-) Lysine Saccharomyces cerevisiae
Wild type (+) Lys Saccharomyces cerevisae
Rubber gloves
Test tubes
Microtubes
Test tube rack
Rite Aid ® Mineral Oil

14. Procedure
Strain of yeast (-) Lys phenotype grown for 2 days in YEPD media.
Sterile yeast pellet washed with SDF to remove any residual nutrients (lysine)
Stock re-suspended and stored in com. (-) Lys media for 2 days
A 100% stock and a 10% sub-stock of the mineral oil were made by diluting the variable with sterile water.
The pellet in SDF was re-suspended.

15. Procedure (cont’d)
The following ingredients were pipetted into sterile microtubules
Water
Variable
Yeast
Total Volume 0%
0.8 mL
0 mL
0.2 mL
1 mL
0.1%
0.7 mL
0.1 mL (of 10% substock) 1%
0.79 mL
0.01 mL
10%
0.1 mL

16. Procedure (cont’d)
7. Cells were allowed to sit for 25 mins mL of each tube plated onto 6 complete (-) Lys plates (necessary to show cells that have reverted via mutation to wild-type (+) Lys) 9. Remaining 0.4 mL of each tube was split into two 0.2 mL aliquots and plated onto 2 complete (-) Lys plates 10. Plates were incubated for 5 days at 32 °C 11. Colonies counted and recorded. Each colony assumed to have arisen from a single cell.

17. UV exposure Procedure (-) Lys Yeast Regular Yeast
0.1 mL of suspended (-) Lys cells were plated on each of 12 complete (-) Lys plates
Plates exposed to UV light in groups of three – at 0, 15, 30, and 45 seconds
Plates were incubated for three days at 32 °Celsius
Colonies were counted, each colony assumbed to have arisen from a single cell
Regular Yeast
0.1 mL of suspended Saccharomyces ceravisae cells were plated on each of 12 YEPD plates
Plates were exposed to UV light in groups of three at 0, 15, 30, and 45 seconds
Plates incubated for three days at 32 °Celsius
Colonies were counted, each colony assumed to have arisen from a single cell

18. (-) LYS UV Exposure Results
UV Light Effects on (-) Lys Yeast
P value =
# of Colonies
Exposure Time (s)

19. Dunnett’s Test Results
T-Critical = 3.75
Test
T Value
Interpretation
0 sec vs. 15 sec
4.8805
Significant
0 sec vs. 30 sec
2.6103
Not Significant
0 sec vs. 45 sec
0.7945

20. REGULAR YEAST UV EXPOSURE RESULTS
UV Light Effects on Yeast Survivorship
P-value =
P value =
# of Colonies
Exposure Time (s)

21. Dunnett’s Test Results
T-Critical = 3.75
Test
T Value
Interpretation
0 sec vs. 15 sec
Not Significant
0 sec vs. 30 sec
Significant
0 sec vs. 45 sec

22. Mineral Oil Exposure REsults
Mineral Oil Effect on Mutagenesis
P Value =
# of Colonies
Mineral Oil Concentration

23. Dunnett’s Test Results
T-Critical = 2.88
Test
T Value
Interpretation
0% vs. 0.1%
Not Significant
0% vs. 1%sec
0% vs. 10%

24. Conclusions
None of the concentrations of mineral oil showed ability to significantly affect the rate of yeast mutagenesis
Null Hypothesis was accepted
Alternate Hypothesis was rejected

25. Limitations and Extensions
Slightly out-of-synced plating which leads to slightly different exposure times to mineral oil
Inability to control the exact amount of cells on each plate (minor difference overshadow by massive amount of cells)
Slight positioning differences in UV Oven Inability to account for cell deaths due UV Light

26. Limitations and Extensions
Different model
Reduce lag time with lab assistants
Trypan Blue Assay to account for cell deaths
A future experiment testing mineral oil's effects on mammalian and cancerous cell lines to see if it promotes uncontrollable growth.

27. Sources
inogens/known-and-probable-human-carcinogens
e0ec51acb6422cbb1a4&keytype2=tf_ipsecsha

28. ANOVA Seconds Exposed 15 30 45 Wild Type Yeast 91 74 69 49 80 46 92 5015 30 45
Wild Type Yeast 91 74 69 49 80 46 92 50 39
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1 3
263
Column 2
228 76 12
Column 3
165 55
151
Column 4
133
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
Within Groups 8
Total 11

29. Mineral Oil Effects on Yeast Mutagenesis
Luke Giannetta
Second Year in PJAS
Central Catholic High School
10th Grade